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The nuclear export of DNA topoisomerase iialpha in hematological myeloma cell lines as a function of drug sensitivity

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Title:
The nuclear export of DNA topoisomerase iialpha in hematological myeloma cell lines as a function of drug sensitivity clinical implications and a theoretical approach for overcoming the observed drug resistance
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English
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Engel, Roxane
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
Etoposide
Mitoxantrone
Leptomycin b
Protein trafficking
Cancer
Dissertations, Academic -- Biochemestry and Molecular Biology -- Doctoral -- USF   ( lcsh )
Genre:
government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemotherapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ultimate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemotherapeutic agents that was independent of altered drug transport. We established that cellular drug resistance in these cells is a function of topo IIalpha subcellular localization and further demonstrate that topo IIalpha translocates to the cytoplasm in a cell-density dependent manner.We provide experimental data that supports the nuclear export of topo IIalpha as the most likely event contributing to drug resistance to topoisomerase II inhibitors, which occurs when transformed cells transition from log to plateau cell density. We provided a plausible nuclear export pathway for topo IIalpha, by identifying two Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export receptor, exportin-1. Thus, topo IIalpha is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIalpha. We confirmed that the nuclear export signals identified in topo IIalpha are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIalpha gene. Furthermore we demonstrate that the nuclear export signals can be abolished by site-directed mutagenesis of specific amino acids residues found in the nuclear export signal.Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo IIalpha. The potential implications of a functioning nuclear enzyme located in the cytoplasm of cells and theoretical mechanisms for overcoming the observed drug resistance are considered.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2005.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Roxane Engel.
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Title from PDF of title page.
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Document formatted into pages; contains 291 pages.
General Note:
Includes vita.

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aleph - 001709510
oclc - 68623515
usfldc doi - E14-SFE0001358
usfldc handle - e14.1358
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ABSTRACT: The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemotherapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ultimate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemotherapeutic agents that was independent of altered drug transport. We established that cellular drug resistance in these cells is a function of topo IIalpha subcellular localization and further demonstrate that topo IIalpha translocates to the cytoplasm in a cell-density dependent manner.We provide experimental data that supports the nuclear export of topo IIalpha as the most likely event contributing to drug resistance to topoisomerase II inhibitors, which occurs when transformed cells transition from log to plateau cell density. We provided a plausible nuclear export pathway for topo IIalpha, by identifying two Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export receptor, exportin-1. Thus, topo IIalpha is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIalpha. We confirmed that the nuclear export signals identified in topo IIalpha are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIalpha gene. Furthermore we demonstrate that the nuclear export signals can be abolished by site-directed mutagenesis of specific amino acids residues found in the nuclear export signal.Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo IIalpha. The potential implications of a functioning nuclear enzyme located in the cytoplasm of cells and theoretical mechanisms for overcoming the observed drug resistance are considered.
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The Nuclear Export of DNA Topoisomerase II in Hematological Myeloma Cell Lines as a Function of Drug Sensitivity: Clinical Implications and a Theoretical Approach for Overcoming the Observed Drug Resistance by Roxane Engel A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemist ry and Molecular Biology College of Medicine University of South Florida Major Professor: Daniel M. Sullivan, M.D. Duane C. Eichler, Ph.D Kenneth Wright, Ph.D Edward Seto, Ph.D Srikumar Chellappan, Ph.D Date of Approval August 29, 2005 Keywords: etoposide, mitoxantrone, leptom ycin B, protein trafficking, cancer Copyright 2005, Roxane Engel

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Acknowledgments I thank my mentor, Dr. Daniel Sull ivan, for funding my doctoral work and providing me with generous opportunities to present my data at several international scientific meetings. A heartf elt thank-you goes to Jana Gump for all of her teachings, but especially for her help with the soft agar cytotoxicity assays. I thank Dr. Nikola Valkov for his data on the clinical samples, log-pl ateau work, and teaching me everything that I needed to know about microscopy. My sinc ere gratitude goes to the very talented, Jennifer Derderian, for the long hours spent on th e micromanipulator. I also thank Joel Turner for his adept work on the transfecti on experiments. I am appreciative to Dr. Dalton, for his critical reviews of my work, but most of all, for approving the purchase of a second Micromanipulator. I also thank Dr Teresita Muoz and Dr. Richard Jove for the time that they served on my doctoral committee. Dr. Jove's cheerful optimism never waned and his experimental advice was mo st helpful. I thank Dr. Feldhoff for sequencing my peptides. I thank Dr. Gene Ness for his insightful tutorials and for preparing me for the Written Qualifying Exam ination. A warm thank-you goes to Kathy Zahn for her guidance throughout my graduate career and for coordinating my successful graduation. I am grateful to Dr. Nilanjan Ghosh and Brenda Flam for their friendship and support. However, my loving parents, sister and fianc, Alan Durkin, deserve my deepest thanks, for it was their love a nd support that made writing th is dissertation possible. And last but not least, I thank Brutus Enge l Durkin for his unconditional loyalty.

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Note to Reader Note to Reader: The original of this docum ent contains color that is necessary for understanding the data. The or iginal dissertation is on file with the USF library in Tampa, FL.

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i Table of Contents List of Tables................................................................................................................. ...vii List of Figures................................................................................................................ ....ix ABSTRACT.....................................................................................................................xi v Chapter One. Introduction..................................................................................................1 1.1 Cancer Trends and Statistics....................................................................................1 1.2 Discussion Points.................................................................................................... .4 Chapter Two. Molecular Basis of Cancer Pathogenesis.....................................................5 2.1 What is Cancer....................................................................................................... ..5 2.2 Cancer Biology of Multiple Myeloma.....................................................................6 2.3 Conclusion .......................................................................................................... ....8 Chapter Three. Biochemistry of DNA Topoisomerases.....................................................9 3.1 General Introduction to th e DNA Topoisomerase Family of Enzymes...................9 3.2 Classification of Mammalian DNA Topoisomerase..............................................11 3.3 Type IB: Human Mitochondrial DNA Topoisomerase I (Topo Imt)....................14 3.4 Type IB: Human DNA Topoisomerase I (Topo I).................................................15 3.5 Type IA: DNA Topoisomerase III.........................................................................18 3.6 Conclusions to Topoisomerase I............................................................................20 3.7 General Introduct ion to the Type II Enzymes........................................................20 3.8 Type IIA: Similarities between Topo II and Topo II .........................................21 3.9 Differences between DNA Topoisomerase II and Topo II ................................25 3.10 Conclusions......................................................................................................... .31

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ii Chapter Four. DNA Topoisomerase Targeted Cytotoxic Agents.....................................32 4.1 Introduction to Topo Targeting Agents.................................................................32 4.2 Topo I and Topo II Poisons...................................................................................33 4.3 Topo I Suppressors and Topo II Catalytic Inhibitors.............................................34 4.4 DNA Topoisomera se I Targeting Agents .......................................39 4.5 DNA Topoisomerase II Targe ting Agents: Etoposide and Mitoxantrone.............41 4.6 Dual Inhibitors of DNA Topoisomerase................................................................44 4.7 Conclusions.......................................................................................................... ..46 Chapter Five. Non-topoisom erase Interacting Agents......................................................47 5.1 Introduction......................................................................................................... ...47 5.2 Cisplatinum.......................................................................................................... ..48 5.3 Paclitaxel........................................................................................................... .....49 5.4 Cytarabine (Ara-C)................................................................................................51 5.5 Carmustine (BCNU)..............................................................................................52 5.6 Conclusions.......................................................................................................... ..53 Chapter Six. In vitro Mechanisms of Topoisomerase Associated Drug Resistance.........54 6.1 Introduction......................................................................................................... ...54 6.2 Pre-target Even ts: Altered Drug Transport............................................................55 6.3 Pre-target Even ts: Altered Drug Distribution........................................................62 6.4 Pre-target Event: Drug Metabolism.......................................................................63 6.5 Drug-target Events: Altered Qu antity of Topoisomerase I or II Protein...............65 6.6 Drug Target Interactions: Altered Quality of Topoisomerase I or II Protein........66 6.7 Drug Target Interactions: Topoisomerase Gene Mutations...................................69 6.8 Drug Target Interactio ns: Altered Chromatin Structure........................................71 6.9 Post-target Events: DNA Repair............................................................................72 6.10 Post-target Events: Al terations in Cell Cycle Progression..................................74 6.11 Post-target Events: Altered Cell Death Pathways................................................74 6.12 Conclusions......................................................................................................... .75

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iii Chapter Seven. In vivo Mechanisms of Topoisomerase Associated Drug Resistance.....76 7.1 Introduction......................................................................................................... ...76 7.2 Gene Mutations......................................................................................................7 7 7.3 Topoisomerase I and II Gene and Protein Expression...........................................78 7.4 Tumor Microenvironment......................................................................................79 7.5 Conclusions ......................................................................................................... ..81 Chapter Eight. Nuclear-cytopl asmic Trafficking of Proteins...........................................82 8.1 Introduction......................................................................................................... ...82 8.2 The Nuclear Pore Complex....................................................................................82 8.3 Nucleoporins......................................................................................................... .83 8.4 Nuclear and Cytoplasmic Targeting Sequences....................................................84 8.5 Nuclear Localization Signals.................................................................................85 8.6 Nuclear Export Signals..........................................................................................86 8.7 Retention Signals...................................................................................................8 7 8.8 Bi-directional Shuttling Signals.............................................................................89 8.9 Signal-mediated Nuclear Transport.......................................................................89 8.10 Regulating Nuclear-cytoplasmic Transport.........................................................93 8.11 Conclusions......................................................................................................... .94 Chapter Nine. Experimental Objectives and Rationale....................................................96 Chapter Ten. Materials and Methods................................................................................98 10.1 Materials........................................................................................................... ...98 10.2 Cell Culture........................................................................................................ ..98 10.3 Log, Plateau, and A ccelerated-plateau Cell Model...........................................100 10.4 Bone Marrow Samples.......................................................................................101 10.5 Clonogenic Cytotoxicity Assays........................................................................101 10.6 Flow-cytometric Cell Cycle Analysis................................................................102 10.7 Gel Electrophoresis and Immunodetection........................................................104

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iv 10.8 Nuclear-cytoplasmic Separation........................................................................105 10.9 Immunofluorescence Microscopy......................................................................106 10.10 Quantitative Measuremen t of the Immunofluorescence of Topo II ..............107 10.11 Equilibrium concentrations of [3H]-VP-16......................................................107 10.12 Band Depletion Assay......................................................................................108 10.13 Comet Assay....................................................................................................109 10.14 Putative NES Peptides.....................................................................................110 10.15 Generation of BSA-Peptide-FITC Conjugates................................................111 10.16 Microinjection..................................................................................................114 10.17 Topoisomerase II Cloning and Site Directed Mutagenesis...........................115 10.18 Transfection Protocol.......................................................................................116 10.19 Immunofluorescence of FLAG-Topo II ........................................................117 10.20 Western Blot of FLAG-Topo II .....................................................................117 Chapter Eleven. Experimental Results:..........................................................................119 Part I: Cell density-dependent VP-16 Sens itivity of Leukemic Cells is Accompanied by the Translocation of Topo II from the Nucleus to the Cytoplasm..................................................................................................119 11.1 Preliminary Results.......................................................................................119 11.2 Resistance Phenotype of Plateau-phase Tumor Cell Lines...........................119 11.3 Cell Cycle Distri bution and [3H]-thymid ine Incorporation..........................123 11.4 Cellular Content of Topo in Log and Plateau-phase Cell Lines...................125 11.5 Subcellular Distribution of Topo in Log and Plateau-phase Cell Lines.......128 11.6 Conclusions...................................................................................................1 30 Chapter Twelve. Experimental Results:.........................................................................131 Part II: The Cytoplasmic Trafficking of DNA Topoisomerase II Correlates With Etoposide Resistance in Human Myeloma Cells..................................131 12.1 Introduction................................................................................................... 131 12.2 Accelerated-plateau Human Myeloma Cell Line Model..............................131 12.3 Drug Sensitivity of Hu man Myeloma Cell Lines in Accelerated-plateau....133 12.4 Cell-cycle Analysis.......................................................................................136

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v 12.5 Cellular Amount of Topoisomerase by Immunoblotting..............................137 12.6 Drug Transport in Tumor Cell Lines............................................................139 12.7 Subcellula r Distribution of Topoisomerase II ............................................140 12.8 Topoisomerase II Enzyme Activity..............................................................146 12.9 Subce llular Distribu tion of Topo II in Malignant Plasma Cells.................152 12.10 Conclusions.................................................................................................15 3 Chapter Thirteen. Experimental Results.........................................................................157 Part III: Human Topoisomerase II Contains Two Leptomycin B Sensitive Nuclear Export Signals.................................................................................157 13.1 Introduction................................................................................................... 157 13.2 LMB Modulation of Topoisomerase II Trafficking...................................158 13.3 Determination of Consensus Sequence for Nuclear Export Signals.............162 13.4 Transfection of Topo II and Alternative Experiments................................162 13.5 Peptides NES1054-1066 and NES1017-1028 Signal the Nuclear Export of BSA-FITC.................................................................................................1 74 13.6 LMB Blocks ntNES1054-1066 and ntNES1017-1028 Mediated Nuclear Export............................................................................................... 176 13.7 Topoisomerase II Cloning, Site Directed Mutagenesis, and Gene Expression............................................................................................17 8 13.8 FLAG-topoisomerase II Immunofluorescence...........................................179 13.9 Peptide ntNES1054-1066 and ntNES1017-1028 are Conserved..................184 13.10 NES1054-1066 and NES1017-1028 Reside within a Putative Coiled-coil Domain.....................................................................................187 13.11 Conclusions.................................................................................................18 8 Chapter Fourteen. Discussion.........................................................................................192 14.1 Previous Findings..............................................................................................192 14.2 Log and Plateau Cell Lines Drug Sensitivity Phenotype...................................192 14.3 Novel Human Myeloma Cell Line Models........................................................193 14.4 Log and Accelerated-plateau Drug Sensitivity Phenotype................................195

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vi 14.5 Subcellular Distribution of Topo II .................................................................197 14.6 Clinical Relevance.............................................................................................201 14.7 Topoisomerase II as an Alternative Molecula r Target in Human Myeloma...203 14.8 Nuclear Content of Topo II is a Determinate of Topo II Drug Cytotoxicity...205 14.9 Possible Roles of Cytoplasmic Topo II ...........................................................207 14.10 Topoisomerase II Contains Two Functional Nuclear Export Signals...........208 14.11 Nuclear Transport and Oncogenesis.................................................................211 Chapter Fifteen. Future Considerations..........................................................................214 15.1 Phosphorylation.................................................................................................214 15.2 Calmodulin Dependent Kinases.........................................................................216 Chapter Sixteen. Closing Remarks.................................................................................220 List of References............................................................................................................2 21 Appendices..................................................................................................................... ..266 Appendix A. The Complete Amino Acid Sequence for DNA Topoisomerase I...................................................................................2 67 Appendix B. The Complete Amino Acid Sequence for DNA Topoisomerase II ...............................................................................268 Appendix C. Gene Rearrangeme nts in Hematological Malignancies...............269-272 About the Author...................................................................................................End Page

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vii List of Tables Table 1. Characteristics of Human DNA Topoisomerase I, II, and III.............................13 Table 2. DNA Topoisomerase II Protein Interactions..............................................29-30 Table 3. DNA Topoisomerase I and II Inhibitors.............................................................35 Table 4. Comparison of DNA Topoisomerase I nhibitors and Non-Topoisomerase Targeting Anticancer Agents...............................................................................50 Table 5. Drug Transporters and Chemotherapy Substrates..............................................59 Table 6. Nuclear Export Receptors....................................................................................93 Table 7. Drug Resistance of Plateau Phas e Non-transformed and Tumor Cell Lines....122 Table 8. Analysis of S-phase and [3H]-thymidine Incorporation in Log and Plateau Phase Cell Lines............................................................................................... 124 Table 9. Drug Sensitivity of Log and Accel erated-plateau H929 Cells to Cytotoxic Agents......................................................................................................... .....135 Table 10. Determination of a Consensus Sequence from Established Nuclear Export Signals (NES)................................................................................................. .161 Table 11. Putative Nuclear Export Signa ls in Human DNA Topoisomerase II ............164

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viii Table 12. Wild-type and Mutated Nucleotide Sequences of Putative Nuclear Export Signals....................................................................................................... ......181 Table 13. Sequence Alignment of DNA Topoisomerase II NES1017-1028......................185 Table 14. Sequence Alignment of DNA Topoisomerase II NES1054-1066......................186 Table 15. Inhibitors of the Nuclear-cytoplasmic Transport Pathway.............................210 Table 16. Nucleoporin (NUP) Gene R earrangements Associated with Hematological Malignancies...........................................................................213 Table 17. Phosphorylation Sites in DNA Topoisomerase II Established In vitro ........214 Table 18. Predicted Phosphorylatio n Sites in DNA Topoisomerase II ........................220 Table 19. Oncogenes and Fusion Proteins in Hematological Malignancies.................269

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ix List of Figures Figure 1. Topological Conf igurations in DNA that are Resolved by Topo......................10 Figure 2. Structural Domains of Human DNA Topoisomerase I.....................................15 Figure 3. The Catalytic Cycl e of DNA Topoisomerase I.................................................17 Figure 4. The Structural Domains Shared by DNA Topo II and Topo II .....................22 Figure 5. The Catalytic Cycl e of DNA Topoisomerase II................................................23 Figure 6. The Mechanis m of DNA Topo I Poisons..........................................................37 Figure 7. The Mechanism of DNA Topo II Poisons and Catalytic Inhibitors..................38 Figure 8. Camptothecin (CPT)..........................................................................................39 Figure 9. Topotecan (TPT)...............................................................................................39 Figure 10. Podophyllum peltatum also known as the May Apple Mandrake..................41 Figure 11. Etoposide (VP-16)...........................................................................................42 Figure 12. Mitoxantrone (MTX).......................................................................................43

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x Figure 13. Paclitaxel (Taxol), Cytarabine (Ara-c), Cisp latinum (CDDP), Carmustine (BCNU)....................................................................................................... .....49 Figure 14. Alterations in Pr e-target Events Associat ed with Drug Resistance.................56 Figure 15. Altered Drug-target Interactions and Post-target Interactions Associated with Drug Resistance.......................................................................................57 Figure 16. Cytoplasmic Retention Signa ls and Nuclear Export Sequences for Cyclin B............................................................................................... .....89 Figure 17. Nuclear Import Pathway..................................................................................91 Figure 18. Nuclear Export Pathway..................................................................................92 Figure 19. Mechanisms of Nuclear-c ytoplasmic Transport Regulation...........................95 Figure 20. Sulfosuccinimidyl 4-[N-malei midomethyl] cycloh exane-1-carboxylate (Sulfo-SMCC)...............................................................................................1 10 Figure 21. An Illustration of BSA-FITC Peptide Complexes.........................................112 Figure 22. Eppendorf FemptoJet and Micromanipulator on Nikon TE 200 Inverted Microscope.....................................................................................114 Figure 23. Western Blot Analysis of Topo I, Topo II and Topo II from Whole Cell Lysates of Cell Lines at Log and Plateau Densities.......................................127 Figure 24. Immunofluorescent Stai ning and Confocal Microscopy...............................129 Figure 25. Growth Curves...............................................................................................132 Figure 26. Cell-cycle Analysis of L og and Accelerated-plateau H929 Cells.................136

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xi Figure 27. Western blot Analysis of DNA Topoisomerase I and II...............................138 Figure 28. The Subcellular Distribution of Topo II in Log and Accel erated-plateau H929 cells at 16 h and 24 h............................................................................141 Figure 29. The Subcellular Distri bution of DNA, Topo I, and Topo II in Log and Accelerated-plateau H929 cells at 24 h..........................................................142 Figure 30. Method for Selecting Cells by Confocal Microscopy...................................143 Figure 31. Determination of Nu clear-cytoplasmic Ratios (N/C)....................................144 Figure 32. Nuclear-cytoplasmic Separati on and Western blot Analysis of DNA Topoisomerase I and II and LDH...................................................................146 Figure 33. Determination of VP-1 6 Induced Double-strand DNA Breaks.....................148 Figure 34. Illustration of Band Depletion Assay............................................................149 Figure 35. Measurement of Cleavable Complexes.........................................................151 Figure 36. Subcellular Distri bution of Topoisomerase II in Plasma Cells from One Untreated Multiple Myeloma Patient.............................................................155 Figure 37. Western Blot Analys es of Topoisomerase I and II in Two Patients with Multiple Myeloma being Treated with Chemotherapy..................................155 Figure 38. Subcellular Distribution of T opoisomerase in Two Multiple Myeloma Patients Being Treated with Chemotherapy..................................................156 Figure 39. Confocal Microscopy and Nuclear-Cytoplasmic Ratios of DNA Topoisomerase II Immunofluorescent Staining in the Presence or Absence of Leptomycin B............................................................................................16 0

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xii Figure 40. Coomassie Blue Stain of SD S Page gel with NES-peptide BSA Conjugates.................................................................................................. ...168 Figure 41. Absorbance Spectra of a BSA NES-peptide Conjugate at 562 nm...............169 Figure 42. FITC Labeled BSA NES-pe ptide on Size Exclusion Column......................169 Figure 43. Silver-stain Analyses of SD S-Page NES Peptide-BSA FPLC Fractions......170 Figure 44. Silver-stain Analyses of SDS-Page Each NES Peptide-BSA FPLC Fractions......................................................................................171-172 Figure 45. Comparison of H929 Cells and HeLa Cells During Microinjection.............173 Figure 46. HeLa Cells Mi croinjected with Peptid e-BSA-FITC Conjugates..................175 Figure 47. HeLa Ce lls Microinjected with TRITC-BSA................................................176 Figure 48. HeLa Cells were microinjected with wild -type peptide-BSA-FITC conjugates in the presence of 2 ng/ml LMB (leptomycin B).........................177 Figure 49. Western Blot of Full-length FLAG-topo II ..................................................179 Figure 50. FLAG-topo II Immunofluorescence............................................................182 Figure 51. Quantitation of FLAG-topo II Immunofluorescence...................................183 Figure 52. The Complete Amino Acid Sequence of Human DNA Topo II (accession number NP 0010508).....................................................................................189 Figure 53. Predicted Tertiary Structure of Wild-type and Mutant Peptides............190-191

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xiii Figure 54. The Complete Amino Acid Sequence for Human DNA Topoisomerase I (accession number NP 003277).....................................................................267 Figure 55. The Complete Amino Acid Sequence for Human DNA Topoisomerase II (accession number NP 001059).....................................................................268

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xiv The Nuclear Export of DNA Topoisomerase II as a Function of Drug Resistance in Human Myeloma Cell Lines: Clinical Implications and Mechanisms for Overcoming Drug Resistance Roxane Engel ABSTRACT The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemothe rapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ul timate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemot herapeutic agents that was independent of altered drug transport. We established th at cellular drug resistan ce in these cells is a function of topo II subcellular localiza tion and further demonstrate that topo II translocates to the cytoplasm in a celldensity dependent manner. We provide experimental data that supports the nuclear export of topo II as the most likely event contributing to drug resistance to topoisom erase II inhibitors, which occurs when transformed cells transition from log to pl ateau cell density. We provided a plausible nuclear export pathway for topo II by identifying two

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xv Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export rece ptor, exportin-1. Thus, topo II is likely to be exported from the nucleus at plateau ce ll densities when exportin-1 binds topo II We confirmed that the nuclear expor t signals identified in topo II are functional when expressed in human myeloma cells transf ected with an epitope-tagged topo II gene. Furthermore we demonstrate that the nuclear export signals can be abolished by sitedirected mutagenesis of specific amino acids residues found in the nuclear export signal. Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo II The potential implications of a functioning nuclear enzy me located in the cytoplasm of cells and theoretical m echanisms for overcoming the observed drug resistance are considered.

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1 Chapter One Introduction 1.1 Cancer Trends and Statistics. Cancer is the second leading cause of death in the United States, exceeded only by heart disease (American Cancer Soci ety, 2005). Increased life expectancy, environmental factors, genetics, and socioeconomic issues can all contribute to the emergence of cancer around the world (K nudson, 2002; Doll, 1996). According to the American Cancer Society (ACS), approxi mately 1,368,030 new cancer cases will have been diagnosed in the year 2004 and 563,700 Americans are exp ected to have died of cancer, more than 1500 people per day. The AC S anticipates the trend in further cancer cases to continue for the year 2005. The Nati onal Institutes of Health also estimate that medically related cancer costs will exceed the initial 2004 projection of 200 billion dollars (National Institute of Health, 2005). Preventive measures have been insufficient at reducing the incidence of cancer, thus driv ing numerous medical researchers to seek new drugs and drug combinations for more eff ective cancer treatment. The sum of these investigations has lead to the development of new treatme nt strategies, promising anticancer agents, and improved quality of life for many cancer patients. Consider, for instance, the recent advancements in the cl inical management of multiple myeloma, a mostly incurable hematologic malignancy (M organ and Davies, 2005; Rajkumar et al.,

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2 2002). For example, the administration of me lphalan plus prednisone has been the conventional chemotherapy given for the trea tment of multiple myeloma (Rajkuman et al., 2002). The overall response rate with th is regimen is approximately 50%, but the complete response rate is less than 10% and the median surviv al is approximately 3 years (Rajkumar et al., 2002). High-dose th erapy followed by autologous stem cell transplantation is a newer treatment st rategy for multiple myeloma with improved response rates. Response rates with this strategy exceed 75% to 90% and complete response rates range from 20% to 40% (R ajkuman et al., 2002). Despite these improvements however, multiple myeloma remains to be a mostly incurable plasma cell malignancy with a median survival of 34 years (Terpos et al., 2005). Even when conventional therapy or high-dose chemothera py is administered with autologous stem cell transplantation, myeloma pa tients continue to relapse (Rajkuman et al., 2002). These findings can be attributed in large part to unresponsive or drug resistant cellular phenotypes at the time of diagnosis or relapse (Yang et al., 2003). In these instances, a detailed understanding of the mechanisms of drug action and a rationale for their effective application is required before eff ective cancer therapy can be delivered. Thus, medical and scientific investigators have combined efforts to analyze the molecular basis of cellular drug resistance in the laboratory to improve clinical outcome, often referred to as translational or “bench-to-bedside” medi cine (Horousseau et al ., 2004; Goessel, 2003; Fishman and Sullivan, 2001). A group of enzymes that regulate DNA topology, called DNA topoisomerases (topos), are at the center of many of the translational studies that are addressing cellular drug resistance in hematological malignancies.

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3 DNA topoisomerases are a key subject in cancer research because these proteins are the molecular targets of some of the mo st commonly used chemotherapy agents in the treatment of human cancers. Topo targeted agents have b een used in the effective treatment of leukemias, lymphomas, multiple myeloma, breast cancer, and other malignancies. However, their clinical eff ectiveness is often limited by acquired (after drug exposure) or intrinsic cellular drug (befor e drug exposure) resistance, especially in the treatment of multiple myeloma (Valkov a nd Sullivan, 1997). Thus, researchers are investigating acquired and intrin sic mechanisms of drug resist ance at the molecular level, to circumvent drug resistance obs erved in myeloma patients. The focus of this investigation is about DNA topoisomerases, their roles as molecular targets of chemotherapy, and mech anisms of cellular drug resistance to topoisomerase targeting agents. The ultimate goal of this investig ation was to identify mechanism(s) of drug resistance so that a st rategy to overcome drug resistance could be conceived. We established an in vitro cell model that demonstrated de novo drug resistance to commonly used chemotherapeutic agents. We determined that the observed drug resistance was specific to topoisomerase targeting agents. We provide experimental data that supports the nuclear export of topo II as the most likely event contributing to drug resistance in our cell model. We establ ished a plausible nuclear export pathway for topo II by identifying two Leptomycin B sensit ive nuclear export signals, and then demonstrate that these signals are functional in human myeloma cells transfected with a FLAG tagged topo II gene. Hematological cell lines are used throughout this discussion as a cell model for elucidating the role that topoisomerases impart on intrinsic cellular drug resistance to anti-cancer agents.

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4 1.2 Discussion Points. To appreciate the mechanisms of cellu lar drug resistance to topo targeting cytotoxic agents, several concep ts are reviewed. First, the fundamental concepts for the molecular basis of cancer pathogenesis are c overed in chapter two followed by a review of human DNA topoisomerases, including but not limited to, their structure, function, and mechanisms of catalysis. Chapters four and five depicts the physical biochemistry of commonly used anti-cancer agents with particular attention to cytotoxic agents that target DNA topoisomerase I and II. These chapters de tail how specific anti -cancer agents exert their cytotoxic effects, a subject that is necessary to realize how cells evade drug cytotoxicity. Chapters six and seve n cover our current understanding of in vitro and in vivo mechanisms of de novo cellular drug resistance related to multiple myeloma. These chapters illustrate many of th e challenges that clinicians must overcome for therapy to become more effective. The mechanisms of nuclear-cytoplasmic transport of proteins are discussed in chapter eight because the subcel lular distribution of proteins between the nuclear and cytoplasmic compartments ha s recently been established as another mechanism of regulating protein function. Fi nally, the nuclear-cytopl asmic transport of topo is revealed as a novel mechanism of cellular drug resistance in human myeloma cell lines in chapters eleven through thirteen. The significance of these findings in the clinical environment is assessed and future projections are deliberated.

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5 Chapter Two Molecular Basis of Cancer Pathogenesis 2.1 What is Cancer? Cancer is not a single di sease but rather a group of di seases characterized by the proliferation of cells that bypass regulatory checkpoints of the cell-cycle and/or escape induction of apoptosis (reviewed in Bocche tta and Carbone, 2004; Bertram, 2000). In general, the proteins responsible for main taining DNA and the cell cycle can be divided into six categories that include: (1) cytokines/growth factors; (2) growth factor receptors; (3) intracellular signaling molecules; (4) transcription factors; (5) cell-cycle/apoptotic regulatory proteins; and (6) proteins that re gulate DNA topology and metabolism (i.e., DNA topoisomerases). There is a high degree of cross-talk between proteins of the same class and between different classes of pr oteins, resulting in a network of highly synchronized events often referred to as a signal transduction cascad e. Malignant cells usually acquire DNA mutations in one or mo re of these cell cycle or DNA regulatory proteins, which gives them growth or prolif erative advantages ove r other cells. Since proteins function as part of a coordinated ne twork with other protei ns, even a single gene mutation can result in the propagation of additional gene mutations (i.e., if a gene mutation occurs in a DNA repair protein) or proliferative advantages (i.e., if a gene mutation occurs in a growth factor recepto r). These genetic a dvantages disrupt the

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6 balance between cell proliferation and cell d eath. For example, the retinoblastoma protein (pRb) is encoded by the RB tumor suppr essor gene and regulat es the cell cycle by arresting DNA replication when DNA damage is present (Sellers, 1997). Mutations in the RB gene occur in 30 to 40% of all human cancers, thus permitting cells with DNA damage to divide endlessly (Merck, 2004). In addition to genetic mutations, tumor cells can also influence their microenvironment to favor their survival. For example, tumor cells secrete or promote the secretion of cytokines and growth factors to stimulate angiogenesis (Sivridis et al., 2003). Th e new blood vessels supply nutrients and additional cytokines that cont inue to activate cell signaling pathways that further tumor sustenance. To better illustrate some of the points described above, let us take a closer look at one type of cance r, multiple myeloma. 2.2 Cancer Biology of Multiple Myeloma: Multiple myeloma is a type of B-cell neoplasm characterized by the slow proliferation of well-differentiated plasma cells in the bone marrow (Bhawna, 2003; Ho et al., 2002). Although mature plasma cells are the predominant t ype of cell found in multiple myeloma, pre-B, myeloid and T-cells are coexpressed with the mature plasma cell (Barlogie, 1989). The populat ion of malignant cells is collectively referred to as myeloma cells. Multiple myeloma tumor progression is a multistep process that can be described to occur in three distinct phases: immortalization, unch ecked cell proliferation, and dissemination of the malignant plasma cells (Pratt, 2002; Shain et al., 2000). The disease may first present as a monoclonal gammopat hy of undetermined significance (MGUS), a

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7 clinical term used to describe a single plasma cell that has formed a limited number of clones (Tricot, 2002; Dalton et al., 2001). The precise cellular origin is not well understood, but is believed to be an immature Bcell that has been repeatedly exposed to antigen in the germinal centers of the lym phoid tissue. The disease advances when the transformed cells clonally expand and gr ow in the bone marrow (intramedullary myeloma) (Van Riet et al., 1998). During this time, the disease is usually chemosensitive and may enter a dormant phase (Pratt, 2002). The myeloma cells receive growth and anti-apoptotic signals that f acilitate their survival thr ough interactions with the bone marrow microenvironment (Dalton, 2003; Shain, et al., 2000). For example, expression of the adhesion receptors, very late antigen-4 (VLA-4) and very late antigen-5 (VLA-5) (Hata et al., 1993), permits myeloma cells to bi nd fibronectin, whereas the expression of the 2 integrin lymphocyte function associated antigen-1 allows for cell-cell aggregation (Asosingh et al., 2003; Kawano et al., 1991). Such interactions stimul ate the secretion of various cytokines from myeloma and stromal cells in the bone ma rrow microenvironment (Klein et al., 2003). IL-6, the main plas ma cell activating factor, stimulates cell proliferation via the Ras/Mitogen-activate d kinase (Ras/MAPK) signaling cascade (Kawano et al., 1988; Ogata et al., 1997). IL -6 also induces myeloma cell migration and angiogenesis via vascular epit helial growth factor (VEG F) induction (Dankbar et al., 2000). Advanced disease is distinguished by i ndependence from growth factors in the bone marrow microenvironment and proliferation to extramedullary sites (extramedullary myeloma) (Pratt, 2002). Throughout this time, advanced myeloma is characterized by resistance to apoptosis and chemotoxic ag ents (Dalton, 2003). Understanding the bone marrow microenvironment in multiple myeloma disease pathogenesis has had a role in

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8 the development of new cytotoxic agents. For example, bortezomib is the first proteasome inhibitor to be evaluated in clinic al trials for the treatment of hematological malignancies (Adams, 2004; Adams and Kauffman, 2004). 2.3 Conclusions. In summary, cancer is a multifaceted di sease that is propagated by multiple DNA mutations that are acquired vi a hereditary and natural means. The gene mutations can result in altered cellular homeostasis and conf er growth and prolifer ative advantages. In turn, the collection of protei ns that are involved in re gulating the cel l cycle and DNA replication/topology are the fre quent targets of anti-cancer drugs (Damiens, 2000). Thus, understanding the proteins involved in the intracellular cell signaling pathways, DNA replication/topology, and cell cycl e regulation are the cornerst ones of cancer research. The focus of this investigation is about DNA topoisomerases, enzymes that regulate DNA topology. We focus on the ro le of DNA topoisomerases as molecular targets of anti-cancer agents and mechanisms of drug resistance. Although the complete family of human DNA topoisomerases is review ed, the focus of this work centers around type II enzymes, topo II and topo II the current targets of cancer chemotherapy.

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9 Chapter Three Biochemistry of DNA Topoisomerases 3.1 General Introduction to the DNA Topoisomerase Family of Enzymes. The human genome consists of approximate ly 3.2 billion base pairs and would be about 2 meters in length, if stretched out from end to end (Porter et al., 2004). Nevertheless, DNA squeezes into the nucleus, wh ich is approximately 6 M in diameter (Porter et al., 2004). For example, the sma llest human chromosome is 14 mm long but is compacted to just 2 M in length (Ball, 2003; Greeley, 1978). Thus, DNA exists in a tightly compacted and dense form. Conversel y, DNA must also allow itself to become accessible to numerous transcription factor s and regulatory proteins to allow gene transcription and DNA replica tion to occur (Khorasanizade h, 2004). The ability of DNA strands to separate and/or unwind, however, is limited by the torsional constraints of a double helix. DNA topoisomerases are a fam ily of nuclear enzymes that regulate the topological state and relieve the torsional stress of the DNA that arise during gene transcription, DNA replication, and ce ll division (Champoux, 2001; Wang, 1996). DNA can become tangled into knots, supercoiled, a nd form interlocked circles that must be resolved by topo action (Figure 1) (Pu lleyblank, 1997). To regulate DNA topology, all topo enzymes introduce singleor double stra nd breaks in DNA to allow another strand of DNA or duplex DNA to pass through th e newly generated gap (Champoux, 2001). Topo activity has been shown to be impor tant during DNA replication, transcription,

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10 genetic recombination, sister chromatid se gregation, and chromosome condensation and decondensation. For example, the human type I and II topoisomerases relax positive supercoils that are generated in front of the moving replicat ion fork. This is significant because RNA polymerase generates tension in the DNA that must be relieved for gene transcription to occur. Topo II also has an essential role in the proper execution of DNA replication and cell division. Newly synt hesized DNA strands must be completely unlinked from the parent strand and every rep licated chromosome must be disentangled from each other to allow proper segregation of the chromosomes into daughter cells. "Molecular Houdini's" and en zymes with a "double-edged sword" are two terms that are used to describe DNA topoisome rases because the integrity of DNA is susceptible to topo cleavage activity (Osh eroff, 1998; Anderson and Berger, 1994). To ensure the integrity of DNA, all DNA break s generated by topo are accompanied by the Figure 1. Topological Confi gurations in DNA that are Resolved by Topo. (a) Twisting of DNA about its axis results in either positive or negatively supercoiled DNA, (b) DNA knots, and (c) catenated DNA is when two or more circles of DNA interlock. Although the family of topoisomerase enzymes are required for DNA replication, recombination, transcrip tion and chromosome segregation, each type of enzyme makes different contribu tions to each process. For example, human topo I and II are able to relax supercoiled DNA, but only topo II is proficient knotting/unknotting and catenati on/decatenation reactions. This figure was re p roduced from reference Pulle y blank 1997. a. Supercoiled b. Knotted c. Catenated DNA

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11 formation of a covalent phosphotyrosine bond th at tethers topo to the broken strand of DNA while DNA strand passage occurs (Tse et al., 1980). The covalent topo-DNA intermediate, called a "cleavage complex" or a "cleavable complex", is transient and freely reversible (Roca and Wang, 1992). Some anticancer agents exploit the DNA cleavage activity of topo to turn these enzymes into molecular toxins (Nitiss and Wang, 1996). Thus, there has been cons iderable interest in topo be cause they have become the molecular targets of some clinica lly important anticancer agents. In summary, DNA topoisomerase enzyme ac tivity is essential for maintaining the topology of DNA during many DNA processes, including DNA replication, transcription, and chromosome segregation. DNA topoisome rases have also emerged as important molecular targets of anti-cancer agents, esp ecially in hematological malignancies (Wang, 1987; Zijlstra et al., 1990). Th e next topic to be covered de scribes the nomenclature and classification system for the family of DNA topoisomerases. This will be followed by a complete description of each type of human topoisomerases. 3.2 Classification of Mammalian DNA Topoisomerase. To date, six different DNA topoisomerases have been identified in humans and mice (referred to as topo I, topo II, and topo III, etc) and are cl assified as either type I or II enzymes (Champoux, 2001; Wang, 1996). The human type I, type II, and type III topo enzymes have been distinguished by differences in their gene locus, molecular weights, catalytic requirements, mechanism of catalysis and biological role s (table 1) (Champoux, 2001; Wang, 1996). Type I enzymes include topo III, topo I, a nd mitochondrial DNA topo I (topo Imt). All type I enzymes are active as monomers a nd catalyze transient

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12 single strand DNA breaks without ATP. The t ype I enzymes can be further distinguished as IA or IB enzymes based on more specific differences in their reaction mechanisms. For example, the type IA enzymes covalent ly bind to the 5’-end of the cleaved DNA strand while type IB enzymes bind to the 3’-end of the cleaved DNA strand. In contrast to type I en zymes, type II enzymes form dimers and couple doublestrand DNA breaks with ATP hydrolysis. Type II enzymes have two known isoforms referred to as topo II and topo II Several monoclonal and polyclonal antibodies have been used to detect the different enzyme s by Western blot and immunohistochemistry. Monoclonal antibodies are available for human topo I (Beidler and Cheng, 1995), whereas several monoclonal (Kellner et al ., 1997; Negri et al., 1993) and polyclonal antibodies are available for human topo II (Sullivan et al., 1993) and topo II (Austin et al., 1995). One monoclonal antibody ha s been used to detect topo III expression by immunohistochemistry (Lodge, 2000). A review covering human type I and type II enzymes ensues. The type I enzymes (i.e., topo Imt, topo I, and t opo III) are discussed first, fo llowed by a discussion of the type II enzymes (topo II and topo II ). The type II enzymes are the focus of the work presented here and thus, are discussed in greater detail than the type I enzymes.

