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Drug resistance to topoisomerase directed chemotherapy in human multiple myeloma

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Title:
Drug resistance to topoisomerase directed chemotherapy in human multiple myeloma
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English
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Turner, Joel G
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Nuclear Export
Tumor Suppressor Proteins
Cell Cycle Inhibitors
ATP Binding Cassette Protein G2 (ABCG2)
Chromosome Maintenance Protein 1 (CRM1)
Dissertations, Academic -- Biology -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Summary:
ABSTRACT: Human multiple myeloma is an incurable hematological malignancy characterized by the proliferation of plasma cells in the bone marrow. Myeloma represents approximately 20% of all blood cancers. In this research we have explored examples of both intrinsic and acquired drug resistance in myeloma. Topoisomerases are enzymes that are critical for cell division, especially in rapidly dividing cells such as are found in cancer. Topoisomerase poisons are a common group of drugs that are used to treat cancer. Topoisomerase I and II poisons used in the treatment of multiple myeloma include topotecan, mitoxantrone, doxorubicin, and etoposide In order for topoisomerase drugs to be effective, the enzyme must be in direct contact with the DNA. In chapters one and two we examined the export of topoisomerase II alpha from the nucleus as a mechanism of drug resistance.High density cells, similar to those found in the bone marrow, export topoisomerase II alpha from the nucleus to the cytoplasm, rendering the cell drug resistant. We found that blocking nuclear export using the CRM1 inhibitor ratjadone C, or CRM1 specific siRNA, could sensitize high density cells to topoisomerase drugs. Sensitization to topoisomerase inhibitors was correlated with increased topoisomerase/DNA complexes and increased DNA strand breaks. This method of sensitizing human myeloma cells suggests a new therapeutic approach to this disease. In chapter three we examined the role of the molecular transporter ABCG2 in drug resistance in multiple myeloma. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan or doxorubicin. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after drug treatment and at relapse.We found that expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, upregulated in response to chemotherapy, and may contribute to drug resistance.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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Statement of Responsibility:
by Joel G. Turner.
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Title from PDF of title page.
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Document formatted into pages; contains 98 pages.
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Includes vita.

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aleph - 002007632
oclc - 405658015
usfldc doi - E14-SFE0002446
usfldc handle - e14.2446
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ABSTRACT: Human multiple myeloma is an incurable hematological malignancy characterized by the proliferation of plasma cells in the bone marrow. Myeloma represents approximately 20% of all blood cancers. In this research we have explored examples of both intrinsic and acquired drug resistance in myeloma. Topoisomerases are enzymes that are critical for cell division, especially in rapidly dividing cells such as are found in cancer. Topoisomerase poisons are a common group of drugs that are used to treat cancer. Topoisomerase I and II poisons used in the treatment of multiple myeloma include topotecan, mitoxantrone, doxorubicin, and etoposide In order for topoisomerase drugs to be effective, the enzyme must be in direct contact with the DNA. In chapters one and two we examined the export of topoisomerase II alpha from the nucleus as a mechanism of drug resistance.High density cells, similar to those found in the bone marrow, export topoisomerase II alpha from the nucleus to the cytoplasm, rendering the cell drug resistant. We found that blocking nuclear export using the CRM1 inhibitor ratjadone C, or CRM1 specific siRNA, could sensitize high density cells to topoisomerase drugs. Sensitization to topoisomerase inhibitors was correlated with increased topoisomerase/DNA complexes and increased DNA strand breaks. This method of sensitizing human myeloma cells suggests a new therapeutic approach to this disease. In chapter three we examined the role of the molecular transporter ABCG2 in drug resistance in multiple myeloma. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan or doxorubicin. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after drug treatment and at relapse.We found that expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, upregulated in response to chemotherapy, and may contribute to drug resistance.
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Tumor Suppressor Proteins
Cell Cycle Inhibitors
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Chromosome Maintenance Protein 1 (CRM1)
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Drug Resistance to Topoisomerase Directed Chemotherapy in Human Multiple Myeloma By Joel G. Turner A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Biology College of Arts and Sciences University of South Florida Co-Major Professor: Dani el M. Sullivan, M.D. Co-Major Professor: James R. Garey, Ph.D. Brian Livingston, Ph.D. My Lien Dao, Ph.D. Date of Approval: February 18, 2008 Keywords: Nuclear Export, Tumor Suppre ssor Proteins, Cell Cycle Inhibitors, ATP Binding Cassette Protein G2 (ABC G2), Chromosome Maintenance Protein 1 (CRM1) Copyright 2008, Joel G. Turner

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i i Table of Contents List of Tables iii List of Figures iv Abstract v Chapter One: Review of nucl ear export of proteins and chemotherapeutic resistance in cancer 1 Summary 1 Introduction 3 Nuclear export mechanisms 4 Export of drug targets 8 Topoisomerases 8 Topoisomerase II alpha 9 Topoisomerase I 10 Export of tumor suppressors 11 P53 11 APC/ -catenin 13 FOXO family of transcription factors 14 Export of cell-cycle regulators 15 P21cip1 15 P27kip1 16 Nuclear export and potential drug targets 18 Chapter Two: Drug resistance to topoisomerase directed chemotherapy in human multiple myeloma 22 Summary 22 Introduction 23 Materials and Methods 26 Cell lines 25 Cell density and drug treatment 26 Immunofluorescent microscopy 27 Apoptosis assay 28 CRM1 siRNA knockdown and Western blot 28 Band depletion assay 30 Comet assay 30 Results 31 Log and plateau density myeloma cells 31

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ii ii Intracellular trafficking of topo II 33 CRM1 inhibitor and topo II chemotherapeutics 35 Topo II trafficking and CRM1 inhibition 37 CRM1 inhibitor and topo II inhibitor synergy 37 CRM1 siRNA sensitizes myeloma cells to topo II poisons 39 Topoisomerase Western blot 42 Increase in cleavable complex formation by CRM1 inhibition 42 Comet assay 45 Discussion 45 Chapter Three: ABCG2 expre ssion, function and promoter methylation in human multiple myeloma 49 Summary 49 Introduction 50 Materials and Methods 53 Cell lines 53 Clinical trial with high-dose melphalan and topotecan 54 Real-time quantitat ive PCR (QPCR) 55 Western blot for cell lines and patient myeloma samples 56 Flow cytometry/ABCG2 functional assay 57 Immunofluorescent mi croscopy and quantitative measurement of ABCG2 59 Cell density and low dose drug treatment 59 Bisulfite sequencing and demethylation by 5-aza2'deoxycytidine of t he ABCG2 promoter in patient samples and myeloma cell lines 60 Methylation-s pecific quantitative PCR 61 Results 62 Quantitative PCR of ABCG2 62 ABCG2 protein expression determined by Western analysis, flow cytometry and immunofluorescence 65 ABCG2 functional assay: topotecan efflux 67 Effect of the microenv ironment and topoisomerase inhibitors on ABCG2 expression 72 ABCG2 promoter methylation 75 Methylation-specific quantitative PCR of patient myeloma cells 77 Discussion 78 Dissertation Summary 84 References 86 About the Author End Page

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iii iii List of Tables Table 1.1 Drug targets, tumor suppr essors and cell-cycle inhibitors that undergo CRM1-mediated ex port in various cancers. 2 Table 3.1 ABCG2 mRNA expression determined by QPCR in human cancer cell lines 63 Table 3.2 ABCG2 mRNA expression determined by QPCR in CD138 selected human plasma cells from bone marrow aspirates obtained fr om patients with multiple myeloma prior to and dur ing high-dose chemotherapy, and at the time of relapse 64

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iv iv List of Figures Figure 1.1 Nuclear export of proteins 5 Figure 2.1 Intracellular trafficking of topo II in log and plateau density myeloma cells 32 Figure 2.2 Cellular localization of topo II in 8226 and U266 cells under log and plateaudensity growth conditions 34 Figure 2.3 CRM1 inhibitor and topo II chemotherapeutics 36 Figure 2.4 H929 topo II immunofluorescence 38 Figure 2.5 CRM1 inhibitor and topo II inhibitor synergy 40 Figure 2.6 CRM1 knockdown using siRNA makes myeloma cells more sensitive to the topo II poison doxorubicin 41 Figure 2.7 Log and plateau expressi on of topoisomerases in myeloma cell lines 43 Figure 2.8 Band depletion and comet assay 44 Figure 3.1 ABCG2 expression and f unction in myeloma cell lines 66 Figure 3.2 Functional assay in patient myeloma cells 68 Figure 3.3 ABCG2 expression increases in response to doxorubicin exposure 70 Figure 3.4 ABCG2 expression is elev ated in log phase myeloma cells 71 Figure 3.5 ABCG2 expression increases in response to topotecan chemotherapy 73 Figure 3.6 ABCG2 increases in patient plasma cells after high-dose chemotherapy and at relapse 74 Figure 3.7 ABCG2 promoter methylation 76