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13Table 1. Characteristics of Human DNA Topoisomerases I, II, and III* Topo I Topo Imt** Topo II Topo II Topo III Topo III Type IB IB IIA IIA IA IA Chromosome 20q12-13.2 8q24.3 17q21-22 3q24 17p11.2-12 22q11 Molecular Weight (kDa) 91 60-70 170 180 ~110 ~96 mRNA (kb) 4.2 nda 6.2 6.5 4.0-7.2 2.8-3.8 DNA strand cleavage single nda double double single single Covalent Intermediate 3' nda 5' 5' 5' 5' ATP dependence no no yes yes no no Cell cycle dependence no nda yes, maximal in G2/M no nda nda Knockout mouse early embryonic lethal nda presumed lethal perinatal death early embryonic lethal shortened mean life span Active site Tyr-723 predicted Tyr-559 Tyr-805 Tyr-821 predicted Tyr-337 predicted Tyr-336 Location nucleolus nucleoplasm mitochondria nucleoplasm nuclear matrix nucleoplasm nucleolus nucleusd mitochondriad isoform I: nucleoplasmee isoform II: cytoplasmee Intracellular Targeting Signal(s) 1. basic NLSb 2. acidic NLSc 3. nucleolarc 1. mitochondrial* 1. bipartite NLSf 2. LMB sensitive NESg ( in vitro/in vivo ) 1. bipartite NLSf 2. LMB sensitive NESh, ( in vitro, only ) 1. mitochondriald, ( in vitro/in vivo ) nda Inhibitors topotecan, karenitecin, 9-nitrocamptothecin, camptothecin, CPT-11 CPT sensitive; potential molecular target VP-16, VM-26, mitoxant rone, m-AMSA, doxorubicin, merbarone, daunomycin, ICRF-193, ICRF-187, ICRF159, and XK469 (beta specific) potential molecular target potential molecular target

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14 3.3 Type IB: Human Mitochondrial DNA Topoisomerase I (Topo Imt). There are approximately 1000 mitochondria in every mammalian cell (Zhang et al., 2004a). Each human mitochondrion contai ns 5-10 copies of closed circular mitochondrial DNA (mtDNA) that has16, 569 base pairs (Moraes, 2001; Anderson, 1981; Bibb, 1981). Human mtDNA encodes 22 tRNAs, 13mRNAs, 12S and 16S rRNA (Anderson, 1981). DNA topo enzyme activity re solves the topological constraints of DNA within the mitochondria (Wang et al., 2002). The human TOP1mt gene is located on chromosome 8q24.3 and encodes for a 601 amino acid polypeptide that is highly homologous to DNA topo I (Zhang et al., 2001 ). The protein was identified as mitochondrial DNA topo I (topo Imt) because it contains a mitochondrial targeting sequence and lacks a nuclear localization signal (Zhang et al., 2001). Topo Imt also has different biochemical properties from those of the nuclear enzyme, DNA topo I (Zhang et al, 2001). A MegaBlast search of all Nati onal Library of Medicine databases was conducted and indicated that only vertebrate s contain both mitoc hondrial and nuclear topo I gene (Zhang et al., 2004a). These resu lts raise the question of how non-vertebrates perform mitochondrial DNA metabolic functions. It is possible that one gene encodes two polypeptides that are target ed either to the nucleus or to the mitochondria (Zhange et al., 2004a). Current investigations are unde rway to elucidate gene mutations in mitochondrial topo I that could potentiate human disease. Thus, topo Imt could evolve as a new molecular target in the future. Unle ss otherwise noted, this discussion will focus on nuclear DNA topo I, the current molecu lar target of anticancer drugs.

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15 3.4 Type IB: Human DNA Topoisomerase I (Topo I). Eukaryotic topo I was first described in mammalian cells in 1972 (Champoux and Dulbecco, 1972). Although topo I gene knockouts are lethal in mous e models, yeast topo I gene knockouts are viable (Champoux, 2001). The gene for human DNA topo I was cloned in 1988 and the complete amino acid sequence of topo I protein is shown in Appendix A on page 270 of this manuscript (J uan et al., 1988). The structure topo I includes four functional doma ins (Figure 2): a highly charged N-terminal domain, a conserved core domain and an essential C-te rminal domain (Redinbo et al., 1999; Stewart et al, 1996). The C-terminal domain contai ns the active site ty rosine residue (Tyr723) (Eng et al., 1989) and is essent ial for DNA binding and relaxation in vitro, whereas the amino terminal region may have an important role in regulating the cellular localization of topo I because it contains the nuclear lo calization signal (NLS) and nucleolin binding site. Amino acids 150-156 have been shown to be sufficient for nuclear localization 1 215 636 COOH N-terminal domain Core domain Linker domain C-terminal domain Poorly conserved Highly charged Protease sensitive Nuclear Targeting Highly conserved DNA binding Enzyme activity Poorly conserved Dispensable in vitro Conserved Active site Tyr-723 H2N Figure 2. Structural Domains of Human DNA Topoisomerase I. Topo I has four domains: an N-terminal domain ( ) (amino acids 1-215), a core domain ( ) (amino acids 215-636), a linker domain ( ) (amino acids 636-713), and a C-terminal domain ( ) (amino acids 713-765). The most significan t properties are described below each domain. Figure reproduced from reference (Champoux, 2001). 713 765

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16 (D’Arpa et al, 1988), but a second NLS has b een identified within amino acids 117-146 (Mo et al., 2000). Topo I shows a stable ex pression throughout the cell cycle (Heck et al., 1988). Topo I is post-translationally modified by phosphorylation (D’Arpa and Liu, 1995) and ubiquitination (Desai et al., 1997), which may be important for regulating its activity, localization or degrada tion. Topo I has an important role in transcription, rRNA synthesis, DNA replication and transcription initiation and is the specific target for camptothecin (CPT) and its derivatives (topot ecan and CPT-11) (D’Arpa, 1990). DNA topo I catalyzes transient single st rand DNA breaks to relax DNA supercoils (reviewed in Wang, 2002). Topo I is capable of relaxing both negatively and positively supercoiled DNA without the presence of a cofactor, but metal cations such as Mg2+ stimulate topo I activity (Liu and Miller, 1981). Figure 3 illustrates th e catalytic cycle of topo I. To cleave DNA, topo I catalyzes the tr ansesterfication of the active site tyrosine (Tyr-723) to form a 3'-phosphot yrosine covalent enzyme DNA intermediate (D'Arpa et al., 1988). The 3'-phosphotyrosine bond is form ed when the O-4 oxygen of tyrosine at position 723 (Tyr723) completes a nucleophilic attack on the scissi le phosphate in the DNA backbone (D'Arpa e al., 1988). The formation of the 3'-phosphotryosine bond is significant because it forms a "proteinaceous" bridge between topo I and DNA, and thus ensures the integrity of DNA. The covalent t opo-DNA intermediate is also referred to as a "cleavage complex" (Pommier, 2003) or "cl eavable complex" (Tewey et al., 1984).

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17

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18 After DNA unwinding, the reaction reverses wh en the 5' OH of the cleaved DNA strand reattacks the phosphotyrosine re sidue in a secondtransest erfication reaction. Under normal cellular conditions, topo I cleavage complexes are transient and usually undetectable. The steady-state concentra tion of the covalent 3' phosphotyrosine intermediate is kept low becau se the rate of DNA religation is faster than the rate of cleavage (Pommier et al., 2003). However, t opo I cleavage complexes are also produced by other endogenous and exogenous factors, such as UV-induced base modifications, DNA oxidation by oxygen free radicals, mismatch repair deficiencies, DNA alkylating agents, and some anticancer agents (Pom mier., 2003; Pourquier and Pommier, 2001). Such topo I cleavage complexe s can result in DNA damage and cell death, if left unrepaired. The objective of so me cancer chemotherapy, however, is to capture topo I cleavage complexes by administer ing a drug (ie, a DNA topo I inhibitor) that traps topo I on DNA in a cleaved form (Nitiss and Wang, 1996). 3.5 Type IA: DNA Topoisomerase III. The gene for eukaryotic DNA topo III wa s discovered in 1989 (Wallish et al., 1989) and later identified in humans on ch romosome 17p11.2-12 (Hanai et al., 1996). There are two topo III homologues that ar e expressed in mammalian cells, topo III and topo III and each of these has isoforms generated by alternative mRNA splicing (Seki et al., 1998). Human topo III has a putative mitochondrial targeting sequence and localizes to both the nucleolus and mitochond ria (Wang et al, 2002). In contrast, human DNA topo III is found only in the nucleus (Kobaya shi and Hanai, 2001). Both topo III and III can relax negatively supercoiled DNA, but the biological functions of the

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19 different topo III enzymes have not been determined (Goulaouic et al., 1999). Yeast studies demonstrate that Top3 gene mutants grow very slowly, display hyperrecombination and have defects in meiotic recombination (Gangloff et al., 1999). Furthermore, targeted disruption of the mouse topo III gene shows that topo III expression is essential in early embry ogenesis (Li and Wang, 1998). Topo III gene expression occurs in multiple somatic tissues and is differentially regulated during different developmental stages, which is a ssociated with binding of the upstream stimulatory factor-2 (USF-2) to an E-box elem ent located in the topo III gene promoter (Han et al., 2001). The YY1 transcription fact or may also have a role as a cell type specific topo III transcriptional activator (Park et al., 2001). Both topo III and III interact with the RecQ/SGS 1 family of DNA helicases, which has been shown to occur in yeast and Escherichia coli (Kim et al., 1998). It has been suggested that RecQ helicases may func tion to recruit two molecules of topo III to DNA structures during genetic recombination to facilitate the cleav age and passage of double strand DNA (Park et al., 2001). Human topo III is shown to associate with the Bloom syndrome protein (BLM), another memb er of the RecQ family of DNA helicases, in somatic and meiotic human cells (Shima moto et al., 2000). A mutation in the BLM gene is associated with Bloom’ s syndrome, a rare genetic disord er that is associated with hyperrecombination between sister chromatids and increased propensity for cancer (Ellis et al., 1995). An association of human topo III with BLM could be significant in regulating the frequency and fide lity of recombination events by either disrupting nascent joint molecules or by regulating the migration of Holiday structures (H armon et al, 1999). Thus, topo III may have a role as a tumor s uppressor gene, but addi tional investigations

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20 are necessary to further elucidate its role in recombination and suppression of cancer. Furthermore, defining the physiological roles of topo III enzymes could lead to the development of new anti-cancer agents th at specifically target these enzymes. 3.6 Conclusions to Topoisomerase I. In summary, type I enzymes cleave single strand DNA in an ATP independent manner. Topo Imt and topo III are two of the most recent type I enzymes to be identified in humans and these enzymes could be the future molecular targets of anti-cancer drugs. However, topo I is the current molecular target of chemotherapeutic agents. In chapter 4, we will learn more about how drugs, called cam ptothecins, target t opo I and exert their cytotoxic effects in cancer cells. Before that, however, a discussion of the type II enzymes must be covered because these too are the molecular targets of commonly used anti-cancer agents. 3.7 General Introduction to the Type II Enzymes. In the preceding section, we reviewed the type I topoisomerases that are expressed in humans. Now, we will turn our at tention to topo II, a type II enzyme. In humans, topo II has two isoforms, topo II and topo II that share many similar properties. Although many difference in st ructure, tissue distribution, and gene expression have been described for topo II and topo II differences in their functional roles, drug action, and relative contributi ons to cancer chemotherapy are less clear (Austin and Marsh, 1998). Topo II has been the most extensiv ely investigated isoform, whereas the role of topo II is drug resistance is not well understood (Beck et al., 1999).

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21 Immediately following in section 3.8, is a description of common principles and biochemical properties that are ge nerally applied to both topo II and topo II Then, in section 3.9, the features that distingu ish the two isoforms are given. 3.8 Type IIA: Similarities between Topo II and Topo II The type II enzyme, called DNA gyrase, was isolated from Escherichia coli and was the first type II topoisomerase to be char acterized (Gellert et al., 1976). Bacterial DNA gyrase is unique because it can introdu ce negative supercoils in DNA or convert positive supercoils into nega tive supercoils (Wang, 1996). DNA gyrase is the target for clinically important quinolone antimicrobial agents, such as Ciproflaxin (Hooper et al., 1993). However, DNA gyrase should not be confused with mammalian topo II enzymes, the current molecular target s of anticancer agents. In contrast to bacterial DNA gyrase, e ukaryotic topo II is unable to generate supercoils in vitro (Liu et al., 1983). Topo II activity is required to separate replicated chromosomes prior to cell division, and unlike topo I and topo III, is able to decatenate intertwined double strand DNA molecules (Marini et al., 1980). All type II topoisomerases form dimers (Champoux, 2001) and each topo II monomer can be divided into three domains (Jensen et al., 1996) an N-terminal domain that contains the ATP-binding region (Bjerbaek et al., 2000; Ga rdiner et al., 1998), the central domain containing the active site tyrosine residue (Oka da et al., 2000) and the C-terminal domain that contains the nuclear lo calization sequences (Figure 4) (Mirski et al., 1997, 1999). The C-terminal domain has also been shown to be important for interactions with other proteins (Messenger et al., 2002; Kroll, 1997).

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22 All topo II enzymes share common mechanisms for DNA-cleavage and religation reactions (Champoux, 2001; Wang, 1996). A description of the catalytic cycle of topo II is important in understanding how drugs targ et topo II. The cata lytic cycle of topo II involves several distinct st ages including: DNA binding, st rand cleavage, strand passage, religation, and enzyme turnover (Figure 5) (Champoux, 2001; Wang, 1996). Topo II dimers first bind to double strand DNA, and then each monomer cleaves opposite DNA strands to generate a 4-base stagger. Topo II mediated DNA breaks occur through. Figure 4. Structural Doma ins Shared by DNA Topo II (shown in A) and topo II (shown in B). Topo II and topo II have three domains as shown. The ATPase domain (white box) is at the amino termi nus and contains the ATP binding site. The DNA domain (grey box) contains the activ e site Tyr residues and the carboxyl terminus (dark grey box) is important for intracellular localization because it contains the nuclear localization ( NLS ) and nuclear ex p ort si g nals ( NES ) H2N COOH ATPase DNA Binding Carboxyl terminus Active site NLS NES H2N ATPase COOH DNA Binding Carboxyl terminus Active site NES A. Topo II B. Topo II NLS

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23 Mg2+ 1 2 3 4 ATP ADP + Pi 5 6 Figure 5.Catalytic Mechanism of DNA Topo II. Th e "two-gate model" of topo II catalysis is shown. According to this model, topo II has two lobes called A' (colored red) and B' (colored blue). The DNA strand that is cleaved is called the "gate DNA" or "G-segment". The DNA strand that is transported is ca lled the "transport DNA" or "T-segment". (1) Topo II binds gate DNA (orange rod) and undergoes conformational changes. (2) Topo binds ATP (yellow star) and the transport DNA molecule (gre en rod). (3) The ATPase domains on topo II dimerize and topo II cleaves the gate DNA. The presence of a metal cation is required. (4 ) The transport DNA is transported through the break and into the central ca vity of the B' lobe. Gate DNA is held within the A' lobe. (5) Following DNA strand passage th e transport DNA is released from the enzyme through a gate formed in the B' lobe. The gate DNA is resealed. (6) ATP is hydrolyzed and ADP and inorganic phosphate (Pi) are released to complete enzyme turnover. This figure was reproduced from references Champoux, 2001 and Wang, 1996. Cleavable complex Cleavable complex

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24 transesterfication reactions, in which a DNA phosphoester bond is transferred to the active site tyrosine residue in each monomer. Thus, each monomer remains covalently attached to the 5' end of DNA through a phosphotyrosine bond. A conformational change in the enzyme pulls the cleaved DNA duplex apart to create an opening that is called, "the gated or G-segment DNA". Stra nd passage occurs either from the same DNA molecule (relaxation, knotting, or unknotting) or different molecules (catenation/decatenation). The DNA segment th at is passed through the opening is called the "transported or T-segment DNA". En zyme turnover requires ATP hydrolysis and divalent cations (Mg2+) (Osheroff, 1987). The mechanis m of topo II enzyme catalysis is an important concept for unders tanding how topo poisons and ca talytic inhib itors target topo II. Topo II can also catenate /decatenate interlocked circles of DNA (Champoux, 2001). Topo II decatenation activity is essentia l at the end of DNA replication for the proper separation of daughter chromosomes. Cells lacking topo II are unable to decatenate sister chromatids or carry out ch romosome condensation reactions prior to mitosis, which results in non-disjunction a nd chromosome breakage (Holm et al., 1989). Topo II catalytic activity is measured in vitro by the decatenation of kinetoplast DNA (kDNA) isolated from the mitochondria of Crithidia fasiculata. kDNA consists of numerous interlocked minicircle s and maxicircles. Unlike th e type I enzymes, topo II is able to decatenate the interlocked circles to generate free miniand maxicircles that can be separated from the network of kDNA by centrifugation (Sahai and Kaplan, 1986; Marini et al., 1980). Thus, the ability to decatenate kDNA is an important distinction

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25 between topo I and topo II enzymes because it allows us to estimate topo II enzyme activity independent of topo I activity in vitro 3.9 Differences between DNA Topoisomerase II and Topo II Topo II has two isozymes, topo II and topo II with different molecular masses and gene loci (Lang et al., 1998; Drake et al ., 1987) (Table 1, page 13). The presence of two different gene loci suggests that these isoforms may be di fferentially regulated at the level of transcription. The tissue distribution of topo II and topo II mRNA and protein have been determined in mouse, rat, and humans (Bauman et al., 1997). Topo II mRNA is highest in many proliferating cells such as thymus, spleen, bone marrow, intestine, and testis. Topo II mRNA is detectable in a wider ra nge of tissue types; including those with few proliferating cells (C arpanico et al., 1992). Topo II is 90 amino acids longer than topo II and the two isoforms are 68% homologous in amino acids (Austin and Marsh et al., 1998). The greatest vari ability in amino acids between topo II and II occurs in the C-terminal doma in, and thus the C-terminus may impart specific differences in the functional roles of these isozymes. Topo II is expressed in a cell cycle dependent manner with peaks in the S-phase and G2/M-phase of the cell cycle and is the target of ubiquitin-mediated proteolysis (Meyer et al., 1997; Naka jima et al., 1996). Topo II protein expression is more uniform throughout the cell cycle, suggesting that it may have a role as a "housekeeping gene" (Drake et al., 1989). Topo II is also highly expressed in many rapidly proliferating human tumors (Holden et al, 1990, Turl ey, et al, 1997), thus making topo II a frequent molecular target of anti-cancer drugs. In contrast, topo II expression is constant

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26 throughout the cell cycle with limited fluctuations in the amount or stability of the protein in either highly replicating or non-re plicating cells (Woessner, 1991). Topo II is emerging as a new molecular target in tumors that are refractory to cytotoxic agents that target topo II (Gatto and Leo, 2003). Both topo II and topo II are differentially phosphor ylated throughout the cell cycle with maximal phosphor ylation occurring at G2/M phase (Burden et al, 1993; Burden and Sullivan, 1994). Thus, maximum topo II phosphorylation correlates with chromosome condensation and segregation. As cells transition from M phase to G1 phase of the cell cycle, 75% of topo II becomes dephosphorylated, whereas 25% of topo II is dephosphorylated (Wells and Hickson, 1995; Burden and Sullivan, 1994) The kinases known to phosphorylate topo II in vitro include protein kinase C (PKC) (Wells et al., 1995), casein kinase 2 (CK2) (Is hida et al., 1996; Alghisi et al., 1994), and a proline directed kinase (Wells and Hick son, 1995). The phosphor ylation of precise amino acid residues by specific kinase s have not been mapped in topo II The expression of the different topo II is oforms seems to be necessary to fulfill distinct physiological roles, but what those roles are remains unclear (Wang, 1991; Wang, 2002). Both enzymes are able to unknot DNA, relax supercoiled plasmid DNA, and catalyze ATP-dependent DNA strand passage activity in vitro (Austin and Marsh, 1998). Topo II has a significant role in ce ll proliferation, whereas topo II is suggested to have a role in cell differentiation (Aoyama et al., 1998). Topo II has an essential role in chromosome condensation and segregat ion during mitosis (Uemura et al., 1987). Recent findings, however, suggest that topo II is able to substitute for topo II in chromosome condensation and segregation, but that topo II has a crucial role in

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27 shortening of chromosome axes (Sakaguchi and Kikuchi, 2004). Distinguishing the physiological roles of topo II from those of topo II could lead to a better understanding of the molecular mechanisms conferring differe nces in drug sensitivity. A transgenic cell line lacking topo II was recently established and may lead to a better understanding of the differential roles between topo II and II (Errington et al., 1999). To date, there are no established cell lines that lack topo II I searched PubMedCentral, the U.S. Na tional Institutes of Health free digital archive of biomedical and life sciences journa l literature, and identi fied several proteins that have been reported to interact with topo II in vitro and/or in vivo (PubMedCentral, 2005). I have included a brief description of the protein part ners that interact with topo II and speculated on the potential implication(s) of such prot ein-protein interactions in conferring drug resistance. The protein partners that asso ciate with topo II spans a variety of protein classes with roles in vari ous cellular processes, including chromatin remodeling, apoptosis, mRNA splicing, RNA ex port, and proteasome-mediated protein degradation. A complete discussion of each pr otein interaction identified in table 2 is beyond the scope of this investigation. Brie fly however, the data presented in this investigation suggests that the nuclear export of topo II may be regulated by the export receptor, exportin-1. Our laboratory has also collaborated with ot her laboratories to investigate topo II interactions with HDAC (Tsai et al., 2000) and 14-3-3 proteins. For example, histone deacetylases (HDAC) modi fies protein histones and has a role in regulation of gene transcription. Topo II was shown to complex with HDAC1 and HDAC2 in vivo (Tsai et al, 2000). Histone deacety lase activity coupled with topo II DNA cleavage has been implicated in etoposide i nduced apoptosis. We also investigated

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28 a potential interaction between 14-3-3 epsilon with topo II in human myeloma and leukemia cell lines at different cell densiti es because we thought that 14-3-3 may modulate the subcellular localization of topo II in these cells. However, we did not observe 14-3-3colocalization with topo II by confocal microscopy (data not published). The 14-3-3 protei n, however, has been shown to interact with topo II in HL-60 and CCRF human leukemia cells (Kurz et al., 2000). This interaction was shown to abrogate topo II cleavable complex formation in the presence of the topo II poison, etoposide. Although several protein-protein interact ions have been described for topo II specific protein-pr otein with topo II have not been described. The only observation of a protein interaction with topo II has been the observation that topo II can heterodimerize with topo II Although topo II usually forms homodimers, a subpopulation of / heterodimers has been reported to occur, but the biological significance of this interaction is not clearly established (G romova et al., 1998; Biersack et al., 1996).

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29Table 2. DNA Topoisomerase II Protein Interactions Name In vitro or In vivo Function(s) of Protein Partner Function(s) of Topo-Protein Interaction (Observed or Predicted) Role of Topo-Prote in Interaction in Cellular Drug Sensitivity or Resistance (Observed or Predicted) References Topo II In vitro DNA topology Possible role in DNA metabolism; potential therapeutic target Potential molecular targ et for new drugs that inhibit the topo II / heterodimer Biersack et al., 1996 Gromova et al., 1998 p53 In vivo In vitro Tumor suppressor protein; regulation of G2/M transition Interaction with topo II protein may be necessary for detection/repair or activation of apoptotic response following DNA damage. p53 mediated topo II gene suppression blocks cells in G2/M, allowing cells to repair damaged DNA. p53 has been reported to have both proand antiapoptotic effects depending on the cell type and cytotoxic agent investigat ed. For example, mouse fibroblasts expressing wild-type p53 are more sensitive to etoposide and doxorubicin, but less sensitive to UV light and alkylating agents when compared to p53 deficient mouse fibroblasts. These results are in contrast to lymphoblastoid cells that are more sensitive to UV light and alkylating agents than their p53 mutant counterparts. Cowell et al., 2000 Bernd, K. 2003 Caspase activated DNase (CAD) In vitro Deoxyribonuclease involved in apoptosis and nuclear disassembly CAD enhances topo II decatenation activity in vitro CAD may initiate at or near topo II binding sites to initiate chromosomal condensation and nuclear disassembly during apoptosis. Could be involved in apopt otic response to druginduced DNA damage Durrieu et al., 2000 Interleukin enhancer binding factor 2 In vivo Transcription factor; also associated with pre-mRNA splicing complex May be involved in regulating IL-2 gene transcription during T-cell activation To date, there is no litera ture to describe ILF2topo II effects on cellular drug sensitivity. Ajuh et al., 2000 Marcoulatos et al., 1996 Peptidyl-prolyl isomerase (Pin1) In vitro An isomerase that interacts with several phosphoproteins during the cell cycle Inhibits casein kinase II (CK2) phosphorylation of Thr1342 on topo II but especially during G2/M; may regulate CK2 phosphorylation of topo during the cell cycle. Alterations in the phosphorylation status of topo II could regulate topo II enzyme activity, protein degradation, or s ubcellular localization and confer alterations in drug sensitivity. Messenger et al., 2002 Table 2 continued on next page.

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30 Table 2 continued. Name In vitro or In vivo Function(s) of Protein Partner Function(s) of Topo-Protein Interaction (Observed or Predicted) Role of Topo-Prote in Interaction in Cellular Drug Sensitivity or Resistance (Observed or Predicted) References RNA helicase A (RHA) In vivo Unwinds doublestranded DNA and RNA; nuclear export of retroviral RNA RHA may facilitate the access of topo II to chromatin and work together to maintain chromatin structure during transcription. RHA may be a transcriptional co-activator (i.e. topoII may activate RNA polymerase when complexed with RHA) Sumoylation could signal RHA to interact with topoII to initiate transcription of specific genes in response to cellular demands (i.e. during apoptosis or when DNA damage is present) Lee et al., 2004 Zhou et al., 2003 SUMO1/Ubc9 In vivo In vitro An E2-type ubiquitin-conjugating enzyme involved in sumoylation (SUMO) and proteasome mediated degradation; SUMO could regulate topo II degradation, subcellular localization and/or enzyme activity. Ubc9 is also necessary for the assembly of RNA helicase-A topo II complexes to form. Sumoylation may alter drug sensitivity by modifying the amount, location or activity of topo II Sumoylation may be a step in chromosome condensation or DNA cleavage during apoptosis. SUMO-topo II is observed after teniposide induced DNA damage; SUMOtopo II is observed after exposure to the catalytic inhibitor ICRF-193. Mao et al., 2000 Isik et al., 2003 Argasinska et al., 2004 Histone deacetylase1 Histone deacetylase -2 In vivo In vitro Chromatin remodeling Regulation of chromatin structure and gene transcription. The histone deacetylase inhibitor, sodium butyrate (NaB) results in a 2-fold increase in topo II protein and increased sensitivity to etoposide in human leukemic cells. Tris et al., 2000 Johnson et al., 2001 Kurz et al., 2001 14-3-3epsilon In vivo Adapter molecule involved in signal transduction pathways Interaction with 14-3-3 may modulate topo II protein activity or subcellular localization. 14-3-3 epsilon abrogates topo II cleavable complex formation to etoposide as a result of reduced DNA binding activity. Kurz et al., 2000 Exportin 1 In vivo In vitro Protein receptor for nuclear export Export topo II from the nucleus to the cytoplasm Decreased drug sensitivity to etoposide has been associated with a subcellular distribution of topo II from the nucleus to the cytoplasm in human myeloma cell lines. Engel and Turner et al., 2004 Mirski et al., 2003 c-Jun In vivo Transcription factor; proto-oncogene c-Jun stimulates topo II decatenation activity Etoposide induces c-jun gene expression in HL60 and U-937 myeloid leukemia cell lines and may be an apoptotic event in etoposide-induced DNA damage Kroll et al., 1993

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31 3.10 Conclusions. Thus far, cancer pathogenesis and the family of DNA topoisomerases have been reviewed. Some key points to remember are that topo I and topo II are the major nuclear enzymes that regulate DNA topology duri ng DNA replication, transcription, and cell division. Topo I catalyzes single-strand DNA br eaks in an ATP-independent manner and is not expressed in a cell-cycle dependent manner, whereas topo II enzymes cleave double-strand DNA in an ATP-dependent ma nner. Topo II has two isoforms, topo II and topo II Topo II protein expression is cell cy cle dependent, whereas topo II expression is more constant throughout th e cell cycle. Topo I and topo II are the molecular targets of clinically important ch emotherapeutic agents especially in the treatment of hematological malignancies. Ne xt, the drugs that ta rget topo I and topo II are reviewed.

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32 Chapter Four DNA Topoisomerase Targeted Cytotoxic Agents 4.1 Introduction to Topo Targeting Agents. Topo proteins are the molecular target s of commonly used anti-cancer agents (Topcu, 2001). Cytotoxic agents that target topo can be classified as poisons, catalytic inhibitors, or suppressors (Pommier et al ., 1998; Andoh and Ishida, 1998). Some poisons are specific to topo II (i.e., et oposide and teniposide), whereas other poisons target topo I (i.e., topotecan and irinotecan). The poisons are further subclassified as either DNA intercalators or non-intercalators, but severa l drugs have multiple mechanisms of action, and thus do not fit discreetly in to one specific drug category (T able 3) (Ross et al., 1984). DNA intercalators are able to bind to DNA, whereas the non-int ercalators do not bind to DNA. Some drugs, such as Actinomycin D, can act as a poison ag ainst both topo I and topo II, and yet other compounds can act as a poison against one class of topo enzymes, but as a catalytic inhibitor of another cl ass of topo enzymes (van Hille et al., 1999). The mechanism of drug action of the poisons is distinctly differe nt from that observed with the catalytic inhibitors and suppressors. The main differe nce between catalytic inhibitors and suppressors is their mol ecular target. Catalytic inhib itors inhibit topo II activity, whereas the suppressors inhi bit topo I activity. The t opo I and topo II poisons are described in a separate section from th e catalytic inhibito rs and suppressors.

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33 4.2 Topo I and Topo II Poisons. To comprehend how anti-cancer agents target DNA topoisomerases, it is important to recall that, wh en all topo enzymes cleave DNA, they remain bound the cleaved DNA via a phosphotyrosine bond. In th is manner, topo bridges the gap between the cleaved DNA strands, thus ensuring that the correct strands of DNA are resealed back together. Under normal physiological conditions, the complex between topo and the cleaved DNA strands, called a "cleavage" or "c leavable complex", is transient and freely reversible. Some anti-cancer agents, however, stabil ize the form of topo that is bound to cleaved DNA and are called, topoisomerase poisons. Topo II poisons include the epipodophyllotoxins (i.e., etopos ide, teniposide) and anthra cyclines (i.e., doxorubicin), whereas the topo I poisons include the campt othecins (i.e., topotecan, irinotecan). The topo poisons act by increasing the steady-st ate concentration of covalently bound DNAtopo cleavage complexes by blocking the re ligation of cleaved DNA and stabilizing the DNA-drug-enzyme intermediate (Osheroff et al., 1994). However, it is not clear why cells die, since the topo II-DNA covalent co mplexes trapped by topo poisons are freely reversible so that removal of the drug allows the DNA to re turn to an undamaged state (Nitiss and Wang, 1996). It is possible that cells accumulate DNA damage to a point of no return, such that apoptotic pathways are triggered and cell death occurs even if DNA damage is repaired. It is generally belie ved that the drug stabilized cleavage complexes become lethal when either DNA helicases (Howard et al., 1994) or an advancing replication fork collides with the drug-t opo-DNA ternary complex (Hsiang et al., 1989). Such collisions are believed to be lethal b ecause they disrupt the proteinaceous bridge between topo and the cleaved DNA, and thus the transient singleor doublestrand DNA

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34 break that was being held together by topo becomes a permanent double-stranded break (Osheroff, 1995; Chen and Liu, 1994). The double-stranded DNA break becomes a target for gene recombination and repair pa thways (Osheroff, 1998), resulting in sister chromatid exchanges, large insertions and deletions, and chromosomal translocations (Chen and Liu, 1994; Anderson and Berger 1994; Corbet and Osheroff, 1993). Therefore, at least some of the double-strand br eaks that are generated by collision with the replication fork can not be repaired by the enzyme (Nitiss and Beck, 1996; Zhang et al., 1990). So, although the covalent t opo-DNA complex is transient and freely reversible, collision with the replication fork can produce DNA lesions that persist after the drug is removed (Nitiss and Beck, 1996). Though not well understood, these cellular events eventually trigger cell death pathwa ys (Gupta et al., 1995, Pommier, 1993). One additional caveat is that genera tion of a lethal collision is dependent on the orientation of the cleavable complex relative to the advanci ng replication fork. Co llision is lethal only if the cleavable complex is formed on th e lagging-strand of DNA (Pourquier et al., 1999l Tsao et al., 1993). 4.3 Topo I Suppressors and Topo II Catalytic Inhibitors. In contrast to the topo poisons, the suppre ssors of topo I and catalytic inhibitors of topo II do not stabilize DNA-enzyme complexes, but rather work at another step of enzyme catalysis (Pommier et al., 1998). Th e catalytic inhibitors include the topo II targeting agents, merbarone and the bi sioxopiperazines (ICRF-193, ICRF-187).

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35 Class Drug Poisons Catalytic Inhibitors DNA Intercalators Free Radical Formation Epipodophyllotoxi ns etoposide (VP-16), teniposide (VM-26) +++ + Isoflavinoids genistein ++ Anthracyclines doxorubicin, daunomycin ++ + ++ + Actinomycins actinomycin D +++ Anthracenediones mitoxantrone ++ + + Acridines m-AMSA +++ + + Topo II Agents Miscellaneous fostriecin, merbarone, ICRF-193, ICRF-187, ICRF-159 +++ Topo I Agents Alkaloids topotecan, camptothecin (CPT), irinotecan (CPT11), 9-nirocamptothecin (9-NC), karenitecin +++ Table 3. DNA Topoisomerase I and II Inhibitors

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36 Topo suppressors and catalytic inhibito rs may inhibit binding between DNA and topoisomerase, stabilize noncovalent DNA topoi somerase complexes, or inhibit ATP binding (Larsen et al., 2003; Burden and Osheroff, 1998). For example, catalytic inhibitor, called ICRF-193, has been shown to lock the enzyme in a closed form on DNA, preventing DNA cleavage (Roca et al., 1994, Fo rtune et al., 1998). Recall that topo II cleavage activity is described to occur in a two-gate model. The enzyme undergoes two forms, open and closed, in the absence or presence of ATP, respectively. ATP binding triggers a conformational change in topo II, resulting in t opo II gate closure around DNA. ATP hydrolysis results in opening of the topo II gate. Therefore, ICRF-193 stabilizes topo II in a closed formation around DNA and prevents topo from opening again. In general, the drugs that target topo II come from a range of classes with differing structures and mechanism of action. Conversely, most of the antitumo r agents that target topo I are poisons (refer to Table 3), and thus are the focus of this investigation. A summary of the mechanisms of drug action of topo II targ eting agents are illustrated in figure 7. In general, topois omerase poisons can be distinguished from catalytic inhibitors by their cytotoxic profiles (Froelich-Ammon and Osheroff, 1995 Nitiss, 1994). Increased ce llular content of topo protei n corresponds with cellular hypersensitivity to topo poisons, but decreased drug sensitivity to catalytic inhibitors (Bjornsti et al., 1989; Ishida et al., 1995). Conversely, decr eased cellular content of topo protein corresponds with decrea sed sensitivity to topo poiso ns but hypersensitivity to catalytic inhibitors (Bjornsti et al., 1989; Ishida et al., 1995; Nitiss and Wang, 1988).

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37 Figure 6.The Mechanism of DNA Topo I Poisons. (1) Topo I monomer binds to supercoiled DNA (2) Topo I cleaves single strand of DNA .The cleavable complex is stab ilized in the presence of topo I poison (shown as orange box) (3) Advancing replication fork collides with cleavage complex, resulting in double strand DNA break. 1 2 3 Supercoiled DNA Cleavable Complex

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38 Mg2+ 1 2 3 4 ATP ADP + Pi 5 6 Figure 7. The Mechanism of DNA Topo II Poisons a nd Catalytic Inhibitors. This is the same figure illustrated on page 23, but now the steps of enzyme inhi bition by topo II poisons and catalytic inhibitors are indicated. Briefly, (1) Topo II binds DNA (orange rod) (2) Topo II binds as second DNA strand, and then binds ATP (yellow star) (3 ) The ATPase domains on topo II dimeri ze, and then topo II cleaves DNA.(4) Strand passasge occrs through the gap (5) transport DNA is released, and then ga te DNA is resealed. (6) ATP is hydrolyzed and ADP and inorganic phosphate (Pi) are released to complete enzyme turnover. Topo poisons act on the cleavable complex (forms d and e) and prevent reaction 4 from proceeding forward. The catalytic inhibitors act on any other step of the catalytic cycle (forms a, b, c, and f). Catalytic inhibitors prevent topo binding to DNA, ATP bi nding, and ATP hydrolysis. This fi gure was modified from references Champoux, 2001 and Wang, 1996. a. b. c. d. e. f. Cleavable com p lex Cleavable com p lex Poisons Catalytic Inhibitors

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39 4.3 DNA Topoisomerase I Targeting Agents. Camptothecin (CPT) (Figure 8.) is a plant alkaloid isolated from Camptotheca acuminata (Wall et al., 1986) Initial studies showed that CPT displayed potent antitumor activity against a wide ra nge of tumors, but the drug was discontinued because of toxic side effects (Pommier, 2005). There was a renewed interest in CPT when subsequent findings identified topo I as the main molecular target (Hsiang et al., 1985). Sin ce CPT was the only known drug to target topo I, finding less toxic and more water sol uble CPT derivatives became a subject of numerous investigations (Kollmannsberger, et al., 1999; Wani et al., 1987, 1986; Kunimotto et al., 1987). Topotecan (TPT) (Figure 9.) and irinotecan (CPT-11) are two water soluble CPT derivatives that are currently being used for the treatment of hu man cancers (Lackey et al., 1996; Luzzio et al., 1995). CPT-11 has been used in the treatm ent of colon, breast, gastric and small-cell lung cancers as well as leukemia (Rosen, 1998) whereas topotecan has been used against recurrent small cell lung cancer ovarian cancer and endometri al cancer (Treat et al., 2004; Ahmad and Gore, 2004; Traina et al., 200 4). CPT-11 must be converted to the active compound, SN-38 (7-ethyl-10-hydr oxycamptothecin B-glucuronide) by carboxylesterase (Satoh et al., 1994). Thus increased drug sensitivity to irinotecan N N O O O OH C H3N+O-O Fi g ure 8. Cam p tothecin ( CPT ) Figure 9. Topotecan (TPT) N N N CH3C H3O H O O O OH C H3

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40 correlates with carboxylesterase expression and activity (Wierdl et al., 2001). Cisplatin is also a substrate of the ABC drug transport protein, multidrug resistance related protein-3 (MRP3). CPT and its derivatives interact reve rsibly with a DNA-enzyme complex and usually have no detectable bindi ng to isolated topo I or DNA (Fan et al., 1998; Hsiang et al., 1985). However, the x-ray crystal stru cture of human topo IDNA-topotecan ternary complex shows that topotecan can mimic a D NA base pair (Staker et al., 2002). These findings may partially explain how topotecan can intercalate in DNA in the absence of topo enzyme in some instances. In the pres ence of camptothecins, topo I is unable to reseal nicked DNA because the drug blocks the active site of the covalently bound enzyme (Woo et al., 2002). However, single strand DNA breaks alone are not sufficient for cell death to occur and instead must be converted into permanent DNA double strand breaks by collision with the moving replica tion fork (Mosseso, 2000). Conversely, it has also been reported that CPT induced DNA strand-breaks can occur in unstimulated leukocytes where little or no re plication is occurring (Daza et al., 2002), and thus other cellular events besides the collision of cleav able complexes with replication forks may have a role in CPT induced DNA damage. The molecular basis of CPT induced cell death is not completely understood, since topo I cleavable complexes are readily reversible after drug removal (Pommier, 2005). It is generally accepted that collision of the moving replication fork with the t opo I cleavable complexes leads to three biochemical events: formation of a double-stra nd DNA break, irreversible arrest of the replication fork, and formation of an irreve rsible topo I-linked DNA break (Tsao et al.,

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41 1993; Hsiang et al., 1989). It is thought that one or more of these events initiates S-phase cell death and G2 phase cell cycle arrest. 4.4 DNA Topoisomerase II Targeting Agents: Etoposide and Mitoxantrone. In contrast to the limited number of drug classes that target to po I, topo II is the molecular target for a number of diverse drug classes, including the epipodophyllo toxins, isoflavinoids, anthracyclins, actinomycin s, anthracenediones and acridines (Table 3). Etoposide (VP-16) and teniposide (VM-26) are two semi-synthetic epipodophyllotoxins. Etoposide and teniposide are derived from 4'-demethylepipodophyllin benzylidene glucoside (DEPG), a naturally occurring compound synthesized by Podophyllum peltatum the North American May Apple Mandrake (Figure 10) (Imbert, 1998; Hande, 1998; Sinkule, 1984). The May Apple was first described in 1731 and can be found gr owing from the coasts of Northern Florida to Maine (Catersby, 1731). Native American Indians and American pioneers reportedly used the plant for medicinal purposes, such as for the treatment of skin cancer (Hande, 1998). In 1844 the first alcohol extraction of the plant was re ported and the isolated resin was called podophyllin (King, 1844). For over twenty years, numerous isolation procedures were conducted, fronting an inte nse search for the active compound. These Figure 10. Podophyllum peltatum also known as the May Apple Mandrake. The plant shown here is bearing fruit, the richest source of podophyllotoxins. This photograph was reproduced with written permission from the Maryland Conservation Council.

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42 O CH3OH O C H3O O O O O O OH OH O O C H3 Fi g ure 11. Eto p oside ( VP-16 ) investigations lead to th e development of VP-16 and VM-26, which were renamed, etoposide and teniposide, respectively (Stahelin and Von Wartburg, 1989). Unlike topo I targeting agents topo II targeting drugs are able to kill cells at all points in the cell cycle (Dubrez et al., 1995). Topo II inhibitors such as VP-16 (Figure 11) and mitoxantrone (Alberts et al., 1980; Koeller and Eble, 1988) (Figure 12) stabilize the complex formed by topo II and the 5’ cleaved ends of the DNA, thus forming stable protein-linked double strand DNA breaks (Meresse et al., 2004). VP-16 and mitoxantrone inhibit topo II DNA religation activity, leading to an incr ease in DNA scission (Liu, et al., 1983). The DNA breaks are believed to interfere with the progress of the moving replication fork (Qiu et al., 1996). Cells recognize drug-induced DNA damage and eliminate the damaged cells by apoptosis, but the pathway from DNA damage to cell death remain s uncertain (Valkov and Sullivan, 2003). Etoposide mediated ce ll death is reported to occur when DNAdependent protein kinase (DNA-PK) recognizes an accu mulation of DNA double strand breaks and then phosphorylates p53 (Karpinich et al., 2002). Activated p53 then results in increased transcription of the pro-apoptotic protein Bax, which then translocates into the mitochondria and induces the re lease of cytochrome c. Mitoxantrone (MTX) is a synthetic anth racenediones that was developed as an analogue of doxorubicin (Fox, 2004). Mitoxantro ne has been shown to have multiple mechanisms of action. In addition to being a topo II inhibitor (as previously described

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43 OH OH O O N H N H NH OH NH OH Fi g ure 12. Mitoxantrone ( MTX ) above), mitoxantrone also interferes with RNA synthesis, and intercalates with DNA.(Duff, 1984; Isabella et al., 1993) DNA intercalation results in aberrant DNA-protein crossli nks and DNA strand breaks (Hagemeister et al., 2005). Mitoxantrone has also been shown to i nhibit antigen presenta tion and decrease the secretion of proinflammatory cytokines, which has importa nt roles in modulating the immune response (Fox, 2004). MTX has also been shown to alter prostaglandin biosynthesis and calcium release (Ehninger et al., 1990). In summary, MTX has been shown to have multiple biological effects but the drug's antitumor activity is mainly attributed to its inte raction with DNA topoisomerase (Koe ller and Eble, 1988). However, it is possible that these other cellular events could contribute to drug sensitivity to MTX. Mitoxantrone was approved by the Food and Drug Administration for the treatment of adult acute myeloid leukemia and sympto matic hormone-refractory prostate cancer. Mitoxantrone also has demonstrated beneficial activity in preclinical lymphoma models and appears clinically ac tive against both aggressive and follicular lymphomas (Hagemeister e., 2005). Topo II targeting agents have b een used effectively in the treatment of non-Hodgkin’s lymphoma, leukemi as, small-cell lung can cer and soft-tissue sarcomas. However, cancer cells have ma ny mechanisms for evading topo II mediated drug cytotoxicity. Mechanisms of drug resistan ce to topo II targeting agents are reviewed in chapter six.

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44 4.5 Dual Inhibitors of DNA Topoisomerase. Dual inhibitors of topo I and topo II ar e emerging as a new class of cytotoxic agents with promising anti-tumor activity (Denny and Baguley, 2003). The dual topo inhibitors have activity agains t both topo I and topo II enzymes and can be classified into three groups: DNA intercalators, non-DNA inte rcalators, and hybrid molecules (Denny and Baguley, 2003). The DNA intercalators phy sically intercalate between DNA bases, whereas the non-DNA intercalator s recognize structural motifs present in topo I and II proteins. Two examples of dual topo I and II D NA intercalating agents in clude are N-[2-(dimethylamino) ethyl] acridine-4-car boxamide dihydrochloride (DACA) and the benzophenazine referred to as XR11576 (Mistry et al., 2002). I n vitro experiments using purified topo and DNA substrates show that DACA induces DNA breakage by both topo I and II (Finlay et al., 1996). However, the mechanism of DACA inhibition on topo I and II is still in question. The data suggests th at DACA may act as a topo poison at a low drug concentration (0.5-5 M) and as catalytic inhibitor at a highe r drug concentration ( 10M) (Spicer et al., 2000; Bridewell et al., 1999). Thus, topo-DNA cleavage complexes are not observed at the higher drug concentr ations. This may be possible, if the intercalation of DACA into DNA at higher dr ug concentrations inhi bits the binding of topo to DNA. DACA has completed phase I and II clinical trials (D ittrich et al., 2003; Caponigro et al., 2002; Twelves et al., 1999) and has been shown to overcome mechanisms of in vitro drug resistance in several multidrug resistant cell lines that express drug efflux pumps (i.e., P-glyc oprotein, multidrug resistance protein, and multidrug resistance related protein) (Davey et al., 1997). DACA has also been shown to be active against solid tumors in mice (Bridewell et al., 1999).

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45 Four examples of non-DNA intercalating ag ents have been described and include NK109, acetyl Boswellic acid, BN 80927, and F11782. Acetyl-Boswellic acid (acetylBA) is isolated from the gum resin of Boswellia serrata, a tree that grows in Asia. Acetyl-BA inhibits topo I and II by binding directly to topo enzymes, rather than DNAtopo complexes, and is believed to co mpete with DNA for binding sites on topo (Syrovets et al., 2000). BN 80927 is a camptothecin analogue that has a seven-membered -hydroxylactone ring, in contrast to other camptothecins that have a six membered -hydroxylactone ring (Huchet et al., 2000, Demarquay et al., 2000). The -hydroxylactone ring in BN 80927 is modified by the insertion of methylene (-CH2) group. When compared to camptothecin, topotecan, and irinotecan, BN 80927 has improved plasma stability because the a dditional methylene group undergoes slow and irreversible hydrolytic ring-ope ning (Demarquay et al., 2000). In vitro and in vivo experiments show that BN 80927 inhibits t opo-I mediated supercoiled-DNA relaxation because it stabilizes topo Icleavage complex (Lavergne et al., 1999). BN 80927 was also shown to inhibit topo II medi ated supercoiled-DNA relaxation and DNA decatenation activity (Demarquay et al., 2000). In contrast to etoposide, BN 80927 does not stabilize topo II-DNA cleavage complexe s (Demarquay et al., 2000). Thus, BN 80927 is a topo I poison and a topo II catalytic inhibitor. The fourth example of a nonintercalating dual topo I and II inhibitor mentioned above was F11782. Treatment of P388 murine leukemia cells with F11782 resu lts in dose-dependent DNA fragmentation and activation of apoptotic pathways through a caspase 3/7 dependent mechanism. F11782 has also been shown to have improved in vivo anti-tumor activity, relative to three other cytotoxic agents (Kruczynski et al., 2004).

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46 The third group of dual topo inhibitors is known as "hybrid molecules" because they consist of a topo I inhi bitor biochemcially linked with a topo II inhibitor. The hybrid molecules are still in early stages of drug development and have not been formally reported in the literature (Denny and Bagul ey, 2003). The dual topo inhibitors are promising new drugs because they may evad ing toxicity by compensating one enzyme for another enzyme. 4.6 Conclusions. Topo I and topo II are the molecular targets of several antitumo r agents used for the treatment of hematological maligna ncies (Wang, 1996). Topo I is the major molecular target of the camptothecins (i.e., topotecan and irinotecan). The camptothecins are topo I poisons, and thus stabilize t opo I-DNA cleavage complexes Topo II poisons, such as VP-16 and VM-26, stabilize topo II cl eavable complexes, wh ereas the catalytic inhibitors of topo II do not stabilize cleavab le complexes. The catalytic inhibitors prevent topo binding to DNA, or trap topo II in a closed form ation. Dual inhibitors of topo I and topo II are promising new cytotoxic ag ents, but the focus of this investigation is on topo I and topo II poisons and topo II cata lytic inhibitors. Dr ugs that act on other molecular targets besides topoisomerases ar e briefly described in following chapter.

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47 Chapter Five Non-topoisomerase Interacting Agents 5.1 Introduction. Although some drugs interfere with DNA metabolism, they may do so without associating with a topoisomerase and should not be confused with the topoisomerase interacting agents described above. Thus, th e primary mechanism by which these types of drugs exert their cytotoxic eff ect is independent of topo cont ent or activity. For example, DNA alkylating agents such as Carmustine, a lipophilic nitosourea, undergoes hydrolysis in vivo to form reactive metabolites that produce lethal O6-chloroethylguanine DNA interstrand crosslinks (Egyhazi et al., 1991) The non-topoisomerase interacting agents can be used in laboratory studies to he lp distinguish mechanisms of cellular drug resistance that are a result of alterations in the quality or content of topo from other nontopo related cellular events, such as alterations in drug tran sport. In some instances, exposure to one type of drug can result in cr oss-resistance to many other drugs that are structurally unrelated, a phenomenon known as multidrug resistance. We assessed human myeloma cell lines for cross-resist ance to taxol, ara-c, cisplatinum, and carmustine. These cytotoxic agents were select ed because of their differences in chemical structure and mechanism of acti on (figure 13; table 4). To improve drug sensitivity, the non-topoisomerase drugs are often given in combination with topo targeting cytotoxic

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48 agents. For example, a combination of topotecan ara-c, and cisplatin have reached phase II clinical studies for relapsed or primar y refractory lymphomas (Coutinho et al., 2004). Unfortunately, multidrug resistance to combination chemotherapy is a limitation that needs to be overcome. Cisplati n, paclitaxel, ara-c, and carmu stine are four examples of non-topoisomerase targeting agents described herein. 5.2 Cisplatinum. Cisplatin ( cisdiamminedichloroplatinum; CDDP) and its synthetic analogues are DNA crosslinking agents and platinum contai ning compounds that exert their cytotoxic effects by inducing the formation of various types of DNA lesions (Crul et al., 2002; Iikima et al., 2004). Cisplatin is a DNA cr osslinking agent with pot ent antitumor activity against various kinds of cancers including, non-small cell lung cancer (Wang et al., 2004). However, acquired drug resistance to CDDP is a common observation in these patients. The mechanisms of drug resist ance to cisplatin are unknown, but enhanced DNA repair, altered drug transport, and dr ug inactivation by metallothionine have all been associated with decreased drug sens itivity to CDDP (Wang et al., 2004). Furthermore, sensitivity to DNA-crosslinking reagents are inversely proportional to topo levels and activity, and thus elev ated levels of topo II correlat e with resistance to cisplatin (Tan et al., 1987). This has been explained by the involvement of topo II in processing DNA damage induced by DNA crosslinking agents (Tan et al., 1987). A deficiency in topo II activity correlates with increased drug sensitivity as a result of an increase in drug-induced sister chromatid exchanges. These results suggest that the efficacy of CDDP in cancer chemotherapy can be improved through inhibition of topo II

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49 5.3 Paclitaxel. Microtubules are intracellular organelles, formed from the protein tubulin, involved in chromosome segregation, transpor t, motility, and cell structure (Jordan and O NH OH O2+O O C H3O CH3OH O C H3O O OO HCH3C H3O O O C H3O Pa c litax e l ( Tax o l ) N N O NH2O OH OH O H C y tarabine ( Ara-c ) Pt2-Cl Cl NH3 +N H3 + Cisplatinum (CDDP) N N O NH O Cl Cl Carmustine(BCNU) Figure 13. Paclitaxel (Taxol), Cytarabi ne (Ara-c), Cisplatinum (CDDP) and Carmustine (BCNU). Structures of the nontopoisomerase associ ated agents used in the soft agar cytotoxicity experi ments (see “materials and methods”)

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50 Topoisomerase Associated Non-topoisomerase Associated Topotecan (TPT) Etoposide (VP-16) Mitoxantrone Paclitaxel (Taxol) Cytarabine (Ara-C) Carmustine (BCNU) Cis-platinum (CDDP) Class Alkaloid Epipodophyllotoxins Anthracened iones Mitotic inhibitor Anti-metabolite Nitrosourea (Alkylating) Alkylating MW* 457.9 588.6 517 853.9 243.2 214.06 300 Mechanism of Action Topo I inhibitor Topo II inhibitor Topo II inhibitor Inhibits microtubule formation DNA polymerase DNA crosslinking DNA crosslinking Cell-cycle dependent? yes, S-phase yes, late S and G2 yes yes yes, S-phase some, G1 and G2/M no Examples of In vitro Mechanisms of Drug Resistance Altered cellular content or activity of topo I; altered drug transport Altered cellular content. location or activity of topo II ; altered drug transport Altered cellular content location, or activity of topo II ; altered drug transport Altered drug transport; gene mutations in tubulin Altered drug transport Enhanced DNA repair Drug inactivation; enhanced DNA repair; altered drug transport Therapeutic Uses Ovarian cancer, SCLC, gliomas, AML, CML, MM, MDS, neuroblastoma, ALL, AML, Brain tumors, Ewing’s sarcoma, Histiocytosis X CML, AML, ALL, breast cancer, NHL, ovarian cancer breast cancer, ovarian cancer, head and neck cancer, lung cancer AML, ALL CML, NHL brain tumors, Hodgkin’s lymphoma, NonHodgkin's lymphoma bladder cancer, adrenocortical cancer, lung cancer, ovarian cancer, testicular References Rasheed and Rubin, 2003 Meresse et al., 2004; Hande, 2003 Thomas and Archimbaud, 1997; Center, 1993 Jordan and Wilson, 2004 Wills et al., 2000; Abdel et al., 2000 Linfoot et al., 1988 Ueda-Kawamitsu, et al., 2002; Henderson et al., 1987 Brabec and Kasparkova, 2005; Arbuck, 1994 Table 4. Comparison of DNA Topoisomerase Inhibitors w ith Non-Topoisomerase Targeting Anticancer Agents

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51 Wilson, 2004). Taxol is a tubul in-binding antimitotic agent w ith antitumor activity in a variety of cancers including breast cancer (Yvon et al., 1999; Fountzillas et al., 2004). Cells acquire drug resistance to taxol by d ecreasing intracellular co ncentration through protein transport pumps and by gene mutations in the beta tubulin gene that alter drug binding properties (Zhou et al., 2004; Hadfiel d, et al., 2003; Dumontet and Sikie, 1999). 5.4 Cytarabine (Ara-C). Cytarabine (ara-C) is an antimetabolit e and nucleoside analog of deoxycytidine (Frei et al., 1973; Kufe and Spriggs, 1985). Ara-c differs from deoxycytidine by the addition of a hydroxyl group on the 2' position of the sugar moiety. Once inside the cell, ara-C is converted into an active form, cyta rabine-5’-triphosphate (a ra-CTP) (Frei et al., 1973). The rate limiting step of ara-C activ ation is catalyzed by deoxycytidine kinase (Coleman et al., 1975). Ara-CTP compet es with deoxycytidine triphosphate for incorporation into DNA, and thus is a co mpetitive inhibitor of DNA polymerase (Kufe and Spriggs, 1985; Furth and Cohen, 1968). Incorporation of ara-CTP into DNA eventually results in inhibition of DNA synt hesis and cell death (M ikita and Beardsley, 1988; Kufe et al., 1984). Incorporation of Ar a-c into DNA is also associated with local conformational changes in DNA, inhibition of DNA polymerase, and a decreased ability of transcription factor binding to DNA binding elements (Zhang et al., 2004b; Mikita and Beardsley, 1988). Ara-C is often used in combination therapy for different types of leukemia (Szafraniec et al., 2004). Complete re sponse rates in acute myelocytic leukemia (AML) reach 65-80%; however, patients usually relapse within 2-years and are resistant to subsequent chemotherapy (Cros et al., 2004). Resistance to ara-C has been attributed

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52 to overexpression of the P-glycoprotein dr ug efflux pump, but some patients who are resistant to ara-C do not expr ess P-glycoprotein (Galmarini et al., 2002a). Drug resistance in these patients has been partially attributed to deficiency in the human equilibrative nucleoside transporter 1 (hENT1), a transm embrane drug influx pump (Boleti et al., 1997). A lack of hENT1 presumably results in less intracellular conc entrations of ara-C because of decreased intracel lular drug import (Galamarini et al., 2002a; Lang et al., 2001). 5.5 Carmustine (BCNU). Carmustine (BCNU) is a nitosourea compound with DNA alkylating activity. BCNU forms a chloroethyl adduct on the O6 position of guanine, which spontaneously converts to N1-O6 ethanol-guanine (D’Incalci et al., 1988). The adjacent cytosine covalently binds to the N1-O6 ethanol-guanine to form an intrastrand cross-link that interferes with DNA replication and transcri ption (Erickson et al., 1980). Carmustine has antitumor activity against leukemias and glio mas, and is currently being evaluated in phase II clinical trials for recurrent gl ioblastoma (Brandes et al., 2004). A major limitation to BCNU effectiveness is that acqui red drug resistance usually occurs after the first administration of the drug (Brandes et al., 2002). Acquired chemoresistance to BCNU has been attributed to the expr ession of the DNA repair enzyme, O (6)alkylguanine-DNAalkyltransfer ase (AGT), because this enzyme removes alkyl groups from DNA (Brandes et al., 2002).

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53 5.6 Conclusions. Topoisomerases are not the molecular ta rgets of DNA crosslinking agents (i.e., cisplatin), anti-metabolites (i.e., ara-C), DNA al kylating agents (i.e., carmustine), or antimicrotubules (i.e., paclitaxel). Non-topoisome rase targeting agents are sometimes used in combination with topoisom erase targeting agents in cancer chemotherapy. Cancer cells have several molecular adaptations to evade drug induced cytotoxicity. Mechanisms of drug resistance to topoisomeras e targeting agents are the focus of this investigation and are discussed in chapter five.

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54 Chapter Six In vitro Mechanisms of Topoisomerase Associated Drug Resistance 6.1 Introduction. In the preceding sections, the family of DNA topoisomerases and commonly used chemotherapeutic drugs has been reviewed. The focus has been on the topoisomerase I and II as the molecular targets of topoisom erase targeting cytotoxic agents. In this chapter, the mechanisms of tumor cell resist ance to topo targeting ag ents and especially topo II mediated cellular drug resistance are expl ained (reviewed in Engel et al., 2003). Although the development of new drugs and new drug combinations has resulted in more effective treatment of cancer, a major factor limiting their usefulness is the presence of drug-resistant cancer cells th at can exist prior to (intrinsic or de novo drug resistance) or arise during cancer therapy (a cquired drug resistance) (Bellamy et al., 1990). Whether drug resistance is intrinsic or acquired, mechanisms conferring altered drug sensitivity may be categorized as invol ving a pre-target event, a drug-target interaction, or a post-target event (Larsen and Skladanowski, 1998). Mechanisms of drug resistance that result in a modification to the drug itself are called pre-target events because the cellular insult occurs before the drug reaches its intende d molecular target. Drug metabolism is one example of a pre-ta rget event because the drug becomes.

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55 modified before reaching its intended molecu lar target. Modifications to the drug's intended target, however, can also occur and re sults in altered drug-target interactions. For example, sequestration of the drug's in tended target into a cellular compartment different from the drug results in an inability of the drug to interact with its target. In other instances, neither the dr ug nor the drug's target is m odified. Post-target events occur after the drug-target interaction occurs, and thus do not directly involve the drug or the drug's target. For example, altered cell-cycl e progression can be a po st target event, if the drug's target is not a cell-cycle regulator y protein. Pre-target events, drug-target interactions, and post-target events are discusse d herein as they pertain to mechanisms of drug resistance to topoisomerase targeting agen ts. Figure 14 and figure 15 summarize the pre-target events, drug-target in teractions, and post-target even ts that can lead to altered drug sensitivity to topo targeting agents. Each type of mechanism of drug resistance that is illustrated in these two figures is expl ained separately in the following sections. However, mechanisms of drug resistance are not mutually exclusiv e events and thus, there is a fair amount of overlap between the types of drug resistance mechanisms. 6.2 Pre-target Events: Altered Drug Transport. Alterations in drug transpor t can either be a result of decreased drug import or increased drug efflux. Some hydrophilic drugs n eed to be transported across the plasma membrane and into cells by sp ecific drug transporters (Cass et al., 1998). For example, cytarabine

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56 Figure 14. Alterations in Pre-target Events Associated with Drug Resistance. Mechanisms of cellular drug resistance to topoisomerase inhibitors can be categorized as pre-target events, drug-target interac tions, and post-target events (see referen ce Larsen and Skladanowski, 1998). Multiple cellular factors can be involved in conferring altered drug sensitivity. Altered drug transpo rt is shown in (1) as the expression of drug transport protein pumps located on the plasma membrane th at can result in either decreas ed drug import or increased drug efflux from the cell. Once inside a cell, a drug can be sequestered into intracellular vesicles resulting in altered drug distribution as shown in (2). Some drugs undergo metabolic modifications, illustrated in (3), that alter their mec hanism of action or result in an inactive form. Other drugs such as irinotecan are a prodrug that must be converted to an active metaboli te. Cytotoxic agents that target topoisomerase must be move into th e nucleus (4) where drug-target in teractions occur. Mechanisms of drug resistance by alterted drug-target and pos t-target events are illustrated in figure 15. Drug Effluxed drug

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57 Figure 15. Alterted Drug-target Interacti ons and Post-target Events Associated with Drug Resistance. Altered drug-target inte ractions include (1) altered subcellular localization of nuclear enzyme that results in decreased amount of topo present in the nucleus (2) decreased amount of active enzyme in the nucleus is associated with resistance to topo poisons (3) increased amount of active topo enzyme is usually atttirbuted to decr eased drug sensitivity to catalytic inhibitors of topo and (4) altered chromatin struct ure can result in altered topo-DN A binding activity. Post-target events associated with drug resistance to topo targeting agents include (5) decreased amount of replicating cells, (6) DNA-repair of cleavable complexes, a nd (7) decreased ability to initiate apoptosis. Topo is shown as yello w circles, the drug is shown as blue/grey ci rcles, and DNA is shown as a red double helix except in (4) where DNA is shown in a relaxed conformation. In diagrams (5-7) the area of the cytoplasm is omitted because these events occur in t he nucleus. For a review of this topic see reference (Larsen and Skladanowski, 1998). 2 4 3 7 6 5 1 Post-target Events Dru g -tar g et Interactions

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58 (ara-C) is a hydrophilic nucleosid e analogue that is transpor ted into cells by the human equilibrative nucleoside transport protein 1 (hENT1), a drug influx pump that is expressed in the plasma membrane of so me human cells (Boleti et al., 1997). In vitro cellular drug resistance to ara-C has been associated with lack of hENT1 mRNA expression in human T-lymphoblast CCRF-CEM ce ll lines selected for resistance to araC (Lang et al., 2001). Furthermore, shorter disease-free survival correlates with the absence hENT1 gene transcription in patients with acute myeloid leukemia (Galmarini et al., 2002b). Topoisomerase targeting agents must also be taken up by cells and transported to the nucleus to be effective as cytotoxic agen ts. Therefore, reduced drug accumulation is a chief mechanism conferring drug resistance to poisomerase targeting agents in several drug-resistant cell lines (Matsumoto et al., 2005; Kamath et al., 1992; Hendricks et al., 1992). In general, topoisomerase ta rgeting agents are presumed to enter cells by passive diffusion and are rarely found to be substrat es of drug influx pumps. However, both active and passive transport of camptothecin ha s been found to occur in human intestinal cells (Gupta et al., 200). Influx of topotecan and SN-38 has also been suggested to occur by active transport in ovarian cells (Ma et al ., 1998). Drug resistance in these cells was attributed to decreased drug accumulation independent of P-gp and MRP expression. Nevertheless, topo targeting agents are mo re commonly shown to be substrates for several drug efflux pumps. Therefore, tumo r cells that express drug transport proteins involved in the extrusion of topo interacting agents are found to be drug resistant. Table 5 summarizes the chemotherapy related subs trates for drug transport proteins.

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59Table 5. Drug Transporters an d Chemotherapy Substrates rug Name Drug Transporters* Member of ABC-Transporter Superfamily? Drug Influx or Efflux? References Etoposide (VP-16) P-gp/MDR1, MRP1, MRP2, MRP3, Yes Efflux Lorico et al., 1995 Neuhaus et al., 1995 Teniposide P-gp/MDR1, MRP3 Yes Efflux Kool et al., 1999 Mitoxantrone MRP2, BCRP Yes Efflux Neith and Lage, 2005 Topotecan (TPT) BCRP Yes Efflux Yang et al., 2000 MRP2, MRP3 Yes MRP2 and MRP3: drug efflux Cis -platinum (CDDP) CTR1 No, CTR1 is a plasma membrane carrier for nucleoside analogs. CTR1: drug influx Kool et al., 1997 Kawabe et al., 1999 Ishida et al., 2002 Sn-38 (active metabolite of CPT-11/ irinotecan) BCRP Yes Efflux Nakatomi et al., 2001 Paclitaxel (Taxol) P-gp/MDR1, MDR2, BSEP Yes Efflux Gerloff et al., 1998 Childs et al., 1998 Reinecke et al., 2000 Cytarabine (Ara-C) hENT1 No, hENT1is an integral membrane proton belonging to the equilibrative nucleoside transport protein family Influx Boleti et al., 1997 Cass et al., 1998 Lang et al., 2001 Galmarini et al., 2002b Carmustine (BCNU) Not a substrate for known drug transporters BCNU enters the cell by diffusion Carter et al., 1972 Balcerczyk et al., 2003 Abbreviations used in this table: ABC-Tr ansporter, ATP-binding cassette transporter; P-gp, P-glycoprotein; MDR, multidrug resis tance; MRP, multidrug resistance-related protein; BCRP, breast cancer resistance protein; CTR1, copper transpor ter 1; BSEP, bile sa lt export pump; hE NT1, human equilibrative nucleoside transporter1. *Reviewed in reference, Ambudkar et al., 2003

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60 Under normal conditions, ATP-binding cassette (A BC) transport proteins pump cytotoxic agents and xenobiotics out of the cells (van der Does and Tampe, 2004). Expression of ABC transporters in cancer cells results in decreased intracellular levels of the administered chemotherapeutic agent that is associated with the multi-drug resistance (MDR) phenotype (Ling, 1997). Multi-drug re sistance describes the phenomenon that occurs when cells initially e xposed to one class of cytotoxi c agents become resistant to other unrelated drugs with various struct ural and chemical properties (Larsen and Skladanowski, 1998) The ABC transport proteins identified in humans include, multidrug resistance protein-1/P-glyc oprotein (MDR-1/P-gp) (Hof fmann and Kroemer, et al., 2004), multi-drug resistance-associated protei n (MRP) (Hoffmann and Kroemer, et al., 2004), and breast cancer resistan ce protein (BCRP) (Doyle an d Ross, 2003; Allen et al., 1999). The expression drug efflux pumps in cancer cells can redu ce the intracellular accumulation of chemotherapeutic agents. P-gp is a 170 kDa transmembrane glycoprotei n that is able to expel a structurally heterogenous group of chemotherapeutic agents including anthracyclines, vinca alkaloids and epipodophyllotoxins (Shustik et al., 1995) P-gp can extrude chemotherapeutic agents out of cells against a concentra tions gradient (Lankelma et al., 1990). Overexpression of P-gp is estimated to occur in approximately 50% of all human tumors, and thus P-gp activity likely has a role in tr eatment related failure of some chemotherapy (Gottesman, 1993; Sikic, B.I. 1993). Overe xpression of P-gp has been observed in many intrinsically drug resistant tumors derived fr om kidney, liver, adrenals, colon, and rectum (Goldstein et al., 1989). However, many drug resistant cells do not express P-gp,

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61 suggesting a role for other drug efflux pumps in cellular drug resistance (Mirski et al., 1987, Zijlstra et al., 1987). Some drug resistant cell lines express the multidrug resistance-associated protein (MRP) (Cole et al., 1992). MRP has seven fa mily members conferring different substrate specificities. For example, methotrexate is a substrate for MRP1, but not MRP2 (Konno et al., 2003). Both P-gp and MRP are able to transport glutathione-S-conjugated chemotherapeutic agents (Mller et al., 1994). MRP expression is found in a wide range of tissues, but varies by family member. Fo r instance, MRP1 expression is found in most tissues, whereas MRP6 expression is limited to the liver and kidney. The breast cancer resistance protein is another t ype of drug efflux pump that is found to be overexpressed in some drug resistant cell lines (Doyle and Ro ss, 2003). BCRP is expressed in breast tissue, placenta, intestine, and liver. Overe xpression of BCRP results in cross-resistance to mitoxantrone and topotecan, but not to microtubular inhibitors, such as paclitaxel (Litman et al., 2000). The lung resistan ce related protein (LRP) is a major vault protein localized on lysosomes and in the nuclear pore complexes (Scheffer et al., 1995). LRP was initially described in drug resistant nonsmall cell lung cancer cell lines that were deficient in P-gp expression (Scheper et al., 1993). LRP modulates the transport of drugs between the nucleus and the cytoplasm. E xpression of LRP is associated with the multidrug resistance and intrin sic drug resistance to doxorubicin, vincristine, and cisplatin (Mossink et al., 2003; Scheffer et al., 2000; Izquie rdo et al., 1996). Drug uptake of [3H-VP-16] is one method that is used in the laboratory to de termine the cellular uptake of etoposide (or other cytotoxic agents) and drug transport protein activity.

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62 6.3 Pre-target Events: Altered Drug Distribution. After cellular drug uptake, cells may sequester drugs in to intracellular vesicles and thus, withhold the drug from reaching its molecular target. Dr ug sequestration into cytoplasmic vesicles has been described in vitro in several drug re sistant cell lines by following fluorescently labeled compounds, such as mitoxantrone (Schuurhuis et al., 1991; Gervasoni et al., 1991). Altered intr acellular distribution of doxorubicin has also been described in leukemic cells obtained from patients with acute myeloid leukemia (Schuurhuis et al., 1995). Altere d drug distribution is usually described as a shift in the amount of fluorescence from the nucleus to the cytoplasm. The pattern of fluorescence in the cytoplasm is consistent with vesicle form ation. The apparent shift in fluorescently labeled compounds from the nucleus to the cyt oplasm may be a result of LRP, the major vault protein found on the nuclear membrane that modulates nuclear-cytoplasmic drug transport. However, vesicle formation is not well understood (Slapak et al., 1992). One mechanism has been proposed for the seque stration of DNA topoisomerase targeting agents inside acidic vesicles (Larsen and Skladanowski, 1998). Accordingly, the cytoplasm of most cells is mildly alkaline, a requirement for the proper functioning of the trans -Golgi network and endocytot ic secretory pathways (Tarakoff et al., 1983). Since some topo targeting agents such as anthr acyclines, anthracenediones and amascarine act as weak bases, they can be protonated and reta ined inside acidic vesicles that are present in the cytoplasm (Larsen and Skladanowski, 1998). Drugs retained inside the lysosomes are then extruded from the cell via exocytos is (Tartakoff et al, 1983). Protonation and retention of topo targ eting agents inside cytoplasmi c lysosomes would theoretically inhibit drug transport into the nucleus. This hypothesis is supported by adriamycin-

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63 resistant MCF-7 cells that se quester protonated compounds w ithin vesicles, and secrete the drugs by endocytotic secretory pathwa ys (Schindler et al., 1996). Hence, drug resistant tumor cells are able to maintain a pH gradient between the acidic environment of lysosomes and the alkaline environment of th e cytoplasm, which ensures the entrapment of toxic substances inside acidi c vesicles and subsequent extrusion of drugs out of the cell via the trans -Golgi network. Conversely, drug sens itive cells fail to establish a pH gradient, which disrupts proper formation of acidic vesicles and functioning of the trans Golgi-network (Schindler et al., 1996). In summary, sequestration of chemotherapeutic agents inside vesicular compartments may have a role in altered drug sensitivity by reducing the amount of drug available to interact with its molecular target. 6.4 Pre-target Event: Drug Metabolism. Once inside a cell, some topo I and II targeting agents can be modified by metabolic enzymes. Modifications to chem otherapeutic drugs may be necessary for activation of a prodrug or can result in a less effective cytotoxic agent. For example, CPT-11 is an inert drug that must be converted to the active compound SN-38 by carboxylesterases. Thus, tumor cells expr essing low levels of carboxylesterases are resistant to CPT-11 (Danks et al., 1998). Furthermore, SN-38 can be detoxified when modified by one of several enzyme s belonging to the family of UDPglucuronosyltransferases (UGT) (Ciotti et al ., 1999). The UGT enzymes can convert SN38 into SN-38 glucuronide. Glucuronidation of SN-38 is associated with increased drug efflux from colon cancer cells and decreased dr ug sensitivity in colon, breast, and lung cancer cells (Cummings et al., 2002). Modification of DNA alkylating agents has also

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64 been attributed to altered drug sensitivity. DNA alkylating agents can be detoxified when modified by the enzyme glutathione S-transfer ase (GST). GST belongs to a family of enzymes that catalyzes the conjugation of gl utathione (GSH) to cancer drugs (Hayes and Pulford, 1995). Chemotherapeutic agents that are conjugated to glutathione are usually less active than their unconjuga ted counterparts. Glutathione conjugated drugs can also become substrates for the drug efflux pump, multidrug resistance protein 1 (MRP), and thereby pumped out of the cell (Paumi et al., 2001). Cellular drug resistance to anthracyclines is also associ ated with the intracellular amount of glutathione present because glutathione neutralizes free radicals. Some topo targeting agents, including the anthracyclines, exert th eir cytotoxic effect at least in part by producing free radicals. Glutathione is implicated in modulation of free radical formation by decreasing OH production. This is significant, because so me human tumor cells have significantly higher levels of GSH. In summary, acquired drug resistance to DNA alkylating agents is attributed to deactivation by GST and extr usion from the cell by MRP, whereas drug resistance to anthracyclines is attributed to neutralization of free radicals by GSH. Additional mechanisms of cellu lar drug resistance to topo I inhibitors has also been associated with induction of hepatic cytoch rome P-450, activation of nuclear factor kappa B (NFB), and activation of DNA-tyrosine phosphodi esterase (DTP1) (Frei et al., 2002; Yang et al., 1996; Harris and Hoc hhauser, 1992). DTP1 is an enzyme able to cleave the phosphodiester linkage between DNA and the active site tyrosine of topo I (Debethune et al., 2002). Yeast cells expressing DTP1 gene mutations are hypersensitive to CPT and thus, DTP1 may modulate CPT activity and affe ct cell sensitivity to topo I inhibitors (Cheng et al., 2002).

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65 6.5 Drug-target Events: Altered Quantity of Topoisomerase I or II Protein. Generally, topo targeting agents turn t opo into a lethal DNA damaging agent, so that the more target enzyme that is available to induce DNA damage, the more effective the agent. Thus, the amount of cleavable complex formation should increase as a function of increasing enzyme levels (L arsen and Skladanowski, 1998). This is supported by findings that the cytotoxicity of topo II poisons frequently correlates with topo II levels and the prolifer ative status of the cells in vitro (Sullivan et al., 1987a, 1987b; Chow and Ross, 1987). Since cancer ce lls often have a higher level of topo II protein and activity at all st ages of the cell cy cle, topo II is a fr equent target of antineoplastic agents. Tumor ce lls that are undergoing high ra tes of DNA replication are more sensitive to topo inhibitors because topo II is preferentially tr anscribed in S-phase cells and cells that are in S and G2/M have higher levels of topo II enzyme than nonproliferating cells. This has been demonstrat ed in breast cancer cell lines that are drug resistant to etoposide because of lower amounts of topo II protein relative to the parental cell line (Potmesil et al., 1988). Colorectal tumors show a 5-35 fold increase in topo I protein levels compared to normal colorectal cells. Topo I levels ar e also increased 2-10 fold in prostate tumors compared to benign pr ostate tissues. Altera tions in the expression of topo protein may be due to loss or gain of gene copies or due to altered gene transcription. For example, methylat ion of the topo I gene may account for downregulation of topo I protein content in resistant cells (Fujimori et al., 1996).

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66 6.6 Drug Target Interactions: Altered Quality of Topoisomerase I or II Protein. The phosphorylation status may contri bute to alterations in cellular drug sensitivity. Hyper and hypophosphorylation of t opo II has been associated with reduced cleavable complex formation and decreased dr ug sensitivity to topo II targeting agents (Ritke et al., 1994). Changes in topo II phos phorylation are also associated with both increased and decreased enzyme activity and a decrease in the cellula r content of topo II protein (Kroll, 1997). For exampl e, hyperphosphorylation of topo II by casein kinase II is associated with decreased protein levels and decreased drug sens itivity to etoposide in breast cancer cells (Matsumoto et al., 1997). However, topo II phosphorylation does not always result in attenuation of topo II content. HL60 cells selected for resistance to doxorubicin were found to be cros s-resistant to eto poside because of a reduction in the formation of VP-16 stabilized cleavable complexes, when compared to the parent cell line that were sensitive to both doxorubici n and etoposide (Ganapathi et al., 1996). However, the attenuation of cleavable complex formation was not a result of either a reduction in the amount or activity of topo II pr otein in these cells. The reduction in VP16 stabilized cleavable complex formation wa s attributed to a 3-fold decrease in the phosphorylation status of topo II Further investigations determined that, unlike topo II there is a concomitant hype rphosphorylation of topo II in the HL60 cells selected for doxorubicin resistance (Grabowski et al., 1999). Furthermore the changes in the phosphorylation status of both topo II and II correlated with an increase in drug sensitivity to m-AMSA a DNA intercalating agent that inhibits topo II. The increased drug sensitivity to m-AMSA was attribut ed to a 2-fold increase in topo II stabilized cleavable complex formation as compared to VP-16 -topo II cleavable complex

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67 formation. These results suggest that the phos phorylation status of the different topo II isoforms may have a role in drug sensitivity that depends on the specific mechanisms of drug action. Although topo I has been shown to be differentially phosphorylated during mitosis (D'Arpa and Liu, 1995), whether the changes in topo I phosphorylation have a role in conferring differences in drug sensit ivity to topo I targeti ng agents has not been established. Human purified topo I enzyme activity is inactivat ed by phosphatases and can be reactivated by casein kinase II or protein kinase C, suggesting that phosphorylation regulates topo I enzyme activit y (Kaiserman et al., 1988; Pommier et al., 1990). However, differently phosphorylated fo rms of topo I have been shown to cleave chromosomal DNA and relax supercoiled DNA w ith about equal topo I activity (D'Arpa and Liu, 1995). Alternatively, sumoylation fo llowed by redistribution of topo I from the nucleoli to the nucleoplasm in response to t opotecan or camptothecin exposure has been reported (Mo et al., 2002; Rallabhandi et al., 2002). It is not clear whether the translocation of topo I from the nucleoli to the nucleoplasm results in altered drug sensitivity to topotecan or camptothecin. A nother cellular mechanism that could confer cellular resistance to topo I poisons appears to be the degradation of DNA-linked topo I, which is mediated by ubiquitination and small ubiquitin relate d modifiers (SUMO) (Desai et al., 1997). Proteins modified with ubiquitin molecules are frequently destroyed by the proteasome, whereas sumoylation has be en shown to modulate gene transcription, protein-protein interactions, s ubcellular localization, and protei n stability. When Chinese hamster ovary cells are treated with campt othecin, topo I-DNA cleavable complexes are multi ubiquitinated and destroyed by the 26S proteasome mediated pathway. However,

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68 only 5-10% of the total amount of cellular topo I that wa s trapped in the cleavable complex after CPT treatment becomes conjuga ted to ubiquitin. Thus, it appears that a fraction of topo I cleavable complexes are ma rked for ubiquitin mediated proteolysis. Additional investigations are needed to address whether ubiquitin modified topo I cleavable complexes can result in altered drug sensitivity to camptothecin and whether proteasome mediated degradation of topo I cl eavable complexes is specific to CPT and topo I. Camptothecin also induces sum oylation of topo I on Lys-117, located in the amino terminal region of topo I (Rallabhandi et al., 2002). Sumoylation of Lys-117 is associated with the clearing of topo I from th e nucleoli to the nucleoplasm in response to CPT treatment. There are numerous examples found in th e National Library of Medicine that describe posttranslational modifications of t opo I and II occurring in cell lines that have been selected for resistance to topo targeti ng drugs (Yu et al., 2004; Matsumoto et al., 2001, 1999; Yanase et al., 2000; Yu et al., 1997; Kasahara et al., 1992) However, it is beyond the scope of this investigation to review the results of all of these in vitro studies. A reasonable conclusion that can be inferred fr om the collective resu lts of all of these investigations is that post-tra nslational modification of topo I or II likely has a role in conferring alterations in drug sensitivity by alte ring the amount or activ ity or the enzyme, cleavable complex formation, or the st ability of cleavable complexes. In vivo drug resistance is likely to be manifested by any one or more of these methods that is dependent on the drug class, en zyme targeted, and cell type.

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69 6.7 Drug Target Interactions: Topoisomerase Gene Mutations. In some cell lines, cellular drug resistan ce to topo I and II targeting agents has been attributed to both point mutations a nd deletions. Mutations in the topo I gene usually confers in vitro cellular drug resistance to one class of topo I targeting agents (Chang et al., 2002; Yanase et al., 2000). This is in contrast to topo II gene mutations that usually result in cells being cross-resist ant to all classes of topo II targeting agents (Larsen and Skladanowski, 1998). However, some point mutations in the topo I gene, including tyrosine 723 to phenylalanine (T723F) and tyrosine 727 to phenylalanine (T727F), result in cells that are resistant to CPT and the indolocarbazo le derivates of the antibiotic rebeccamycin (Woo et al., 2002). T opo I gene mutations conferring resistance to the camptothecins have been found in drug resistant human cell lin es, yeast cells, and in tumor tissue from patients treated with ir inotecan (Tsurutani et al., 2002). Mutations in the topo I gene appear to cluster either around the active s ite tyrosine residue (Tyr723) or around amino acids 361 through 364 (Lar sen and Skladanowski, 1998). Gene mutations that involve amino acids 361, 362, 363, and 364, confer drug resistance to CPT and are believed to alter the DNA-enzyme binding properties of CPT (Rasheed and Rubin, 2003; Larsen and Skladanowski, 1998). Point mutations that occur in the core domain of topo I have been attributed to cel lular drug resistance to camptothecin because of a reduction in topo I-DNA interactions (Kin gma et al., 1999). Camptothecin resistant P388 murine leukemia cells contain both gene rearrangements and hypermethylation in one allele of the topo I gene, resulting in a reduction of topo I enzyme activity and mRNA transcripts (Tan et al., 1989).

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70 Mutations in the topo II gene that confer altered ce llular drug sensitivity to topo targeting agents have been studies in both yeast and mamm alian cell lines (Larsen and Skladanowski, 1998). Point mutations in yeas t cells have been shown to confer drug resistance to etoposide (Kingma et al., 1999) A point mutation in yeast topo II that results in histidine 1012 changed to a tyrosi ne (H1012Y) residue corr esponds with a 3-4 fold increase in drug resistance to etoposide as compared to yeast cells expressing nonmutated topo II enzyme (Kingma et al., 1999) The observed drug resistance in these cells was explained by a decreased affinity of etoposide-enzyme binding as compared to non-mutated enzyme (Kingma et al., 1999). In human cell lines, selection for drug resistance to etoposide or teniposide has been shown to result in point mutations in the topo II gene (Patel et al., 1993; Bugg et al., 1991). Mutations found in human topo II in drug resistant cell lines tend to cluster either around ATP binding domain or the active site tyrosine residue (Nitiss and Beck, 1996). Furthermore, since topo II forms dimers, it is possible that heterodimers exist that consist of one drug-sensitive and one drug resistant subunit. Many cell lines that have been selected fo r resistance to topo II poisons in vitro have been shown to carry mu tations in the gene for topo II but mutations are rarely reported in patient samples (Wessel et al., 2002). Alterations in topo gene copy number can also occur in human cancers. For example, the topo II gene is coamplifed with the HER-2/neu oncogene in breast, ova rian, and bladder cancer cells because the two genes are in close proximity to each ot her (Simon et al., 2003; Hengstler et al., 1999). This is significant because an increase in topo II gene copy number is associated with cancers that have increased sensitivity to topo II poisons such as doxorubicin, while a deletion of one copy of the topo II gene is associated with resistance to doxorubicin.