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v v Drug Resistance to Topoisomerase Directed Chemotherapy in Human Multiple Myeloma Joel G. Turner ABSTRACT Human multiple myeloma is an incurable hematological malignancy characterized by the proliferation of pl asma cells in the bone marrow. Myeloma represents approximately 20% of all blood cancers. In this research we have explored examples of both intrinsic and acquired drug resistance in myeloma. Topoisomerases are enzymes that are critical for cell division, especially in rapidly dividing cells such as are found in cancer. Topoisomerase poisons are a common group of drugs that are used to treat cancer. Topoisomerase I and II poisons used in the treatment of mu ltiple myeloma in clude topotecan, mitoxantrone, doxorubicin, and etoposide In order for topoisomerase drugs to be effective, the enzyme must be in direct contact with the DNA. In chapters one and two we examined the export of topoisomerase II alpha from the nucleus as a mechanism of drug resistance. High density cells, similar to t hose found in the bone marrow, export topoisomerase II alpha from the nucleus to the cytoplasm, rendering the cell drug resistant. We found that blocking nuc lear export using the CRM1 inhibitor ratjadone C, or CRM1 specific siRNA, could sensitize high density cells to topoisomerase drugs. Sensitization to t opoisomerase inhibitors was correlated

PAGE 7

vi vi with increased topoisomerase/DNA co mplexes and increased DNA strand breaks. This method of sensitizin g human myeloma cells suggests a new therapeutic approach to this disease. In chapter three we examined the role of the molecular transporter ABCG2 in drug resistance in multiple myeloma. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan or doxorubicin. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after drug treatment and at relapse. We found that expression of ABCG2 is regulated, at leas t in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myel oma cells, regulated by promoter methylation, affected by cell density, upregulated in response to chemotherapy, and may contribute to drug resistance.

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1 1 Chapter 1 Review of nuclear export of proteins and chemotherapeutic resistance in cancer SUMMARY Expression levels of intact tumo r suppressor proteins and molecular targets of antineoplastic agents are critical in defining c ancer cell drug sensitivity; however, the intracellular location of a specific protein may be equally as important. Many tumor suppressive protei ns must be present in the cell nucleus in order to perform their policing acti vities or for the cell to respond to chemotherapeutic agents. Ex amples of nuclear proteins needed to prevent cancer initiation or progression, or to optimize chemotherapeutic response, include: tumor suppressor proteins p53, APC/ -catenen and FOXO family genes, negative regulators of cell cycle progression and survival such as p21 and p27, and chemotherapeutic targets such as DNA topoisomerase I and topoisomerase II ( Table 1) Mislocalization of a nuclear protei n into the cytoplasm can render it ineffective as a tumor suppressor or as a target for chemotherapy. It is possible that blocking the nuclear export of any or all of these proteins may restore tumor suppression, apoptosis, or in the case of topoisomerase I and topoisomerase II reversal of drug resistance to inhibitors of these enzymes. During the course of

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2 2Protein Functional significance Export receptor Modification for nuclear export. Cancer type where protein is exported to the cytoplasm Topoisomerase II DNA topology, drug target CRM1 Phosphorylation by Casein Kinase 2. Multiple myeloma Topoisomerase I DNA topology, drug target CRM1 Unknown Anaplastic astrocytoma Neuroblastoma p53 Tumor suppressor CRM1 Ubiquitinylation by MDM2 E3 ubiquitin ligase. Colorectal cancer Breast cancer APC Tumor suppressor CRM1 Mutation causing frame-shift or premature termination. Colorectal cancer FOXO Tumor suppressor CRM1 Phosphorylation by Akt kinase. Breast, prostate, and thyroid cancer Glioblastoma Melanoma p21Cip1 Cell-cycle inhibitor CRM1 HER2/neu mutation and phosphorylation by Akt. BCR-ABL translocation and phosphorylation by Akt. Phosphorylation by PKC Ovarian and breast cancer Chronic myeloid leukemia (CML) p27Kip1 Cell-cycle inhibitor CRM1 Phosphorylation by human kinaseinteracting stathmin (hKIS) Breast cancer Acute myelogenous leukemia (AML) Table 1.1: Drug targets, tu mor suppressors and cell-cycle inhibitors that undergo CRM1-mediated export in various cancers.

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3 3 disease progression or in response to the tumor environment, cancer cells appear to acquire intracellular mechani sms to export anti-cancer nuclear proteins. These export mechanisms generally involve modification of nuclear proteins that cause the proteins to re veal leucine-rich nuclear export signal protein sequences, with subsequent export m ediated by CRM1. In this review we will define the general processes invo lved in nuclear export mediated by CRM1/RanGTP (Exportin/XPO1), examine the functions of individual tumor suppressor nuclear proteins and nuclear targets of chemotherapy, and explore the potential mechanisms that are used by the cancer cell to induce export of these proteins. In addition, we will briefl y discuss experimental therapeutics that could potentially counteract nuclear export of specific proteins. INTRODUCTION Drug resistance is the single greates t impediment to t he treatment of cancer. Cancer cells can be intrinsicall y drug resistance due to the breakdown of many normal cellular processes during cancer development or cancers may acquire drug resistance in response to selection by chemotherapeutic treatment. Acquired resistance may develop into crossresistance to many other drugs that have very different mechanisms of action (Gottesman, 2002; Longley and Johnston, 2005). Specific types of drug re sistance include, chemical inactivation of the drug(s) such as cis -platinum, irinotecan and me thlytrexate (Ishikawa and Ali-Osman, 1993; Xu and Villalona-Ca lero, 2002), alteration of repair

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4 4 mechanisms for drug induced damage to DNA in response to platinum drugs (Dabholkar et al., 1994; Durant et al., 1999; Fink et al., 1998), evasion of apoptosis induced by 5-fluor ouracil (Longley and Johnston, 2005), drug efflux of mitoxantrone, VP-16, doxorubicin, vinblas tine, anthrilamide and flavorpiridol by ATP binding cassette (ABC) transporters (Ambudkar et al., 1999; Goldman, 2003; Gottesman et al., 2002; Krishna and Mayer, 2000; Roe et al., 1999; Thomas and Coley, 2003), down-regulati on of pro-apoptotic factors or drug targets to vinca alkaloids and other micr otubule inhibitors (Dumontet and Sikic, 1999; Kavallaris et al., 2001; Longley et al., 2003; Xu and Villalona-Calero, 2002), modification of specific drug targets to 5-fluorouracil, oxaplatin, irinotecan, and campothecan (Boyer et al., 2004; Li et al., 1996), and mislocalization of either drug targets or tumor suppressive proteins (Fabbro and Henderson, 2003; Kau and Silver, 2003; Yashir oda and Yoshida, 2003). This latter mechanism, the export of drug targets, tumor suppresso rs, and cell-cycle inhibitors from the nucleus, is the primary focus of this review. NUCLEAR EXPORT MECHANISIMS Once an mRNA is translated into a pr otein, the protein is directed to a specific intracellular compar tment either for further modi fication or to perform its function within the cell. Movement of prot eins is governed by specific amino acid signaling sequences contained withi n the particular protein.

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5 5 Figure 1.1, Nuclear export of proteins. Nuclear export of a cargo protein by association of the nuclear export signal (NES) with CRM1 and Ran-GTP and transport through the nuclear pore complex.