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71 The only mutation in topo II that has been reported to date is derived from drug-resistant cell lines which contain topo II gene mutations that completely abate the activity of the enzyme (Dereuddre et al., 1995). No study has been reported that a ssesses mutations in topo II from clinical specimens. 6.8 Drug Target Interactions: Altered Chromatin Structure. The topological structure of DNA modulates the leve l of topoisomerase binding (Zechiedrich and Osheroff, 1990; Howard et al., 1991). In general, eukaryotic topoisomerase I and II type enzymes pref er positive and negatively supercoiled DNA over relaxed DNA substrates (Osheroff, 1986). Modifications such as glycosylation and methylation have been shown to influen ce DNA conformation; however, the molecular mechanism leading from DNA modification to cellular drug resistance is not well documented (Stopper and Boos, 2001; Lopez-Ba ena et al., 1998). In mammalian cells, DNA methylation of cytosine in CpG islands is an importa nt regulatory mechanism of gene expression. DNA methylation has al so been shown to influence local conformational changes in the structur e of DNA (Zacharias et al., 1988; Diekmann, 1987) DNA methylation is associated with remodeling of euchromatin to heterochromatin, suggesting that methylated DNA somehow alters th e accessibility of DNA to transcription factors and regulatory proteins (Hori, 1983). Conformational changes that occur in methylated DNA have been also been suggested to alter the binding and DNA cleavage properties of topo I and II (Boos and Stopper, 2001; Leteurtre et al., 1994). For example, topo I and II cleavage patterns are modified in vitro by CpG methylation in c-myc gene DNA fragments (Leteurtre et al., 1994). Topo I and II

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72 cleavage activity is shown to be dependent on the location of the methylated cytosine on the DNA in relation to the scissile bond. Me thylated cytosine ha s both suppressive and stimulatory activity on topo I, whereas topo II cleavage activity is mostly suppressed by methylated cytosine. DNA met hylation of CpG islands been proposed to have a role in tissue selectivity and efficiency of DNA topo ta rgeting agents (Leteu rtre et al., 1994). DNA methylation has also been shown to a lter topo II decatenation activity in vitro, which may result in genomic instability, but whether this contributes to cellular drug resistance is not know n (Zwelling et al., 1989). 6.9 Post-target Events: DNA Repair. Both topo I and topo II have roles in DNA damage recognition and repair processes, but how cells repair drug-sta bilized DNA cleavable complexes is poorly understood. Sensitivity to DNA-cr osslinking reagents is inve rsely proportional to topo levels and thus, elevated levels of topo II correlate with resistance to DNA cross-linking agents such as melphalan and Cisplatin (Dvor akova et al., 2002; Hi rota et al., 2002). This has been explained by the involvement of topo II in processing DNA damage induced by melphalan (Hirota et al ., 2002). A deficiency in topo II activity correlates with increased drug sensitivity because of an increase in melphalan-induced sister chromatid exchanges (Karpinich et al., 2002). These results suggest th at the efficacy of melphalan in cancer chemotherapy can be improved through inhibition of topo II Similar observations have been reported in H69 small-cell lung cancer cells resistant to radiation and cisplatin, in part because of an in crease in topo II expression (Hennes et al., 2002). The results suggest that increased topo II expression may confer radiation

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73 and drug resistance because of increased DNA repair, and that radiation followed by a topo II targeting agent ma y be a potential strategy for overcoming resistance in small cell lung cancer. DNA repair pathways have also been implicated in conferring sensitivity to etoposide and camptothecin in hu man colorectal cancer cells (Jacob et al., 2001; Aebi et al., 1997). DNA mismatch re pair (MMR) is normally involved in correcting base/base mismatches during DNA re plication but it is also implicated in double strand DNA break repair and for modulat ing the induction of apoptosis as a result of various types of drug induced DNA damage Cell lines defective in DNA mismatch repair pathways show increased sensitivity to both CPT and etoposide as compared to MMR proficient colorect al cell lines and sensitivity to th ese drugs does not correlate with endogenous levels of topo I or t opo II (Jacob et al., 2001). In summary, differences in the processing and repair of DNA lesions are likely to have a role in drug sensitivity to topo targeting agents. It is plausible that the way cells remove and repair drug-stabilized cleavable complexes could influence cellu lar sensitivity to DNA damaging agents. 6.10 Post-target Events: Alterations in Cell Cycle Progression. The cellular content of topo II protein is highest during G2/M and lowest during early G1 of the cell cycle (Meyer et al., 1997; Nakajima et al., 1996). Topo II protein is also markedly decreased in growth-arrested cells as compared to proliferating cells (Holden et al, 1990, Turley, et al, 19 97). In contrast, topo I and topo II have a relatively uniformed expression throughout the ce ll cycle as compared to topo II (Drake et al., 1989). The cell-cycle and proliferati on dependent fluctuations of topo II gene expression may also explain why some sl ow growing cancers may be intrinsically

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74 resistant to topo inhibitors. Therefore, the application of drugs specifically directed against topo II could be useful for tumors that express a mutant form of topo II or have a large fraction of cells in Go/G1. In these cases, topo II could be a preferred molecular target because unlike topo II topo II expression is not depe ndent on the cell cycle or proliferative stage of the ce ll. XK469 is a new anticancer agent that is reported to specifically target topo II although it may also have an an ti-proliferative role involving cell-cycle related proteins (Snapka et al ., 2001; Gao et al., 1999). XK469 exerts its cytotoxic effect by arresting cells in prophase as a result of an irreversible accumulation of cyclin B1 in the G2/M phase of the cell cycle (Di ng et al., 2001). XK469 has been shown to have activity against a broad spectru m of murine solid tumors, including tumors that express the multi-drug resistance gene (LoRusso et al., 1998-99). 6.11 Post-target Events: Altered Cell Death Pathways. Certain cellular conditions such as glucose starvation, hypoxia, and DNA damage, are associated with drug resistance to topo II inhibitors, such as etoposide and adriamycin, because of a decrease in topo II gene expression. A decrease in transcription of topo II gene correlates with increas ed levels of Sp-3, YB1, and p53 status. Several studies have investigated the role of p53 in res ponse to topo mediated DNA damage (reviewed in Valkov and Sulliva n, 2003). The p53 protein has been shown to interact with topo II in vitro and stimulates the decatenatio n activity of topo II (Cowell et al., 2000). The p53 protein can arrest ce ll cycle progression a nd can commit cells to programmed cell death (Bladoskl onny, 2002). Since p53 is mutated in a large percentage of cancers, this could suggest that a failure to induce apoptosis results in resistance to

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75 DNA-damaging agents (Bunz et al., 1999). Since topo-targeting agents kill cells by inducing DNA damage, p53 deficient cells coul d be resistance to topo poisons. Other alterations in the apoptotic pathway may al so confer cellular drug resistance to topo inhibitors. For example, an elevated level of the anti-apoptotic protei n bcl-2 is associated with decreased drug sensitivity in cancer cells (Real et al., 2004). 6.12 Conclusions. In summary, cancer cells have multiple different ways to evade the cytotoxic effects of topo targeting drugs. Tumor cells with de novo resistance fail to respond to initial drug treatment.. In contrast, tumor ce lls with acquired drug re sistance are initially sensitive to drug exposure, but then become resistant with future drug exposures. The most commonly observed mechanisms of in vitro drug resistance to topo targeting agents usually describes alterations in drug transport or alterations in the amount or activity of topo II protein that may or may not be accompanied by topo II gene mutations.

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76 Chapter Seven In vivo Mechanisms of Topoisomerase Associated Drug Resistance 7.1 Introduction. The mechanisms of drug resistance desc ribed for topo I and topo II inhibitors in model human cell lines have been well established, but the actual in vivo situation in patients with solid tumors and hematologic malignancies is still uncertain. Mechanisms of in vivo drug resistance to topoisomerase targ eting agents are usually related with alterations in drug transpor t and the quantity of topo pr otein or mRNA in clinical preparations. However, in vivo drug resistance to topo inhi bitors is likely to be a phenomenon influenced by a network of physio logical factors, such as drug dosage, presence of the molecular target, the tumo r microenvironment, and presence or absence of specific genetic aberrations (reviewed in Lehnert, 1996). The c linical activity of chemotherapy is also dependent on hepati c drug metabolism, extracellular drug concentration, intracellular drug transport, a nd ability of the drug to access the cancer cells. Furthermore, antitumor agents differ in their ability to diffuse through different tissues. For example, scar tissue from surg ery or radiotherapy reduces response rates with chemotherapy, especially in head and neck cancers (Rooney et al., 1985). Furthermore, some tumors can be poorly vasc ularized and some drugs are unable to cross

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77 the blood brain barrier. Therefore, various in vivo factors can influence how much active drug reaches the molecular target withincancer cells. Cellular drug resistance in humans is complex and appears to be both multif actorial and variab le amongst individual patients. The work presented herein definitively describes an in vitro mechanism of drug resistance observed to topo II targeting ag ents in a human leukemia and myeloma cell lines. Although clini cal applications are speculated from the in vi tro data presented in this work (refer to pages 201-203 he rein), a complete description of in vivo mechanisms of cellular drug resistance in the clinical environment was outside the limits of this investigation (reviewed in Enge l et al., 2003). The focus here is to briefly describe our current understanding of clin ical drug resistance to topoisomerase targeting chemotherapeutic agents. This chapter focu ses on gene mutations, cellular content of topo I and II, and the tumor microenvironment as mechanisms of clinical drug resistance to topo targeting chemotherapeutic agents. 7.2. Gene Mutations There has been a constant search for point mutations, deletions, truncations, and atypical isoforms of topo in a wide variety of clinical samples and diverse tumor types. Several genetic aberrations, such as allelic inactivation, deletions and point mutations, have been reported to occur in the gene fo r topo I and II in human cell lines. However, topo I and II gene mutations are rarely detect ed in patient samples. Thus far, the bronchial aspirate of one patient with small cell lung cancer treated w ith either etoposidecontaining chemotherapy is the only reported case to have identified two point mutations

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78 in the topo II gene (Kubo et al., 1996). DNA sequence analysis of this tumor show two transversions at codons 486 (G to A) and 494 (A to G), resulting in two missense mutations (arginine to lysine and glutamate to glycine, respectively). More frequently, chemotherapy with topo II inhibitors leads to gross genomic rearrangements, including gene amplifications and deletions of the topo II gene (Jrvinen and Liu, 2003). For example, a recent clinical trial has detected topo II gene amplification by fluorescence in situ hybridization (FISH) in clinical samples from primary breast cancer tumor samples, which correlates with response to topo II inhibitors (Jr vinen et al., 1999). Breast cancer seems to be one disease wher e amplification or deletion of the topo II gene frequently occurs (Jrvinen and Liu, 2003). Furthermore, topo II has been shown to be a marker of tumor aggressiveness and invasive potential in breast cancer, but the complexity of the response of breast tumors to chemotherapy is usually determined by evaluation of several mark ers (Cardoso et al., 2001; Nakopoulou et al., 2000). There have been a limited number of clinical studies reporting topo I gene mutations (Ohashi et al., 1996). One patien t with large cell lung carcinoma who did not respond to cisplatin or irinotecan was found to have a missense mutation at codon 737 in the topo I gene (Tsurutani et al., 2002). Ho wever, it was not determined if the missense mutation altered CPT induced topo I cytotoxici ty. Colorectal carc inomas have a high expression of topo I protein that is due to an increased topo I gene copy number, reaching up to eight copies (Boonsong et al., 2002, 2000). For this reason, irinotecan, a topo I inhibitor, is being evaluated in clinical tria ls for the effective tr eatment of colorectal carcinoma. Additional clinical studies are needed to determine if gene mutations found in drug resistant cell lines are clinically relevant.

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79 7.3 Topoisomerase I and II Gene and Protein Expression. The amount of topo protein has been investigated as a predictive factor for clinical response to t opo I and topo II targeting chemot herapeutic agents. All studies point to a substantial interpatient variability in the amount of topo content and often fail to correlate the amount of topo enzymes with either the prognosis or with the clinical response to topo I and topo II inhibitors used in different combinations (McKenna et al., 1994). For example, in acute lymphoblasti c leukemia (ALL), Western blot analysis, polymerase chain reaction (PCR), Northern blot analysis and immunohistochemistry demonstrate that topo I and topo II gene and pr otein expression are variable (Stammler et al., 1994; McKenna et al., 1994). In these studies, a total of 60% of the blasts are shown to be positive for topo II while the levels of topo I are in the range of 25-85% of the control cell line. However, no correlation is found between the level of topo and clinical response. 7.4 Tumor Microenvironment. Additional physiological conditions exist in vivo that contribute to decreased drug sensitivity. For example, under physiological conditions in the bone marrow, tumor cells aggregate to form tumor cell masses that c onfer drug resistance. This is supported by in vitro studies demonstrating that when cancer cells are cultured in monolayers they are more susceptible to cytotoxic agents than when cultured as 3D aggregates, a phenomenon referred to as multicellular drug resistance (Jia nmin et al., 2002; De soize and Jardillier, 2000)). Cell adhesion mediated drug resi stance (CAM-DR) is another phenomenon whereby tumor cells bind stromal cells or extr acellular matrix prot eins (Dalton, 2003).

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80 CAM-DR represents an intrinsic mechanism of drug resistance used by malignant cells (Dalton, 2003). Collectively, these findings suggest that the resi stance to anticancer drugs that are often observed in the clinic is likely to be a result of many physiological conditions that act simultaneously (Matte rn and Volm, 1993). This hypothesis is supported by in vitro findings that cells selected for drug resistance have multiple mechanisms conferring decreased drug sensitivity (Hazlehurst et al., 1999; Harker et al., 1995). An example of an in vitro cell model that supports this theory comes from the mitoxantrone resistant 8226/MR4 8226/M20 my eloma cells (Hazlehurst et al., 1999). When compared to the drug-sensitive parental cell line, the 8226/MR 4 cells were 10-fold resistant to mitoxantrone a nd 4-fold resistant to etoposid e, whereas the 8226/M20 cells were 37-fold resistant to mit oxantrone and 15-fold resistant to etoposide. Drug resistance in both cell lines was associated with d ecreased drug accumulation in a P-gp and MRP independent mechanism. Decreased drug accumulation in these cells was attributed to an ATP dependent ouabain-insensitive drug transpor t mechanism. Drug resistance could be reversed by the chemosensiti zer, fumitrenogin C. However, drug resistance in the 8226/M20 cells was also attributed to a 70-88% reduction in topo II and topo II protein content and reduced topo II enzyme activit y, which was not observed in the 8226/MR4 cells (Hazlehurst et al., 1999). Thus, can cer cells have numerous mechanisms for overcoming the drug cytotoxicity.

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81 7.5 Conclusions. Resistance to chemotherapy is a common problem in patiens with cancer and a major obstacle that must be overcome for cancer therapy to be effective. In vivo drug resistance is likely to be a multifactorial problem. Tumor cells can manipulate their tumor microenvironment to gain growth and proliferative advantages and some tumor cells may have gene mutatins that contribute to altered drug sensitivity. Therefore, clinicians must consider nu merous biological variables th at may differ between patients to optimize the effectiveness of cancer chemotherapy.

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82 Chapter Eight Nuclear-cytoplasmic Trafficking of Proteins 8.1 Introduction. The separation of the nucleus from the cy toplasm plays a centra l role in regulating the subcellular locali zation of proteins, but is also a variable in drug delivery and cytotoxicity. Generally, topo poisons conve rt the enzyme into a lethal DNA damaging agent, such that the more active enzyme that is present in the nucleus, the more effective the poison (reviewed in Engel et al., 2003). Without the co-existen ce of the ternary drugDNA-topo complex, topo inhibitors are less effective as cytotoxic agents. Thus, investigating the selective movement of macromolecules across th e nuclear envelope represents a recent area of drug development (Dean et al., 2003). The potential of the DNA topoisomerases to shuttle between the nucle us and cytoplasm has been one area of recent investigations (Engel et al.2004; Valkov et al., 2000). 8.2 The Nuclear Pore Complex. Recent investigations have elucidated se veral molecular pathways for the nuclear import and export of proteins across trans port passageways or nuclear pore complexes (NPCs) (Weiss, 2003). The NPCs are located in the nuclear envel ope where the inner

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83 and outer membranes are fused, providing a lin k between the cytoplasm and the interior of the nucleus (Davis, 1995; Doye and Hurt 1997; Grlich and Mattaj, 1996). The NPC is a large (125 MDa) (Reichelt et al., 1990) multimeric protein structure that perforates the nuclear envelope and channe ls proteins greater than 40 kD a into or out of the nucleus (Pante and Kann, 2002; Hinshaw et al., 1992). The NPC consis ts of a central ring-spoke structure flanked by filaments emanating fr om its nuclear and cytoplasmic surfaces (Hinshaw and Milligan, 2003; Akey and Rade rmacher, 1993). The pores are 25 nm in diameter and form 9 nm long channels (Peter s, 1986). The number of NPCs per nucleus varies with the organism, cell type, and gr owth conditions (Maui, 1977). It is common for cells to have between 102 and 5 X 107 NPCs present per nucleus, depending on the metabolic or differentiation state of the cell. Mammalian cells typically have 3,000-5,000 NPCs, whereas yeast cells have approximately 120 NPC per nucleus (Winey et al., 1997). 8.3 Nucleoporins. The NPCs are composed of approximately eight copies of 30 different proteins, the nucleoporins (Nups) (Rabut et al., 2004). A number of membrane and nonmembrane anchored Nups have been described in yeast and mammalian cells by mass spectroscopy (Cronshaw et al., 2002). Nup210 and glycopr otein 121 (gp121) are believed to play critical roles in anchoring the NPC to the nuclear envelope (Wozni ak et al., 1989). The Nups are phosphorylated in a cell-cycle dependent manner and are hyperphosphorylated during M-phase (Favreau et al., 1996). During mitosis, the nuclear membrane breaksdown and the NPC is disassembled. Phos phorylation of nucleoporins is associated with the disassembly of the NPC as well as reformation of the NPC when cell division is

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84 complete. Nucleoporins contain characterist ic domains featuring multiple repeats of short peptide sequences e nding in the amino acids phe nylalanine and glycine (FG repeats) (Bayliss et al., 2000). The FG repeat s provide docking sites fo r proteinreceptor cargo complexes (Radu et al., 1995). Binding of transport receptors to the FG domains on the nucleoporins triggers the pore to dilate (Shahin et al., 2005; Shulga and Goldfarb, 2003). One model suggests that multiple FG ri ch repeats in the NPC channel form a meshwork through weak hydrophobic interact ions, which can function as a sieve (Ribbeck and Grlich, 2001). Hydrophobic r eceptor-cargo complexes can transiently interact with the hydrophobic re peats, and thus dissolve the meshwork and translocate thorough the NPC. In contrast, hydrophilic pr oteins remain excluded, explaining the high selectivity of the NPC. Tr ansport through the NPC machinery appears to be regulated by mitogenic signals, phosphorylation, and Ca2+ concentrations within the endoplasmic reticulum and nuclear lumen (Harreman et al., 2004; Greber and Gerace, 1995). For example, Ca++ stores in the nuclear envelope of Xenopus laevis oocytes, is suggested to regulate NPC gating (Perez-Terzie et al., 1996). In these cells, conformational changes in the NPC require calcium, such that when the Ca++ stores are depleted, a central plug occludes passage of nuclear transport processe s. Similar results have been observed in HeLa cells grown in the presence of the calcium pump inhibitor, thapsigargin, which blocks signal-mediated protein import (Grebe r and Gerace., 1995). Th ese results suggest that calcium stores present in the lume n of the endoplasmic reticulum, which is contiguous with the nuclear envelope, may ha ve a role in regulating nuclear–cytoplasmic trafficking.

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85 8.4 Nuclear and Cytoplasmic Targeting Sequences. Since 1999, when Gnter Blobel received the Noble Prize in Physiology and Medicine for discovering that proteins have intrinsic signals that govern transport and localization in cells, there have been significant advancem ents in understanding the mechanisms of nuclear-cytoplasmic transpor t of proteins (Raju, 2000; Shields, 2001). Investigative reports now desc ribe many different types of nuclear-cytoplasmic targeting sequences, including nuclear localization se quences (NLS), nuclear export sequences (NES), nuclear retention sequences (NRS), cytoplasmic retention sequences (CRS), and bi-directional shuttling sequen ces (Jans et al., 1998). Prot eins targeted for receptormediated transport across the NPC must eith er contain an NLS or NES. However, some proteins that do not contain a nuclear-targe ting sequence still traverse the NPC by piggybacking with another protein containing a NLS or NES. 8.5 Nuclear Localization Signals. The first NLS, P KKKRK was identified in the Simian Virus 40 (SV 40) large T antigen (Kalderon et al., 1984). The NLS f ound in the SV40 large T antigen was later referred to as a monopartite sequence to di stinguish it from the bipartite sequence, KR PAATKKAGQA KKKK LD, found in Xenopus nucleoplasmin (Rob bins et al., 1991). The monopartite sequence is often referred to as the “classical” NLS and consists of a short cluster of basic amino acids, often pr eceded by an acidic amino acid or proline residue. Human DNA topo I has a classical monopartite NLS between amino acids 150156 (Stewart and Champoux, 1996). The amino ac id sequence of the monopartite NLS in topo I is KKIKTED. Recently, a second non-cl assical monopartite signal was identified

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86 in topo I between amino acids 117-146 (Mo et al., 2000). In contrast to the basic amino acid residues found in a classical NLS, this NL S is unique because it is characterized by two clusters of acidic ami no acids at amino acids 117-146 and 151-156. The first cluster of acidic amino acids is KDEPEDDG followe d by a spacer region of 16 amino acids, and then the second cluster of acid amino aci ds is DEDDAD. Thus, topo I has two independent NLS with opposite charged ami no acids. The precise amino acid sequence that targets topo II to the nucleus has not been determ ined, but the last 116 amino acids found in the C-terminal domain in necessary and sufficient for nuc lear localization of topo II in mammalian cells (Cowell et al., 1998). The C-terminal domain is presumed to have a bipartite NLS, which has been im plied by the presence of several bipartite NLS type motifs found in the C-terminal domain of topo II Bipartite NLS, a second type of NLS, c onsists of two clusters of basic amino acids separated by a spacer region of approxi mately ten amino acids, often flanked by a neutral or acidic amino acid (Robbins, et al., 1991). Topo II contains a bipartite NLS located in the carboxy-terminus between amino acids 1454-1497 that consists of a spacer region of 21 amino acids (Mirski et al., 1997). The amino acid sequence for the NLS in topo II is KPDPA K T K N RRKRK PSTSDDSDSNFEKIVSKAVTS KK S K GESDD (Mirski et al., 1997). The two clusters of basic amino acids are shown in bold text. Computer software programs are available that can scan amino acid sequences for potential bipartite NLS (Shelagh et al., 1999) Additionally, previously described NLS are annotated in SWISS-Prot and PIR and can be retrieved at the NLS database located at the Predict NLS server (Nair et al., 2003).

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87 8.6 Nuclear Export Signals. Classic protein NES are hydr ophobic rich sequences that have a characteristic spacing of leucine, isoleucine valine, and/or phenylalanine amino acid residues (Elfgang et al., 1999). The Center for Bi ological Sequence Analysis at the Technical University of Denmark has prepared an NES predictor, call ed the NetNes 1.1 server, accessible to the public and found on the worldwide web (la Cour et al., 2004). The NetNes 1.1 server uses a prediction algorithm based on previously described NES, but also uses a “hidden Mark model” to identify non-hydrophobic residu e that are not included in the consensus sequences for classical NES. To date, approxi mately 80 experimentally validated proteinNES have been identified and compiled on the NESbase version 1.0 database, which is also accessible on the worldwide web (la C our et al., 2003). NES have been identified based on the fact that mutation or deletion of the NES renders it inactive in mediating nuclear export of the protein ca rrying it, or that the NES is active in effecting export from the nucleus of a carrier protein such as beta-galactosidase (Jans et al., 1998). 8.7 Retention Signals. Some proteins contain nuclear retention or cytoplasmic retention signals, which do not specifically target a protein to th e nuclear or cytoplasmic compartments, but assures their retention once ther e (Jans et al., 1998). The nucle ar retention signals usually consist of an arginine rich motif and may include an arginine rich -helical structure. One example of a NRS has been described in an SR protein. The SR proteins are RNA splicing factors that contain RNA recognition motifs and a C-terminal domain rich in alternating serine (S) and arginine (R) amino acids, known as the RS domain. The RS

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88 domain can become phosphor ylated and direct the s ubcellular localization and nucleocytoplasmic shuttling of individual SR proteins (Cazalla et al., 2002). The human SC35 SR protein, however, contains a dominant nuclear retention sign al located in the RS domain, and thus is not shuttled between the nucleus and cytoplasm. The nuclear retention signal for the SC35 SR protein is, PPPVSKRESK SRSRS KSPPKSPEEEGAVSS (Cazalla et al., 2002). The adenovirus E4orf6 protein also contains an amphipathic arginine rich -helical region with the amino acid sequence ARRTRRLMLRAVRIIAE, which has been associated with E4orf6 nuc lear retention. The cytoplasmic retention signals are less well characterized (Satoshi et al., 2003). For ex ample, cyclin B1 contains a cytoplasmic retention signal that consists of a stretch of 45 amino acids located in the amino terminus (Pines and Hunter, 1994). The CRS is situated within a CRM1 dependent NES of cyclin B1 (Yang et al., 1998). During interphase, cyclin B1 is exported to the cytoplasm via the NES and is retained in the cytoplasm through its cytoplasmic retention signal. Cyclin B1 is then imported back into the nucleus by NLS phosphorylation (Hagting et al., 1999; Li et al., 1997). Cy toplasmic retention signals have been identified in human and Xenopus cyclin B1 and Xenopus cyclin B2 (Yoshitome et al., 2003; Pines and Hunter 1994). The amino acid sequence alignments for CRS and NES for human and Xenopus cyclin B are shown in Figure 16.

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89 Human cyclin B1 142 PVKEEKLSPEPI LVDTASPSPMETSGCAPAEED LCQAFSDVIL AVN 154 Xenopus cyclin B1 91 EP SSPSPMETSGCLPDE LCQAFSDVLIHVKDV DADD 126 Xenopus cyclin B2 89 PSPVPMDVSLKEEE LCQAFSDALTSVEDI DADD 121 Figure 16. Cytoplasmic Retention Si gnals and Nuclear Export Sequences for Cyclin B. The CRS in human cyclin B1 consists of a stretch of 45 amino acids (residues 142-154) and a NES between amino acids 142151. The CRS for Xenopus cyclin B1 and B2 contains amino acid residues 91-126 and 89-121, respectively. All of the NES (shown in bold text) that have been found in either cyclin B1 or B2 are nested within the CRS. (Yoshitome et al., 2003; Pines and Hunter, 1994). 8.8 Bi-directional Shuttling Signals. Another type of shuttling signal is characterized by the M9 sequence of heterogeneous nuclear ribonucleoprotein A1, wh ich consists of a 38-amino acid stretch that is enriched in aromatic residues and glycine (Pollard et al., 1996). The M9 sequence functions in both nuclear import and nuclear ex port and is now referred to as a “shuttling sequence” to distinguish it from the unidirect ional targeting sequences (NLS and NES). 8.9 Signal-mediated Nuclear Transport. Active transport between the nucleus and cytoplasm involves primarily three classes of macromolecules: substrates, adaptors and receptors. In general, protein import occurs when the transport receptors, importinand importin, form a complex with the protein-NLS and escort the protein cargo ac ross the NPC into the nucleus (Figure 17) (Nigg, 1997; Grlich and Mattaj, 1996; Moroia nu et al., 1996). The protein cargo is released into the nucleus when the guanine nucleotide exchange factor, RCC1/RanGEF, replaces Ran bound GDP with GTP. In the presence of GTP. importinbinds the export receptor, cellular apoptosis susceptibility prot ein (CAS), and is transported back into the cytoplasm. Importinremains bound to GTP and is recycl ed back to the cytoplasm.

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90 Once in the cytoplasm, RanBP1 and RanGAP1 hydrolyze the bound GTP to GDP (Gasiorowski and Dean, 2003). Protein export occurs when RanGTP and the nuclear export receptor, chromosome region maintenance-1 (Crm-1 or exportin-1), bind to a protein bearing an NES and transports the protein cargo into th e cytosol (Figure 18) (Fukuda et al., 1997). The protein cargo is released into the cytoplasm when Ran-GTP is hydrolyzed by RanBP1 and RanGAP1, small GTPase activatin g proteins (Yamada et al., 2004; Grlich et al., 2003). In this manner, continue d nuclear-cytoplasmic shuttling occurs by maintaining a gradient of Ran-GTP in th e nucleus and Ran-GDP in the cytoplasm (Grlich et al., 1996). Several notable exceptions to this general mechanism have been reported (Miyamoto, 2002). For example, HIV-1 Re v proteins are recognized by importinrather than the importin/importinheterodimer (Tiganis et al., 1996). Moreover, the accessory protein Tat of HIV-1 Rev is not recognized by either importin or (Efthymiadis et al., 1998), but rather, by other receptors related to importin At least 21 different importin family members have been identif ied in humans. Furthermore, Crm1 belongs to the growing Kap family of proteins (table 6) (Moroianu, 1998). Crm-1 was identified as a general receptor for protei ns bearing hydrophobic rich NES, but table 6 establishes that recepto r mediated nuclear export may be mo re specific (Jans et al., 2000). Many more import and export receptors are believed to exist, so it is possible that nuclear import and export pathways show sufficient sp ecificity to be considered targets for therapeutic intervention. The pr ecise nuclear transport pathwa ys for topo enzymes has not been elucidated (Engel and Turner et al., 2004; Mirski et al., 2003).

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91Figure 17. Nuclear Import Pathway. Protei n cargo containing a nuclear localization signal binds to the nuclear import receptor importin Importin -bound to Ran-GDP forms a complex with the importinprotein cargo and escorts the entire complex across the nuclear pore complex. Once inside the nucleus, the nucleotide exchange factor, RCC 1, replaces Ran-GDP w ith Ran-GTP. Importin-Ran-GTP is transported back to the cytoplasm.where GTPases recycle Ran-GTP back to Ran-GDP. Importinis exported from the nucleus by the export r eceptor, cellular apoptosis susceptibility protein (CAS).

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92 RanGAP RanBP1 Nucleus Cytoplasm CRM-1 CRM-1 Cargo CRM-1 Recycled CRM-1 CRM-1 Ran-GDP Cargo Figure 18. Nuclear Export Pathway. Th e presence of guanine nucl eotide exchange factors in the nucleus maintains a high concentration of Ran-GTP in the nucleus and Ran-GDP in the cytoplasm. In the nucleu, Ran-GTP binds the export receptor, Crm-1 and a NES-containing pr otein cargo. Crm-1 escorts the complex across the nuclear por complex. In the cytoplasm, Ran-GTPases hydrolyze GTP to GDP. Crm-1 and Ran-GDP are recycled. Recycled Ran-GDP Cargo Ran-GDP Ran-GTP Ran-GDP Ran-GTP RCC1/RanGEF

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93Table 6 Nuclear Export Receptors. Mammalian Kap Family Export Receptors Protein Receptor Export Cargo CRM-1 (exportin-1) general export receptor for leucine rich export sequences CAS importinImportin-13 eukaryotic translation factor-5A Exportin-4 hypusine-modified eukaryote translation factor-5A* Exportin-5 dsRNA binding proteins Exportin-6 actin export Exportin-7 p50RhoGAP Non-Kap Family Nuclear Export Receptors Mex67p (yeast) mRNA TAP/NXF (metazoans) mRNA *hypusine, [N(epsilon)-(4 amino-2-hydroxybutyl )lysine], is a rare amino acid. Hypusine is formed when a lysine residue is post-tran slationally modified by the transfer of a butylamine from spermidine followed by hydroxylation reaction (Mehta et al., 1994; Shiba et al., 1971). 8.10 Regulating Nuclear-cytoplasmic Transport. Signal mediated transport has been show n to be regulated by several cellular processes that are summarized in Figure 19. Phosphorylation (Engel et al., 1998), ubiquitination (Lohrum et al., 2001), and sum oylation (Salinas et al., 2004) are the most widely reported mechanisms of transport re gulation. Like ubiquitination, sumoylation is a three-step process involving an EI-activ ating enzyme heterodimer Aos/Uba2, the E2conjugating enzyme Ubc9 and substratespecific E3 ligases (Gill, 2004). The nucleoporin Nup358 (or RanBP2) is an example of an E3 ligase involved in modifying nucleo-cytoplasmic shuttling proteins with small ubiquitin-like modifiers (SUMO)

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94 (Azumo and Daso, 2002). This finding illust rates the intimate association of SUMO modification in regulating nuclear-c ytoplasmic protein transport. Several drugs and molecular agents have been used to decipher transport processes in the laboratory (T able 15 appearin g on page 213) LMB is an unsaturated branched-chain fatty acid isolated from Streptomyces pombe (Hamamoto et al, 1983a). LMB is an antifungal antibacterial agent w ith anti-tumor activity (Hamamoto et al., 1983b) and was also shown to be an in hibitor of the nuclear export receptor, chromosome region maintenance protein-1 (C rm-1) (Kudo et al., 1998). LMB attacks the sulfydryl groups on cysteine-529 of Crm-1 in a Michael-type reaction (Kudo et al., 1999). In this way, LMB blocks all Crm-1-mediated nuclear export. The ratjadones represent a new class of natural compounds which inhibi t proliferation in eukaryotes by blocking nuclear export (Meissner et al ., 2004). Like LMB, the ratjad ones covalently bind to Crm1. Additional compounds are being screened for their ability to bloc k nuclear-export of proteins at different point s in the export pathway 8.11 Conclusions. The nuclear-cytoplasmic traffick ing proteins have potenti al applications in drug delivery and drug-target inte ractions. The nuclear pore complexes are the gateway between the nuclear anc cytoplasmic compar tments. Protein transport across the NPC requires the presence of nucle ar localization or nuclear e xport signals. Crm-1 has been identified as the major export receptor that tr ansports NES containing proteins out of the nucleus. Leptomycin B specifically inhibits Crm-1 mediated nuclear export of proteins and is being investigated fo r its therapeutic potential in the treatment of cancer.

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95 P Proteincar g o Protein car g o P Phos p hor y lation Examples FKHR, (import) (Zhang et al., 2002) MAPKAP, (Engel et al., 2004) cyclin B1, (Yang et al., 2001) Dephosphorylation Proteincar g o Proteincar g o P FKHR (export), (Rena eta l., 2002) NLS Protein-protein interactions ( ex. p i ggy b ackin g) Proteincargo Proteincar g o MDM2/p53 (export) (Roth et al., 1998) Hepatitis B X protein/NF B (import) (Weil et al., 1999) Proteincargo Complex Disassembly (ex. exposure of NLS or NES) N L S NF B/I B (Malek et al., 2001) p53 (export) (Stommel et al., 1999) Proteincar g o Nucleolin (Schmidt-Zachmann et al., 1993) Protein -cargo Nuclear Retention NLS Nuclear import NLS Proteincargo NRS Ub SUMOylation or Ubiquitination Proteincar g o Proteincar g o Sp100 (Sternsdorf et al,. 1999) p53 (Lohrum et al., 2001) SUMO Figure 19 Mechanisms of Nuclear-cytoplasmic Transport Regulation. NLS, nuclear localization signal; NES, nuclear export signal; NRS, nuclear retention signal; CRS, cytoplasmic retention si gnal; FKHR, fork head transcription factor; Ub, ubiquitin; and SUMO, sma ll ubiquitin-like modifier (reviewed in Jans et al., 2000). NES CRS Nuclear export Cytoplasmic Retention Cyclin B1 (Pines and Hunter, 1994) Protein -cargo NES Proteincargo NF B (Chen et al., 2001) Acetylation/Deacetylation Proteincar g o Proteincar g o O C C O N L S

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96 Chapter Nine Experimental Objectives and Rationale In vitro experiments demonstrate that cancer cells may exhibit multiple different mechanisms to evade drug cytotoxicity. We were interested in identifying mechanisms of cellular drug resistance to DNA topoiso merase targeting agents. Altered drug transport and alterations in the quantity or quality of topo protein, which may or may not be a result of topo gene mutations, have alrea dy been described in cell lines selected for drug resistance. In culture, most cell lines also display a differential sensitivity to topo II inhibitors, which depends on cell density (S ullivan et al., 1986, 1987; Chow and Ross, 1987; Markovits, 1987). It is well established th at when cell lines become confluent and maintain extensive cell-cell interactions they become intrinsically drug resistant (Croix and Kerbel, 1997). How cell adhesion controls cell growth, apoptotic signal, cell cycle progression and drug resistance remains to be elucidated. One mechanism of in vitro drug resistance to topo II inhi bitors has been described to occur when cells transition from log to plateau cell density. For ex ample, in non-transformed cell lines, the transition from log to plateau density may lead to an attenuation of cellular topo II amount and activity, with concomitant drug resi stance (Sullivan et al ., 1987). However, tumor cell lines do not necessarily downregul ate topo protein cont ent at plateau cell density, yet the cells are resistant (Sulliv an et al., 1986, 1987). Therefore, tumor cell lines must have other ways besides the de gradation of topo II protein for evading drug

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97 cytotoxicity. Thus, we investigated the differences in drug sensitivity of several leukemia and myeloma cell lines at three levels of incr eased cell densities, called log, plateau, and accelerated-plateau, and then explained any observed drug resistance experimentally. Therefore, we investigated po ssible alterations in the cell cycle, drug transport, topo II enzyme activity, cleavable complex formation, and subcellular distri bution of topo I, topo II and topo II Much of our current understanding of dr ug resistance is derived from cell lines that have been selected for drug resistan ce by applying a continuous drug pressure, and then correlating the total cellu lar content of topo or expressi on of drug efflux pumps with drug resistance (Davis et al., 1998). Our da ta establish a novel mechanism of drug resistance in human myeloma cell lines that is detectable prior to any drug exposure and independent of protein expression or P-gp medi ated drug resistance. We demonstrate that topo II translocates to the cytoplasm in a cel l-density dependent manner and that the nuclear export of topo II has a role in decreased drug se nsitivity to VP-16 and other topo targeting agents, but less so with non-topoisomerase inhibitors. The cytoplasmic localization of topoisomerase II has thus far been attri buted only to C-terminally truncated proteins that have lo st a critical nuclear localizatio n signal (Wessel et al., 1997). Our data are the first to describe a nuclear export signal of topo II by cells expressing full-length topo II -GFP. We also demonstrate that a cytoplasmic transport of endogenous topo II in human myeloma cells can be abrogated in the presence of the nuclear export inhibitor, leptomycin B. The clinical significance of these findings is the treatment of multiple myeloma is suggested.

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98 Chapter Ten Materials and Methods 10.1 Materials. Etoposide (VP-16), phenylmethyl sulphonyl fluoride (PMSF), cis -platinum, paclitaxel (taxol), carmustin e (BCNU), ara-C (cytosine 1-D-arabinofuranosyl hydrochloride), dimethyl sulphoxide (DMSO) 2-mercaptoethanol, and leptomycin B, NonidetP-40 (NP-40), phenylmethylsulphonyl (PMSF), adenosine triphosphate (ATP), dithiothreitol (DTT), ethylenediaminetetroacte tic acid (EDTA), MTT, and vincristine, were all obtained from Sigma Chemical (St. Louis, MO). Mitoxantrone was generously provided by Immunex Corp (Seattle, WA). Topotecan was generously provided by SmithKline Glaxo (Philadelphia, PA). 10.2 Cell Culture. All cells were obtained from the Ameri can Type Culture Collection (Manassas, VA). The RPMI-8226 cell line is from the peri pheral blood of a 61 year old male with multiple myeloma and the NCI-H929 cell line is from the malignant effusion of a 62 year old Caucasian female with myeloma. The HL-60 cell line was obtained from a 36-year old Caucasian female with acute promyelocytic leukemia. This cell line is positive for the myc oncogene. The KG-1a human acute myelogenous leukemia ce lls are a subline variant of the KG-1 cell line. The parental KG-1 cells were obtained from a 59-year old Caucasian male with erythroleukemia that evolved into acute myelogenous leukemia.

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99 After 10 passages, the KG-1 cells were split and cultured by two different laboratories in the same department and unde r identical conditions. After 35 passages, one of the cultures exhibited different mor phological characteristic s (reviewed in ATCC) and were called KG-1a cells. The CCRF ce ll line was obtained from the peripheral blood buffy coat of a 4-year old Caucasian fema le with lymphoblastic leukemia. The L1210 cells were derived from a female mouse w ith lymphocytic leukemia. HeLa cells are epithelial adenocarcinoma cells that were obtai ned from the cervix of a 31 year old black female. HeLa cells were grown in Alpha Minima l Essential Media (Gibco) containing 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 g/ml streptomycin and 10% fetal bovine serum (FBS) (Hyclone). The HL60, KG1a, RPMI8226, and CCRF cells were grown in RPMI me dium containing100 U/ml penicillin and 100 g/ml streptomycin from Gibco BRL (Ga ithersburg, MD) and 10% FBS (Hyclone). The NCI-H929 cells were grown in RPMI me dium containing 100 U/ml penicillin, 50 M -mercaptoethanol (Sigma) and 100 g/ml streptomycin from Gibco BRL (Gaithersburg, MD). The L1210 cells were grown in RPMI medium containing 100 U/ml penicillin, 100 g/ml streptomycin fr om Gibco BRL (Gaithersburg, MD) and 20% FBS. The Chinese Hamster Ovary (CHO) ce lls were grown in Alpha Minimal Essential Media (Gibco) containing 100 U/ml penicillin, 100 g/ml streptomycin, and 5% fetal calf serum (FCS). Flow fibroblast 2000 cells were grown in Alpha Minimal Essential Media (Gibco) containing 100 U/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum.