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6 6 Signals exist for trafficking to the nucle olus (Guo et al., 2003; Liu et al., 2006; Nakamura et al., 2003)nuclear localiz ation (Hodel et al., 2001) and nuclear export signals (Bogerd et al., 1996; Hender son and Eleftheriou, 2000; Ikuta et al., 1998; Kanwal et al., 2002), in addition to cytoplasmic compartment localization signals to the endoplasmic reticulum (A ndres et al., 1990; Munro and Pelham, 1987), golgi (Zeng et al., 2003), mito chondria (Anandatheerthavarada et al., 2003), lysosomes (Bonifacino and Traub, 2003), and peroxisomes (Gould et al., 1989).Nuclear export signals (NES) in par ticular are comprised of hydrophobic amino acid residues including leucine and isoleucine-rich sequences (Kutay and Guttinger, 2005). Proteins larger than 40-60 kDa cannot enter or exit the nucleus through the nuclear pore complex without the assi stance of soluble tr ansport receptors called karyopherin proteins, which bind to the export or import signal peptides (Bednenko et al., 2003). The nuclear pore of a cell is a very large (125 MDa) protein made of approximatel y 100 nucleoporins (Lim et al., 2008). The nuclear pore has a central iris-like transporter region through which the proteins are transported. In addition, the nuclear por e has eight large cytoplasmic filaments and a nuclear basket structure. The nucl ear pore complex is imbedded in the nuclear envelop bilayers (Terry et al ., 2007). The exact mechanism of transport of proteins through the cent ral channel is not known. However, it is known that for nuclear export of a car go protein to occur, it must bind to a transport receptor protein called an exportin which in turn is regulated by a small GTPase molecule

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7 7 referred to as Ran-GTP (Arnaoutov et al ., 2005). Nuclear export is controlled by the concentration gradient of Ran-GT P and Ran-GDP; Ran-GTP is highly concentrated in the nucleus and therefore drives nuc lear export, whereas RanGDP is concentrated outside the nuclear membrane in the cytoplasm (Arnaoutov et al., 2005). Stable export complexes are formed in the nucleus with Ran-GTP, the exportin transport recept or and the cargo protein (Weis, 2007). After the protein complex is exported into the cyt oplasm, Ran-GTP is hydrolyzed to RanGDP by Ran-GAP and the export complex is dissociated, releasing the cargo protein into the cytoplasm. The expor tin receptor protein and Ran-GDP are subsequently recycled back into the nucleus through the nuclear pore complex to undergo another round of export (Figure 1). The primary export receptor protein is chromosome maintenance protein 1 or CRM1 (Daelemans et al., 2005; Yoneda et al., 1999). CRM1, with the assistance of Ran-GTP, binds to the nuclear export signal peptide (NES) of the cargo protein. Howe ver, the NES must be present and accessible to CRM1 (Arnaout ov et al., 2005; Black et al., 2001). The NES of a cargo protein can be expos ed by changes in the three-dimensional conformation of the protein whic h are generally caused by protein phosphorylation or dephosphorylat ion (Vogt et al., 2005), although other protein modifications such as acet ylation (Vogt et al., 2005), sumoylation (Pichler and Melchior, 2002), ubiquitination (Bonifacino and Traub, 2003; Vogt et al., 2005) or the binding of protein-specific co-facto rs can reveal export signals and induce export (Kutay and Guttinger, 2005; Poon and Jans, 2005; Yoneda et al., 1999).

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8 8 EXPORT OF DRUG TARGETS Topoisomerases Topoisomerases (topo) ar e major targets in canc er chemotherapy due to their specific cellular activities. Topoiso merases that are useful as cancer drug targets in human cells are primarily of two types, type IB (topo I) and type II enzymes (topo II and topo II ) Topo I is a 100 kDa protein which pr oduces a transient nick in one strand of the DNA double helix in an ATP indep endent manner and essentially allows the supercoiled DNA to unwind in a c ontrolled manner. Topo II isoforms form a homodimer that makes a transient DNA do uble strand break, which requires the hydrolysis of an ATP molecule and allows the passage of an entire DNA strand through the strand break, thus maki ng topological isomer s of the DNA. Topo molecules of both types are e ssential to organize the approximately two meters of DNA contained within each cell during transcription, DNA replication and recombinatio n. There are two isofo rms of topo II, topo II (170 kDa), and topo II (180 kDa). Topo II is constitutively expressed, present in the nucleus and nucleolus and may allow localized unwinding of DNA during DNA repair (Emmons et al., 2006). Topo II is highly express ed in proliferating cells and is essential for transcription, DNA replication, chromatin condensation and chromatid separation. Topo II -/mice are embryoni c lethals that cannot progress past the 4 to 8 cell stage, whereas topo II -/mice die at birth due to neurological defects (Lyu and Wang, 2003; Sakaguchi and Kikuchi, 2004).

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9 9 Topoisomerase II alpha Previous studies hav e revealed that topo II has a bipartite nuclear localization signal (NLS) in the carboxyl terminus of the protein (Mirski et al., 1997; Wessel et al., 1997). Drug resistanc e to the topo II poison etoposide has been described in cells that ex press low levels of topo II or which were truncated in the carboxyl terminal region of topo II (Feldhoff et al., 1994; Wessel et al., 1997; Yu et al ., 1997). Truncated topo II lacking a functional NLS, remains in the cytoplasm and theref ore cannot produce DNA damage. Two nuclear export signals for topo II have been described in the Cterminal domain of the topo II molecule (Mirski et al., 2003; Turner et al., 2004). This was determined by site-directed mutag enesis of export signal motifs of fulllength topo II protein (Turner et al., 2004). Topo II nuclear export is mediated by CRM1, and is likely to be controlled by phosphorylation (unpub lished data). In multiple myeloma cell lines, topo II is actively exported into the cytoplasm in cells grown at high densities; consist ent with patient bone ma rrow cell densities. In high density myeloma cell cultures, w here topo II has been exported into the cytoplasm, the cells were found to be appr oximately 10-fold mo re resistant to topoisomerase poisons such as doxorub icin and etoposide (Engel et al., 2004). Drug sensitivity was not due to differenc es in cell cycle, drug uptake or topo protein levels, but was due primarily to enzyme trafficking to the cytoplasm (Engel et al., 2004). Blocking CRM1 nuclear export of topo II using leptomycin B

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10 10 (Engel et al., 2004), ratjadone C, or a CRM1 specific siRNA knockdown was found to make drug-resistant human myel oma cells sensitive to the topo II poisons doxorubicin and VP-16. Topoisomerase I Topo I contains two motifs that are nearly identical to the published NES in topo II (Turner et al., 2004), and it is likel y that topo I may be exported by a CRM1 mediated mechanism. Topo I is normally located in the nucleus and the nucleoli of cells, but has been found to be ex ported to the cytoplasm in specific cancer cell lines. In addition, cytoplasmic lo calization of topo I occurs in direct response to the topo I inhibitory drugs topotecan and CPT-11. A fter treatment of anaplastic astrocytoma cells with the topo I inhibitor topotecan for one hour, the amount of cytoplasmic t opo increased in the cytoplasm 50-100% and nuclear topo I decreased by 25% after topotecan treatment (Danks et al., 1996). Post topo I export the anaplastic astrocytoma cells became more resistant to the effects of topotecan. Cytoplasmic topo I was found to be fully active enzymatically, however the protein was sm aller (68 kDa) than nuclear topo I (100 kDa) and therefore may be a degraded form of the enzyme. Xenograft models of neuroblastoma treated with CPT-11 (Irinotecan) caused the cells to export topo I from the nucleus to the cytopl asm (Santos et al., 2004). Neuroblastoma xenograph cells atta ined an initial response rate to CPT11 treatment of nearly 100% based on hist ological differentiation, however the

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11 11 cells were completely refractory to any subsequent treatment by CPT-11. This was true even after in vivo passage of the cells into a different host animal. The drug-resistant cells demonstrated an ov erall decrease in topo I expression as well as in redistribution to the cytopl asm. In this model the cytoplasmic topo I were of two types, an enzymatically acti ve 68 kDa topo I and an inactive 48 kDa form. EXPORT OF TUMOR SUPPRESSORS P53 Additional examples of the importance of nuclear export in cancer can be found in the tumor suppressor proteins p53, APC/ -catenen and FOXO family genes. The critical function of p53 is to m onitor the fidelity of genomic replication at the G1/S phase cell cycle checkpoint. W hen wild-type p53 is activated it relocates from the cytoplasm to the nucleus where it functions as a transcription factor that induces expression of genes involved in DNA repair, cell cycle arrest, and apoptosis. P53 is a very important tumo r suppressor that must be inactivated for cancer progression. Most of the liter ature has focused on p53 inactivation by mutation or deletion, including dominantnegative p53 mutants. Several recent reviews have described a litany of genetic alterations found in the p53 gene in various cancers (Bourdon, 2007; Soussi, 2007; Soussi and Wiman, 2007; Valkov and Sullivan, 2003). Genetic mutation of p53 accounts for 70% of colon cancers and 50% of breast cancers. Therefore, in the remaining per centage of these