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100 10.3 Log, Plateau, and Accelerated-plateau Cell Model Cell density and viability were determined by staining cells in a 1:1 dilution with trypan blue and counting them with a hemacy tometer. Growth curves were performed on all cell-lines described above (HL60, KG1a, RPMI, 8226, CCRF, L1210, CHO) to determine log and plateau pha se cell density. Log-phase was defined as 3.0 X 105 to 4.0 X 105 cells/ml with 85% viability. The log cell de nsities for the CHO and Flow fibroblasts were 2.0 X 104 cells/ml for 10 ml of media in a 25-cm2 flask, whereas plateau phase was reached at 8.0 X105 cells/ml for CHO cells and 2.0 X 105 cells/ml for the Flow fibroblast cells. When the log growing cells reached confluen ce, they were referred to as “naturalplateau”. Natural-plateau is defined as cell viability 85% (by trypan blue dye exclusion) and no net gain in cell number. The plateau density was different for each cell line: 9.0 X 105 cells/ml for RPMI 8226, 1.0 X 106 cells/ml for H929, 1.6 X 106 cells/ml for HL-60, 2.0 X 106 cells/ml for CEM-CCRF and L1210, and 8.0 X106 cells/ml for KG1a cells. For convenience, the “accelerated-p lateau” model was established by concentrating log-phase H 929, 8226, HeLa, CCRF, or HL-60 cells to a super-confluent cell density for 16 h or 24 h before experi mentation (Valkov et al ., 2000; Engel et al., 2004). Log-phase cells were centrifuged at 100 x g for 5 min at room temperature and resuspended in fresh media to obtain a s uperconfluent cell density and grown with 5% CO2 at 37C. The super-confluent cell density was between 2.0 x 106 and 2.5 x 106 cells/ml for RPMI-8226, H929, and HeLa cells. The super-confluent cell density for HL60 and CEM-CCRF was set between 3.0 X 106 cells/ml and 4.0 X 106 cells/ml. KG-1a,

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101 Chinese hamster ovary, and Flow Fibroblast cells were not used in the acceleratedplateau cell model. 10.4 Bone Marrow Samples. Bone marrow samples were obtained after informed consent from patients with multiple myeloma on a high-dose chemotherapy study, approved by the Institutional Review Board. Approximately 5 ml of bone marrow aspirate was collected in EDTA tubes and diluted to 30 ml with PBS. This was then layered over 15 ml of Ficoll-Paque Plus (Pharmacia Biotech) and centrifuged at 400 x g in a swinging bucket rotor at 4C for 30-min. The mononuclear cell interface was collected and washed twice with cold PBS. The cell number was determined and 105 cells were cytospun onto double cytoslides. The cells were fixed with 4% fo rmaldehyde and stored at -85C. Aliquots of cells were preserved in freezing media c onsisting of 40% RPMI-1640, 50% FCS, and 10% DMSO in liquid nitrogen. Where noted, samples were also obtained in a similar manner but from two untreated myeloma patie nts. Mononuclear fractions of these samples were obtained by centrifuging th e bone marrow aspirates at 700 x g over a Ficoll-Hypaque density gradie nt (Valkov et al., 2000). 10.5 Clonogenic Cytotoxicity Assays. Cytotoxicity assays were performed by treating 1.0 X 105 cells from either log, plateau, or accelerated plateau cell suspen sions (Valkov et al., 2000; Engel, 2004). Drug treatment was for 1 hour with various con centrations of etoposide, mitoxantrone, topotecan, and cis-platinum (diluents were DMSO, water, MeOH, and DMSO,

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102 respectively). Cells were treated with paclit axel for 24 hours and the drug was diluted in DMSO. Cells were treated with carmustin e for 4 hours and the drug was diluted in ethanol. Cells were treate d with ara-C (cytosine 1-D-arabinofuranosyl hydrochloride) for 20 hours and the drug was diluted in water. Cells were exposed to -irradiation (Cs137 gamma irradiator) (0-800 rads). Controls had the same c oncentration of dilutent. A total of 2500 drug-treated cells we re plated in triplicate in 0.3% select agar (Gibco BRL) in RPMI-1640 containing 15% FBS for 14-17 days at 37C in the presence of 5% CO2. Colonies > 50 cells were counted manually. 10.6 Flow-cytometric Cell Cycle Analysis. The cell cycle was analyzed by measuri ng BrdU incorporation and total amount of DNA by propidium iodide staining (E ngel et al., 2004). To measure BrdU incorporation, one million log and accelerated-plateau cells each were treated with 30 g/ml BrdU (Becton Dickinson, San Jose, CA) in serum free media for 30 min. Cells were washed twice in PBS and resuspended in 4 ml of ice cold PBS. While vortexing, 6 ml of ice cold 100% ethyl al cohol was slowly added and th e cells fixed overnight at 20C. At least 1 X 106 cells were centrifuged at 100 x g for 5 min and resuspended in pepsin solution (0.04% pepsin in 0.1% HCl) fo r 1 h at 37 C with gentle shaking. Whole cells were pelleted at 100 x g for 5 min, resuspended in 3 ml of 2 N HCl, and then incubated for 30 min at 37C. To this solu tion, 6 ml of 0.1M sodium borate was added, vortexed for 20 seconds and then recentrifuged. Cells were washed once with a wash buffer containing 0.5% BSA a nd 0.5% Tween-20 in PBS and brought to a final volume of 200 l FITC-conjugated anti-BrdU anti body (Becton Dickinson, San Jose, CA) was

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103 added in a 1:20 dilution and allowed to in cubate in the dark for 60 min at room temperature. These same cells were washed once with wash buffer and resuspended in 10 g/ml PI in wash buffer to achieve 1 X 106 cells/ml. Cells were treated with 250 g of RNase (Sigma) from a 10 mg/m l solution in PBS for 30 min at 37C. The sample was passed through an 18 G needle and transferre d to a Falcon 2054 vial and read by FACSScan (Becton Dickinson, San Jose, CA). Th e experiment was repeated three times and the mean values were plotted using Pris m 2.01 (GraphPad Software, San Diego, CA). In addition to the above, DNA content was also determined by measuring propidium iodide staining alone in three se parate flow cytometry experiments. To measure DNA content by propidium iodide staining, one million log and acceleratedplateau cells each were centrifuged at 1000 r .p.m. for 5 min at 4C and resuspended in 100 l of ice cold PBS. The cells were fi xed with 70% ice cold ethanol by slowly trickling the ethanol into th e cell suspension with constant vortexing. The cells were fixed overnight at -20C, cen trifuged at 1,400 r.p.m. for 5 min at room temperature, resuspended in 200 l of PBS, and transfe rred to a 10 X 75 mm tube (Fisher Scientific, Pittsburgh, PA). An RNase (Sigma) stock, equivalent to 100 K units/mg solid, was diluted to 10 mg/ml in PBS and added to th e cell suspension in a 1:4 dilution. A 1 mg/ml propidium iodide stock solution was prepared in ethanol and added to the cells in a 1:10 dilution immediately before reading and DNA histograms were determined by FACSScan (Becton Dickinson, San Jose, CA). The experiment was repeated 3 times.

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104 10.7 Gel Electrophoresis and Immunodetection. Log-phase H929 and 8226 cells were seeded at 2.0 X 106 cells/ml, returned to the incubator and then lysed e ither 4h, 16h, or 24 h later (E ngel et al., 2004). Whole cell lysates were prepared from log and accel erated-plateau cells by centrifuging 2.0 X 106 cells at 100 x g. for 5 min at 4 C. The pellet was washed once with ice cold PBS and then resuspended in 250 l of H2O (pH 9.0; adjusted with 0.1 M borate buffer). Cells were sonicated on ice with 15 pulses from a Branson sonifier (output 4.0, duty cycle 30%), and then boiled for 3 min in 1X sample buffer (2% SDS, 0.1M DTT, 10% glycerol, and 0.025% bromphenol blue). Protei n concentration was determined by using the BCA protein assay kit (Pierce, Rockford, IL). A total of 50-100 g of protein were loaded onto a 7.5% SDS-page gel, electropho resed under 7 watts and transferred at 65 volts overnight onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). Membranes we re blocked in 0.05% dry milk in TBST buffer containing 0.05% Tween-20 0.5M NaCl, 20 mM Tris, pH 7.5. Primary antibodies used were polycl onal antibody 454 against topo II diluted 1:5000 (Hochhauser et al., 1999), polycl onal antibody JB1 against topo II diluted 1:5000 (Valkov et al., 2000; Austin et al., 1995), or C-21 murine monoclonal IgM antibody directed against the C-terminus of topo I (a generous gift from Dr. Y.-C. Cheng, Yale University Medical School, New Haven, CT ) diluted 1:2500, or polyclonal anti-LDH (Research Diagnostics, Flanders, NJ) diluted 1: 500 in blocking buffer. Membranes were blocked for 1 h, and treated with either the polyclonal antibody 454 against topo II or JB1 against topo II for 1 h or blocked overnight at 4 C and treated with the monoclonal antibody C21 for 2 h. Membranes were washed in TBST every 15 min for 1 h and then

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105 treated with secondary antibody for 1 h at r oom temperature. Secondary antibodies used were either donkey anti-rabbit (topo II and topo II ), sheep anti-mouse (topo I) (Amersham, Arlington Heights, IL) or mouse anti-goat (LDH) (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated to horseradish peroxidase. The topo II and II secondary antibodies were diluted 1: 3000 and the LDH secondary antibody was diluted 1:500 in blocking buffer. The si gnal was detected with ECL (Amersham) and exposed to film. The bands were qu antified with Adobe PhotoShop 5.0 imaging software. The experiment was done one time for topo II and repeated 3 times for topo II and topo I in NCI-H929 and RPMI8226 cell lines (Engel et al., 2004). 10.8 Nuclear-cytoplasmic Separation. Subcellular fractions wa s prepared from 1.0 X 107 cells as previously described (Oloumi et al., 2000) with the following modi fications (Engel et al ., 2004; Valkov et al., 2000). Cells were washed in PBS and resusp ended into cold Buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 300 mM sucrose, 1 mM EDTA (pH 8.0), 1 mM DTT, 0.2% NP-40, 1 mM phenylme thylsulphonyl fluoride, 10 g/ml protease inhibitors mixture (Sigma), and 0.1 mM Na3VO4). The cells were incubated in Buffer A on ice for 15 min., sheared with a pipette 20 times, and then centrifuged for 10 min at 830 x g at 4C. The supernatant was reserved and th e nuclear pellet was prepared for protein determination and gel electrophoresis as desc ribed above (refer to "Gel Electrophoresis and Immunodetection") except that the DNA was sheared with 10 pulses from a Branson sonifier after being resuspended in Buffe r B (250 mM Tris-HCl (pH 7.9), 5 mM MgSO4,

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106 250 mM sucrose, 2 mM NaTT, 1% th iodiglycol, 1% NP-40, 1mM PMSF, 10 g/ml PI, and 0.1 mM Na3VO4). 10.9 Immunofluorescence Microscopy. Slide preparations from log and accelerate d-plateau cells were immunostained as previously described (Engel et al., 2004; Valkov et al ., 2000). Log and accelerated plateau cell suspensions were diluted to 5.0 X 104 cells/ml with PBS and cytocentrifuged for 3 min at 500 r.p.m. onto double cytoslides (Shandon, Pittsburgh, PA). The cells were fixed in 4% paraformaldehyde for 4 min at room temperature and permeablized with 0.5% Triton X-100 and 1% glycin e in PBS overnight at 4 C. After permeablization, the slides were washed in fresh PBS every 15 min for 1 h. The slides were treated with the following combinations of primary anti body: either rabbit polyclonal antibody 454 against topo II (Hochhauser et al., 1999) was us ed simultaneously with a mouse monoclonal antibody against histone (Roche Mo lecular Biochemicals) or a C-2 murine monoclonal IgM antibody against topo I (a ge nerous gift from Dr. Y.-C. Cheng, Yale University Medical School, New Haven, CT) was used simultaneously with the polyclonal antibody JB1 against topo II (Austin et al., 1995). The primary antibodies were diluted 1:100 with staining solution containing 0.1% NP-40 and 1% BSA in PBS and the incubation period was for 1 h at room temperature. Slides were washed in PBS every 10 min for 1 h and then incubated with goat anti-rabbit IgG-TRITC labeled antibody (Sigma) diluted 1:80 and goat anti -mouse IgG FITC-labeled antibody (Sigma) diluted 1:64 in staining solution as above for 35 min in the dark at room temperature. The slides were washed in PBS every 15 min for 2 h, air dried and covered with

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107 antifade/DAPI (Vector, Burlingame, CA). Immunofluorescence was observed with a Leitz Orthoplan 2 microscope and images were captured using a CCD camera with Smart Capture program (Vysis, Downers Grove, IL). 10.10 Quantitative Measu rement of the Immunofluorescence of Topo II Slide preparations from log and accelerate d-plateau cells were immunostained as previously described (Valkov et al., 2000; Engel et al., 2004). Topo II immunofluorescence was analyzed as previous ly described (Valkov et al., 2000; Engel et al., 2004) using Adobe PhotoShop 5.0 (Adobe Sy stems, San Jose, CA) and Prism 2.01 (GraphPad Software, San Diego, CA). The nuclear/cytoplasmic ratios were calculated for each cell by dividing [the number of pixels X (mean intensity background)] of the nuclear compartment by [the number of pixels X (mean intensity – background) of the cytoplasmic compartment. At least 50 cells per experiment were analyzed and the experiment was repeated 3 times. 10.11 Equilibrium concentrations of [3H]-VP-16. [3H]-VP-16 was obtained from Moravek Bi ochemical (Brea, CA). Equilibrium concentrations of [3H]-VP-16 were measured as previ ously described (Sullivan et al., 1986, 1987). Briefly, equal numbers of log and accelerated-plateau cel ls were incubated in the presence of 5 to 50 M of [3H]-VP-16 diluted with cold etoposide for 1 h at 37C. Whole cells were centrifuged at 100 x g for 10 min and washed with cold PBS. Drug uptake for log and accelerated-plateau cells was determined as cpm/mg of cells.

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108 10.12 Band Depletion Assay. Cleavable complex formation was measured in log and accelerated-plateau H929 cells as previously described (Engel et al ., 2004) with few modifications (Kaufmann et al., 1997). Briefly, 2.0 X 107 cells were centrifuged at 1,000 r.p.m. for 5 min in a swinging bucket rotor. The pellet was resuspended in 10 ml of buffer A (10 mM HEPES, pH 7.4 in serum free media). 2.0 X 106 cells were transferred to a 1.5 ml microcentrifuge tube and treated with VP-16 in DMSO for 45 min in a 37C wate r bath and mixed by inverting the tubes every 15 mi n. After the incubation period, the cells were centrifuged at 3200 X g for 1 min. Proceeding one tube at a time, the supernatant was aspirated and 1 ml of lysis buffer (6M guanidine HCl, 0.25 M Tris, pH 8.5 with HC l, 10 mM EDTA, 1% 2-BME, and 1X protease inhibitors cont aining (20 mM PMSF, and 20 g/ml each of antipain, aprotinin, chymostatin, leupeptin, soyb ean trypsin inhibitor, benzamidine, and pepstatin A) was added with brisk vortexi ng. The cells were incubated in lysis buffer overnight at 20C. Genomic DNA was sheared with 20 pulses from a Branson sonifier (output 4.0, duty cycle 35%). To each sample, 100 l of 150 mM iodoacetamide in lysis buffer without 2-BME was added and allowed to incubate in the dark for 1 h at room temperature. After incubation, 10 l of 2BME was added to each sample. The samples were dialyzed at 4C in the dark as follows : 3 X 90 min in 1 L of 4M Urea, 2 X 90 min in 0.1% SDS, and 1 X overnight in 0.1% SDS. Samples were lyophilized and stored at 20C. Immediately before gel electrophores is, protein was solublized by heating the samples in sample buffer (4M Urea, 2% SD S, 62.5 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.03% bromphenol blue and 1% 2-BME) for 20 mi n at 65-70C. Proteins were separated

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109 by gel electrophoresis on a 10% bis-acrylamide gel for western analysis. The experiment was repeated two times. 10.13 Comet Assay. DNA damage was analyzed by the comet assa y as previously described with some modifications (Engel et al., 2004; Ke nt et al., 1995). Briefly, 2.5 X 105 cells/ml were treated with VP-16 for 1 h at 37C. Af ter treatment, 5,000 cells were removed and washed in cold PBS. The supernatant was aspirated and 1.5 ml of 1% agar (Nusieve GTG) was added to 500 l of PBS, mixed with the pellet, and spread onto a frosted glass microscope slide. The slides were solidifie d at room temperature for 2 min and then 4 min at 4C. Cells were lysed for 1 h at 4C in batches (6-8 slides) in 200-250 ml lysis buffer containing 0.5% SDS (w/v), 30 mM ED TA, pH 8.0, and Proteinase K was added (375 units/100 ml) immediately before use. Af ter 1 h, the slides were incubated in the same lysis buffer overnight at 37C for 1216 h. The lysis buffer was washed off with 200 ml of 1X TBE (pH 8) for 2 h, cha nging buffer every 15 min. The cells were electrophoresed in 1X TBE under 25 volts fo r 20 min. The DNA was stained with a 1:10,000 dilution of Syber Green (Molecular Probes ) in 1X TBE for 25 min in the dark at room temperature. Slides were washed two times with 1X TBE for 5 min each, stored in a dark humidified box at 4C, and viewed within 24 h with a Leitz Orthoplan 2 microscope. Images were captured using a CCD camera with Smart Capture program (Vysis, Downers Grove, IL). Fifty single ce lls with no overlapping tails were captured per drug dosage per condition and the experime nt was repeated three times. The comet

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110 moments were captured and analyzed with Op timas and calculated by using the formula: 0 n (Intensity X Distance) /Total Intensity. 10.14 Putative NES Peptides. The complete amino acid sequence for human topo II (accession number NP 001058) was downloaded from the National Center for Biotechnology Information database and searched for matches to the NES consensus sequence from table 12 (Engel and Turner et al., 2004). Six amino acid sequences in topo II matched the NES consensus sequence (refer to Table 11, page 163), and were synthesized as native (nt) or mutated ( ) peptides. The mutated peptides contained alanine in place of those hydrophobic residues suspec ted of being critical for nuclear export (leucine, isoleucine, or valine). To facilitate conjugation with preactivated sulfosuccinimidyl 4-[ N -maleimidomethyl] cyclohexan e-1-carboxylate (SMCC-BSA) (Pierce) (Figure 20), the NES-peptides were designed w ith a cysteine residue at the amino terminus. The peptides obtained from the Biopeptide Company (San Diego, California) were as follows: (ntNES80-91), C80GLYKIFDEILVN91; ( NES80-91), C 80GA YKA FDEAAA N91; (ntNES230-241), C230SLDKDIVALMVR241; ( NES230-241), C 230SA DKDAA AA MA R241; (ntNES467-476), C467TLAVSGLGVVG477; ( NES467-477), C 467TA AA SGA GAA G477, (ntNES1017-1028), C1017DILRDFFELRLK1028; ( NES1017-1028), N O O O O N O O S O O ONa+Figure 20. Sulfosuccinimidyl 4-[ N -maleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC). Sulfo-SMCC is a water soluble non-cleavable crosslinkin g a g ent.

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111 C 1017CDIA RDA FEA RA K1028. Peptides (ntNES569-580), C569FLEEFITPIVKV580; ( NES569-580), C 569AA EEAA TPAA KA 580; (ntNES1054-1066), C1054FILEKIDGKIIIE1066; and ( NES1054-1066) C 1054FIA EKA DGKA IA E1066 were obtained from the University of Florida Protein Chemistry Core Facility (Gai nesville, FL). All peptides were HPLC purified to 95% and analyzed by mass spectroscopy. In addition, peptide sequences and purity were confirmed by Rick Feldhoff, Ph.D at the University of Louisville, School of Medicine (Louisville, KY). 10.15 Generation of BSA-Peptide-FITC Conjugates. Peptides were crosslinked to BSA and FITC. Figure 21 illustrates the plausible binding of peptides and FITC to BSA. First, a total of 2 mg Imject Maleimide activated sulfosuccinmidyl 4-( N -maleimidomethyl) cyclohexan e-1-carboxylate bovine serum albumin (Sulfo-SMCC BSA) (Pierce, Rockford, IL) was reconstituted in 200 l distilled water for a final concentration of (0.1 M sodium phosphate buffer, 0.15 M NaCl, 0.1 M EDTA, pH 7.2). Then, a molar excess (1 -2 mg) of native or mutated topo II NES peptide in 400 l of conjugation buffer (83 mM sodium phosphate buffer, 0.1 M EDTA, 0.9 M NaCl, 0.002% sodium azide, pH 7.2) wa s mixed with the Sulfo-SMCC BSA and reacted for 30 min at room temperature.

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112 The reaction was quenched by adding 40 mM cyst eine solution in deionized water to the peptide-SMCC-BSA solution to obtain a mola r excess of cysteine to peptide sample (approximately 7 nmoles cysteine/nmole of SMCC-BSA-peptide). The conjugates were purified by size exclusion chromatography at room temperature using the Pharmacia PFigure 21. An Illustration of BSA-FITC-P eptide Complexes. Multiple peptides covalently bind BSA-SMCC ( )via disulfide bonds (red) formed by the placement of a cysteine residue (blue) at the carboxyl terminus of th e peptide. Here the peptide is illustrated as the backbone of any amino acid residue (shown in black brackets [ ] FITC is shown in green. FITC covalently binds to any primary amine, whether located on the BSA or peptide molecules. Theoretically, multiple FITC and peptides will be crosslinked per mole of BSA SO N H2SNH R1O N H R2O OH C H3 O O O OH O H O O O OH O H O O O OH O H SO NH2SNH R1O NH R2O OH C H3 SO N H2SN H R1O NH R2O O H C H3 1 2 3 . n1 2 3 ... n1 2 3 . n BSA-SMCC

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113 500 FPLC system with LKB control Unit UV1. The high resolution column (10 mm inner diameter and 30 cm length) (Amersham Pharmacia, Piscataway, NJ) was packed at 2.0 ml/min with Superdex 200 prep grade (Amersham Phar macia) in filtered and degassed PBS, pH 7.4. The peptide conjugate s were loaded onto the column using a 500 l Superloop and were run at 0.5 ml/min in degassed dH2O. The 500 l fractions were collected with a Fraction-100 collector (Pharm acia Biotech) and stor ed at 4C overnight. Total protein was estimated in peak samp les by measuring absorbance at 562 nm using the Fisherbrand Protein A ssay. Approximately 25 g of protein from eluted fractions were loaded onto a 10% SDS-page gel a nd electrophoresed with 7 watts for 2-3 h. Peptide conjugation was confirmed by silver stain analysis. Similar fractions of crosslinked BSA-peptide were pooled and c oncentrated on a microsep 30k filter (Pall Corporation) by centrifuging at 5,000 x g in an SS-34 rotor until dry. The samples were eluted with 400 l of PBS, pH 7.4 to obtain approxi mately 2 mg/ml peptide-conjugate solution. FITC was solubilized in DMSO to 1 mg/ml and added to the peptide sample in four 5 l aliquots until a total of 20 l of FITC (Sigma) was added. FITC was reacted with the peptides for 6 h at 4C, and then 23 l of 1 M NH4Cl in PBS, pH 7.4 was added to the sample and incubated for 2 h at 4C. FITC-BSA-peptide conjugates were separated from unincorporated la bel by FPLC. The ratio of fluorescein to protein was determined by measuring the absorbance at 495 nm and 280 nm. The nuclear control, tetramethylrhodamine-bovine serum albumin (T RITC-BSA), was obtained from Sigma.

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114 Figure 22. Eppendorf FemptoJet and Micromanipulator on a Nikon TE 2000 Inverted Microscope. 10.16 Microinjection. To promote cell adherence, Fisherbrand gla ss coverslips were pretreated with 1N HCl for a minimum of 4 hours at 50C and then rinsed extensively with deionized water. Coverslips were washed in 100% ethanol a nd dried between pieces of Whatman paper. Subconfluent HeLa cells growing in Al pha Minimal Essential Medium (Gibco) containing 0.1 mM nonessential amino acids 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 g/ml streptomycin and 10% FBS (Hyc lone) were plated onto the center of glass coverslips in NUNC brand petr i dishes and incubated at 37C for 24-48 h preceding microinjection. Prior to microinjecti on, cells were gently rinsed with sterile PBS warmed to 37C and replaced with Le ibovitz’s L-15 Medium containing no phenol red. FITC labeled BSA-NES peptide conjuga tes were centrifuged at 13,000 x g for 30 min at 4C, and the supernatant then load ed into Eppendorf Femptotips (diameter of 0.5 m 0.2 m). All cells were injected using the semi-automated Eppendorf Injectman NI2 and Femtojet microinjector on a Nikon TE 2000 inverted microscope (Figure 22) under exactly the same conditions (injection pressure (Pi), 100 hPa; compensation pressure (Pc), 30 hPa; injection time (It) 0.2 sec, atomospheric conditions). Following injection, the cells were incubated at 37C for up to 90 min, washed with Leibovitz’s L-15 Medium, and then fixed with 4%

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115 paraformaldehyde for 3 minutes at room temp erature, rinsed in PBS and mounted onto Shandon microscope slides with mounting me dium containing DAPI. Fluorescence was observed with a Leitz Orthoplan 2 microsc ope and images were captured using a CCD camera with Smart Capture program (Vysis, Downers Grove, IL). 10.17 Topoisomerase II Cloning and Site Directed Mutagenesis. Topo II primers were designed which c ontained the eight amino acid FLAG peptide preceded by a start codon and a Kozak motif (5’-ATG GAC TAC AAA GAC GAT GAC GAC AAG GAA GTG TCA CCA TTG CAG CCT GTA AAT GAA AAT ATG-3’ forward primer, 5’-ATG C GG CCG CTT AAA ACA GAT CAT CTT CAT CTG ACT CTT C-3’ reverse primer). Amp lification was performed with an enzyme mixture of Taq and Pyrococcus species GB-D thermostable DNA polymerases (Elongase, Invitrogen), in 60 mM Tris-SO4 (pH 9.1), 18 mM (NH4)2SO4, 2 mM MgSO4, 200 M dNTP mixture and 200 nM each primer. Forty cycles were performed (94 C for 30s, 60 C for 30s, and 5.5 min at 68 C), and the PCR products were agarose gel-purified and ligated to a pcDNA3.1 vector using a TO PO-TA cloning system (Clontech). The 5’ end of the new FLAG-topo II fusion protein vector was sequenced to ensure that the DNA was in-frame. Site-directed mutagenesi s was performed using a Quickchange XL site-directed mutagenesis k it (Stratagene). Briefly, 100 ng of template dsDNA were mixed with 125 ng of each oligonucleotide primer, 2.5 units of PfuTurbo DNA polymerase, in a reaction mixture containi ng 2 mM dNTPs. Eighteen cycles were performed (95 C for 50 s, 60 C for 50s, and 68 C for 20 min), after which the parental plasmid was digested with methylation specific enzyme Dpn-I Ultra-competent cells

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116 were transformed with mutated plasmid and clones were sequenced to determine the presence of desired mutations. DNA sequencing was performed at the H. Lee Moffitt Cancer Center Molecular Biology Core Facility. 10.18 Transfection Protocol. Human myeloma H929 and human leukemia HL-60 cells (ATCC) were plated at log phase density (2x105 cells/ml) two days prior to transfection. Transfection was performed as previously described (V an den Hoff et al., 1992). Briefly, 40 g of wildtype or mutated topo II plasmid in 300 l tris-EDTA (TE) buffer was precipitated by the addition of 30 l of 5 M NaCl and two volumes of 95% ethanol on ice for ten minutes. Plasmid was pelleted by centrifugation fo r fifteen minutes at 20,000 x g at 4 C, washed with 75% ethanol and re-centrifuged. All remaining ethanol was removed by pipette and the DNA immediately resuspended in 50 l of cytomix buffer containing 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl2, 2 mM ATP, and 5 mM glutathione, pH adjust ed to 7.6 by the addition of KOH. ATP and glutathione were made fresh and adde d prior to each transfection (26). Two days prior to transfection, human myeloma H929 cells and le ukemia HL-60 cells were placed in fresh growth media (RPMI/10% FBS/pen-st rep) at a concentration of 2 x105 cells/milliliter. Cells were collected and 1.6x107 cells pelleted by centrifugation at 1500 x g for 5 minutes. Cell pellets were washed twice in 10 ml of sterile PBS, resuspended in 350 l of cytomix buffer (4 C), mixed with prepared DNA, and placed in a 4 mm electroporation cuvettes. Elect roporation was at 250V/750 cap acitance, after which cells

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117 were split into even groups and plated at log and plateau growth conditions for twenty hours in 5 % CO2 incubator at 37C with RPMI medium containing a 5% FBS. 10.19 Immunofluorescence of FLAG-Topo II Twenty hours post-transfection, live cells were isolated by centrifugation for 20 minutes at 20 C on a ficoll gradient, and then washed with PBS. Transfected cells were plated on a glass microscope slide usi ng cytospin funnels and fixed with 4% paraformaldehyde at 20oC for ten minutes. The fixati on was stopped by washing in PBS and cells were permeabilized for twenty-four hours in a solution c ontaining 1% glycine and 0.25% Triton X-100 in PBS. Slides we re stained with anti-FLAG M2 monoclonal antibody-FITC conjugate (Sigma) diluted 1: 100 with 0.1% NP-40 and 1% BSA in PBS, and incubated one hour at room temperature. Slides were washed in PBS, dried briefly and counterstained with V ectashield mounting media an tifade/DAPI (1 :1) (Vector Laboratories Inc., Burlingame, CA). I mmunofluorescence was observed with a Leitz Orthoplan 2 fluorescent microscope and im ages were captured by a CCD-camera with Smart Capture program (Vysis, Downers Grove IL). Quantitation of FITC fluorescence was performed using the Adobe Photoshop 7.0 program. 10.20 Western Blot of FLAG-Topo II Hela cells grown in RPMI media contai ning 5% FBS were transfected directly on 100 cm2 tissue culture plates. Plasmid DNA (10 g) was mixed with 60 l of Superfect transfection reagen t (Qiagen) in 300 l of serum-free media for 10 minutes, followed by 600 l of serum containing media, and the entire mixture was then added directly to cell

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118 culture plates. Transfection was allo wed to proceed for three hours at 37 C in a 5 % CO2 incubator, and terminated by the removal of transfection solution and the addition of 15 ml of 5% FBS containing RPMI media. Afte r incubation for twenty -four hours, the cells were harvested by the addition of 0.53 mM EDTA, washed with cold PBS, and lysed in SDS buffer (2% SDS, 10% glycerol, 0.06 M Tris, pH 6.8). Protein from 2x105 cells per lane was separated on an 8% SDS-PAGE gels and electrobloted (Biorad) onto nitrocellulose membranes (Amersham). The blots were blocked for one hour at ambient temperature in a blocking buffer containing 0.1M Tris-HCl buffered saline, 0.5% Tween20, and 5% non-fat milk. Blots were staine d by the direct addition of anti-FLAG M2 (Sigma) antibody and incubated overnight at 4 C. Membranes were washed three times for ten min with 0.1M Tris-HCl buffered saline and incubated with anti-mouse IgG antibody (Sigma) in blocking buffer containi ng 0.1M Tris-HCl buffered saline, 0.5% Tween-20, and 5% non-fat milk for sixty mi nutes at room temperature. Antibody binding was visualized by ECL (Amers ham) on autoradiography film (Kodak).

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119 Chapter Eleven Experimental Results Part I: Cell density-dependent VP-16 Sensitivity of Leukemic Cells is Accompanied by the Translocation of Topo II from the Nucleus to the Cytoplasm 11.1 Preliminary Results. The results of the data described immedi ately below were obtained prior to my entering the laboratory, but are important be cause they established a foundation for my doctoral work on mechanism of drug resistance to topoisomerase targeting agents. A complete description of the data is given in Valkov et al., 2000. I became a contributing author of Valkov et al., 2000 for my imm unostaining and microscopy work on log and plateau 8226 and CCRF cell lines, whic h included the development of an in vitro cell model for investigating the nuclea r-cytoplasmic distribution of topo II 11.2 Drug Resistance Phenotype of Pl ateau-phase Tumor Cell Lines. Most cell lines display a di fferential sensitivity to t opo II targeting agents such that when cells reach confluence they become drug resistant to topo II poisons (Sullivan et al., 1987, 1986; Chow and Ross, 1987; Mark ovits, 1987). Drug resi stance to topo II poisons in non-transformed plat eau cell lines have been attri buted to attenuations in the amount and activity of topo II protein. However, the transition from log to plateau cell density does not necessarily lead to a decreased cellular content of topo II protein in

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120 transformed cell lines (Sullivan et al., 1987). Thus, we were interested in explaining the observed drug resistance in transformed cel l lines at plateau density, which is independent of the cellular content of topo II First, the differences in drug sensitivity between log and platea u cell densities of several transformed and non-transformed cell lin es were investigated. To do this, the cytotoxic effects of two topoisomerase II poisons (i.e., VP-16 and MTX), one topo I poison (TPT), and two non-topoisomerase target ing agents (i.e., cispla tin and vincristine) in several log and plateau cell lines was determined. A comp arison of the drug sensitivity of four leukemic cell lines (CCRF, HL60, KG-1a, L1210), one myeloma cell line (RPMI-8226) and non-transformed human Flow 2000 fibroblasts and Chinese hamster ovary (CHO) cells shows that, at plateau cell density, all ce ll lines become resistant to VP-16 (Table 9). The magnitude of crossresistance to another topo II inhibitor, mitoxantrone, was uniformly less in all cell lines, but followed a similar pattern of resistance as that observed with VP-16. The mouse leukemia L1210 and Chinese hamster ovary (CHO) cell lines showed signifi cant plateau phase drug resistance to the topo I inhibitor topotecan. Resistance to dr ugs that act by mechanisms other than the inhibition of topo was low, except for the re sistance of human leukemia KG-1a cells to vincristine. These data suggest that the log to plateau phase transition involves specific changes in topo in these leukemic and nontransformed cell lines. Altered drug sensitivity to VP-16 has been shown to resu lt from a cell-cycle dependent degradation of topo II protein. It is well established that the cellular content of topo II is maximal in G2/M of the cell cycle, and thus alterations in the cell cycl e can result in altered drug sensitivity to topo II poisons. The specific contributions of topo II in conferring drug

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121 resistance to topo II poisons are less well unde rstood. Attenuation in the cellular content of topo I protein, however, has also been attrib uted to decreased drug sensitivity to topo I targeting agents. Furthermore, topotecan is reported to be S-phase specific cytotoxic agent. Thus, we examined the cell cycle dist ribution by flow cytometric analysis and the cellular content of topo I, topo II and topo II by Western blot an alysis in log and plateau cells.

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122 VP-16 IC50 (mol/l) Rf ** MTX IC50 (mol/l) Rf TPT IC50 (mol/l) Rf CDDP IC50 (mol/l) Rf Vincristine IC50 (mol/l) Rf Fibroblasts 11.56.6a. 5.20.2a 17, 17.1b 2.6 2.40.6 3.30.1 ND ND ND ND CHO 4.0** 15.0d 25.0†† 8.0 0.180.15 274168 3.0c 1.0 ND ND CRRF 0.630.61 39.011.5e 1.370.66 7.73.9g 0.370.28 16.812.6 0.710.29 1.90.05 1.250.91 1.30.4 HL-60 0.550.04 7.60.3 18.0c 2.2 0.060.02 1.00 ND ND ND ND 8226 0.570.26 10.42.1 8.50.5 2.60.2 0.950.55 2.01.0 1.150.35 1.10.1 1.8 1.3 L1210 1.330.29 17.76.6f 7.50.5 4.90.5 0.300.29 282156 0.550.55 1.30 3. 550.05 1.30.1 KG-1a 5.671.7 3.51.4 22.312.2 1.10.1 1.250.75 2.50 3. 10.1 1.10.1 1.0, 1.1b 27.5, 25.3b Table 7. Drug Resistance of Natural Plateau Phase Non-transformed and Tumor Cell Lines IC50, drug concentration that inhibits growth by 50% as determined by the colony fo rming assay; values reported are for log phase c ells **Rf, the resistance factor or ratio of IC50 values of plateau and log phase cells aMean SEM bn = 2, the experiment was done two times and both values are shown cn = 1, the experiment was done one time and the individual value is shown dFrom Sullivan et al., 1986 eSimilar results found with MT T assay (n = 3), where Rf = 26.0 4.9. fSimilar results found with MT T assay (n = 3), where Rf = 10.5 0.5 gSimilar results found with MTT assay (n = 3), where Rf = 6.0 4.3

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123 11.3 Cell Cycle Distribution and [3H]-thymidine Incorporation. The drug resistance to topotecan observe d in the L1210 and CHO cell lines may be explained in part by the decrease in th e number of S-phase cells at plateau density (Table 10), as topotecan is thought to be an S-phase specific agent. However, the magnitude of the decrease in S-phase cells at confluence did not parallel the degree of resistance. The fibroblast cells were only mi nimally resistant to topotecan, and yet had the second greatest decrease in number of Sphase cells at conflu ence. Although the HL60 cells showed no decrease in the number of S-phase cells at confluence, they had 6.5 fold less [3H]-thymidine incorporation, suggesting a significant decrease in the number of cycling cells. The sensitivity of HL-60 cells to topotecan, however, paralleled the flow cytometry results (no apparent change in S-phase ). Factors other than a simple change in percentage of S-phase are proba bly involved in the sensitivity to topotecan in this log and plateau cell model.

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124Table 8. Analysis of S-phase and [3H]-thymidine Incorporation in Log and Natural Plateau Phase Cell Lines* BrdU Incorporation Log S (%) BrdU Incorporation Plateau S (%) Log [3H] thymidine (cpm/106 cells) Plateau [3H] thymidine (cpm/106 cells) Flow fibroblasts 29.6 6.9a 10.1 10.3 NDb ND CHO 37.4 5.1 14.9 9.2 ND ND CCRF-CEM 34.7 8.9 25.9 8.2 20760 371 (P < 0.0001)c 2089 256 HL-60 30.0 8.9 30.5 4.9 2289 401 (P = 0.003) 352 33 RPMI-8226 25.4 7.8 15.5 2.3 6819 351 (P = 0.41) 6504 39 L1210 37.3 9.5 27.9 8.1 10570 750 (P = 0.0004) 2306 188 KG-1a 21.6 7.1 11.7 7.9 2860 120 (P <0.0001) 695 50 *Cell cycle distribution was determined by flow cytometry of log and plateau phase cells in three to five independent experimen ts, while incorporation of [3H] thymidine was measured in appropriate cells in three or four separate experiments. aMean SEM bNot determined cP-values relative to uptake in plateau phase

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125 11.4 Cellular Content of Topo in Log and Plateau-phase Cell Lines. The overall quantity of topo was determin ed by Western blot analysis of whole cell lysates of log and plateau phase leukemic, myeloma a nd non-transformed cell lines. A notable difference between malignant and non-transformed cell lines was the near disappearance of topo II from CHO and fibroblasts cells at a time when they reach confluency and lose their prolif erative potential (Figure 23 ). In contrast, all tumor cell lines preserved their total cellular topo II content, independent of cell density. Topo II did not show any substantial changes in the cell lines studied and was not downregulated in either normal or neoplastic cells. The content of topo I was not found to be significantly changed by the l og to plateau phase transiti on in fibroblasts, 8226, HL-60, KG-1a, CCRF, or L1210 cells. Furthermore, a reduction in the total cellular amount of topo II could not account for the decreased sensitivity to VP-16 and mitoxantrone in transformed cell lines. The relatively high leve l of resistance of pl ateau phase L1210 cells to topotecan also does not appear to result from a downregulation of cellular topo I content. Thus far, we have been able to attrib ute the drug resistance to topo VP-16 and MTX observed in non-transformed cell lines to attenuation of topo II protein. However, we have not been able to fully describe the mechanism(s) of drug resistance to VP-16 or mitoxantrone in the transformed cell lines at plateau cell density. A lthough alterations in the cell cycle may have a role in the decrease drug sensitiv ity of L1210 cells to topotecan, the data obtained from analysis of the cell cycle was not alwa ys consistent with the drug sensitivity. Furthermore, there were discre pancies between the per cent of S-phase cells as determined by flow cytometric analysis a nd the number of cycling cells determined by

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126 [3H] thymidine incorporation. For example, plateau HL-60 cells have minimal resistance to topotecan, which is consistent with the analysis of S-phase by flow cytometry. However, HL-60 cells demonstrated rela tively large decrease in the amount of [3H] thymidine incorporation. Thus, we specula ted that other cellular events must be occurring that have a role in drug sensitivity to topoisomerase targeting agents. We questioned whether transformed cell lines may alter their subcellular distribution of topo II protein to evade drug-target interactions with VP-16 or mitoxantrone. To address this possibility, we compared the s ubcellular distribution of topo II in 8226 and CCRF cells at log and plateau density by immunofluorescence microscopy.

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127 CHO to p o II topo II A B Flow fibroblasts topo II topo II topo I A B HL-60 to p o II topo II topo I A B 8226 to p o II topo II topo I A B Kg-1a topo II topo II topo I A B Figure 23 Western Blot Analysis of Topo I, Topo II and Topo II from Whole-cell Lysates of Cell Lines at L og and Plateau Densities. One million cells were loaded in all lanes, and the ECL signal was quantified by densitometry. These results are repres entative of experiments performed a minimum of three times for each cell line. Lane A, log-phase cells; lane B, plateau-phase cells. CCRF to p o II topo II topo I L1210 to p o II topo II topo I A B A B

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128 11.5 Subcellular Distribution of Topo in Log and Plateau-phase Cell Lines. At log density, topo II was strictly nuclear, while at plateau cell density, the tumor cell lines demonstrated an increase in topo II present in the cytoplasm (Figure 24). In order to evaluate the amount of topo II at the single cell level statistically, the pixel intensity of nuclear and cytoplasmic topo II in 200 individual cells (50 cells/experiment four times) at log, midl og and plateau densities was determined (complete data not shown here, refer to Valkov et al., 2000). CHO cells had a proportional decrease in the nucl ear/cytoplasmic ratio, such that at plateau phase the ratio was less than one (<1). This suggested that for the remaining undegraded topo II there was relatively more in the cytoplasm than in th e nucleus. From early log phase to plateau phase, there was a significan t decrease in the topo II nuclear/cytoplasmic ratio for all cell lines except KG-1a. Thus, at confluent densities, all cell lines (except KG-1a) had significantly more topo II in the cytoplasmic compartment. The immunolabelling of three cells lines (CCRF, L1210, and HL-60) for topo I and topo II demonstrated no significant trafficking of thes e topoisomerases to the cytopl asm during the transition from log to plateau phase (data not shown).