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12 12 cancers p53 must be inactivated by some additional means. There is a growing body of literature that descr ibes p53 being kept out of the nucleus by cytoplasmic sequestration (Nikolaev et al., 2003; Wadhw a et al., 2002) or hyperactive nuclear export by MDM2 (Cuny et al., 2000). P53 contains four NES which bind CRM1 for nuclear export (Stommel et al., 1999; Zhang and Xiong, 2001), and three NLS which are involved in nuclear import. Once p53 is imported into the nucleus it forms a tetramer which blocks access to the nuclear export signals (Stommel et al., 1999). MDM2 protein is a negative-r egulator of p53 but at the same time MDM2 expression is transactivated by the p53 tetramer. MDM2 possesses an E3 ubiquitin ligase which binds and ubiqu itinates p53, changing its protein conformation and revealing the p53 NES's (Stommel et al., 1999). P53 is exported by the CRM1 nuclear receptor and is degraded in the cytoplasm by proteasome proteolysis. MDM2 expression is controlled by p53 but its activity is modulated by the tumor suppressor prot ein p14ARF which bi nds to MDM2 and inactivates it, thereby stabilizing p53 function in the nucleus (O'Brate and Giannakakou, 2003). There are also studies indicating the contribution of P13K/AKT, c-ABL, and CBP/p300 to both MDM2 and p53 activity. In addition, p53 contains a number of phos phorylation events that pr event nuclear export and increase nuclear accumulation. Specific kinases that phosphorylate p53 include checkpoint kinase 1 (Chk1) and Chk2. T herefore alteration of p53 associated proteins, ubiquitination and phosphorylation could be used to maintain p53 in the nucleus and induce apoptos is in cancer cells.

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13 13 APC / -catenin Human adenomatous polyposis coli ( APC) is a tumor suppressor gene that is mutated in both inherited and sporadic human co lorectal cancers (Powell et al., 1992). APC is a large (312 kDa) mu lti-domain protein which binds to and negatively regulates -catenin, a Wnt signaling effe ctor (Senda et al., 2007). APC forms a multiprotein complex with the scaffold protein axin, glycogen synthase kinase-3 (GSK3 ) and -catenin. GSK3 phosphorylates -catenin leading to its proteosomal degradation, and ultimately blocks the Wnt signaling pathway (reviewed in Senda et al 2007) (Senda et al., 2007). Mutations in APC lead to nuclear export and allow -catenin mediated tumorigenesis. APC has been observed to translocate between the nucleus and cytoplasm in several cancer cell lines. APC contains both an NLS sequence in the central domain of APC, and multiple NES sequences in the N-termi nus and central region of the protein (Neufeld et al., 2000). APC export is m ediated by CRM1 binding as shown by site-directed mutation of the NES amino acid signal and also by inhibition of export by leptomycin B (Neufeld et al., 2000). Mutations usually occur only in particular regions of the APC gene causi ng frame-shift mutations and pre-mature termination of translation. Tr afficking of nuclear APC into the cytoplasm has been directly correlated to an increase in nuclear -catenin and subsequent oncogenic transcriptional activity. The rate of nuclear export of APC, not the rate of import, has been directly correlated to the rate of -catenin transcriptional activity and tumor progression (Rosin-Arbesfeld et al ., 2003). Maintaining APC in the nucleus

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14 14 could potentially inhibit colorectal cancer progression. FOXO transcription factors The FOXO or forkhead family of tran scription factors are protein regulators that control apoptosis, DNA repair, def ense against oxidative molecular damage and cell cycle progression. FOXO proteins must be in contact with the DNA in the nucleus in order to perform their antineoplasm activities. Post-transcriptional modifications of FOXO proteins include multiple phosphorylations by various kinases, acetylation and ubiquitination (V ogt et al., 2005). However it is the phosphorylation by the serine/threonine ki nase Akt (or protein kinase B) that disrupts binding to DNA and leads to nuc lear export of FO XO. Once exported into the cytoplasm FOXO proteins undergo proteosomal degradation. Akt kinase is inappropriately activated by phosphoinositide 3-kinase (PI3K) in cancers where the tumor suppr essor PTEN is mutated. Therefore, mutation of PTEN in breast cancer, prostate cancer, thyroid cancer, glioblastoma, and melanoma leads to nucl ear export of FOXO. In studies where PTEN activity was restored, FOXO proteins localized to the nucleus and induced cell-cycle arrest and apoptosis (Nakamur a et al., 2000). In addition, recent studies to discover small molecule inhi bitors of PI3K, Akt, and CRM1 have been performed to keep FOXO proteins in t he nucleus as a potential anti-cancer treatment (Kau et al., 2003; Kau and Silver, 2003).

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15 15 EXPORT OF CELL CYCLE REGULATORS P21Cip1 P21Cip1 (p21) is a cell cycle inhibitor which binds to cyclin-cyclin dependent kinase 2 (cdk2) or cdk4 molecular co mplexes and prevents cell cycle progression at G1. P21 transcriptional expression is r egulated by wild-type p53 in response to cellular stress, activated DNA-repair mechanisms, apoptosis or cellular senescence. Cellular growth is arrested when p21 binds to cyclin-cdk complexes in the cellular nucleus, therefore nu clear localization of unmodified p21 (phosphorylation or ubiquitination) is e ssential for its anti-cancer functions. Nuclear export of p21 protein is induc ed by multiple ways. In some human breast and ovarian cancers the cell memb rane receptor tyrosine kinase, HER2/ neu, is overexpressed. HER-2/ neu activates the phosphatidylinositol-3 kinase (PI-3K)/Akt pathway. After activation by HER-2/ neu, Akt, a serine/threonine kinase, is released from the inner surf ace of the cell membrane, enters the cell nucleus and phosphorylates p21 at thr eonine 145 which results in cytoplasmic localization of p21(Zhou et al., 2001a). Threonine residue 145 of p21 is in the NLS region, therefore phosphor ylation inhibits nuclear localization and promotes cytoplasmic localization of p21 and allowi ng cell proliferation. In addition, cytoplasmic p21 has anti-apoptotic properti es by associating with apoptosissignal-regulating kinase 1 (ASK1) (Asada et al., 1999). Two nuclear export signals are nece ssary for export of p21 from the nucleus to the cytoplasm (Henderson and Ele ftheriou, 2000; Hwang et al., 2007).

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16 16 Reactive oxygen species induce nucleo-cyt oplasmic translocation of p21, along with ubiquitination and subsequent proteos omal degradation. This process is blocked by leptomycin B or site-direct ed mutation of the two nuclear export signals, therefore nuclear export is by a CRM1-mediated mechanism. Phosphorylation of p21 at serine 153 in the carboxy-terminus by protein kinase C is also sufficient to induce nuclear export. When this si te is blocked by calmodulin protein binding to p21, p21 accumulates in the cell nucleus (Rodriguez-Vilarrupla et al., 2005). In chronic myeloid leukemia (CML), cells eventually develop an aggressive, drug resistant form of the di sease during the "blast crisis" phase. Curiously, p21 protein expression is upregul ated in these cells, however, it has been observed that the p21 is predominantly localized to the cellular cytoplasm (Keeshan et al., 2003). CML is characterized by the presence of the oncogenic chimeric protein, Bcr-Abl. Bcr-Abl has been shown to upregulate p21 expression and activate the serine/threonine kinase Ak t in a PI3-K indepen dent manner. Akt then phosphorylates p21 and prom otes nuclear export of p21, ultimately resulting in an aggressive, drug-resistant CML phenotype. P27 Kip1 P27Kip1 (p27) is a potent cell-cycle inhibi tor which is expressed at its highest levels in the G0-G1 transition of the cell-cycle. P27 forms a heterotrimer complex with cyclin E-CDK2 and inhibits it s activity, effectively preventing cell-

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17 17 cycle progression out of the G0 cell-cycle phase. P27 func tion is inactivated by several mechanism including nuclear export, cytoplasmic sequestration and proteosome-ubiquitin degradation (Susaki and Nakayama, 2007). Nuclear export of p27 is mediated by the CRM1 export receptor and p27 must be phosphorylated at serine 10 (S10) for CRM1 binding to occur (Ishida et al., 2002). Substitution of S10 with al anine by site-directed mutagenesis prevented co-immunoprecipitation of p27 with CRM1 and prevented nuclear export of p27. In addition, nuclear ex port of p27 was block ed by incubation with the CRM1 inhibitor leptomycin B (Foster et al., 2003). Before the cells can leave G0 and enter G1 p27 must be depleted or expor ted from the nucleus. In G0 nuclear import proceeds CRM1 mediated nucl ear export after phosphorylation at S10. Once exported, p27 undergoes two addi tional phosphorylations at threonine 157 (T157) and threonine 187 (T187). Akt m ediates phosphorylation of T157 in the cytosol which functions to prevent nuc lear re-entry by blocking the nuclear localization signal (Shin et al., 2005). High-frequency of Akt-mediated phosphorylation of p27 with subsequent cyt oplasmic localization has been found in breast cancer and may be elicited by estrogens (Foster et al., 2003). Cytoplasmic localization of p27 and phosphorylation by Akt is an indicator of poor prognosis in acute myelogenous leukemia (Min et al., 2004). Phosphorylation at T187 also occurs in the cytosol but is mediated by cyclin E-Cdk2. Cdk2 phosphorylation of p27 at T187 allows it to form a complex with a ubiquitin ligase, it is then degraded by proteolysi s in the cytoplasm during G1 to S phase (Connor