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129 Figure 24. Immunofluorescent Staining and Confocal Microscopy. Immunofluorescent staining for topo II (A, D, G, J, histones (B,E, H, and K), and merged images (C, F, I,and L) in log and plateau phase CCRF and 8226 cells. Log phase 8226 cells (A-C) were at a density of 2.0 X 105 cells/ml; plateau-phase 8226 cells (D-F) at 9.0 X 105 cells/ml; log phase CCRF cells (G-I) at 2.0 X 105 cells/ml; and plateau phase CCRF cells (J-L) at 1.6 X 106 cells/ml. Cell viability in all experiments was 95% and the immunofluorescence was observed with a Leitz Orthoplan 2 microscope and captured dig itally using the Smart Capture program. Topo II Histones Merged 8226 Log Phase 8226 Natural Plateau CCRF Log Phase CCRF Natural Plateau

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130 11.6 Conclusions. The resistance of several leukemia and myeloma cell lines (CCRF, L1210, HL-60, KG1a, and RPMI-8226) to VP-16 was found to increase with cell density and to be maximal (3.5 to 39-fold) in plateau phase cell cultures, as measured by clonogenic and MTT assays. Non-transformed confluent Flow 2000 human fibroblasts and Chinese hamster ovary (CHO) cells were also five a nd 15-fold resistant to VP-16, respectively. The transition from log to plateau phase was accompanied by a drastic decrease in topoisomerase II content in CHO cells and human fi broblasts, while the leukemic cells maintained constant cellular levels of topo II and topo II The nuclear-cytoplasmic ratio of topo II may be critical in determining the se nsitivity of leukemi a cells to topo II inhibitors.

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131 Chapte Twelve Experimental Results Part II: The Cytoplasmic Traffi cking of DNA Topoisomerase II Correlates with Etoposide Resistance in Human Myeloma Cells 12.1 Introduction. The data described in Part I, suggests that the nuclear-cytoplasmic ratio of topo II may be critical in determining the sensit ivity of myeloma and leukemia cells to topo II inhibitors. However, many questions rema in. Does a nuclear-cytoplasmic trafficking of topo II contribute to drug resistance in vitro ? Are plateau leukemia and myeloma cells cross-resistant to other classes of an titumor agents besides topo targeting agents? How does a cytoplasmic topo II contribute to altered drug sensitivity? Is the cytoplasmic distribution of topo II a result of deceased nuclear import or increased nuclear export? Can we manipulate the nuclear-cytoplasmic trafficking of topo II by either inducing its cytoplasmic location or by blocking the cytoplas mic relocation of topo II in plateau cells? These question, and othe rs established the purpose of my doctoral work. 12.2 Accelerated-plateau Human Myeloma Cell Line Model. To more precisely define the role that the nuclear-cytoplasmic shuttling of topo II has in modulating drug sensitivity to topo in hibitors and other classes of anti-cancer

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132 agents, we developed a human multiple myeloma cell line model. The data presented inthe log and plateau cell models suggest that the nuclear-cytoplasmic trafficking of topo II could be cell-density dependent and thus influenced by cell-cell contact. To exploit cell-cell contact, log-phase (defined as 3.0 x 105 cells/ml) human myeloma cell lines (NCI-H929 and RPMI-8226) and the human leuke mia HL-60 cell line were concentrated to a superconfluent cell density (2.0 x 106 cells/ml) and grown at this density for up to 24 h before experimentation. After growing at the superconfluent density for as long as 24 h, the accelerated-plateau cells were still > 85% viable by trypan blue exclusion. The superconfluent cell density was initiall y determined by obtaining growth curves for NCI-H929 (Figure 25) and RPMI8226 (data not shown) cells to establish the cell-density 0 20 40 60 80 100 120 140 160 180 200 220 0 2 4 6 8 10 12 14 16 0 20 40 60 80 100 Log PlateauTime (h)Cell Count x 105 cells/ml ( )Percent Viability ( ) Figure 25. Growth Curves Growth curves for human myeloma H929 and 8226 (data not shown) cells were plotted to determine log and plateau cell densities. H929 cells enter logarithmic growth approxima tely 2 days after being seeded to an initial cell density of 2.0 x 105 cells/ml in fresh media. After growing continuously for 2 days, H929 cells reach “natural-plateau” near 1.2 x 106 cells/ml The graph illustrates the relatively long plateau phase (~4-5 days) characteri stic of these cell lines. Cell density as cell count x 105 cells/ml (o); Percent viability, ( ).

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133 of “naturally-plateaued” H 929 and 8226 cells. For example, after being seeded at 2.0 X 105 cells/ml, H929 cells require approximately 48 hours to reach log-phase, and then The remain in log-phase for an additional 3648 hours before reaching plateau phase. H929 cells remained in plateau phase for approximately 2 days. The H929 cells reach confluence when their cell density nears 1.2 X 106 cells/ml (natural plateau). Thus, the superconfluent or accelerated-plateau was se t to approximately double the natural-plateau cell density (2.0 x 106 -2.5 x 106 cells/ml).growth curve also illustrates the relatively long natural plateau-phase (approxi mately 4-5 days) characteri stic of H929 human myeloma cells. Other cell densities were also analyzed (1.0 X 106 cells/ml, 5.0 x 106 cells/ml, and 1.0 x 107 cells/ml) however, 2.0 x 106 cells/ml was determined to be an optimal celldensity that preserved cellular vi ability and demonstrated topo II export in a measurable period of time. 12.3 Drug Sensitivity of Human Myel oma Cell Lines in Accelerated-plateau. To determine if topo II protein trafficking correlates with cellular drug resistance to other topo targeting agents as well as other classes of antineoplastic agents, the toxic effects of several classes of antineoplastic agents were ex amined in log and acceleratedplateau H929 and 8226 cells. We expanded the drug sensitivity data that was described for log and plateau cells (in Valkov et al., 2000 ) to include an antimetabolite (ara-C), a nitosourea DNA crosslinking agent (BCNU), a platinum containing DNA crosslinking agent (cisplatin), an antim icrotubule (Paclitaxel), and -irradiation. A comparison of drug sensitivity of log-phase and accele rated-plateau phase H929 and 8226 (data not shown) cells shows that the log-phase cells ar e more sensitive to topo inhibitors, such as

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134 VP-16 and mitoxantrone, compared to cells s eeded at the plateau-phase density for 16 h or 24 h, as determined by colony forming assays (similar results were obtained with 8226 cells) (Table 11). The accelerated -plateau cells were most re sistant to VP-16, 10-fold at16 h and 24-fold at 24 h). The accelerated-plateau H929 cells demonstrated less resistance to the non-topo inhibitors such as ara-C, taxol, BCNU, and -irradiation, but were found to be 3-fold resistant to topotecan. Thus, the observed drug resistance appears to be more specific for topo inhibitors than other classe s of chemotherapeutic agents. These data suggest that the principal mechanism of drug resistan ce involves quantitative or qualitative changes in topoisomerase. However, other cellular even ts, such as alterations in either drug transport or cell-cycle can also contribute to a decrea se in drug sensitivity to topo poisons. To explain the mechanism(s) of observed drug resistance to topo poisons in accelerated-plateau cells, changes in tota l amount of topo protein, drug uptake, cell cycle distribution, and subcellula r localization were examined.

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135 Resistance Factord (RF) Antitumor Drugb Log-phase IC50 c 16 h 24 h VP-16 ( DMSO, 1 h) 0.360 0.08e 9.6 3.3 23.5 5.1 Mitoxantrone (water, 1 h) 0.0009 0.0001f 5.3 0.2 5.1 0.2 Topotecan (methanol, 1 h) 3.32 1.0g 3.1 0.4 3.5 0.3 Taxol (DMSO, 24 h) 1.40 0.92 1.2 0.3 2.8 0.2 -Irradiation 75 cGy 8.6 0.33 0.2 1.3 0.1 Cis -platinum (DMSO, 1 h) 7.00h 0.43 0.5 Ara-C (water, 20 h) 1.20h 1.3 5.0 BCNU (ethanol, 4 h) 1.0h 1.5 1.0 aUnless otherwise noted, three to five independent experiments were perfor med for each drug in triplicate. The mean values SEM are shown. bSolvent for drug dissolution and hours of dr ug exposure are shown in parentheses. cIC50 is the concentration of drug that in hibits growth by 50% and the values reported are for logphase cells in M concentrations. dFold resistance (RF) is the ratio of 50% inhibi tion concentration values (IC50’s) obtained from colony-forming assays. eSimilar results were found in 8226 cells at 24 h, IC50 = 0.460 0.06 (n =3), where RF = 10.3 1.6. f Similar results were found in 8226 cells at 24 h, IC50 = 0.003 0.001 (n =3), where RF = 5.0 0.5. g Similar results were found in 8226 cells at 24 h, IC50 = 1.1 0.5 (n =3), where RF = 2.0 1.1. hThe colony-forming assay was perf ormed one time in triplicate. Table 9. Drug Sensitivity of Log and Accel erated-plateau H929 Cells to Cytotoxic A a

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136 12.4 Cell-cycle Analysis. The cellular amount a nd activity of topo II is regulated in a cell-cycle and proliferation dependent manner, and altera tions in the amount and activity of topo II correlate with cellular drug resistance to topo inhibitors. Therefore, it was necessary to separate the contributions of the cell cycle to the observe d drug resistance from other cellular events. The cell cycle was analyzed over a 24 h period for changes in the S-phase cell population from the time that the log-pha se cells were concentrated to 2.0 X 106 cells/ml (time zero) (Figure 26). Analysis of BrdU incorporation in H929 cells indicates that 19.4% 5.0% of log-phase cells are in S-phase compared to 14.4% 1.3% of Figure 26. Percent of Accelerated-plateau Cells in S-phase. BrdU incorporation was measured to determine the percent of NCI-H929 and RPMI 8226 cells in Sphase. The experiment was repeated th ree times and the mean values with standard deviation are shown (o ), 8226 cells; (•), H929 cells. 0 4 8 12 16 20 24 0 10 20 30 40 50 60 70 80 90 100Time (hours)Percent of Cells

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137 accelerated-plateau cells at 16 h. These data suggest that the changes in the cell cycle distribution that occur within 16 h of being plated at 2.0 x 106 cells/ml are not likely to be the main mechanism contributing to the drug resistance observed to topo inhibitors. A greater reduction in the S-phase population of accelerated-plateau cells was observed at 24 h and this difference could account for the increase in drug resistance observed with ara-c (1.3 fold to 5.0 fold) and taxol (1.2 fo ld to 2.8 fold). To more specifically determine the contribution that topo II trafficking plays in drug sensitivity, subsequent experiments were performed within 16 h to mi nimize contributions of cell cycle changes to drug sensitivity in the accel erated-plateau cell model. 12.5 Cellular Amount of Topoisomerase by Immunoblotting. An attenuation of topo enzymes can cont ribute to drug resistance by decreasing the amount of drug target. To determine if there are any changes in the total cellular amount of topo protein in accelerated-plateau cel ls that could contribute to the observed drug resistance, the amount of topo protein in whole cells was determined by Western blot analysis. No significant changes in the amount of topo II prot ein were observed in whole cells when compared to log-phase cells 4-24 h after seeding at the super-confluent cell density (Figure 27). Thus a decrease in the total amount of topo II protein is unlikely to account for the observed drug resistance in accelerated-plateau H929 and 8226 cells to VP-16 and mitoxantrone, while a decrease in topo I (20-30%) may contribute to the resistance to topotecan.

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138 The observed decrease in topo I may also contribute to the decrease in drug sensitivity to ara-C because recent data s hows that topo I cleavable complexes can be trapped by ara-C incorporation into DNA (Pour quier et al., 2000). Inhibition of topo I by ara-C is suggested to occur by structural m odifications that occur in the DNA helix when ara-CTP is incorporated. Ara-c incorporation into DNA results in structural modifications in the DNA helix and inhibition of DNA synthesis (Pourquier et al., 2000) Ara-c incorporation into DNA is believed to result in misalignment of the DNA 5'hydroxyl with the enzyme-DNA tyrosyl phosph odiester bond, which is necessary for nucleophilic attack and DNA religation (P ourquier et al., 1997, 1998, 2000). Thus, it Figure 27. Western blot Analys is of DNA Topoisomerase I and II. The total cellular amount of topo I, topo II and topo II protein in whole cell lysates from 1.0 x 106 log and accelerated-plateau H929 and 8226 cells wa s determined by Western blot analysis. There are no significant differe nces in the amount of topo II or II protein up to 24 h by densitometry. A small decrease in topo I is seen in H929 cells 16h, while a 30% decrease in topo I is se en in 8226 cells at times 4 h. Hours refer to the time after concentrating log-phase cells to super-c onfluent densities. The expermiment was performed one time in duplicate for topo II and repeated three times each for topo I Topo II Log 4 hr 16 hr 24 h r 100% 105% 100% 101% 100% 100% 87% 84% 100% 104% 104%104%Log 4 h r 16 hr 24 h r H929 8226100% 67% 60% 66% 100%8 9% 90% 88% 100% 103% 105% 101% Topo I Topo II

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139 appears that ara-C cytotoxici ty is a result of both inhibi tion of DNA polymerization and topo I poisoning. Inhibition of topo I by ara-C could explai n the 5-fold resistance of plateau cells to ara-C at 24 hours because there is a concomitant decrease in the total cellular amount of topo I prot ein in plateau cells. Thus, it is possible that cell death induced by ara-C in the log growing cells wa s due in part to the formation of topo I cleavable complexes in addition to cessation of DNA synthesis. 12.6 Drug Transport in Tumor Cell Lines. Reduced drug accumulation is a major mechanism conferring drug resistance in several drug-resistant cell lin es, and many important mamma lian drug transport proteins are ATP-dependent proteins that belong to th e ABC superfamily (reviewed in Leonard et al., 2002). Under normal conditions, these pumps protect cells from accumulating toxic substances by actively pumping out cytotoxic substances. Thus, topo I and II inhibitors may be effectively eliminated from cells by a variety of these drug transporters. To determine if the differences in log and accelerat ed-plateau cells in drug sensitivity to topo inhibitors was a function of altered drug transport, cellu lar uptake of [3H]-VP-16 was measured as described in "Materials and Me thods" (Sullivan et al ., 1987). Briefly, H929 cells were incubated in the presence of [3H]-VP-16 for 1 h to achieve steady state, washed, and pelleted by centrif ugation. The pelleted cells we re transferred to drying paper and dried overnight, and then weighed. After weighing, the pellets were dissolved in a buffer, and then 3H content was determined by scin tillation counting. Drug uptake for log and accelerated-plateau cells was dete rmined as cpm per milligram of cells. No

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140 considerable difference was obs erved in the uptake of [3H]-VP-16 between log (1.7 x 104 counts/mg of dry weight) and acc elerated-plateau cells (1.8 x 104 counts/mg of dry weight). We have previously shown that th ere are no differences be tween several log and natural-plateau human myeloma and leukemia cel l lines (Valkov et al ., 2000; Sullivan et al., 1986, 1987). Thus, an alteration in drug tran sport of VP-16 is not likely to be the main mechanism conferring drug resistance to topo inhibitors in the accelerated-plateau model. 12.7 Subcellular Distribution of Topoisomerase II Although the total amount of topo protein does not appear to change in the whole cells, a change in the subce llular distribution of topo coul d alter the amount of topo protein located in the nucleus. Therefore, we examined the subcellular distribution of topo II (28 ) and topo I and topo II (29) was examined by immunofluorescence microscopy and confirmed by sca nning confocal microscopy.

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141 Topo II Histones Merged Plateau (16 h) Plateau (24 h) Log Figure 28. The Subcellular Distribution of Topo II in Log and Accelerated-plateau H929 Cells at 16 h and 24 h. Histones are non-shuttled proteins that define the area of the nucleus. Topo II is nuclear in log-phase cells and has a markedly increased cy toplasmic distribution in accelerated -plateau cells at 16 h and 24 h. Arrows point to cells with cytoplasmic topo II

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142 DNA To p o II To p o I Merged Log Plateau ( 24 h ) Figure 29. The Subcellular Distribu tion of DNA, Topo I, and Topo II in Log and Accelerated-plateau H929 Ce lls at 24 h. DNA was staine d with DAPI which defines the area of the nucleus. Topo I appears nuclear in log and accelerated-plateau cells. Topo II is found in the nucleus and cyt oplasm of log and plateau cells.

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143 A least 50 cells per experimental condition (i.e ., log or plateau) were randomly picked by 4'-6-diamidino-2-phenylindole ( DAPI) staining of the nucleus. Cells were numbered as 1-50 and then, analyzed for the pixel inte nsity of either tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein is othiocyanate (FITC) labeled antibody as described in Figure 30. Topo II is predominately nuclear in log-phase cells, but there is a time-dependent translocation of topo II in the accelerated-plateau cells after 16 h. Similar results were observed in the na tural plateau cells (data not shown). Figure 30. Method for Selecting Cells by Confocal Microscopy. Cells were selected randomly by positive DAPI staining and images captured using a CCD camera as described in “Materials and Methods”. The cells were numbered 1-50. Mitotic cells and cel ls touching the edge of the field of vision were not included in the data. Furthermore, any cell staining positive for DAPI but histone negative were presumed to be cell debris and also were not included in the data.

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144 The subcellular distribution of topo protein was quantifie d in log and acceleratedplateau H929 and 8226 cells by calculating the nu clear/cytoplasmic ratio (N/C) from the pixel intensity of the individual compartments from 50 cells per time point. The staining for nuclear topo I and II was internally cont rolled in each cell by DAPI staining of DNA and histone staining. Figure 31 illustrates the method, as de scribed in "materials and methods", for analyzing the subcellular lo calization of topo by i mmunofluorescence. A least 50 cells per experimental condition (i.e ., log or plateau) were analyzed. These experiments were performed in duplicate a nd repeated three times in both H929 and 8226 cells. The median N/C ratio of topo II is 29.9 9.1 for log-phase H929 cells and decreases to 1.3 1.3 for the accelerated-plate au cells after 16 h and to 0.90 1.1 after 24 h. The median N/C ratio of topo I and II did not change. The median N/C ratio of Figure 31. Determination of Nuclea r-cytoplasmic Ratios (N/C). Using AdobePhotoshop, we were able to zoom-in on each cell. To obtain the value of the pixel intensity between the nuclear and cyt oplasmic compartments for either TRITC or FITC labeled antibody, the nuclear and cytopl asmic compartments were outlined using the "lasso" feature. In the cell shown here the cytoplasm is outlined in blue, whereas the nucleus is outlined in yellow. The N/C ratio was determined from the following equation: number of pixels in nucleus x (mean-background)/number of pixels in cytoplasm x (mean background).

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145 topo I was 27.0 3.2 in log-phase H929 cells and 29.2 4.1 at 16 h accelerated plateau. The median N/C ratio of topo II was 10.1 5.5 in log-phase and 8.9 5.4 at 16 h accelerated plateau. The subcellular distribut ion of histones was monitored as a nuclear control and also does not change in the accele rated-plateau cells. Similar results were obtained in 8226 cells (data not shown). For example, the median N/C ratio in 8226 cells of topo II in log-phase was 9.5 6.3 versus 2.1 1.6 at 16 h accelerated plateau. The subcellular distribution of topo II in H929 cells was confirmed by biochemical separation of the nuclear and cy toplasmic compartments with subsequent western blot analysis (Figure 32). The subcel lular locations of topo I and LDH were used as nuclear and cytoplasmic controls respectively. The amount of topo II found to be distributed to the cytoplasmic compartment in plateau-phase H929 cells varied between 25% and 50% of the total cellular amount. This experiment was repeated seven times and the majority of the experiments demonstrated 40-50% cytoplasmic topo II in plateauphase cells. These results agr ee with the microscopy findings above, which demonstrated a N/C ratio of 1.3 1.3 for 16 h plateau-phase H929 cells. The partial degradation of topo II that occurred during th e nuclear-cytoplasmic separation was not observed when cells were immediately immunobl otted, and was not prevented by the addition of several protease inhibitors (see “Mat erials and Methods”). Simila r immunoblot results were obtained in HL-60 cells (dat a not shown) and 8226 cells. T hus, it appears that topo II but not topo I or II is shuttled to the cytosol when H929 cells are grown at a superconfluent cell density.

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146 12.8 Topoisomerase II Enzyme Activity. Although the total amount of topo II protein does not change between log and accelerated-plateau cells, a redistribution of topo II from the nucleus to the cytoplasm would be expected to decrease the amount of drug target in the nucleus. If the nuclearcytoplasmic trafficking of topo II results in attenuation of nuclear content, then there would be less drug-induced DNA damage a nd fewer cleavable complexes expected. Thus, we hypothesized that a cytoplasmic distribution of topo II would protect cells from drug induced DNA damage by attenuating the amount of lethal drug-enzyme-DNA Figure 32. Nuclear-cytoplasmic Separati on and Western Blot Analysis of DNA Topoisomerase I and II and LDH. Nucl ear-cytoplasmic separation of log and plateau (16h) H929 cells followed by Western blot analysis for topo II topo I, and LDH. Experiment A demonstrates that in plateau phase there is approximately onehalf of the cellular topo II (partially degraded) in each of the nuclear and cytoplasmic compartments. In experiment B, the amount of total cellular topo II is distributed as approximately 75% to th e nucleus and 25% to the cytoplasm in plateau phase growth (determined by dens itometry). The subcellular locations of topo I and LDH were used as nuclear a nd cytoplasmic controls, respectively. WC, whole cells; N, nucleus; C, cytoplasm.

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147 complexes formed. To test this hypothesis the amount of VP-16 induced DNA damage was compared in log and accelerated-plateau phase (16 h) H929 cells by the alkaline comet assay (Figure 33). The mean of thr ee experiments showed that the average comet moment was higher in log-phase cells than accelerated-plateau cells at each drug concentration. For example, the average comet moment for log-phase cells was 80 7 and 50 5 for accelerated-plateau ce lls in the presence of 10 M VP-16. The results indicate that there are less drug-induced double strand DNA break s in cells seeded at the super-confluent cell density.

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148 Figure 33 Determination of VP-16 Induced Double-strand DNA Breaks. Measurement of VP-16 induced double-strand DNA breaks in log and accelerated-plateau H929 cells at 16 h using the comet assay. Cells were treated with 0-50 M VP-16 as descri bed in “Materials and Methods”. The average comet moment of 50 cells at each drug concentration was calculated from three experiments and is shown in panel B. A representative comet is shown in A. (o), log-phase cells; (•), acceleratedplateau cells.

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149 To determine if the decrease in DNA damage can be explained by a reduction in the number of drug-induced enzyme-DNA complexes formed, the amount of topo II drug-stabilized cleavable complex formati on was compared in log and acceleratedplateau (16 h) cells using a modification to the band depletion assay originally described for observing topo I cleavable complex formation in the presence of topotecan. We adapted the same procedure to observe topo II cleavable complex formation in the presence of etoposide. This assay is base d on the observation that topo II covalently bound to DNA migrates more slowly than fr ee topo II on sodium dodecyl sulfate (SDS)polyacrylamide gels (Figure 34). Accordin gly, as topo II forms increasing amounts of cleavable complexes in the pr esence etoposide, ther e is a corresponding decrease in the Figure 34 Illustration of Band Depl etion Assay. The band depletion assay was preformed as described in "m aterials and methods". Briefly, cells treated with increasing concentrations of etoposide (VP-16) are lysed in a buffer containing guanidine hydrochloride, which acts as a strong protein denaturant that traps preexisting covalent topo II-DNA complexes. Cell preparations are then electrophoresed on an SDSpolyacrylamide gel. Topo II -DNA complexes run more slowly than free topo II which is observed as a decrease in the intensity of the topo II by Western blot analysis. Free topo II Trapped topo II VP 16 Stacking gel Separating gel

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150 molecular weight signal of topo II by Western blot analysis (Figure 35) (Kaufmann et al., 1997). Results from the band depleti on assays showed that a 7-fold higher concentration of etoposide was needed to deplete the topo II levels by 50% in accelerated-plateau H929 cells. The drug concen tration to achieve 50% band depletion is 5 M in log-phase cells and 35 M in accelerat ed-plateau cells, sugge sting that there are fewer enzyme-drug DNA complexes formed in accelerated-plateau cel ls relative to logphase cells. These results were further c onfirmed by measuring the catalytic activity (kDNA decatenation) of topo II present in the nuclear extracts from log and acceleratedplateau H929 and HL-60 cell lines (data not shown). Approximately 4-times more nuclear extract protein was needed to decatenate 1 g of kDNA in either acceleratedplateau H929 or HL-60 cells than in log-phase cells.

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151 Figure 35. Measurement of Cleavable Complexes. Differences in the formation of VP-16-stablized enzyme-DNA complexes were examined in log and accelerated-plateau H929 cells using the band-depletion assay.These results show that a 7-fold increase in VP-16 was needed to achieve a 50% reduction in the topo II band in plateau-phase cells relative to log-phase cells. (o), lo g-phase cells; (•), accelerated-plateau cells. To p o II cleava b le com p lexes become tra pp ed in the stackin g g el Topo II

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152 Collectively, these data suggest that th ere are significantly fewer topo catalyzed double-strand DNA breaks in VP-16 treated accel erated-plateau cells than log H929 cells because there are fewer druginduced DNA-protein complexes formed. Thus, a decrease in drug-induced DNA-protein complexes c ould explain in part the observed drug resistance to topo inhibitors; attenuation in the total amount of cleavable complexes formed can best be explained by th e cytoplasmic trafficking of topo II enzyme. Also, nuclear topo II present in plateau H929 cells ap pears to be less drug sensitive. 12.9 Subcellular Distribution of Topo II in Malignant Plasma Cells. We next studied the distribution of topo I, topo II and topo II in clinical samples: bone marrow aspirates from myeloma patients. Representa tive aspirates from two out of 10 patients examined are shown in Figure 20 and figure 21. The aspirates (both with >90% plasma cells ) were obtained before highdose chemotherapy but after standard-dose chemotherapy. Figure 37 show s the subcellular distribution of topo II in one untreated patient with multiple myeloma. In all patients, there was a considerable proportion of topo II in the cytoplasmic compartment of the plasma cells. The percentage of plasma cells that was bri ghtly fluorescent and displayed a nuclear distribution of topo II was always in the range of 10-15% and reflected cells that were probably in the S-or G2/M-phase of the cell cycle. Topo I was found to be strictly nuclear or nucleolar except for one patient (90% of cas es). In the one patient who had relapsed after high-dose chemotherapy, t opo I was found to be exclusively cytoplasmic. The topo II subcellular distribution was mostly a mixe d nuclear and cytoplasmic pattern, and in ~50% of the patients showed nucleolar staining as well. Generally, topo II labeling

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153 demonstrated a low expression by immunofluor escence and was nearly undetectable on Western blots. When the nuclear/cytoplasmic ratios of topoisomerases were examined in the malignant plasma cells from 10 myel oma patients by confocal laser scanning microscopy, only topo II was present with ratios below 1 in the majority of cells. Collectively, the data in this study suggest s that a correlation may exist between the cytoplasmic location of topo II and decreased sensitivity of th ese cells to topo inhibitors. 12.10 Conclusions. In this study, we have investig ated the role of topoisomerase II trafficking in cellular drug resistance. We developed a cell model (called accel erated-plateau) using human myeloma H929 and 8226 cells that reproducibly translocates topo II to the cytoplasm. It was necessary to separate th e influence of the cell cycle, drug uptake, topo protein levels, and enzyme trafficking of drug se nsitivity in this cell model. Compared to log-phase cells, the cytoplas mic redistribution of topo II in the accelerated-plateau cells correlated with a 10-fold resistance to VP -16 and a 40-60% reduction in the number of drug-induced double strand DNA breaks. In ad dition, 7-fold more VP-16 was necessary to achieve 50% topo II band depletion, suggesting that there are fewer drug-induced topo-DNA complexes formed in the accelerated-pla teau cells than the log-phase cells. The total cellular amount of topo II and topo II protein in logand accelerated-plateau was similar as determined by Western blot analysis. There was a 25% reduction in Sphase cell number in plateau cells (d etermined by bromodeoxyuridine (BrdU) incorporation).

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154 The data from this study support th e hypothesis that the nuclear-cytoplasmic trafficking of topo II mediates, at least in part, cellula r drug resistance to topo inhibitors by reducing the amount of topo II in the nucleus and the subsequent number of enzyme drug-induced DNA complexes formed. Thus, we established a novel mechanism of de novo drug resistance that is independent of drug transport and cellular quantity of topo II Furthermore, Western blot analysis demonstrates that the cytoplasmic location of topo II is not likely to be a resu lt of expression of a truncated enzyme that has lost the C-terminal NLS (Wessel et al., 1997). These data may have clinical implications in the treatment of human myeloma, as immunohistochemistry (Figures 36 and 38 ) and Western Blot an alysis (Figure 37) demonstrates that topo II has a cytoplasmic distributi on in malignant plasma cells obtained from the bone marrow aspirates of myeloma patients before (Figure 36) and during treatment (Figures 37 and 38). The data also suggests that topo II and topo I may be more appropriate molecular targets in multiple myeloma because neither their amount nor their subcellular localization were found to be al tered at plateau densities.

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155 Figure 36. Subcellular Distribution of Topoisomerase II in Plasma Cells from One Untreated Multiple Myeloma Patient. Immunofluroescent staining for Topo II and histone. Goat anti-rabb it IgG-TRITC labeled antibody was used for detecting topo II (appears red). Goat anti-mouse IgG-FITC labeled secondary antibody was used to detect histone (appears green) as a nuclear control. Cells were mounted on micr oscope slides containing Vectashield mounting media containing DAPI, wh ich binds DNA (appears blue). Figure 37 Western Blot Analyses of Topoisomerase I and Topo II in Two Patients with Multiple Myeloma being Treated with Chemotherapy. The nuclear and cytoplasmic compartmen ts were separated as described in "Materials and Methods" and the equivalent of 5.0 x 105 cells was loaded in each lane. The CCRF cells were loaded as a positive control for topo I and topo II immunoreactivity. WC, whol e-cell lysates; nu, nuclear fraction; cy, cytoplasmic fraction. Topo II Histone Merged

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156Figure 38. Subcellular Distribution of Topoisomerase in Two Multiple Myeloma Patients being Treated with Chemotherapy. Confocal microscopy was performed on plasma cells obtained from patient 1 (A-F) and Patient 2 (M-R). Cells were stained for topo II (A and M) with polyclonal antibody 454 and histones (B and N). The merged image (C and O) demonstrates that topo II is nuclear ~ 10% of the cells (proliferative fraction) and cytoplasmic or faintly nuclear in the remainin g plasma cells. Histone staining was used to define the nucleus. Patient plasma cells were also stained for topo II (D and P) with the polyclonal JB1 antibody, for topo I with the C-21 monoclonal antibody (E and Q) and the merged images (F and R). Topo II was observed in the nucleus (D), cytoplasm (F) and nucleoli (P), but its amount was relatively low compared with topo II Topo I distribution was typically nuclear (E) or nucleolar (Q). Merged Histones Topo II Topo II Histones Merged Merged Topo I Topo II Topo I Merged Topo II Patient 1 Patient 2

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157 Chapter Thirteen Experimental Results Part III: Human Topoisomerase II Contains Two Leptomycin B Sensitive Nuclear Export Signals 13.1 Introduction. Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase c onditions to high-density plasteau phase conditions exhibit a cell density de pendent translocation of topo II to the cytoplasm. The determination of the mechanism of topo II transport across the nuclear membrane could lead to a better understa nding of how to regulate the le vels of active enzyme in the nucleus, and thus sensitize cells to topo inhibitors. We questioned whether the cytoplasmic distribution of topo II was a result of decreased import or increased export. Initially, we tried to block the import of topo II in log and plateau density cells by using the general nuclear import in hibitor, lectin. Wheat germ agglutin (WGA) is a nonspecific inhibitor of general nuclear import (Duveger et al., 1995). WGA is believed to inhibit nuclear import by binding nucleopor ins and "clogging" the nuclear pore complexes. We did not observe a differen ce in the nuclear-cytoplasmic distribution of topo II in log or accelerated plat eau 8226 or H929 cells treated with lectin as determined

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158 by confocal microscopy (unpublished data). On e possible explanation of these results is that in log-phase cells, topo II was already present in the nu cleus prior to drug treatment and does not shuttle continuously between th e nucleus and cytopl asm of log-growing cells. We also treated accelerated plateau 8226 and H929 cells with lec tin to determine if a greater percentage of topo II protein would become trapped in the cytoplasmic compartment of lectin treated cells comp ared to untreated cells. We observed no significant differences in the nuclea r-cytoplasmic distribution of topo II in the presence of lectin. These results sugge st the possibility that topo II is exported from the nucleus to the cytoplasm, and thus is not inhibited by lectin. Therefore, we investigated the ability of the nuclear export inhibitor, Leptom ycin B, to block the nuclear export of topo II in the accelerated-plateau cells. 13.2 LMB Modulation of Topoisomerase II Trafficking. To determine the mechanism by which topo II accumulates in the cytoplasmic compartment, accelerated-plateau H929 cells were treated with the nuclear export inhibitor, LMB (Figure 39). This Streptomyces metabolite has been shown to inhibit CRM1-mediated nuclear export of proteins with leucine-ri ch nuclear export signals (Engel et al., 1998; Wolff, B. et al., 1997; Nishi et al., 1994; Hama moto et al., 1985). Immunofluorescent microscopy demonstrates that the median N/C ratio of topo II is increased 58-fold in plateau H929 cells tr eated with 0.5 ng/ml of LMB for 16 h when compared to untreated cells. The median N/ C ratio of untreated accelerated-plateau cells is 0.98 2.0 and increases to 57.5 170.2 in th e presence of LMB. A total of 50 cells were examined per condition and the experiment was repeated three times. Similar results

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159 were obtained in HL-60 cells (data not shown) The large standard deviation observed in the LMB-treated cells can be accounted for by the presence of several cells with N/C ratios > 300. Therefore, the median values ha ve been reported as a more representative value of the true N/C ratio. These data indica te that the cytoplasmi c translocation of topo II can be blocked by LMB and suggests that topo II is exported from the nucleus as opposed to retained in the cytoplasm in untr eated cells. Furthermore, the average total pixel density of topo II in whole cells decreased fr om 113,929 51,530 in control cells to 38, 053 41, 040 in LMB-treated cells, a 67% reduction in pixel density of whole cells. These data suggest that topo II may be downregulated in the presence of LMB because there is a greater re duction in total pixel density of treated cells relative to untreated plateau cells. Th e pixel density of topo II immunofluorescence in the nuclear compartment is reduced from an averag e pixel density of 52,241 21,716 to 868 1226 pixels. One possible explanati on is that LMB blocks the export of topo from the nucleus and topo becomes degraded by nuclear protea somes. The degradation of topo has been shown to occur in the nucleus, bu t Western Blot analysis of topo II protein content in LMB treated plateau cells would be necessary to confirm these results. These results suggest that topo II trafficking in the log-plateau cell model could be a result of increased nuclear export.

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160Pixel Density Pixel Density 0 100000 200000 0 100000 200000 y Figure 39. Confocal Microscopy and Nuclea r-cytoplasmic Ratios of DNA Topoisomerased II Immunoflurescent Staining in the Presence or Absence of Leptomycin B. Subc ellular localization of topo II and pan-histone in 16 h accelerated-plateau H929 cells 0.5 ng/ml LMB (panel A, 0 ng/ml LMB; panel B, 0.5 ng/ml LMB). The nu clear-cytoplasmic ratio per cell is shown for 50 plateau-phase cells ( LMB) in panel s C and D. tetramethylrhodamine isothi ocyanate (TRITC) staining (red) is for topo II and FITC staining (green) is for histones, in the immunofluorescence figures. The experiment was repeated three times in H929 cells and once in HL-60 cells (data not shown). Cytoplasm ( ), nucleus ( ). Plateau (16 h), 0 ng/ml LMB A. Plateau ( 16 h ), 0.5 n g /ml LMB B.

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161 Table 10. Determination of a Consensus Sequence from Established Nuclear Export Signals (NES) -actin (Wada et al., 1998 ) A L* P H A I M R L D L A -actin (Wada et al., 1998) A L P H A I L R L D L A TFIIIA (Fridell et al., 1996) L P V L E N L T L PKI (Wen et al., 1995 ) E L A L K L A G L D I N MAPKK (Fukada et al., 1996) A L Q K K L E E L E L D RevHIV (Fischer et al., 1995) Q L P P L E R L T L D Ran BP-1 (Zolotukhin and Felber, 1997) K V A R K L E A L S V R C-Abl (Taageoera et al., 1998) L E D N L R E L Q I C hZyxin (Nix and Beckerle, 1997) L T M K E L E E L E L L MdM2 (Roth et al., 1998) S L S G D L S L A L C P53 (Strommel et al., 1999) F R E L N E A L E L K D Consensus H X1-4H X2-3H X H (X = Leu, Ile, Val, or Phe) *Bold letters represent hydrophobic residues thought to be crucial for nuclear export

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162 13.3 Determination of Consensus Sequence for Nuclear Export Signals. Since we observed a 58-fold increase in the nuclear-cytoplasmic ratio of topo II immunofluorescence in accelerated -plateau H929 cells treated with leptomycin B when compared to untreated cells, we hypothesized that topo II may contain a LMB sensitive nuclear export signal. Thus, we aligned seve ral proteins with esta blished nuclear export signals to determine a consensus sequence (t able 10). We determined the consensus sequences as HX1-4HX2-3HXH (where H represents the hyd rophobic amino acids, leucine, isoleucine, valine, or phenylalanine). Th e hydrophobic amino acids are separated by a characteristic spacer region containing any amin o acids (represented as the letter "X"). Using the consensus sequence we identified si x matching nuclear export sequences in the amino acid sequence for human topo II (Table 11 ), which was downloaded from the National Center for Biotechnology Informa tion database (accession number NP 001058). We initially expected to express exogenous topo II in human myeloma cells to investigate the nuclear cytoplasmic distributi on between log and plateau cell-density. 13.4 Transfection of Topo II and Alternative Experiments. For one year, I attempted to transfect a number of human myeloma and leukemia cell lines with a full-length topoisomerase II green fluorescent (GFP-topo II ) fusion protein expression vector. The expression vector that I work ed with was obtained from a laboratory that described a me thod for cloning the GFP-topo II gene that produced high levels of expression of the topo II -GFP chimera in the nucleus of human cells (Mo et al., 1998). During this time, I worked with the combined efforts of Dr. Terresita Muoz and her laboratory personnel. However, we f ound that in most cases we were unable to

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163 obtain a positive clone. The cl ones that were achieved ofte n failed to grow exponentially, suggesting that the bacteria were resi stant to the uptake of the large topo II gene. Thus, many of my initial experiment s resulted in a low yield of plasmid with the topo II gene making subsequent transfection procedures unsuccessful. We used several different methods for transfecting human myeloma cell lines including cationic lipids and electroporation. We also atte mpted to express topo II -GFP under the regulation of an inducible promoter, but also had negative results. The most successful experiments demonstrated transient expression of the topo II -GFP chimera, but at very low transfection efficiency. For example, I tr ied to transfect human myeloma H929 cells, HL-60, and 8226 cells by electroporation. However, electroporation also produced negative results that included a high rate of cell death and low transfection efficiency. Of the cells that were transf ected, I observed gross mor phological changes in their appearance. In addition, tr anslocation of GFP-topo II to the cytoplasm in plateau density cells was extremely limited when compared to endogenous topo II (data not shown). I found that expression of GFP-topo II recombinant protein was cytotoxic, inducing cell death in all cell lines tested (CCRF, H929, HeLa, HL-60, 8226, Flow fibroblast 2000) within 12-16 hours after tran sfection. This was a significant problem that impeded our ability to monitor the nuc lear cytoplasmic trafficking of the topo II GFP fusion protein because according to my accelerated-plateau cell model, the transfected cells would need to grow for 16-24 hours at a high-cell density.