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18 18 et al., 2003). NUCLEAR EXPORT AND POTE NTIAL DRUG TARGETS Preventing nuclear export of tumor s uppressors and drug targets could be clinically useful in the tr eatment of cancer. Protein trafficking can be modulated by phosphorylation which reveal NES amin o acid sequences, therefore, blocking protein modification especially by phosphoryl ation could prove useful. In our lab we were able to block nuclear export of topo II by preventing phosphorylation of a specific serine residue in the carboxyl te rminal region. Using a specific inhibitor of casein kinase II, 4,5,6,7-tetrabromobenz otriazole (Calbiochem), we were able to prevent nuclear export of topo II and sensitize multiple myeloma cells to the topo II poisons doxorubicin and VP-16. In addition, we were able to sensitize cells using siRNA knockdown of casein ki nase II (data not shown). To prove that phosphorylation of a specific amino acid led to nuclear export we mutated serine 1524 in a FLAG-tagged topo II vector fr om a serine to an alanine which prevented nuclear export in transfected ce lls. Small molecule kinase inhibitors may provide similar effects for other tu mor suppressor protein targets such as Akt phosphorylation of FOXO (Brunet et al., 1999) and p21Cip1(Zhou et al., 2001a), -catenin phosphorylation by GSK3 (Rosin-Arbesfeld et al., 2003; Senda et al., 2007), and hKIS phosphorylation of P27Kip1 (Boehm et al., 2002). To date no chemical agents have been shown to inhibit nuclear import receptors such as importin, however, a number of substances have been found

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19 19 to inhibit the function of the nuclear export receptor CRM1. A recent study performed on ovarian cancer biopsies, it was found that CRM1 protein expression was upregulated in aggressive, late-stage cancers (Noske et al., 2008). Increased CRM1 ex pression was found to be a negative prognostic indicator in ovarian cancer and may prove to be a good therapeutic target. Leptomycin B was the first specific CRM1 inhibitor to be discovered. Leptomycin B was isolated from Streptomyces bacteria by investigators who were searching for new antibiotic reagent s (Hamamoto et al., 1983). Leptomycin B modifies CRM1 at a reactive site cyst eine residue (Cys-529) by a Michael-type covalent addition which blocks bindi ng of the NES to CRM1 at nanomolar concentrations (Kudo et al., 1999). Leptomycin B was tested in a phase 1 clinical trial but was not found to be clinically usef ul due to severe toxi cities (Newlands et al., 1996). Additional CRM1 inhibitors have been found, including the ratjadone A-D compounds (Falini et al., 2006; Kalesse et al., 2001; Koster et al., 2003). Ratjadones, which have a different chemic al structure than Leptomycin B, also modify CRM1 at Cys-529 (Meissner et al., 2004). We found that ratjadone C inhibited nuclear export of topo II and sensitized myeloma cells to the topo II inhibitors doxorubicin and VP-16 when us ed at single nanomol ar concentrations (data in press). Knockdown of topo II by siRNA abrogated this effect, demonstrating that it was topo II specif ic. However, we found that ratjadone also

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20 20 sensitized cells to several additional chemotherapeutic agent s tested including topotecan and cis -platinum (unpublished data). This may indicate that blocking CRM1 may sensitize cancer cells by pr eventing export of additional tumor suppressors or cell-cycle inhibitors. In a study performed by Kau et al, a biological screening regimen was used to identify additional inhibitors of CRM1. The original pur pose of this study was to maintain FOXO family transcripti on factors in the nucleus as a potential cancer therapy (Kau et al., 2003). From a library of small molecules, 19 were identified as general nuclear export inhi bitors. Cells were transfected with an HIV Rev/GFP fusion protein, tr eated with the CRM1 small mo lecule inhibitors and assayed by fluorescence microscopy for nuclear export. The HIV Rev/GFP fusion protein contains a very strong nuclear ex port motif which binds to and is exported by CRM1. Of the 19 small molecule inhi bitors identified, 11 were found to covalently modify CRM1 at Cys-529 by a Michael-type reaction, similar to Leptomycin B and ratjadone. Others were fo und to modify Cys-529 by either nucleophilic attack, substitution by a good leaving group or because of an undetermined chemical rearrangement. In conclusion, CRM1 inhibitors may or may not be useful as a single agent, but their usefulness could be to potentially enhance other drugs when used in combination. Sequestering a variet y of drug targets, tumor suppressors, cell cycle inhibitors or apoptosis inducing proteins in the nucleus and restoring

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21 21 their normal anti-proliferative cell activi ty may sensitize cells to a number of different anti-cancer agents or treatments.

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22 22 Chapter 2 Density-dependent drug resistance to t opoisomerase II inhibitors in human multiple myeloma cells is abrogated by CRM1 inhibition SUMMARY We have previously demonstr ated that topoisomerase II is exported from the nucleus of human multiple myelom a cells by a CRM1-dependent mechanism at densities similar to those in patient bone marrow. This results in resistance to topoisomerase II poisons since the enzyme is trafficked to the cytoplasm where it is not in contact with the DNA, and thus unable to produce DNA cleavable complexes and cell death. We inhibited topoisomerase II nuclear export to determine whether nuclear localization of this enzyme woul d sensitize cells to topoisomerase II poisons. Three methods were used to block topoisomerase II nuclear export, the CRM1-specific inhibitor ratjadone C, CRM1 targeted siRNA knockdown, and knockdown of CRM1 expression by anti-sense oligonucleotides. Immunofluorescence microscopy showed that both ratjadone C and CRM1 siRNA effectively inhibited nuc lear export of topoisomerase II CRM1 specific siRNA produced an 81.5% knock down of CRM1 protein. Three human myeloma cell lines, (8226, H929 and U266), were treated with ratjadone C or CRM1 specific siRNA and exposed to either doxorubicin or etoposide at high cell

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23 23 densities. CRM1 treated cells we re four-fold more sensitive to topoisomerase II poisons as determined by an apoptosis assay. Cell death was correlated with increased DNA double strand breaks, as shown by the comet assay. Band depletion assays of myeloma cells expos ed to the CRM1 inhibitor increased topoisomerase II covalently bound to DNA. These results suggest that blocking topoisomerase II nuclear export sensitizes myeloma cells to topoisomera se II inhibitors. This method of sensitizing human myeloma cells suggests a new therapeutic approach in this disease. INTRODUCTION DNA topoisomerases are ubiquitous en zymes that function to relieve the torsional strain in DNA for several crit ical intracellular processes (Wang, 2002). Topoisomerase II (topo II ) is an important anti-neoplas tic drug target. Clinically useful topo II poisons include eto poside (VP-16), doxorubicin, daunomycin, epirubicin and mitoxantrone (Burden et al., 1993; Gatto and Leo, 2003; Nitiss, 2002; Pommier et al., 2003; Sordet et al ., 2003). Topoisomerase II poisons are agents that stabilize the covalent DNA-topo II complexes. During DNA replication the stabilized cleavable complexes are c onverted into DNA strand breaks, the accumulation of which ultimately resu lts in cell death (Bertrand et al., 1991). Resistance to topo II poisons is a major obstacle in the treatment of multiple myeloma (MM). Topo II poisons that are used in the treatment of MM