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164Table 11. Putative Nuclear Export Signals in Human DNA Topoisomerase II Topo II Amino Acids Consensus Sequence H X1-4H X2-3H X H (X = Leu, Ile, Val, or Phe) 80-90 G L* Y K I F D E I L V 230-241 S L D K D I V A L M V R 467-476 T L A V S G L G V V 569-580 F L E E F I T P I V K V 1017-1028 I L R D F F E L R L K 1054-1065 F I L E K I D G K I I I *Bold letters represent hydrophobic residues t hought to be crucial for nuclear export

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165 The lack of success with expressing exogenous topo II into human cells led us to look for an alternative method for defining a nuc lear export signal. Different methods for defining a nuclear export signal have been described (Koster et al., 2005; Henderson and Eleftherion, 2000; Bogerd et al., 1996). We decided to crosslink the putative nuclear export signals found in topo II to bovine serum albumin (BSA ), a large carrier molecule that is unable to be transported across the nucleus, and then microinject the NES-peptide BSA conjugates into the nucleus of cells. By labeling the NES-peptide BSA conjugates with a fluorescent molecule (ie FITC or TRIC) we were able to visualize the subcellular localization of the microinjec ted peptides. Nuclear export was observed when the NESpeptide was able to transport BSA from the nucleus to the cytoplasm. BSA is a suitable molecule for investigating nuclear-cytoplas mic transport pathways because it does not contain a functional NES. Th erefore, putative nuclear expor t signals can be crosslinked to BSA to determine if they are sufficient to transport BSA across the nuclear membrane and into the cytoplasm. Although the objec tive of transfecting human myeloma cells with topo II was not abandoned, my specific efforts were redirected towards the microinjection experiments. The six putative NES shown in table 11 were synthesized and their sequences confirmed as described in "materials and me thods". The peptides were synthesized as native (nt) or mutated ( ) sequences. The mutated peptides contained alanine in place of those hydrophobic residues suspected of be ing critical for Crm-1 mediated nuclear export. Crm-1 is a nuclear export recept or that recognized sp ecific types of NES containing a characteristic spacing of hydr ophobic amino acids. Although other export receptors are likely to exist, crm-1 is the only export receptor for NES containing proteins

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166 that has been described to date. To faci litate conjugation with preactivated SMCC-BSA, the NES peptides were designed with a cysteine residue at the amino terminus. In this manner, SMCC-BSA forms a disulfide bond with the cysteine residue in the peptide sequences. We did an initial experiment to determine the optimal duration required to incubate the NES peptides with SMCC-BS A and the approximate quantity of total protein needed for gel electrophoresis. Fi gure 40 shows that after five minutes of reaction time, detection of NES peptides cr osslinked with SMCC-BSA are noticeable as a shift in the apparent molecular weight of SMCC-BSA (compare lanes 4 and 5). We determined that 20-30 minutes are optimal fo r NES peptide crosslinking to SMCC-BSA. Proceeding one peptide at a time, the peptid es were reacted with SMCC-BSA, and then the conjugated peptides we re purified by size exclusion chromatography on a using a Pharmacia P500 FPLC system. As peptides passed through the LKB control Unit UV-1, the absorbance spectra was recorded. An ex ample of the absorbance spectra obtained for peptide containing amino acids 80-90 is s hown in figure 41. Three main peaks are evident and correspond with the size of the eluted fractions The largest compounds elute first, whereas the smallest molecules become impeded within Superdex gel matrix beads and elute last. FPLC fractions 4-7 (Fi gure 41) are likely to be large SMCC-BSA molecules crosslinked with each other, fractions 16-18 represent conjugated BSA peptides, and fractions 30-32 represent unreact ed peptide molecules. Peptide conjugation was confirmed by running FPLC eluted frac tions on a 10% SDS-PAGE gel, and then visualizing the proteins by s ilver stain analysis (Figure 43 and 44). Similar conjugated BSA peptide fractions were pooled and concen trated before reacti ng with fluorescein isothiocyanate (FITC). FITC labeled BSA-pe ptides were again purified by gel filtration

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167 chromatography (Figure 42) and a large singl e peak was noticeable on the absorbance spectra (data not shown). The type of ce ll to be microinjected was an additional experimental parameter that we had to dete rmine before beginning our experiment. We initially wanted to microinject human myeloma or leukemia cell lines with the crosslinked peptide, but we ra n into two problems. First, the myeloma and leukemia cell lines are suspension cells. To microinject suspension cells, th ey must be held in place by suction with one capillary while being pricked with a needle with the opposite hand. The human myeloma and leukemia cell lines proved to be too small, and instead became trapped inside the capillary (Figure 45). So, we tried using the adhe rent cell lines, HeLa and FlowFibroblasts2000. We found that pre-treating gl ass coverslips with HCl optimized the adhesion of HeLa cells to the co verslips. The HeLa cells spread out across the coverslips and gave us an easily identi fiable nucleus to in ject (Figure 45). Furthermore, microinjection of the HeLa cell di rectly onto the glass coverslips facilitated their visualization by fluorescence microscopy.

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168 Figure 40. Coomassie Blue Stain of SDS PAGE ge l with NESpeptide BSA Conjugates. Lanes 1 & 2 are molecular weight markers; lane 3, bovine serum albumin; lane 4, preactivated SMCCBSA; lane 5, NES peptides incubated with SMCC-BSA for 5 minutes; lanes 6 and 7 are NES peptides incubated with SMCC-BSA for 20 minut es; lames 8-10 are NES-peptides conjugated with SMCC-BSA for 30 minutes; lanes 11-13 are NES peptides conjugated with SMCC-BSA for 1 h. Approximately 25g of protein were loaded in lanes 4-8; 50 g were loaded into lane 9, and 10 g were loaded into lanes 10-13. La ne 4 represents free SMCC-BSA (lower smear) and SMCC-BSA crosslinked with itself (upper smear). Lane 5 shows that after 5 min, some peptide already began to crosslink with SMCC-BSA (com pare lanes 4 and 5, notice upward shift). Arrow indicates conjugated peptides. We dete rmined that 20-30 minutes was optimal for peptide conjugation to SMCC-BSA. 1 2 3 4 5 6 7 8 9 10 11 12 13

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169 Figure 41. Absorbance Spectra of a BSA NES-peptide Conjugate at 562 nm. Three main peaks are evident and represent elution of different sized molecules. SMCC-BSA cross linked with itself is likely to be the largest complex to el ute first (fractions 4-7) followed by BSA-peptide conjugates (fractions 16-18) and then unreacted peptides (fractions 30-34). An absorbance spectra was obtained for each BSA crosslinked peptide. Figure 42. FITC-Labeled-BSA NESpeptide on Size Exlusion Coulmn. Arrows indicate the separati on of FITC-conjugates from unreacted FITC. Unreacted FITC FITC-BSA-Peptides

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170Lane 1. MW Lane 2. Free Peptide Lane 3. SMCC-BSA Lane 4.Peptide pre-FPLC 10 12 14 16 18 20 22 24 26 28 46 206, 000 132,000 78,000 7,600 Figure 43. Silver-stain Analyses of SDS-Page BSA-NES-Peptide FPLC Fractions. FPLC Peptide fractions were collected and electrophoresed as described in Materials and Methods. Gels were stained with Silver staining. Lanes 20-28 show a shift in the appare nt molecular weight of free peptide (lane 2) and free SMCC-BSA (lane 3). La ne 3 also shows that some SMCCBSA crosslinks with itself. FPLC Fractions

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171 1 2 5 6 3 Figure 44. Silver Stain Analysis of each BS A Conjugated Peptide. SDS-PAGE gels were scanned into AdobePhoto Shop 6.0. (1) native peptide 80-91 (2) muta ted peptide 80-91 (3) native peptide 230-241. Please note that mutated pe ptide 230-241 data not available because gel was shattered during the drying process. Since the native peptide did not export, SDS-PAGE was not repeated for the mutated peptide. (5 ) native pepide 569-580 (6) mutated peptide 569580. Arrows indicate FPLC fractions containi ng SMCC-BSA crosslinked to peptides Figure 44 continued on next page.

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172 9 10 7 8 12 11 Figure 44 continued. (7) native peptide 569-580 (8) mu tated peptide 569-580 (9) native peptide 1018-1024 (10) mutated peptide 1018-1024 (11) native pept ide 1054-1065 (12) mutated peptide 1054-1065 Arrows indicate FPLC fractions containing SMCC-BSA crosslinked to peptides.

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173 Figure 45. Comparison of H929 Cells with HeLa Cells for Microinjection. (A) H929 cells (low magnification) (B) H929 cells (High magnification) (C) HeLa cell. H929 cells are suspension cells, wh ereas the HeLa cells are adherent. The suction capillary is necessary to hold suspension cells in place while being microinjected. A B C Microinjection capillary Suction capillary Microinjection capillary Suction capillary

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174 13.5 Peptides NES1054-1066 and NES1017-1028 Signal the Nuclear Export of BSA-FITC. The data suggest that topo II may be translocated from the nucleus to the cytoplasm under specific conditions, and this may result in altered drug sensitivity. Of the six putative NES identified in topo II two peptides, NES1054-1066 and NES1017-1028, exported BSA into the cytoplasm when micr oinjected into the nuc lei of HeLa cells (Figures 46). BSA-NES1054-1066 showed strong cytoplasmic st aining and were seen in the cytoplasm within 15 minutes of microinj ection, as compared to TRITC-BSA alone, (Figure 47), or the mutated BSA-NES1054-1066 conjugate. BSA-NES1017-1028 also appeared cytoplasmic within 15 min of being microinjec ted into the nucleus, but complete nuclear clearing (like the ntNES1054-1066) was not observed even after 90 min. The mutated BSA-NES1054-1066 was nuclear in all cells observed even 90 min after microinjection. BSA-NES80-90 (Figure 47) mutated BSA-NES80-90 (Figure 47), BSA-NES230-241, mutated BSA-NES230-241, and BSA-NES467-476, BSA-NES569-580, and mutated BSA-NES569-580 all remained in the nucleus even 4 h af ter microinjection (data not shown).

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175 Native Peptide Mutated Peptide NES 1054-1066 NES 1017-1028 NES 80-90 Figure 46. HeLa cells Microinjected with Pe ptide-BSA-FITC Conjugates. HeLa cells microinjected with either w ild-type (left column) peptideBSA-FITC conjugates (green), and then counterstained with DAPI (blue). A total of 20-50 cells were successfully microinjected per peptide and similar results were seen in all cells.

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176 13.6 LMB Blocks ntNES1054-1066 and ntNES1017-1028 Mediated Nuclear Export. LMB is a specific inhibitor of Crm-1 me diated nuclear export of proteins. To determine if the nuclear export of BSA conjugated to peptides NES1054-1066 and NES10171028 was Crm-1 dependent, LMB pretreated HeLa cells were microinjected in the presence of 2 ng/ml LMB in ethanol. Fluor escence microscopy demonstrated that LMB blocked the export of BSA c onjugated to peptides NES1054-1066 and NES1017-1028 (Figure 48). Peptide NES1054-1066 had a strong perinuclear stai ning, suggesting that the protein cargo is docking at the NPC. Although the NES defined by microinjection are sufficient to transport a non-shuttling prot ein to the cytoplasm, these leucine rich sequences may or may not serve a role in exporting topo II .TRITC-BSA DAPIMerged Figure 47. HeLa Cells Micr oinjected with TRITCBSA. HeLa Cells were microinjected with BSATRITC in the nucleus and counterstained with DAPI.

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177 Figure 48. HeLa Cells were microinjected with wild-type peptide-BSA-FITC conjugates in the presence of 2 ng/ml LMB (leptomycin B). LMB NES 1054-1066 LMB NES 1017-1028

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178 13.7 Topoisomerase II Cloning, Site Directed Mutagene sis, and Gene Expression. While obtaining results from the microinj ection of the BSA-peptides, we began to obtain promising results with the tran sfection of a FLAG-epitope tagged topo II in HeLa cell lines. Although my focus in the laborator y was concerned with the microinjection of conjugated BSA-peptides, I had a vested in terest in the project and maintained a significant involvement with the transfection studies. As such I shared responsibility for certain experimental procedures such as, de sign of the experiments, selection of the mutated nucleotides (Table 12), a nd immunostaining of the FLAG-topo II chimeras. In the earlier stages of the work, I transformed bacteria and performed a number of plasmid preparations. FLAG peptide is an eight amino acid protein (NYKNNNNK) that does not occur in nature. FLAG does not contain any putative nuclear export signal and its small size limits any secondary protein structure problem s. Hela cells transfected with a FLAGtopo II plasmid vector expressed a full-length (170 kDa) topo II recombinant protein (Figure 49 ) Using a specific transfection protocol previously described by VandenHoff et al., 1992, we were able to transfect H 929 and HL-60 cells by electroporation with a high degree of efficiency for these cell line s (2-20%). The succe ss of the transfection experiments is attributed to the buffer desc ribed by Van den Hoff et al., 1992. The buffer contains ATP and glutathione to promote th e rapid repair of cellular membranes.

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179 13.8 FLAG-topoisomerase II Immunofluorescence. BSA-peptide microinjection data indicated that the putative nuclear export sites at 1017-1028 and 1054-1066 may function to signal export of topo II To confirm these data with a fulllength topo II protein, H929 human myelom a cells were transfected with FLAG-topo II expression vectors possessing mutated hydrophobic residues in the nuclear export sites at 1017-1028 and 1054-1066 (Table 14). Twenty hours posttransfection, live cells were isolated by cen trifugation on a ficoll-paque gradient and plated on glass microscope slides usi ng cytospin funnels. After fixing and permeabilization, slides were stained with anti-FLAG M2 monoclonal antibody-FITC conjugate and counterstained with mounting me dia containing DAPI to show the location of the nuclei. Images were acquired using a fluorescent microscope (Figure 50), with quantitation of FITC fluores cence using Adobe Photoshop 7.0 (data expressed in Figure 51). Figure 50 establishes that the wild-type (non-mutated) FLAG-topo II protein is Figure 49. Western blot of full-length FLAG-topo II HeLa cells were transfected with plasmid containing FLAG-topo II plasmid via cationic lipid, and harvested after 20 hours. Protein extracts from 2.0 x 106 cells per lane were separated on SDA-PAGE gel, blotted onto nitrocellulose and probed with FLAG M2 antibody. Lane A is protein from cells transfected with non-mutated FLAG-topo II plasmid, lane B is FLAG-topo II 1054-1066, lane C transfected with FLAG-topo II plasmid 1017-1028, and lane D is a non-transfected control.

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180 present in the nucleus of the cells plated at log density, whereas FLAG-topo II protein is located in the cytoplasm in cel ls plated at plateau densit y. Quantitation of fluorescence revealed a statistically significant shift ( P =0.00001) for log cells with a nuclear:cytoplasmic ratio of 5.9:1, to a ratio of 0.42:1 in plateau cel ls (Figure 51) when using the wild-type FLAG-topoII plasmid.

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181Table 12. Wild-type and Mutated Nucleotide Se quences of Putative Nu clear Export Signals. Wild-type sequence 1017-1028 TTG GAT ATT CTA AGA GAC TTT TTT GAA CTC AGA CTT AAA TAT TAT L D I L R D F F E L R L K Y Y Mutated sequence 1017-1028 AGC CTG GAC AAA GAT GCT GTT GCA GCA ATG GTC AGA AGA GCA D I L R D A F E A R L K Y Y Wild-type sequence 1054-1065 CGC TTT ATC TTA GAG AAA ATA GAT GGC AAA ATA ATC ATT GAA AAT R F I L E K I D G K I I I E N Mutated sequence 1054-1065 CGC TTT ATC TTA GAG AAA GCA GAT GGC AAA GCA ATC ATT GAA AAT R F I L E K A D G K A I I E N

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182 Mutated 1017-1028 Mutated 1054-1066 Figure 50. FLAG-topo II Immunofluorescence. Human multiple myeloma H929 cells were transfected by electroporation with full-length wild-type and mutated toto II and plated for 20 hours at log and plateau cell densities. Cytospins containing fixed cells were stained with FITC-labeled anti-FLAG M2 antibody (green), counterstained with DAPI for nuclear staining, and assayed by immunofluroescent microscopy.. Wild-type Wild-type Mutated 1017-1028 Mutated 1054-1066 FITC Log DAPI Merged FITC Plateau DAPI Merged

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183 1053-1066 plateau 1053-1066 log 1016-1027 plateau 1016-1027 log Wild-type plateau Wild-type log *p = 0.00001 Nucleus Cytoplasm Figure 51. Quantitation of FLAG-topo II Immunofluoresence. Human myeloma H929 transfected cells (n = 20) stained with anti-FLAG M2 monoclonal antibody-FITC conjugate were assayed for nuclear and cytoplasmic immunofluorescence. Quantitation of FITC fluorescence was performed using Adobe Photshop 7.0 program. Wild-type FLAG-topo II was export to the cytoplasm in cells at plateau density (P = 0.00001), whereas topo II mutated at the putative export sit es, 1016-1027 and 1053-1066, did not demonstrate statistically significant levels of export to the cytoplasm.

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184 13.9 Peptide ntNES1054-1066 and ntNES1017-1028 are Conserved. To determine if peptides NES1054-1066 and NES1017-1028 are conserved, a BLAST search of the SWISS PROT database was pe rformed to identify homologous sequences in topo II Tables 15 and 16 summarize a list of representative species containing homologous topo II sequences. The data show that the characteristic spacing of hydrophobic residues in peptides NES1054-1066 and NES1017-1028 are highly conserved in a broad range of species. For example, le ucine residues appearing in human topo II NES are often substituted with the hydrophobic amino acids isoleucine or valine. Furthermore, Phe1054 and Ile1055 in peptide NES1054-1066 are highly conserved from mammals to the most primitive eukaryotic organism, Giardia lamblia unlike Leu1056. This suggests that the presence of phenylalanine and isoleuci ne are critical for nuclear export of this peptide, and thus an omi ssion of these two hydrophobic amino acids from the peptide sequence could explain why a previous report failed to identify NES1054-1066 as the dominant nuclear export si gnal (Mirski et al., 2003)

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185Table 13. Sequence Alignment of DNA Topoisomerase II NES1017-1028 Homo sapiens (human) NES1017-1028 D I* L R D F F E L R L K Sus scrofa (pig), Mus musculus (mouse), Rattus norvegicus (rat) D I L R D F F E L R L K Cricetulus griseus (Chinese hamster) D I L D F F E L R L K Gallus gallus (chicken) D I L F F E L R L Saccharomyces cerevisia (yeast) I L F V R L Aspergillus niger (fungus), Penicillium citrinum (fungus) D I L F F L R L Nicotiana tabacum (tobacco) D I L F V R L Encephalitozoon cuniculi (protozoan) I L F L R L Bombyx mori (silkmoth) I L R F L R L *Bold letters represent hydrophobic residues t hought to be crucial for nuclear export.

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186Table 14. Sequence Alignment of DNA topo II NES1054-1066 Homo sapiens (human) NES 1054-1066 F I L E K I D G K I I I E Cricetulus griseus (Chinese hamster), Mus musculus (mouse), Rattus norvegicus (Rat) F I L E K I D G K I I I E Caenorhabditis elegans (nematode) F I L E K I D G K I V I E Saccharomyces cerevisiae (yeast) F I L K I I V I E Candida glabrata (yeast) F I K I L I Aspergillus candidus (fungus) F I K I L I I Trichophyton rubrum (fungus) F I K I G I V I Giardia lamblia (protist) F I K I I I Bold letters represent hydrophobic residues t hought to be crucial for nuclear export.

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187 13.10 NES1054-1066 and NES1017-1028 Reside within a Putative Coiled-coil Domain We were interested in predicting the stru ctural features of the region containing NES1054-1066 and NES1017-1028. Each topo II monomer can be divided into three domains, an N-terminal domain that contains th e ATP-binding region, th e central domain containing the active site tyrosine residue, a nd the C-terminal domain that contains the nuclear localization sequences. Both of the NES are situated upstr eam of the bipartite NLS (Figure 52) and downstream of the active-site tyrosine residue (Tyr805). Furthermore, several CK-2 phosphorylation sites downstream of both NES have been identified in vitro and could be important for regula ting the subcellular localization of topo II Although NES1017-1028 alone is predicted to form an -helix, we were interested in predicting the motif of the complete amino acid sequence stretching from NES1017-1028 to NES1054-1066. According to EMBOSS and Predict Protein, two programs designed to predict protein motifs, amino acids 1017-1066 ar e characterized by a high potential to form -helices and also contai n five 4-3 hydrophobic repeats, a typical feature of a coiled-coil motif. Such a repeating pattern of hydrophobic amino acids has been shown to form a hydrophobic core, which is critical for dimerization. In this manner, the hydrophobic amino acids are predicted to align on the same interface which facilitates DNA binding or protein-protein interactio ns. Interestingly, amino acids 1014-1057 in topo II have previously been shown to form a stable two-stranded -helical coiled-coil in solution. Furthermore, the Advanced Chemistry Development ChemSketch 5.12 software was used to predict the 3-D stru cture of native and mutate peptides 1054-1066 and 1017-1028. The secondary structures of each peptide sequence was cleaned and then, visualized with the 3-D viewer. Alt hough each mutated peptide differed from their

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188 native peptides by just two amino acids, figure 53 establishes that each mutated peptide appears more globular than the native peptid e. These results s uggest that the native peptides may have more accessible binding si tes available to bind CRM-1, whereas the mutated peptides are more compact with fewer extensions for CRM-1 binding. These results, however, are limited to the microinj ection data and do not necessarily reflect the tertiary structure of full-length topo II in living cells. However, the results suggest that the larger amino acids such as isoleucine, le ucine, and phenylalanine, have the potential to form extensions that w ould facilitate CRM-1 binding. 13.11 Conclusions In order for topoisomerase targeted ch emotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. We have identified nuclear export signals for topo II at amino acids 101701028 and 1054-1066 using FITC-labeled BSAexport signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals was provided by transfectin of human myeloma cells with plasmids containing the gene for a fu ll-length human FLAG-topoiosmerase fusion protein. We futher showed that export by both signals was blocked by treatment of cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export.

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189 MEVSPLQPVN ENMQVNKIKK NEDAKKRL S V ERIYQKKTQL EHILLRPDTY IGSVELVTQQ 61 MWVYDEDVGI NYREVTFVPG LYKIFDEILV NAADNKQRDP KMSCIRVTID PENNLISIWN 121 NGKGIPVVEH KVEKMYVPAL IFGQLLTSSN YDDDEKKVTG GRNGYGAKLC NIFSTKFTVE 181 TASREYKKMF KQTWMDNMGR AGEMELKPFN GEDYTCITFQ PDLSKFKMQS LDKDIVALMV 241 RRAYDIAGST KDVKVFLNGN KLPVKGFRSY VDMYLKDKLD ETGNSLKVIH EQVNHRWEVC 301 LTMSEKGFQQ ISFVNSIATS KGGRHVDYVA DQIVTKLVDV VKKKNKGGVA VKAHQVKNHM 361 WIFVNALIEN PTFDSQTKEN MTLQPKSFGS TCQLSEKFIK AAIGCGIVES ILNWVKFKAQ 421 VQLNKKCSAV KHNRIKGIPK LDDANDAGGR NSTECTLILT EGDSAKTLAV SGLGVVGRDK 481 YGVFPLRGKI LNVREASHKQ IMENAEINNI IKIVGLQYKK NYEDEDSLKT LRYGKIMIMT 541 DQDQDGSHIK GLLINFIHHN WPSLLRHRFL EEFITPIVKV SKNKQEMAFY SLPEFEEWKS 601 STPNHKKWKV KYYKGLGTST SKEAKEYFAD MKRHRIQFKY SGPEDDAAIS LAFSKKQIDD 661 RKEWLTNFME DRRQRKLLGL PEDYLYGQTT TYLTYNDFIN KELILFSNSD NERSIPSMVD 721 GLKPGQRKVL FTCFKRNDKR EVKVAQLAGS VAEMSSYHHG EMSLMMTIIN LAQNFVGSNN 781 LNLLQPIGQF GTRLHGGKDS ASPR Y IFTML SSLARLLFPP KDDHTLKFLY DDNQRVEPEW 841 YIPIIPMVLI NGAEGIGTGW SCKIPNFDVR EIVNNIRRLM DGEEPLPMLP SYKNFKGTIE 901 ELAPNQYVIS GEVAILNSTT IEISELPVRT WTQTYKEQVL EPMLNGTEKT PPLITDYREY 961 HTDTTVKFVV KMTEEKLAEA ERVGLHKVFK LQTSLTCNSM VLFDHVGCLK KYDTVLDILR 1021 DFFELRLKYY GLRKEWLLGM LGAESAKLNN QARFILEKID GKI IIENKPK KELIKVLIQR 1081 GYDSDPVKAW KEAQQKVPDE EENEESDNEK ETEK S DSVTD SGPTFNYLLD MPLWYLTKEK 1141 KDELCRLRNE KEQELDTLKR KSPSDLWKED LATFIEELEA VEAKEKQDEQ VGLPGKGGKA 1201 KGKKTQMAEV LP S PRGQRVI PRITIEMKAE AEKKNKKKIK NENTEG S PQE DGVELEGLKQ 1261 RLEKKQKREP GTKTKKQTTL AFKPIKKGKK RNPWSDSESD RSSDESNFDV PPRETEPRRA 1321 ATKTKFTMDL DSDEDFSDFD EK T DDEDFVP SDA S PPKTKT S PKLSNKELK PQKSVV S DLE 1381 ADDVKGSVPL SS S PPATHFP DETEITNPVP KKNVTVKKTA AKSQSSTSTT GAKKRAAP KG 1441 TKRDPALNSG VSQKPDPAKT KNRRKRKP S T SDD S DSNFEK IVSKAVTSKK SK GESDDFHM 1501 DFDSAVAPRA KSVRAKKPIK YLEE S DEDDL Figure 52.The Complete Amino Acid Sequence of Human DNA Topo II (accession number NP 001058). The active-site tyrosine resiude (805) is indicated (*). NES10171028. 1017DILRDFFELRLK1028 and NES1054-1066 1054FILEKIDGKIIIE1066 are shaded; bipartite NLS is single underscored; Ser/Thr phosphorylation sites are bold and underscored ; the predicted coiled-coil region is double underscored ; the active-site tyrosine residue is indicated with a star, ( ).

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190 Mutant sequence (1054-1065) RFILEK A DGK A IIE Wildtype sequence (1054-1066) RFILEKIDGKIIIE N H O N H NH2N H2O NH O NH C H3CH3O NH CH3CH3O O O H NH O NH NH2O NH CH3OH NH O O O NH O NH NH2O NH CH3O NH CH3CH3O NH CH3CH3O O O H OH NH N H O NH N H2NH2O NH O NH CH3C H3O CH3NH O O O H NH O NH NH2O NH CH3C H3O H NH O O O NH O NH NH2O NH CH3C H3O N H C H3C H3O NH CH3CH3O O O H OH NH Figure 53. Predicted Tertiary Structure of Wild-type and Mutant Peptides. The 3-D structure of the wild-type and mutant peptid es was determined using the Chem-Sketch 5.12 3-D viewer. Arrows indicated amino acids protruding at or away from the viewer. Figure 53 continue on next page.

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191 Wildtype sequence (1017-1028) LDILRDFFELRLK Mutant sequence (1017-1028) LDILRD A FEA R LKO N H2CH3C H3OH NH O O O NH C H3CH3O NH CH3CH3N H O N H NH2NH O H NH O O O NH O NH O O OH NH O NH CH3CH3N H O N H NH2NH O NH CH3C H3O NH NH2OH O N H2CH3C H3OH NH O O O NH C H3CH3O NH CH3CH3N H O N H NH2NH O H NH O O O NH CH3O NH O O OH NH O NH CH3N H O N H NH2NH O NH CH3C H3O NH NH 2 OH Figure 53. Continued. The 3-D structure of the wild-type and mutant peptides was determined using the Chem-Sketch 5.12 3-D viewer. Arrows indicated amino acids protruding at or away from the viewer.

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192 Chapter Fourteen Discussion 14.1 Previous Findings. It is well established that most cell lines display a differential se nsitivity to topo II inhibitors, which depends on cell density (Sullivan 1986, 1987; Chow and Ross, 1987, Markovits, 1987). When non-tr ansformed cell-lines reach confluence there is a concomitant decrease in drug sensitivity to t opo targeting cytotoxic ag ents that has been attributed to an attenuation in the amount of topo II content (Sullivan, 1987). However, in the tumor cell lines studied, confluence doe s not necessarily lead to a decrease in cellular topo II content, yet the cells are re sistant (Sullivan 1986, 1987). We investigated several possible mechanisms of drug resi stance in human myeloma and leukemia cell lines to explain the discrepancy in the cellu lar content of topo en zymes in transformed and non-transformed cell lines. 14.2 Log and Plateau Cell Lines Drug Sensitivity Phenotype. Findings from this laboratory suggest that there are two general types of growthdependent mechanisms of drug resistance that are unrelated to prior drug exposure. The first is demonstrated by non-transformed Chinese hamster ovary and human Flow 2000 fibroblasts cells lines which are 5-15 fold resi stant to VP-16 in plateau-phase relative to log-phase cells (Table 7, page 122). This is due to an 80-90% reduction in total cellular

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193 topo II protein (Figure 23, page 127), with a co ncomitant decrease in enzyme activity (data not shown). The second mechanism is seen in several hematological cell lines (mouse leukemia L1210, human myeloma RPMI-8226, human leukemia HL-60, human leukemia KG-1a, and human lymphoblastic leukemia CCRF cells), which show significant resistance to VP16 at plateau phase (7.3-55-fold) (Table 7), but show no decrease in total cellu lar amounts of topo I, II or II protein when co mpared to logphase cells (Figure 23). Immunochemistry experiments demonstr ated that in these cells, topo II is found in the nucleus of log-phase ce lls, but had a significant cytoplasmic distribution in plateauphase cells (Figure 24, page 129). It appeared from this data that altered nucleocytoplasmic trafficking of topo II but not topo I or topo II occurs in transformed cell lines at plateau-density. This was in contrast to other non-transformed cell lines that degrade topo II at plateau phase. 14.3 Novel Human Myeloma Cell Line Models. We questioned whether the nuclear-c ytoplasmic trafficking of topo II had a role in conferring altered drug sensitivity to topo targeting agents as we ll as cross-resistance to other non-topoisomerase associated cytotoxi c agents. Alterations in the subcellular localization of topo enzymes have been previ ously associated with a decrease in drug sensitivity, but the weight of the data descri be alterations in the amount of activity of drug resistant cell lines as a result of a truncat ed enzyme that has lost its C-terminal NLS (Wessel et al., 1997). There are two notable ex ceptions that correlate altered subcellular distribution of topo enzymes with altered dr ug sensitivity. First, multicellular spheroids

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194 and Xenograft tumor cells are protected from etoposide-mediated DNA damage due to the cytoplasmic distribution of topo II (Oloumi et al., 2000). Secondly, adhesion of human myelomonocytoid U937 cells to fibronectin by means of 1 integrins protects cells against mitoxantrone and etoposide mediated DNA da mage and is accompanied by an altered sub-nuclear relocalization of topo II to the nucleolus. Thus, changes in the nuclear localization or binding propert ies of the nuclear pool of topo II protein is suggested to have a role in cel lular drug resistance to etoposi de and mitoxantrone in these cells (Hazlehurst et al., 2001). The lack of data to describe th e subcellular dist ribution of topo II especially the nuclear export of the enzyme, as a predictor of drug responsiveness could be attributed to the unavailability of in vitro cell models to study the nucle o-cytoplasmic distribution of topo enzymes, that would otherwise make it po ssible to thoroughly inve stigate the role of topo trafficking in drug sensitivity. Molecular analysis of the topo ge ne has been largely unachievable because overexpression of topo II is lethal to cells, as well as topo II gene knockouts. Most reports ar e successful at transfecting cells with truncated topo or topo peptides (Ernst et al., 2000). In either case, the exogenous fu ll-length topo is not expressed in the nucleus because of a missing C-terminal NLS. These experiments fail to correlate a cytoplasmic distribution of topo II with drug resistance because endogenous topo is still present in the nucleus to cause drug-induced DNA damage. Furthermore, the expression of an exogenous cytoplasmic topo ma kes it ineffectual at studying the nuclear export of the protein (since it never enters th e nucleus). In addition to these laboratory complications, it has proved very difficult to culture cells from a pl asma cell dyscrasia, such as multiple myeloma. Much of our understanding of the biology of multiple

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195 myeloma has been obtained by studying multiple myeloma derived cell lines. Unfortunately, human myeloma cell lines have proven difficult to tran sfect (refer to page 162). We established an in vitro cell model system, referred to as accelerated-plateau, using established human myeloma cell-lines and overcame many of the experimental complications described above. Since log a nd plateau-phase cells represent low and high cell densities relative to one a nother, we postulated that cel l-cell contacts may trigger a cytoplasmic relocation of topo enzymes. This model was used to describe the mechanism of growth dependent drug resi stance described above (Table 9, page 135), and ultimately, to better characterize the m echanism of the nucleo-cytopl asmic trafficking of topo II (Figure 39, page 160). We realize that the plateau density of cell cultures is far below the cell density existing in the tissues and biol ogical fluids of patients. Howe ver, we consider that the changes that occur in the transition from the pr oliferative state in log culture to quiescent plateau densities are a reasonable model syst em to study the molecular mechanisms of acquiring drug resistance without a drug selection pressure. 14.4 Log and Accelerated-plateau Dr ug Sensitivity Phenotype. Accelerated-plateau cells had a similar drug resistance phenotype as the naturalplateau cells described above, but the accelera ted-plateau cells were also analyzed for cross-resistance to a range of drugs with diffe rent mechanisms of cytotoxicity (Figure 5 Table 11). The accelerated-plateau cells were 10-fold resistant to VP-16, but less than 1.5 fold resistant to Ara-c, taxol, BCNU, cis-platinum, and -irradiation. Similar results

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196 were obtained in human myel oma RPMI-8226 cells. These data area not consistent with the multidrug resistance phenotype, but instead suggests that the observed drug resistance is specific for topo II interacting agen ts. Furthermore, drug resistance to DNA crosslinking agents, such as cisplatinum and -irradiation, has been found to be inversely proportional (Gornati, 1997; Barre t et al., 1994) to topo II amount as a result of enhanced DNA repair processes by topo II. The dr ug resistance observed here, in the human myeloma cells, is not likely to be a result of enhanced DNA repair because there was minimal resistance to cisplatinum and -irradiation. Furthermore, cells were hypersensitive to both cisplatinum and -irradiation at 16 h, sugge sting that there was a decrease in the total cellular content of topo II enzyme. Ho wever, Western blot analysis demonstrated minimal changes in the levels of topo protein afte r 16 hours, suggesting another mechanism exists for the observed hypersensitivity to the DNA crosslinking agents. The cells grown at plateau-phase for 24 h continued to be hypersensitive to cisplatinum but, there was a 4-fold decrease in sensitivity to -irradiation. Cell lines resistant to CDDP have not been found to be crossresistant to -irradiation, suggesting that there are different m echanism of resistance to -irradiation and cisplatinum (Caney et al., 2004). For example, there may be diffe rent DNA repair processes for radiation induced DNA damage as compared CDDP induced DNA damage (Caney et al., 1999; Wilkins et al., 1996). Furthermore, an asso ciation between the acet ylation status of DNA histones and sensitivity to radiation has b een previously reported (Zhang et al., 2004; Camphausen et al., 2004; Paol uzzi and Fig, 2004). This sugg ests that histones may be hypoacetlyated in plateau-phase myeloma cells as compared to the log-growing cells.

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197 Exposure to an HDAC inhibitor coul d sensitize the plateau-cells to -irradiation, by maintaining the acetylation of histone proteins. To elucidate other mechanisms conf erring drug resistance to VP-16 and mitoxantrone, log and accelerated-plateau cell s were examined for differences in cell cycle distribution, drug uptake, and subcel lular distribution of topo protein. No considerable difference was obs erved in the uptake of [3H-VP-16] between log and accelerated-plateau cells, sugges ting that altered drug transpor t is unlikely to account for the observed drug resistance. Furthermore, cross-resistance to non-topoisomerase targeting agents would be expected, if pl ateau-phase human myeloma cells overexpressed MRP2 or MRP3 because both VP-16 and CDDP are substrates for MRP2 and MRP3. However, accelerated-plateau cells were not cr oss-resistant to CDDP. Plateau-phase cells were minimally resistant to the non-topoisome rase targeting agents providing additional support of the results from the drug uptake studies. Since flow cytometric analysis indicate that there are minima l changes in the levels of topo protein and in the number of cells in S-phase after 16 hours re spectively, these cellu lar events are also not likely to be the principle mechanisms of drug resistance. 14.5 Subcellular Distribution of Topo II Immunofluorescence microscopy demonstrated that topo II was nuclear in logphase cells but had a cytoplasmic distribution in the accelerated-plateau cells at 16 h or 24 h. Cytoplasmic topo II was not a result of a C-termin ally truncated protein that has lost the NLS, since there we re single bands at the expect ed molecular weights for both topo II and topo II by Western blot analysis. The cytoplasmic distribution of topo II

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198 observed in the accelerated-plateau cell model was similar to that observed in the naturalplateau cells, but was observed in a shorter pe riod of time (16 h for accelerated-plateau versus 4-5 days in natural-plateau cells). This suggests that the nuclear-cytoplasmic trafficking of topo II is a function of time and stimula ted by cell-cell contact. The ratio of topo I and II did not change over the same peri od of time. Topo I remained nuclear in log, plateau, and accelerated-plateau cells. Since Western blot analysis demonstrated a reduction of topo I content in the both 8226 and H929 accelerated-plateau cells (16-34% reduction; Figure 27, page 138), this suggests that topo I may be degraded by nuclear proteasomes or that its degradation in the cy toplasm is rapid, and thus its cytoplasmic location is below the threshold of immunofluorescence detection. Topo II was distributed approximately equally between th e nuclear and cytoplasmic compartments in log and plateau cells. Theref ore, the subcellular distri bution of topo I or topo II did not correlate with drug responsiveness. Rather it appeared that an alteration in the subcellular locali zation of topo II could be the principle m echanism contributing to the decrease in drug sensitivity to topo II inhibitors. While it may appear unusual that DNA replication continues despite having a significant proportion of topo II in the cytoplasm, cells can survive with a predominantly cytoplasmic form of a catalytic ally active enzyme, which exists in several drug resistant cell lines. For example, in the drug resistant human small cell lung carcinoma H69-VP cell line, a cataly tically active yet truncated topo II enzyme is located in the cytoplasm while topo II is expressed in the nucleu s (Grue et al ., 1998). In these cells, chromosomal formation is dependent on cytosolic topo II entering the chromatin during mitosis when the nuclear memb rane disappears rath er than nuclear topo

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199 II performing the mitoti c functions of topo II (Grue et al., 1998). Similar findings have been reported in drug resistant Chinese Hams ter V511 cells that s how minor alterations in cell growth despite having drastic reductions in the total amount topo II protein in both log growth and quiescence (Hashimoto et al., 1995). The expression of topo II remained constant and topo II was presumed to compensate for topo II activity in the V511 cells. This is supported by recent findi ngs of HeLa cells tr ansiently transfected with topo II or topo II siRNA, which demonstrated that topo II can partially substitute for topo II condensation and segregation in HeLa cells (Sakaguchi and Kikuchi, 2004). These findings could explain why there was only a minimal change in S-phase population of cells, but not a complete cessation of DNA replication. Western blot analysis of topo II protein in the nuclear and cytoplasmic co mpartments of log and accelerated-plateau H929 cells indicates that topo II was reduced by 25-50% (by densitometry) compared to the log cells (Figure 32, page 146). Part of the topo II content appears to be degraded in both the nuclear and cytoplasmic compartmen ts and was attributed to experimental artifacts. Topo II degradation was likely to occur during the shearing of the nucleus from the cytoplasm as a similar degradation do es not occur in the w hole cells. Results of the comet assay confirmed that seeding cells at plateau density for 16 hours protects them from etoposide induced DNA damage and addi tional experiments demonstrated that etoposide treatment resulted in less DNA damage because th ere are fewer protein-drugDNA complexes in the cells seeded at plateau density than log-phase cells. The data establishes that the nuclear-cyt oplasmic trafficking of topo II mediates cellular drug resistance to topo inhibitors by reducing the amount of topo II in the nucleus and thus, reduces the number of enzyme-drug-DNA co mplexes formed. A redistribution of a

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200 population of topo II to the cytoplasmic compartment would have a similar effect as to drug sensitivity as an ov erall decrease in topo II content, by decreasing the amount of drug target in the nucleus. Furthermore, these results may partially explain the hypersensitivity of accelerated-plateau cells to CDDP and -irradiation observed at 16 h. As previously mentioned, drug sensitivity to DNA crosslinking ag ents is inversely proportional to topo protein le vels (Gornati, 1997; Barret et al., 1994). Although, the total cellular amount of topo II is not reduced, a shift from th e nucleus to the cytoplasm could account for the observed drug sensitivity. Several mechanisms of drug resistance to topo inhibitors have been defined in vitro and include: (1) over expr ession of drug transport pr oteins (P-glycoprotein, multidrug resistance-associated protein, lung resistance-relate d protein, or breast cancer resistance protein), that re sult in either increased drug efflux or decreased drug uptake (Shustik et al., 1995), (2) a cell-cycle-dependent decrease in the amount of topo II protein (Sullivan et al., 1986, 1987), (3) the expression of a truncated protein that has lost its Cterminal nuclear localization signal such that it remains in the cytoplasm (Wessel et al., 1997), and (4) mutations in the topo gene that result in an enzyme w ith altered catalytic or cleavage activity (Matsumo to et al., 2001). The data presented here demonstrates a fifth mechanism of drug resistance in vitro : (5) nucleocyt oplasmic-trafficking of topo II is a mechanism of de novo drug resistance to topo inhibitors in human multiple myeloma cell lines (Engel and Turner et al., 2004; Engel et al., 2004; Valkov et al., 2000).

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201 14.6 Clinical Relevance. The data establishes that a measurable difference in the nuclear and cytoplasmic distribution of topo II a chief molecular target of co mmonly used anti-cancer therapy, occurs in human myeloma cell-lines at plateau densities. The nuclear content of topo II is critical for topo targeting agents to be effective as cytotoxi c agents because topo cleavage activity is necessary for drug-i nduced DNA damage to occur. The data presented here indicate that a cytoplasmic pool of topo II correlates with cellular drug resistance to etoposide (Engel et al., 2004; Valk ov et al., 2000). This is also supported by evidence that multicell sphero ids and xenograft tumor cells are protected from etoposidemediated DNA damage due to the cy toplasmic distribution of topo II (Oloumi et al., 2000). These findings have potential clinical implications in the treatment of human myeloma as immunohistochemistry demonstrated that topo II has a cytoplasmic distribution in the malignant plasma cells obtained from the bone marrow aspirates of some multiple myeloma patients (Figure 36 and 38). The magnitude of cytoplasmic immunofluorescent staining of topo II was significantly more pronounced in the malignant plasma cells found in the clinical preparation than that observed in the huma n myeloma cells lines at plateau density. Many of the tumor cells demonstrated complete nuclear clearing of topo II that was less frequently observed in the cell lines. The plateau and accelerated-plateau cell models showed a shift (decrease) in the nuc lear: cytoplasmic ratio of topo II immunofluorescence in most cells, rather than complete nuclear cl earing (ratios below 1.0). The data suggest that other physiologi cal conditions besides cell-cell contact are involved in topo II trafficking that exist in the tumor microenvironment of the bone

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202 marrow. For example, the cell line models demonstrate a cytoplasmic distribution of topo II de novo but the cytoplasmic dist ribution observed in the clinical preparations could be influenced by prior exposure to DNA damaging agents. Multiple myeloma is characterized by karyotypic instability a nd many chromosomal aberrations are acquired after exposure to DNA damaging agents (refer to Appendix C on page 269 for a summary of hematological related chromosomal abe rrations). The amount of chromosomal aberrations correlates with di sease stage and prognosis. It is possible that certain chromosomes are susceptible to genetic tran slocations. For example, many chromosomal gene translocations that are observed in hematological malignancies involve the nucleoporins (Nup) (Arai et al., 2000, 1997; Nakamura et al., 1996; Boer et al., 1998). I have summarized a list of gene mutations th at have been described in nucleoporins (refer to Table 8 on page 217 and all reference cont ained therein). Thus, exposure to various chemotherapeutic agents could somehow disr upt the homeostasis of nucleo-cytoplasmic protein trafficking. This is supported by ev idence that multiple gene aberrations in Nup98 occur in some hematological disorders. For example, Nup98 gene translocations have been reported in treatment related AML and myelodysplastic syndrome (MDS). Also, the chromosomal translocation t(11;20 )(p15;q11) results in a Nup98/Topo I fusion protein in de novo acute myeloid leukemia (AML) (P otenza et al., 2004; Iwase et al., 2003; Chen et al., 2003). These observations establish that a significant modulation occurs in topo I, which could theoretically alter topo enzyme stability, enzyme activity, and subcellular distribution. Similar chromo somal translocations involving topo II have not been found. Additional studies to analy ze the subcellular dist ribution of topo II in

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203 untreated patients compared to treated or re lapsed patients combined with cytogenetic analysis are needed 14.7 Topoisomerase II as an Alternative Molecular Target in Human Myeloma. Another interpretation of the data is that topo II could be an alternative molecular target in the treatment of human mu ltiple myeloma. It is well documented that topo II protein expression is rath er constant over the cell cycle and less influenced by cell-cycle changes than topo II Western blot analysis demo nstrated no reduction in the total cellular content of topo II in plateau-cells or accelerated-plateau phase cells when compared to log-phase cells. Topo II was detectable in both the nuclear and cytoplasmic compartments of the plasma cell in clinical samples as well as, in the human myeloma cells lines. However, there was no measurable difference or shift in the amount of topo II immunofluorescence detected in the cytoplas m at plateau density as compared to log density. This suggests th at the balance of topo II nuclear import is influenced in the same manner or by cell-cell c ontact as observed with topo II Recently, microinjection studies identified an NES in topo II (Mirski et al., 2003) and the data presented here demonstrates a possible shuttling of topo II between the nucleus and cytoplasm. A determination of whether leptomycin B can shift the nuclear:cytoplasmic ratio of II toward to the nucleus in pl ateau-phase cells could addr ess the mechanism of topo II trafficking. Nevertheless, the data sugge sts the factors result ing in cytoplasmic distribution of topo II in tumor cells do not alter topo II net trafficking in the same manner.

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204 Several new cytotoxic agents that are specific for topo II are being investigated for the effectiveness in the treatment of solid tumors that are refractory to topo II containing therapy (Hazeldine et al., 2005, 2002; Barthelmes et al., 2001; Gao et al., 1999) Many of these drugs are still in development and are derivatives of XK469, a topoisomerase II selective poison that is reaching phase I clinical trials (Gatto and Leo, 2003). Tumors with large populations of cells in the G0/G1 phase are believed to be more sensitive to topo II specific inhibitors because, unlike topo II topo II protein expression is not cell-cycle dependent. Thus the low proliferativ e index of malignant plasma cells observed in multiple myeloma makes topo II an attractive molecular target. Furthermore, XK469 results in upregulation of the topo II protein (Mensah-Osman et al., 2002). Based on these findings a plausi ble regimen for multiple myeloma patients would contain a topo II specific cytotoxic agent (ex. XK469) combined with a nuclear export inhibitor (leptomycin B) followed by a topo II targeting cytotoxic agent (i.e..., etoposide). Theoretically, the topo II specific cytotoxic agent would induce cell death via topo II activity while also elevating the cellular content of topo II protein. A nuclear export inhibitor would be nece ssary to inhibit the export of topo II from the nucleus and thus, provide a molecular target in the nucleus for etoposide mediated DNA damage to occur. This sequence could only be effective if topo II activity and levels remained sufficient to induce DNA damage. Inhibitors of the pr oteasome represent a new class of cytotoxic agents with promising results in the treatment of hematological malignancies. A proteasome inhibitor may be useful for maintaining the total cellular content of topo protein.

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205 Sequestering proteins in the nucleus is gaining acceptance as a potential mechanism for inducing apoptosis in cancer cells (Yashiroda and Yoshida, 2003). One recent study treated human chronic myelogenous leukemia cells with a tyrosine kinase inhibitor (STI571) to induce th e nuclear import of BCR-Abl, a chimeric oncoprotein that activates mitogenic and anti-a poptotic cell signaling pathways when it is in the cytoplasm of cells (Vigneri and Wang, 2001). The cells we re then exposed to leptomycin B to block the export of BCR-Abl into the cytoplasm, t hus trapping the protein in the nucleus. A general weakness of this drug combination is that leptomycin B inhibits all CRM-1 mediated nuclear prot ein trafficking, but in vitro the drug combination selectively killed transformed cells expressing BCR-Abl chimera. Furthermore, a phase I clinical trial of LMB demonstrated that the drug caused pr ofound anorexia and malaise in patients (Newlands et al., 1996). The clinical trials with LMB were discontinued but, the discovery that CRM is the molecular target of LMB instigated new efforts to reevaluate LMB for potential use in chemotherapy. Sa fer leptomycin B analogues are currently being developed and investigated for their antitumor activit y (Kau and Silver, 2003). Further investigations into the mechanisms of topo trafficking are needed to develop drugs specific for topo that may be less toxic in humans. 14.8 Nuclear Content of Topo II is a Determinate of Topo II Drug Cytotoxicity. There has been one report to describe the role of the nuclear-cytoplasmic trafficking of topo II in de no vo drug resistance (Oloumi et al., 2000). The weight of the data has been limited to measuring the total cellular content of topo protein found either in drug-resistant cell lin es (Mirski et al., 2000’ Davis et al., 1998; Kusumoto et al.,

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206 1996; Chen and Beck, 1995;) or in clinical sp ecimens (Bauman et al., 1997). Traditional practice has relied heavily on in vivo topo quantification as a prognostic indicator (Schrader et al., 2004; Lohri et al., 1997). As such, there have been numerous reports to describe a correlation between topo protein expression with a positive response to topo inhibitors (Lohri et al., 1997; Kaufmann, et al., 1994). Ho wever, there is a growing awareness in both scientific and clinical forums that monitoring the subcellular distribution of topo is also important when predicting drug responsiveness (Oloumi et al., 1993). This is supported by several reports unable to explain a lterations in drug sensitivity to topo targeting agents with a lterations in either topo mRNA, topo protein expression or activity, or P-gp expression (Yamazaki et al., 1997; Davis et al., 1998; Yamazaki et al., 1997; Boege et al., 1993). For ex ample, eight breast epithelial cell lines, including six derived from breast cancers, were analyzed for mechanisms of drug resistance to camptothecin and etoposide (Davis et al., 1998). Drug resistance to camptothecin was attributed to low levels of topoisomerase I and activity. However, topo II levels and activity did not co rrelate with drug sensitivity or resistance to etoposide. These cells were analyzed for alterations in S-phase, doubling time, expression of mdr-1, p53, Bcl-2 and Bax proteins, but none of thes e parameters correlated with drug resistance to etoposide (Davis et al., 1998). Only one cell line (BT 47 cells) exhibited a high bcl2/Bax ratio and mutant p53 protein that was believed to contribute to decreased sensitivity to etoposide. The BT47 cells also displayed slow growth characteristics similar to that observed in the human myel oma cell line models, but the subcellular distribution of topo enzyme wa s not investigated in any of the cells explored. A shift toward the cytoplasm in the nucleo-cytoplas mic shuttling of topo II could have similar

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207 effects as an overall decrease in protei n levels, by decreasing the amount of active enzyme in the nucleus. A similar study wa s performed that analyzed 14 unselected human lung cancer cell lines, including sm all-cell lung cancer and non-small cell lung cancer, for drug sensitivity to doxorubicin a nd etoposide, two topo II targeting agents (Yamazaki et al., 1997). There was varia tion in drug sensitivity to etoposide and doxorubicin in all cell lines, but neither topo II content nor enzyme activity correlated with observed drug resistance. Other cellular pa rameters were not investigated, including the subcellular distribution of topo II This could be significant because a shift in the subcellular distribution of topo II would not be detectable by Western blot of topo II protein in whole cells. Furthermore, the observation of cytoplasmic topo II in patient samples described here suggests that there is a clinical role for the nucleo-cytoplasmic trafficking of topo that could explain de novo drug resistance prev iously described in other tumor types. Thus, examining the nucle ar and cytoplasmic content of topo protein by immunocytochemistry, especially topo II is an important fact or to consider when attempting to explain cellular drug resistance in the laborator y and clinical preparations. 14.9 Possible Roles of Cytoplasmic Topo II. One of the potential consequen ces of exporting a pool of topo II to the cytoplasm is a decrease in sensitivity to topo inhibitors. This could result from cytoplasmic topo II serving as a drug sink by trapping VP -16 in this compartment. This is supported by data demonstrating that the bi nding of VP-16 to topo II can occur in the absence of DNA (Burden et al., 1996). For this to occur, the amount of drug binding to

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208 topo II would need to be sufficient to resu lt in a decrease in druginduced DNA damage. One question that has not been addressed is whether topo II is recruited to the cytoplasm to perform a specific function. If topo II were acting on cytoplasmic substrates, there are at least tw o theoretical possibilities. Firs t, topo II could be involved in maintaining the secondary structure of RNA or be involved in splicing RNA. To date, there has been no direct evidence of topo II binding RNA. A second possibility is that topo II could be recruited to the cytoplasm as an early apoptotic event. I have hypothesized that a link exists between th e calcium/calmodulin pathway and nuclearcytoplasmic trafficking of topoisomerase II during apoptosis in chapter 15, page 220.. 14.10 Topoisomerase II Contains Two Functional Nuclear Export Signals. One question that we addressed was whet her the cytoplasmic distribution of topo II was a result of increased nuc lear export or decreased nucl ear import. Identification of the direction and mechanism of transport ac ross the nuclear membrane could lead to a better understanding of how to re gulate the levels of active enzyme in the nucleus and thus, sensitize cells to topo inhibitors. Human topo I (Mo et al., 2000), II (Mirski, et al., 1999), and II (Mirski, et al., 1999) have NLS that ta rget them into th e nucleus, but only recently has the mechanism of nuclear-export of these proteins been investigated (Mirski et al., 2003). Sumoylation followed by redist ribution of topo I from the nucleoli to the nucleoplasm in response to topotecan or campt othecin exposure has been reported (Mo et al., 2002; Rallabhandi et al., 2002) The lack of data describing the shuttling of topo enzymes may be because topo is usually reporte d to occur in the nucleus of cells and a

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209 cytoplasmic distribution of topo II has usually been ascribed to the expression of a truncated protein that has lost its C-term inal NLS (Mirski, et al., 2000). However, proteins that appear predominately nuclear may still shuttle between the nucleus and the cytoplasm if the rate of nuclear import is gr eater than the rate of nuclear export. Thus, demonstrating that a protei n shuttles between the nucle us and cytoplasm requires defining the specific conditions that will shif t the steady state kinetics toward nuclear export (Table 15). Many conditions have been shown in vitro to alter the shuttling of proteins between the nucleus and cytoplas m, including changes in the cell-cycle and oxidative stress (Miyamoto et al., 2004; Kodiha et al., 2004; Greber and Gerace, 1995). I have created a list of molecular and biochemical agents that have been demonstrated to alter specific transport pathways for different proteins. This table demonstrates that certain cellular conditions or exogenous agents can induce a shift in the nuclearcytoplasmic trafficking of specific proteins. In the accelerated-plateau cell model used in these experiments, it is likely that intensive ce ll-cell contact initiates a signal that induces the export of topo II to the cytoplasm. This is supporte d by the findings of others that a cytoplasmic distribution of topo II occurs in the outer-proliferating cells of multicell spheroids in Xenograft tumors when compared to monolayers formed by the same cells (Oloumi et al., 2000).

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210Table 15. Inhibitors of the Nucl ear-cytoplasmic Transport Pathway Biochemical Agent or MolecularFactor Target Mechanism Wheat germ aggultinin (Duverger et al., 1995) N-Acetylglucosamine moieties expressed on nucleoporins Non-specifically blocks protein import by binding sugar moieties on nucleoporins Anti-FG repeat antibodies (Snow et al., 1987) Importins/Nucleoporins Blocks importins from docking to the NPC Trichostatin A (Chen et al., 2001) NF-kB Blocks NF-kB import Cyclosporin A (Liu et al., 1991) Cyclophilin, NFAT Blocks NFAT import; cyclosporin-cyclophilin complex binds calcinuerin and inhibits dephosphorylation of NFAT 3, 5–Bistrifluoromethyl pyrazoles (Trevillyan et al., 2001) NFAT Blocks NFAT import independently of calcinuerin Leptomycin B (Wolff et al., 1997) Myxobacterial cytotoxins (Ratjadones) (Koster et al., 2003) Chromosome region maintenance protein 1 (Crm1) Blocks CRM1 mediated nuclear export Arylene bis(methylketone) compounds (Dubrovsky et al., 1995) Pre-integration complex (PIC) Blocks import of HIV viral genome Arginine-rich RNA molecules (ARM) (Fineberg et al., 2003) HIV-1 Rev BSA molecules with ARM peptides block HIV-1 Rev protein import Transfected importinbinding domain (Kutay et al., 1997); importinP446L mutant protein (Timinszky et al., 2002) ImportinDominant negative competitive inhibitor of importin / mediated import Cellular Stress: UV radiation Oxidative stress Heat shock (Miyamoto et al., 2004; Kodiha et al., 2004) Ran gradient Blocks nuclear export of importinWortmannin (Arcaro and Wymann, 1993) Phosphatidylinositl-3 kinas (PI3 kinase) Blocks nuclear-cytoplasmic trafficking regulated by PI3kinase phosphorylation Okadaic acid Calyculin A (Elrick and Docherty, 2001) Phosphatase inhibitors Blocks some phosphorylation dependent trafficking. Peptide Aptamers (Colas et al., 2000) Peptide containing a NLS or NES directed against any protein; may also contain ubiquitin. Redirects protein to the nucleus (NLS) or cytoplasm (NES); modify any protein with ubiquitin. Energy Regenerating System and Ran Mix (Timinszky et al., 2002) Ran gradient Energy source for continued nuclear-cytoplasmic shuttling. Thapsigargin (Greber and Gerace, 1995) Calcium pump inhibitor Blocks signal-mediated protein import

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211 14.11 Nuclear Transport and Oncogenesis. Alterations in nucleo-cytoplasmic protein transport are strongly implicated in a number of human diseases that include seve ral autoimmune disorders (Wesierska-Gadek et al., 1996) and human cancers (Kau et al ., 2004). The data presented in this investigation describes a mechanism of dr ug resistance observed in human myeloma and leukemia cell lines that involve s alterations in the subcel lular distribution of topo II The data presented herein suggests that altera tions in the nuclear-cytoplasmic trafficking of topo II may have a role in cellular drug re sistance in human myeloma and leukemia cell lines. Several genetic abberations in the nuclear-cytolasmic transport machinery have been described in human leukemia a nd myeloma cells to date (Lam and Aplan, 2001). At least 17 chromosomal rearrangemen ts in acute and chr onic leukemia involve nucleoporin genes (Table 16) (Cronshaw and Matunis, 2004). Nucleoporin 98 (Nup98) is implicated in 15 of those chromosomal rearrangements (Slape and Aplan, 2004; Takeshita, et al., 2004). Furthermore, ch romosomal translocations involving Nup98 and DNA topo I have been observed in some pa tients with therapy related acute myeloid leukemia, and in one patient with de novo AML (Iwase et al., 2003; Chen et al., 2003). Leukemias associated with nucleoporin gene rearrangements have been reported to be more refractory to therapeutic intervention. (Kakazu et al., 2001) suggesting that these gene rearrangements are involved in drug se nsitivity. Nup98 gene rearrangements have also been implicated in therapy related myelodysplasia (MDS) (Ahuja, et al., 1999), a group of bone marrow disorders that resemb le hematologic malignancies. MDS is characterized by the clonal expansion of stem cells and is most prevalent in individuals over 60 years of age. Nup gene rearrangements in multiple myeloma have not been

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212 reported. In addition to the Nup family of proteins, the major vault protein (MVP) referred to as the lung resist ance related protein (LRP), has been associated with decreased sensitivity to chemotherapy in multiple myeloma. The MVP is a ribonucleoprotein complex that localizes to the nuclear pore complexes (Chugani et al., 1993). Results of a clinical study showed th at expression of MVP in myeloma patients was associated with the multidrug resist ance phenotype not mediated by MDR-1/P-gp (Rimsza et al., 1999). Collectively, these stud ies illustrate the underlying role of nucleocytoplasmic trafficking and the NPC in oncoge nesis. Furthermore, the significance of Nup98/topo I fusion proteins in conferring alte red drug sensitivity represent one area of research yet to be explored

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213Table 16. Nucleoporin (NUP) Gene Rearrangement s Associated with Hematological Malignancies* Nucleoporin Gene Rearrangement FUSION GENE Protein Characteristics Disease References t(7;11)(p15;p15) NUP98/HOXA9 Homeodomain transcription factor AML, CML, MDS Nakamura et al., 1996 t(2;11)(q31;p15) NUP98/HOXD13 Homeodomain transcription factor AML Arai et al., 2000 t(1;11)(q23;p15) NUP98/PMX1 Homeodomain transcription factor MDS/AML Nakamura et al., 1999 t(7;11)(p15;p15) NUP98/HOXA11 Homeodomain transcription factor CML, AML Suzuki et al., 2002 t(11;12(p15;q13) NUP98/HOXC11 Homeodomain transcription factor AML Gu et al., 2003 t(7;11)(p15;p15) NUP98/HOXA13 Homeodomain transcription factor AML Taketani et al., 2002 t(2;11)(q31;p15) NUP98/HOXD11 Homeodomain transcription factor AML Terui et al., 2003 t(11;12)(p15;q13) NUP98/HOXC13 Homeodomain transcription factor AML Panagopoulos et al., 2003 inv11(p15;q22) NUP98/DDX10 RNA helicases AML, MDS Arai et al., 1997 t(11;20)(p15;q11) NUP98/TOP1 DNA topo I de novo AML; t-AML, tMDS; polycethemia vera Potenza et al., 2004 Iwase et al., 2003 Chen et al., 2003 t(10;20;11)(q24;q11;p15) NUP98/TOP1 DNA topo I t-MDS Panagopoulos et al., 2002 t(4;11)(q21;p15) NUP98/RAP1GDS1 Nucleotide exchange factor T-ALL Hussey et al., 1999 t(10;11)(q25;p15) NUP98/ADD3 Membranecytoskeleton T-ALL Lahortiga et al., 2003 t(5;11)(q35;p15) NUP98/NSD1 SET domain AML La Starza et al., 2004 t(8;11)(p11.2;p15) NUP98/NSD3 SET domain AML Rosati et al., 2002 t(6;9)(p23;q34) NUP214/DEK DNA binding AML, MDS Boer et al., 1998 t(6;9)(p23;q34) NUP214/SET Nucleosome assembly protein family AML Saito et al., 2004 *Reviewed in reference Lam and Aplan, 2001.

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214 Chapter Fifteen Future Considerations 15.1 Phosphorylation Phosphorylation has also been shown to be important in regulating the subcellular localization of many proteins and could have a role in topo II trafficking (Harreman et al., 2004). This is suggested by the finding that the outer proliferating cells of multi-cell spheroids contain a cytoplasmic pool of topo II and have a 10-fold decrease in the phosphorylati on state of the enzyme when compared to monolayers containing nuclear topo II (Oloumie et al., 2000). Phos phorylation has been extensively investigated for yeast DNA topo II and to a lesser extent in topo II Phosphorylation Table 17. Phosphorylation Sites in DNA Topoisomerase II Established In vitro Kinase Residue Reference PKC Serine 29 Wells et al., 1995 Presumed to be Casein Kinase 1 or 2 Serine 1106 Chikamori et al., 2003 Casein Kinase 2 Serine 1377 Serine 1525 Wells et al., 1994 Casein Kinase 2 Threonine 1343 Daum and Gorbsky, 1998 Ishida et al., 1996 Casein Kinase 2 Serine 1469 Escarguiel et al., 2000 One or more proline directed kinases Serine 1213 Serine 1247 Serine 1354 Serine 1361 Serine 1393 Wells and Hickson, 1995

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215 events in topo II in human cells is less well characterized (Austin and Marsh, 1998) Several serine residues in topo II are phosphorylated in vitro by CK2 and PKC (Table 17). Many of the phosphorylation sites are locate d near the NLS in the carboxyl terminus (Figure 52, page 192). For topo II the phosphorylation status at S29, S1213, S1247, S1354, S1361, S1393 vary throughout the cell cycl e. In contrast, phosphorylation at S1377, S1525, and T1343 remains constant thro ughout the cell cycle (Austin and Marsh, 1998). To predict additional protein kinase recognition motifs, the complete amino acid sequence was entered into Scansite, a com puter algorithm developed at Massachusetts Institute of Technology. Scansite 2.0 uses a library of phosphorylation data to predict phosphorylation sites and protei n-protein binding domains in any protein (Obenauer et al., 2003). Table 18. Predicted Phosphorylati on Sites in DNA Topoisomerase II Kinase Group Amino Acid Residue Recognition Sequence Src SH3 Proline 887 MDGEEPLPMLPSYKN Protein kinase C Serine 1426 TAAKSQSSTSTTGAK Protein kinase C Threonine 193 YKKMFKQTWMDNMGR Calmodulin dependent kinase II Serine 230 LSKFKMQSLDKDIVA Calmodulin dependent kinase II Threonine1279 TKTKKQTTLAFKPIK Casein kinase 2 Serine 1337 LDSDEDFSDFDEKTD Casein kinase 2 Serine 1106 DEEENEESDNEKETE

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216 The computer program can recognize 62 protei n kinase or protein binding motifs that includes Ser/Thr or Tyr kinases and SH2 a nd SH3 binding domains. Figure 32 is the graphical output showing pred icted phosphorylation sites that have not been reported in the literature. Table 18 summarizes the prot ein kinase recognition sequences identified under high stringency. As expected, topo II has recognition motifs fo r protein kinase C, casein kinase 2 and proline depe ndent Ser/Thr kinases. Several in vitro studies have reported similar phosphorylation oc currences by these kinases, albeit different amino acid residues 15.2 Calmodulin Dependent Kinases Calmodulin dependent kinase-II (CaMK-II ) is one unexpected result in the Scansite report. CamK-II belongs to the family of calmodulin dependent kinases (CaMK). Calmodulin is a cytosolic protein th at responds to intrace llular calcium levels by activating various calcium-calmodulin dependent enzymes, such as calmodulin dependent kinases (CaMK) (Tombes et al., 1995 ). There are six different CaM kinases but, only CaMK I and CaMK II are consistently expressed in prolif erative cells (Tombes and Krystal, 1997). CaMK II was originally believed to be encoded by a single gene. For example, CaMK-II has for different isozymes, II B, II C, II G, and II H, generated by alternative splice sites. In one study, panels of huma n tumor and non-tumor cell lines were examined for expression of all CaMK -II protein isoforms (Tombes and Krystal, 1997). The tumor cell lines included severa l human breast cancer cell lines, colon adenocarcinoma, human neuroblastoma, a nd CCRF-CEM lymphoblastic leukemia cells, whereas NIH 3T3 mouse fibroblasts, mammar y epithelia, and mous e embryo fibroblasts

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217 were the non transformed cell lines examined. The tumor cell lines expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. All tumor cell lines (including the neural derived cells) lacked CamK-II and expressed several alternative splice variants. CaMKII G and II H were preferentially expressed in tumor cell lines as compared to undifferentiated fibroblast s. This data esta blishes that CaMK-II is expressed in a variety of hu man cancer cell lines, includi ng the hematological cell line, CCRF-CEM. Thus, it is possible that CaMK-II is expressed in other hematological cell lines such as, human myeloma H 929 cells. Expression of CaMK-II in H929 cells would be significant if, topo II is regulated by CaMK-II phosphorylation. Identification of a CaMK phosphorylation site in topo II could be significant factor in regulating topo II subcellular distribution for se veral reasons. First, topo II has been shown to be hypophosphorylated in drug resistant cell lines in a calcium dependent manner (Grabowski et al., 1998; A oyama et al., 1998; Ganapathi et al., 1996). Secondly, the complete amino acid sequence for topo II was entered into Scansite but, does not have a calmodulin dependent kinase recognition motif even when the protein was scanned under low stringency. Th is is significant because topo II subcellular localization is not alte red by cell-density in the same manner as topo II suggesting that different cellular conditions can regulate the differential locali zation of the two enzymes. Thirdly, calcium levels present in the ER cisternae have been reported to regulate nuclear-cytoplasmic trafficking of proteins su ch that, when calcium levels are low, the NPC restricts the nuclear cytoplasmic traffick ing of proteins (Grebe r and Gerace, 1995). It has been suggested that calcium stores si gnal the NPC when the cell senses stress such

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218 as, oxidative stress, DNA damage, or proteins un folding in the ER. Thus, there could be a link between NPC trafficking and early apoptotic events. There is increasing evidence that calci um signaling pathways are intimately involved with apoptotic regulatory pathways BCL-2, Bax, Bak, and Bid are apoptotic regulatory proteins that exer t their effect in part by regulating calcium mobilization between the ER and mitochondria. Bax, Bid, a nd Bcl-2 localize in the ER, the central intracellular calcium stores of the cells. BCL-2 mediates the release of ER calcium pools, which results in a concomitant increase in mitochondrial calcium uptake (Oakes et al., 2003; Foyouzi-Youssefi et al., 2000). Bax and Bak, promote apoptosis in part by increasing calcium transfer from the ER to the mitochondria, which results in cytochrome c release during apoptosis (Nutt et al., 2002) Bax and Bid have also been shown to localize on the nuclear envelope and move fr om the cytoplasm to the mitochondria in response to camptothecin exposure (Gajkowska et al., 2004). This data suggests that camptothecin may initiate apoptosis in a via Bax and Bid in a calcium dependent manner. Calcium release from the ER may be linke d with oxidative stress, which has been one physiological condition demonstrated to m odulate NPC mediated pr otein trafficking. This is significant because the tumor micr oenvironment can contribute to hypoxia. For example, solid tumors that are resistant to topo II containing chemotherapy usually have regions of low oxygen, which are not observe d in normal tissue (Tomida and Tsuruo, 1999). Although multiple myeloma is not considered a solid tumor, the disease is characterized by anemia, which has been show n to induce or hasten hypoxia (Sordet et al., 2004; Mittelma, 2003). Furthermore, wh en Bid is expresse d in hypoxic cells, it results in increased sensitivity to the topoi somerase II inhibitor etoposide (Erler et al.,

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219 2004). Downregulation of Bid was attributed to the decrease sensitivity to etoposide; however, the subcellular localiz ation of topo II enzymes was not investigated. Likewise, the total amount of Bcl2, Bid and Bax were not investigated in the natural-plateau or accelerated-plateau human myeloma cell line models. Thus, topo II could be regulated by calmodulin dependent kinase activity such th at, when calcium is released from the ER under conditions of stress, topo becomes phosph orylated and degraded. In another study, proteasomes were found to accumulate in the nucleus of cells grown under stress conditions (glucose starvation) (Ogiso et al., 2002). This could partially explain how tumor cells evade etoposide toxi city and yet, maintain topo II content for cell divisionby sequestering topo II in the cytoplasm away from the nuclear proteasomes. Furthermore, repetitive hypoxia, which coul d be prevalent in multiple myeloma patients who are chronically exposed to DNA damaging agents, could select for drug resistant cells (Weinmann et al., 2004).

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220 Chapter Sixteen Concluding Remarks Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and myeloma. We have re ported that resistance to topo II poisons, such as VP16 is found to increase dramatically with c oncurrent increases in cell density. The highcell density of myeloma cells in the bone marrow may mimic the high-density plateau phase conditions that we established in vitro, and thus these cells may export endogenous topo II from the nucleus to the cytoplasm in vivo In order for topo targeted chemotherapy to function, topoisomerase must have access to the nuclear DNA. Thus, the nuclear export of topo II must be added to the list of potential mechanisms of resistance to topo poisons. It is unique in th at it does not require drug exposure and may mimic the high cell density microenvironment seen in the bone marrow of patients with multiple myeloma. Further defining this m echanism, and possibly modulating export, may lead to methods to preclude this avenue of resistance. Clinical drug resistance is likely to be a multifactorial phenomeon and enhancing drug activity of available agents will likely require a multifaceted approach.

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266 Appendices

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267 Appendix A: The Complete Amino Acid Sequence for Human DNA Topoisomerase I 1 MSGDHLHNDS QIEADFRLND SHKHKDKHKD REHRHKEHKK EKDREKSKHS NSEHKDSEKK 61 HKEKEKTKHK DGSSEKHKDK HKDRDKEKRK EEKVRASGDA KIKKEKENGF SSPPQIKDEP 121 EDDGYFVPPK EDIKPLKRPR DEDDADYKPK KIKTED TKKE KKRKLEEEED GKLKKPKNKD 181 KDKKVPEPDN KKKKPKKEEE QKWKWWEEER YPEGIKWKFL EHKGPVFAPP YEPLPENVKF 241 YYDGKVMKLS PKAEEVATFF AKMLDHEYTT KEIFRKNFFK DWRKEMTNEE KNIITNLSKC 301 DFTQMSQYFK AQTEARKQMS KEEKLKIKEE NEKLLKEYGF CIMDNHKERI ANFKIEPPGL 361 FRGRGNHPKM GMLKRRIMPE DIIINCSKDA KVPSPPPGHK WKEVRHDNKV TWLVSWTENI 421 QGSIKYIMLN PSSRIKGEKD WQKYETARRL KKCVDKIRNQ YREDWKSKEM KVRQRAVALY 481 FIDKLALRAG NEKEEGETAD TVGCCSLRVE HINLHPELDG QEYVVEFDFL GKDSIRYYNK 541 VPVEKRVFKN LQLFMENKQP EDDLFDRLNT GILNKHLQDL MEGLTAKVFR TYNASITLQQ 601 QLKELTAPDE NIPAKILSYN RANRAVAILC NHQRAPPKTF EKSMMNLQTK IDAKKEQLAD 661 ARRDLKSAKA DAKVMKDAKT KKVVESKKKA VQRLEEQLMK LEVQATDREE NKQIALGTSK 721 LNYLDPRITV AWCKKWGVPI EKIYNKTQRE KFAWAIDMAD EDYEF Figure 54. The Complete Amino Acid Sequence for Human DNA Topoisomerase I. (accession number NP 001059). The NLS is underlined.

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268 Appendix B: The Complete Amino Acid Sequence for Human DNA Topoisomerase II 1 MAKSGGCGAG AGVGGGNGAL TWVNNAAKKE ESETANKNDS SKKLSVERVY QKKTQLEHIL 61 LRPDTYIGSV EPLTQFMWVY DEDVGMNCRE VTFVPGLYKI FDEILVNAAD NKQRDKNMTC 121 IKVSIDPESN IISIWNNGKG IPVVEHKVEK VYVPALIFGQ LLTSSNYDDD EKKVTGGRNG 181 YGAKLCNIFS TKFTVETACK EYKHSFKQTW MNNMMKTSEA KIKHFDGEDY TCITFQPDLS 241 KFKMEKLDKD IVALMTRRAY DLAGSCRGVK VMFNGKKLPV NGFRSYVDLY VKDKLDETGV 301 ALKVIHELAN ERWDVCLTLS EKGFQQISFV NSIATTKGGR HVDYVVDQVV GKLIEVVKKK 361 NKAGVSVKPF QVKNHIWVFI NCLIENPTFD SQTKENMTLQ PKSFGSKCQL SEKFFKAASN 421 CGIVESILNW VKFKAQTQLN KKCSSVKYSK IKGIPKLDDA NDAGGKHSLE CTLILTEGDS 481 AKSLAVSGLG VIGRDRYGVF PLRGKILNVR EASHKQIMEN AEINNIIKIV GLQYKKSYDD 541 AESLKTLRYG KIMIMTDQDQ DGSHIKGLLI NFIHHNWPSL LKHGFLEEFI TPIVKASKNK 601 QELSFYSIPE FDEWKKHIEN QKAWKIKYYK GLGTSTAKEA KEYFADMERH RILFRYAGPE 661 DDAAITLAFS KKKIDDRKEW LTNFMEDRRQ RRLHGLPEQF LYGTATKHLT YNDFINKELI 721 LFSNSDNERS IPSLVDGFKP GQRKVLFTCF KRNDKREVKV AQLAGSVAEM SAYHHGEQAL 781 MMTIVNLAQN FVGSNNINLL QPIGQFGTRL HGGKDAASPR YIFTMLSTLA RLLFPAVDDN 841 LLKFLYDDNQ RVEPEWYIPI IPMVLINGAE GIGTGWACKL PNYDAREIVN NVRRMLDGLD 901 PHPMLPNYKN FKGTIQELGQ NQYAVSGEIF VVDRNTVEIT ELPVRTWTQV YKEQVLEPML 961 NGTDKTPALI SDYKEYHTDT TVKFVVKMTE EKLAQAEAAG LHKVFKLQTT LTCNSMVLFD 1021 HMGCLKKYET VQDILKEFFD LRLS YYGLRK EWLVGMLGAE STKLNNQARF ILEKIQGKIT 1081 IENRSKKDLI QMLVQRGYES DPVKAWKEAQ EKAAEEDETQ NQHDDSSSDS GTPSGPDFNY 1141 ILNMSLWSLT KEKVEELIKQ RDAKGREVND LKRKSPSDLW KEDLAAFVEE LDKVESQERE 1201 DVLAGMSGKA IKGKVGKPKV KKLQLEETMP SPYGRRIIPE ITAMKADASK KLLKKKKGDL 1261 DTAAVKVEFD EEFSGAPVEG AGEEALTPSV PINKGPKPKR EKKEPGTRVR KTPTSSGKPS 1321 AKKVKKRNPW SDDESKSESD LEETEPVVIP RDSLLRRAAA ERPKYTFDFS EEEDDDADDD 1381 DDDNNDLEEL KVKASPITND GEDEFVPSDG LDKDEYTFSP GKSKATPEKS LHDKKSQDFG 1441 NLFSFPSYSQ KSEDDSAKFD SNEEDSASVF SPSFGLKQTD KVPSKTVAAK KGKPSSDTVP 1501 KPKRAPKQKK VVEAVNSDSD SEFGIPKKTT TPKGKGRGAK KRKASGSENE GDYNPGRKTS 1561 KTTSKKPKKT SFDQDSDVDI FPSDFPTEPP SLPRTGRARK EVKYFAESDE EEDDVDFAMF 1621 N Figure 55. The Complete Amino Acid Sequence for Human DNA Topoisomerase II (accession Number NP 001059). Nuclear expor t signal (NES) is double-underscored A specific nuclear localization signal has not been iden tified in topo II but the C-terminus is important for targeting the enzyme to the nucleus (Mirski e al., 2003).

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269 Appendix C: Gene Rearrangements in Hematological Malignancies Table 19.Oncogenes and Fusion Proteins in Hematological Malignacies* Oncogene Symbol Functional Product Diseases Abelson Murine Leukemia Viral Oncogene Homolog 1 ABL1 A nuclear and cytoplasmic non-receptor tyrosine kinase activity implicated in cell differentiation, cell division, and cell adhesion. When expressed as Philadelphia translocation its activity is associated with CML Acute Myeloid Leukemia-1 AML-1 Transcription factor (activator) for various hematopoietic specific genes. Expressed during differentiation and hematopoesis. Associated with acute nonlymphocytic leukemia. Acute Myeloid Leukemia-1 / Myeloid Translocation Gene 8 AML1/MTG8 Fusion protein as a result of t(8;21). Proposed role in preventing cell differentiation and thus, propagating leukemic blast cells Associated with de novo acute myeloid leukemias Receptor Tyrosine Kinase AXL Receptor ty rosine kinase. Hematopoietic cancers B-cell CLL / Lymphoma 2 BCL2 B-cell leukemia/lymphoma protein is localized in the inner mitochondr ial membrane. Blocks programmed cell death. Associated with leukemias; linked with follicular lymphoma when overexpressed as a result of the t(14;18) translocation. Breakpoint Cluster Region BCR GTPase activating protein for RAC1 and CDC42; exhibits serine/threonine kinase activity Translocations t(9;22) BCRABL and t(4;22) PDGFRA-BCR associated with CML Breakpoint Cluster Region / Abelson Murine Leukemia Viral Oncogene Homolog 1 BCR/ABL Fusion protein with constitutive tyrosine kinas activity as a result of 9:22 chromosomal translocation. CML, ALL Cyclin D1 CCND1 Important for cell cycle regulation; Dysregulation of cyclin D1 associated with t(11;14) Multiple myeloma *Huret, J.-L. et al., 2003. Atlas of Genetics and Cy togenetics in Oncology and Haematology. [online] http://www.infobiogen. fr/services/chromcancer/ (accessed 2004).

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270Appendix C: (Continued) Table 19 continued. DEK Oncogene / Nucleoporin 214D DEK/CAN DEK is ubiquitous protein expressed in the nucleus of cells and interacts with histones.; fusion protein formed as a result of t(6;9) translocation. Associated with poor prognosis in AML. Transcription Factor 3 / pre-Bcell leukemia transcription factor 1 E2a/Pbx1 Chimeric transcription factor (activator) resulting from t(1;19) chromosomal translocation. pre-B-cell Acute Lymphoblastic Leukemia Erythroblastosis Virus E26 Homolog ETS-1 Transcription factor involved in extracellular matrix remodeling; cell migration; tumor invasion Lymphomas Monocytic leukemia Fibroblast Growth Factor 3 FGFR3 Receptor for acidic and basic fibroblast growth factors. Chromosomal translocation t(4;14) implicated in multiple myeloma Homeobox-11 HOX-11 Homeobox protein; DNA binding protein Found to be deregulated in acute lymphoblastic T-cell leukemia Interluekin-3 IL-3 Cytokine involved in cell survival, proliferation, and differentiation. Overexpression of protein associated with acute pre B-cell leukemia Lymphoid Blast Crisis LBC guanine nucleotide exchange factor Myeloid leukemias Lymphocyte Specific Kinase LCK src family tyrosine kinase critical for T-cell development and activation T-cell lymphoma

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271Appendix C (Continued) Table 19 continued. Lymphoblastic Leukemia Derived Sequence 1 Lyl-1 Basic helix-loop-helix protein. Involved in T-cell ALL through t(7:19) which involves Lyl-1 and T-cell receptor beta chain genes. Mixed Lineage Leukemia MLL Transcription factor involved in maintenance of HOX gene expression by hematopoietic stem and progenitor cells Associated with poor prognosis in acute myeloid leukemia Mixed Lineage Leukemia / Lymphoid Nuclear Protein 4 MLL/LAF4 Fusion protein resulting from t(4;11) translocation. 70-80% of all ALL and 50-60% of AML infants; Reported in secondary leukemias after topo II inhibitors; poor prognosis Musculoaponeurotic Fibrosarcoma MAF Transcription factor; overexpression implicated in cell proliferation and pathological interactions with bone marrow stroma. Multiple myeloma Myeloblastosis Myb A family of DNA binding proteins containing tandem repeats of a helix-turn-helix motif. Expression in hematopoietic cells is necessary fo proliferation. Chromosomal aberrations at the 6q breakpoint linked with colon carcinoma, leukemias, and lymphomas Myeloctyomatosis c-Myc Transcription factor involved in cell cycle regulation, apoptosis, metabolism, cell differentiation, and cell adhesion. Overexpressed in most hematogenic malignancies. Most well characterized in Burkitts lymphoma. Myosin, heavy polypeptide 11, smooth muscle / Core-binding factor beta subunit MYH11/CBFB CBFB is a transcription factor which masterregulates a host of genes specific to hemtopoesis and osteogenesis. Pericentric inversion Inv(16)(p13;q22) fuses CBFB with MYH11 Acute Myeloid Leukemia

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272Appendix C (Continued) Table 19 continued. Nucleophosmin1 / Anaplasticlymphoma kinase NPM/ALK ALK is a receptor tyrosine kinase; The t(2;5) translocation results in a fusion protein with NPM; accumulates in the nucleoli of neoplastic cells. Non-Hodgkin’s lymphomas; Anaplastic lymphoma; Large cell lymphomas pre-B cell Leukemia Transcription Factor 3 Pbx1/E2A fusion protein that activates WNT-16 expression. WNT-16 expression contributes to t(1;19) pre-B ALL Acute pre B-cell leukemia PIM1 Oncogene PIM-1 serine/threonine kinase T-cell lymphoma Promyelocytic Leukemia / Retinoic Acid Receptor alpha PML/RAR PML is a RING finger and nuclear matrix associated phosphoprotein. Functions in growth, transformation and tumor suppressor activity. Fusion with RAR may abrogate the corepressor function. Acute premyelocytic leukemia Reticuloendotheliosis REL Transcription factor; role in differentiation and lymphopoiesis. Gene amplification associated with Hodgkin’s lymphoma Suppressor of variegation, Enhancer of zeste and Trithroax / Nucleoporin 214 SET/CAN CAN is a nucleoporin that may serve as a docking site in receptor mediated nuclear import; Fusion protein resulting from t(6;9) translocation. Acute myeloid leukemia Stem Cell Leukemia SCL Transcription factor; t(1:14) in leukemia cell lines results in deletion between delta chain diversity regions Acute T-cell leukemia Transforming Acidic Coiledcoil Containing Gene 3 TACC3 Centrosomal proteins that can interact with microtubules. t(4;14) implicated in Multiple myeloma.

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About the Author Roxane Engel was born in St. Petersburg, Florida and received her diploma from Boca Ciega High School. Roxane earned a Bachelors of Science Degree from the University of Tampa with a major in Biology and a minor in Chemistry. Roxane was an academic tutor and a teacher’s assistant in the Genetics Laboratory. She completed a research internship at the V.A. Medical Center, graduated with Honors, and was recognized as the Most Outstandi ng Life Science Graduate. Roxane enrolled in the doctoral progra m in Biochemistry and Molecular Biology at USF in the College of Medicine and worked at the Moffitt Cancer Center. She was a Graduate Student Representative, won a re search travel award, received the Walter Trudeau Award, and presented research at se veral scientific meetings, including ones in New Orleans, San Francisco, New York City, and Amsterdam, Holland. Roxane completed one year at Quest Diagnostics as a Genomics and Esoteric Testing Specialist.