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24 24 include mitoxantrone, doxorubicin, and etoposide (Kraut et al., 1998). Several mechanisms of resistance to topo II i nhibitors have been described (Oloumi et al., 2000; Rasheed and Rubin, 2003; Sullivan et al., 1989; Sullivan and Ross, 1991; Valkov and Sullivan, 1997). One mechanism of drug resistance is overexpression of drug efflux pumps such as p-glycoprotein (PGP), multid rug resistance proteins (MRP) and major vault protein (MVP). Topo II poisons are known substrates for PGP, MRP, and MVP. Previous analyses of patient myeloma cells have demonstrated that PGP and MVP are often overexpressed in pl asma cells, although MRP is infrequently overexpressed (Fishman and Sulli van, 2001; Rimsza et al., 1999; Schwarzenbach, 2002). Other mechanisms of drug resistance in clude topo II mutations that alter enzyme activity and mutations that produce a loss of the topo II nuclear localization signal so that the molecule remains in the cytoplasm (Feldhoff et al., 1994; Yu et al., 1997) These mechanism s have been found to be limited to cell lines and have not been reproduced in vivo Cell adhesion mediated drug resistance (CAM-DR) and stromal cell adherence are important param eters in the local bone marrow environment in MM patients and appear to be major determinant s of drug resistance. Data from several laboratories have shown that the microenvironm ent may play a significant role in the drug resistance to antineoplastic agents. Hazlehurst et al have shown that fibronectinadherent human U937 leukemia cells were resistant

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25 25 to mitoxantrone because of a r edistribution of topoisomerase II to the nucleolus (Hazlehurst et al., 2006; Hazlehurst et al., 2001). In addition, cell adhesion mediated drug resistance was found to block cell cycle progression, induce p27kip1 levels and ultimately result in cell cycle arrest and drug resistance (Hazlehurst and Dalton, 2001). Bone marrow stromal cell contact and stromal cell soluble factors have been reported to induce drug resistance to mitoxantrone in human myeloma cell lines (Nefedova et al., 2003). Our laboratory in collaboration with others has shown that human MM cell density is a determinant of sensitivity to topo II inhibitors (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b). At increased cell densities, a significant fraction of nuclear topo II is exported to the cytopl asm resulting in reduced sensitivity to etoposide, doxorubicin and mitoxantrone. This appears to occur both in human myeloma cell lines and in CD-138 positive cells isolated from patients with MM (Engel et al., 2004). We have previously shown that myeloma cells in transition from low-density, l og phase conditions to high-density, plateau phase conditions exhibit a substantial export of endogenous topo II from the nucleus to the cytoplasm (Valkov et al ., 2000b). We have reported that nuclear export of topo II may contribute to drug resistanc e (Engel et al., 2004) and our data suggest that resistance was not due to differences in drug uptake, cell cycle or cellular topo II protein levels (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b). In a recent report, we have def ined nuclear export signals for topo II at amino acids 1017-1028 and 1054-1066 (Tur ner et al., 2004). Export by both

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26 26 signals was blocked by treat ment of the cells with lept omycin B, indicating that a CRM1 dependent pathway mediates export (Turner et al., 2004 ). In this study we show that inhibiting CRM1 mediated export of topo II may render myeloma cells more sensitive to topo II targeted chemotherapy. MATERIALS AND METHODS Cell lines Human myeloma cell lines, RPMI 8226 (8226) cells, U266B1 (U266) and NCI-H929 (H929) cells were obtained from the American Type Cu lture Collection (Rockville, MD). All cell lines were grown in RPMI-1640 media containing 100 U/ml penicillin, 100 g/ml streptomycin (I nvitrogen, Carlsbad, CA), and 10% fetal bovine serum (Hyclone, Logan, UT) at 37C, 5% CO2. H929 cell media required the addition of 0.025% mercaptoethanol (Sigma Chem ical, St. Louis, MO). Cell density and drug treatment The model used to assay microenvironmental factors involved incubating cells at high and low-density culture conditi ons. We have shown previously that cells grown at different dens ities exhibit specific characteristics such as drug resistance and nuclear-cytoplasmic trafficking of topo II (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b). Human myeloma cell lines (8226,H929,U266) grown at 2x105 cells/ml were defined as low density (log-

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27 27 phase), and cells grown at 2x106 cells/ml were defined as high density (plateauphase). Cell lines were placed at log and plateau density conditions and cultured for 20 hours with and without the CRM1 in hibitor ratjadone C (5 nM) (HZI / Helmholtz Centre for Infe ction Research Department of Chemical Biology, Braunschweig, Germany) or transfected with CRM1 siRNA (200 nM) (Dharmcon, Lafayette, CO). Cells were then treat ed with doxorubicin (2 M) (Sigma Chemical), or etoposide (10 M) (Sigma Chemical, St. Louis, MO). Immunofluorescent microscopy Cell lines (1x105) were plated on double cytoslides (Shandon, Waltham, MA) by cyto-centrifugation at 500 rpm for 3 minutes and fixed with 1% paraformaldehyde (Fisher Scientific, Suwa nee, GA) on ice for 30 minutes. Permeabilization of cells was perform ed with 0.5% Triton X-100 (Sigma Chemical) in PBS at room temperature fo r 60 minutes. Cells were stained with a polyclonal antibody against topo II which was produced in this lab (PAB454). The topo II antibody was diluted 1:100 in a buffer containing 1% BSA (Sigma Chemical), 0.1% NP-40( Sigma Chemical) in PBS an d incubated for 1 hour at room temperature or overnight at 4oC. After three washes with PBS, slides were incubated with a secondary anti-rabbit Alexa Fluor 594 (Invitrogen) in addition to a cytoskeletal protein stai n, phalloidin-Alexa Fluor 488 conjugate (Invitrogen). Each were diluted 1:1000 in 1% BSA, 0.1% NP-40 in PBS and incubated for 40 minutes at room temperatur e. Slides were washed four times in PBS, once in

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28 28 distilled water and the nuclei stained with di amindino-2-phenylindole dihydrochloride hydrate (DAPI, Vector laboratories, Burlingame, CA). Immunofluorescence was observed by a Zeiss microscope Axio Imager Z1 microscope (Carl Zeiss Microimaging, Thornwood, NY) with an Axiocam MRm camera (Carl Zeiss Microimaging). Apoptosis assay Apoptosis was assayed using f our different assays, Annexin VFITC/propidium iodide (BD Pharmingen, San Diego, CA), anti-caspase 3/PE (BD Pharmingen), TUNEL assay (BD Pha rmingen), and mitochondrial membrane potential by DilC1(5) (Invit rogen). Each apoptosis assay was used according to standard manufacturer's protocol. In a ll assays, apoptosis was analyzed using Flow Cytometry on a FACSCalibur benc h top analyzer with FlowJo analysis software (Becton-Dickenson, Franklin Lakes, NJ). CRM1 siRNA knockdown and Western blot All electroporation transfections were performed in a freshly made transfection buffer containing 120 mM pot assium chloride, 0.15 mM calcium chloride, 10 mM potassium phosphate (pH 7.6), 25 mM HEPES, 2 mM EGTA (pH 7.6), 5 mM magnesium chloride, 2 mM ATP (pH 7.6), 5 mM glutathione, 1.25% DMSO, and 50 mM trehalose (Sigma Chemical). Each transfection consisted of 3x106 myeloma cells. Cells were washed two times in phosphate

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29 29 buffered saline and placed in a 200 l volume of transfection buffer. CRM1 specific siRNA or a scramble control siRNA (Dharmacon) was added (200 nM), the sample placed in a 2 mm electroporation cuvette and transfected at 140 V and 975 F in a Bio-Rad GenePulser Xcell electroporation unit (Biorad, Hercules, CA). Transfected cells were incubated in the cuvette for 15 minutes at 37C in a 5% CO2 incubator, transferred to a sterile T25 tissue cu lture flask, and 10 ml of fresh media added. After 48 hours the tr ansfected cells were harvested by centrifugation, washed with cold PBS, and lysed by sonication in SDS buffer (2% SDS, 10% glycerol, 60 mM Tris pH 6.8). Protein from 2x105 cells per lane was separated on 8% SDS-PAGE gels and transferred to PVDF membranes (Amersham, Piscataway, NJ) using a Biorad Mini-Transblot Apparatus (Biorad). Membranes were blocked for one hour at ambient temperature in a blocking buffer containing 0.1 M Tris-HCl, 0.9% NaCl, 0.5% Tween-20 (TBST) and 5% non-fat dry milk. CRM1 was identified by incubation in a 1:1000 dilution of H-300 antibody (Santa Cruz Biotec h, Santa Cruz, CA) in blocking buffer overnight at 4C. Membranes were washed thr ee times for 10 minutes in TBST, and incubated with for one hour with goat ant i-rabbit polyclonal IgG antibody Horseradish Peroxidase linked antibody (S igma Chemical) in blocking buffer at a 1:2000 dilution. Antibody binding was visualized by Enhanced Chemiluminescence (Amersham) on autorad iography film (Kodak, Rochester, NY).

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30 30 Band depletion assay Band depletion assays were performed as described by Xiao et al (Xiao et al., 2003). Briefly, 5x10 5 cells were lysed in 50 l alkaline lysis solution (200 mM NaOH, 2 mM EDTA) and the lysate neutraliz ed by the addition of 4 l of both 1 M HCl and 1.2 M Tris (pH 8.0). The lysa te was then mixed with 30 l 3X SDS sample buffer (150 mM Tris-HCl, pH 6. 8, 6 mM EDTA, 45% sucrose, 9% SDS, 10% -mercaptoethanol) and the lysates were separated on 8% SDS-PAGE gels. Comet assay Log density H929 myeloma cells were plated at a concentration of 2 x 105 cell/ml and plateau density cells were plated at 2x106/ml. All cells were grown in a 24 well plate (Falcon) using 1 ml per we ll. Drug treatment groups were vehicle only (1 l/ml DMSO), 10 M etoposide, 5 nM ratjadone C, or a combination of 10 M etoposide and 5 nM ratjadone C. Cells that were treated with ratjadone C were first plated at log or plateau dens ities and incubated for 20 hours with ratjadone C or vehicle, after which et oposide was added for 1 hour. After a one hour etoposide exposure the comet assay was performed as described by Kent et al(Kent et al., 1995), and modified by Chen et al (Chen et al., 2005). To ensure random sampling, fifty images were captured per slide on a Vysis fluorescent microscope and quantified us ing ImagequantR software (Molecular Dynamics, Sunnyvale Ca). The average comet moment value obtained from

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31 31 vehicle control samples was subtracted fr om the average comet moment of each drug treatment sample. The data show n are means and standard deviations of two separate experiments. An analysis of the data was performed by student's ttest. RESULTS Log and plateau density myeloma cells The myeloma cell lines, 8226, H929, and U266, were grown at log-density (2x105 cells/ml media), and plateau-density (2x106 cells/ml) growth conditions for 20 hours. Cells were then treated with the topo II inhibitor, doxorubicin (1 M) for four hours. Cells were then assayed for apoptosis by caspase 3 expression assay. We found that cells grown at plateau densities, a nd treated with 1 M doxorubicin had extremely low levels of apoptosis as compared to log-phase cells (figure 2.1A). This data confirm ed previous publications where we have shown that cells grown at different densitie s exhibit specific characteristics such as drug resistance and nuclear-cytoplasmic trafficking of topo II (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b).

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32 32 Figure 2.1: Intracellula r trafficking of topo II in log and plateau density myeloma cells 2.1A: H929 and 8226 human myeloma cells grown at plateau-phase (highdensity) export topo II whereas log phase (low-density) cells maintain topo II in the nucleus. Cells were grown 20 hours at log or plateau densities, treated with 1 M doxorubicin for 4 hours (n=2). A poptosis was determined by caspase 3 staining using flow cytometry (10,000 cells). Cells which maintained nuclear topo II were more sensitive to topo II targeted chemotherapy. 2.1B: Log, plateau, or ratjadone C treated cells (100 cells/ex periment) were stained for topo II by fluorescence microscopy (n=2). Myel oma cells grown in log-phase conditions had the majority (>90%) of the topo II in the nucleus, whereas plateau-phase cells exported the topo II into the cytoplasm. The CRM1 inhibitor ratjadone C was found to block export of topo II in cells grown in plateau-phase conditions. 0 10 20 30 40 50 60 70 80 90 100 8226H929U2668226H929U2668226H929U266 LogPlateauPlateau/RatC Topoisomerase II alpha % nuclear %cytoplasmic A B 0 20 40 60 80 100 H929/LogH929/Plat8226/Log8226/PlatU266/LogU266/PlatApoptosis %

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33 33 Intracellular trafficking of topo II Log, plateau, or ratjad one C treated cells (100 cells/experiment) were scored as “nuclear” or “cytoplasmic” if the majority (>90%) of topo II was in that compartment as determined by fluoresc ence microscopy (figure 2.1B). Myeloma cells grown in log-phase conditions had >90% of the topo II in the nucleus, whereas plateau-phase ce lls exported >90% of the topo II into the cytoplasm. CRM1 inhibition by ratjadone C was found to block export of topo II in cells grown in plateau-phase conditi ons. As seen in figure 2.2 (8226/U266 cells) and figure 2.4 (H929 cells), topo II (red) is exported into the cytoplasm in plateau-phase cells, but is maintained in the nucleus in log-phase cells. In these photomicrographs the nucleus is labeled using DAPI (blue) .Log, plateau, or ratjadone C treated cells (100 cells/experiment) were scored as “nuclear” or “cytoplasmic” if the ma jority (>90%) of topo II was in that compartment as determined by fluorescence microscopy (f igure 2.1B). Myel oma cells grown in log-phase conditions had >90% of the topo II in the nucleus, whereas plateauphase cells exported >90% of the topo II into the cytoplasm. CRM1 inhibition by ratjadone C was found to block export of topo II in cells grown in plateau-phase conditions. As seen in figure 2.2 (8226/U266 cells) and figure 2.4 (H929 cells), topo II (red) is exported in to the cytoplasm in plateau-phase cells, but is maintained in the nucleus in log-phase cells. In these photomicrographs the nucleus is labeled using DAPI (blue).

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34 34 Figure 2.2: Cellular localization of topo II in 8226 and U266 cells under log and plateau-density growth conditions 8226 and U266 human myeloma cells were grown at log and plateau densities, fixed with 4% paraformaldehyde, permeablized with 0.25% Triton X-100, and stained for topo II (red), and DNA (DAPI-blue). Results indicate that topo II is present in the nucleus of log density cells and is exported from the nucleus in plateau density cells. 8226/log 8226/plat U266/log U266/plat To p o II Da p i Mer g e d

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35 35 CRM1 inhibitor and topo II chemotherapeutics Figure 2.3 illustrates that at high ce ll density (plateau) drug resistance was reversed by the CRM1 inhibitor, ra tjadone C. The human myeloma cell lines 8226, H929 and U266 were incubated at high-densities (2x106/ml) for 20 hours with CRM1 inhibitor ratjadone C (5 nM). Topo II targeted agents etoposide (10 M) and doxorubicin (2 M), were then added, the cells further incubated an additional 7 hours and then assayed for apoptosis using the TUNEL assay (BD Pharmingen). Blocking CRM1-mediat ed nuclear export increased the effectiveness of the topo II targeted drugs to induce apoptosis in all three cell lines approximately four-f old, as compared to drug only controls. Additional apoptosis assays, caspase 3, annexin V staining, and mitochondrial membrane potential (DILC1(5)) staining demonstrated si milar results (data not shown). In addition, to show whether the ratj adone C /doxorubicin and the ratjadone C /etoposide synergistic activity was due to topo II nuclear localization, cells were transformed with a siRNA to knockdown topo II expression. In all three cell lines and with both topo II inhibito rs (doxorubicin and etoposi de), we found that knock down of topo II protein expression reversed the synergistic effect and reduced apoptosis to control (untreated) levels.

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36 36 Figure 2.3: CRM1 inhibitor and topo II chemotherapeutics Human myeloma cells were incubated at high-densities (2x106/ml) for 20 hours with the CRM1 inhibitor ratjadone C (5 nM). Cell cultures were then exposed to topo II targeted agents etoposide (10 M) n=5, doxorubicin (2 M) n=9, for 7 hours and assayed for apoptosis using TUNEL assay (BD Pharmingen). Cells were also transfected with a siRNA specific to topo II CRM1 inhibition increased the effectiveness of DNA-damaging agents to induce a poptosis; this effect was reversed by topo II siRNA knockdown and therefore is topo II dependent. 0 10 20 30 40 50 ControlDoxorubicinVP-16Apoptosis % Control RatC siRNA 0 10 20 30 40 50 60 70 ControlDoxorubicinVP-16Apoptosis % Control RatC siRNA 0 10 20 30 40 50 60 ControlDoxorubicinVP-16Apoptosis % Control RatC siRNA A B C U266 H929 8226

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37 37 Topo II trafficking and CRM1 inhibition H929 human myeloma cells were gr own at log and plateau densities and stained for cytoskeletal protei n (phalloidin-green), topo II (red), and DNA (DAPIblue). Results indicate that topo II was present in the nucleus of log density cells and was exported from the nucleus in plateau density cells (figure 2.4). Nuclear export was blocked in plateau cells by a CRM1 inhibitor ratjadone C and by transfection with CRM1-specific s iRNA. Under the conditions of this experiment, CRM1 siRNA knockdown was 69%. Ratjadone C treated plateau density cells are shown in figure 2. 1B where the majority of topo II was localized in the nucleus of eac h myeloma cell line. CRM1 inhibitor and topo II inhibitor synergy Plateau myeloma cell lines 8226, H929 and U266 were grown for 20 hours, in the presence of ratjadone C (5 nM). The cells were then treated with increasing doses of doxorubicin (0, 0.5, 1, and 2 M) for four hours and assayed for apoptosis by Caspase 3 staining using flow cytometry. Data in figures 2.5A (8226), 2.5B (H929), 2.5C (U266), demons trate that myeloma cells were rendered more sensitive to topo II inhibitors in a dose-dependent manner by inhibiting CRM1 export with ratjadone C.

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38 38 Figure 2.4: H929 topo II immunofluorescence. H929 human myeloma cells were grown at log and plateau densities, fixed with 4% paraformaldehyde, permeablized with 0.25% Tr iton X-100, and stained for cytoskeletal protein (phalloidin-green), topo II (red), and DNA (DAPI-blue). Results indicate that topo II is present in the nucleus of log density cells and is exported from the nucleus in plateau density cells. However, nuclear export is blocked in plateau cells by a CRM1 inhibitor ratjadone C and by transfection with CRM1 specific siRNA. Under the conditions of this experiment, CRM1 siRNA knockdown was 69%. DAPI Cytoskeleton TopoII Merged Log Plateau/RatC Plateau Plateau/siRNA

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39 39 CRM1 siRNA sensitizes myeloma cells to topo II poisons In addition to CRM1 pharmacologic modification by a chemical agent, we used a CRM1 specific siRNA to dete rmine if we could reproduce the topo II inhibitor synergistic activity in another model system. H929 cells were transfected by electroporation with a CRM1 specific siRN A. After transfection, the cells were incubated at log-phase densities for 20 hours, and then concentrated at plateauphase conditions. At 48 hours post-transfect ion the cells were treated with the topo II inhibitor, doxorubicin (2 M) and assayed for apoptosis by Annexin V staining using flow cytometry, (figur e 2.6A). CRM1 kno ckdown was found to increase the effectiveness of doxorubicin. To demonstrate that we were getting efficient siRNA knockdown, SDS lysates of equal cell numbers were assayed for CRM1 by Western blot (figure 2.6B). Pe rcentage of knockdown, as compared to a control scramble siRNA, was assa yed using Adobe Photoshop by pixel intensity of the CRM1 bands. A maximum knockdown of 81.5% occurred at 72 hours post transfection.

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40 40 Figure 2.5: CRM1 inhibitor and topo II inhibitor synergy. Myeloma cell lines 8226 (A), H929 (B), U266 (C), at 2x106 cells/ml were grown in culture for 20 hours. Cells were incubated with the CRM 1 inhibitor ratjadone C (5 nM) for 20 hours. Cell were then treated wit h doxorubicin (0, 0.5, 1, and 2 M) for four hours and assayed for caspase 3 staining by fl ow cytometry. Myeloma cells are made more sensitive to topo II inhibitors in a dose-dependent manner by inhibiting CRM1 export. 0 15 30 45 No DrugDox 0.5Dox 1.0Dox 2.0 Dox Concentration% Apoptosis Dox Dox/RatC 0 5 10 15 20 No DrugDox 0.5Dox 1.0Dox 2.0 Dox Concentration% Apoptosis Dox Dox/RatC 0 10 20 30 40 50 No DrugDox 0.5Dox 1.0Dox 2.0 Dox Concentration% Apoptosis Dox Dox/RatC C B A

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41 41 Figure 2.6: CRM1 knockdown using siRNA makes myeloma cells more sensitive to the topo II poison doxorubicin 2.6A: H929 cells were transfected with siRNA, incubated at log-phase for 20 hours, and concentrated at plateau-phase conditions. At 48 hours cells were treated wit h the topo II inhibitor doxorubicin (2 M) and assayed for apoptosis by Annexi n V staining using flow cytometry, n=2. 2.6B: Western blot data for siRNA trans fection. Percent knockdown was compared to control siRNA (scrambl e). CRM1 knockdown renders plateau density cells more sensitive to topo II inhibitors. 48 72 H929 8226 H929 8226 Control siCRM1 Control siCRM1 Control siCRM1 Control siCRM1 100% 35% 100% 40% 100% 31% 100% 18.5% 0 10 20 30 40 50 60 ScramCRM1 siRNAScram-doxCRM1 siRNAdoxPercent Apoptosis (AnexinV/PI) A B

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42 42 Topoisomerase Western blot Cell nuclei contain three different topoisomerases; topoisomerase I, II and II To determine whether cell density c onditions (log/plateau) affected the levels of endogenous topoisomerases, we assayed whole cell lysates by SDSPAGE analysis. Topoisomerase Western blot assay based both on cell number and total protein were found to be nearly id entical (data not shown). In all three myeloma cells lines topoisomerase I, II and II protein levels were relatively unchanged or changed very slightly in log and plateau density growth conditions (figure 2.7). Increase in cleavable complex formation by CRM1 inhibition The band depletion assay indicated t hat a combination of ratjadone C and etoposide produced more DNA/topo II complexes, depleting the topo II band in the Western blot analysis (figure 2.8A ). These data indicates that blocking nuclear export of topo II will increase the effectiveness of etoposide and induce apoptosis by increased cleavable complexes.

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43 43 Figure 2.7: Log and plateau expr ession of topoisomerases in myeloma cell lines. All three human myeloma cell lines were assayed for endogenous topoisomerase protein expression by Western blot. Ce lls were compared in the same blots under log and plateau-phase growth conditi ons. No relative differences were found when equal protein loading was co mpared to equal cell numbers (data not shown). The data suggests that topoisomerases I, II and II did not change or changed very slightly in log vs. plateau cells. Topo II Topo I Topo II 8226 H929 U266 Lo g Plat Lo g Plat Lo g Plat

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44 44 Figure 2.8: Band depletion and comet assay. 2.8A: Band depletion The combination of ratjadone C and et oposide produced more DNA/topo II complexes, depleting the topo II band in the Western blot analysis. This data indicates that blocking nuclear export of topo II will increase the effectiveness of etoposide and induce apoptosis. 2.8B : Comet assay. Plateau density H929 cells were treated with ratjadone C (5nM) 20 hours and then with etoposide (10 M) for 60 minutes. DNA fragmentation was measured by the comet assay. The CRM1 inhibitor ratjadone C, increased DNA fragmentation from the topo II inhibitor etoposide. Control VP16 50M RatC/ VP16 50M RatC/ VP16 25M VP16 25M RatC A B 0 10 20 30 40 50 60controlRatCVP-16VP-16/RatCComet Moment Control VP16 50M RatC/ VP16 50M RatC/ VP16 25M VP16 25M RatC Control VP16 50M RatC/ VP16 50M RatC/ VP16 25M VP16 25M RatC Control VP16 50M RatC/ VP16 50M RatC/ VP16 25M VP16 25M RatC A B 0 10 20 30 40 50 60controlRatCVP-16VP-16/RatCComet Moment

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45 45 Comet assay Plateau density H929 cells were tr eated with 5 nM ratjadone C for 20 hours and then with 10 M etopos ide for 60 minutes. DNA fragmentation was measured by neutral comet assay. The CRM1 inhibitor ratjadone C, increased DNA fragment ation induced by the topo II inhibitor, etoposide (figure 2.8B). Increased DNA fragmentation led to increased apoptosis in etoposide/ ratjadone C treated cells. DISCUSSION Topo II inhibitors function by stabilizing cleavable complexes resulting in DNA strand breaks. Accumulation of DNA strand breaks will eventually result in cell apoptosis. In order for DNA strand breaks to be produced, the enzyme must be in the nucleus and in contact with the genomic DNA. Ther efore, preventing nuclear export of topo II by CRM1 inhibition may improve the function of topo II inhibitors. The intracellular location of a protein may be at least as important as its expression. Diseases as dissimilar as cystic fibrosis (Welsh and Smith, 1993), schizophrenia (Karpa et al., 2000), nephrogenic diabetes insipidus (Edwards et al., 2000) and many types of cancers [revie wed in (Davis et al., 2007)] may be caused by intracellular mislocalization of individual proteins. Specific examples of proteins that must be in the nucleus to prevent cancer initia tion, progression or chemotherapeutic response include, p53 [reviewed in (Fabbr o and Henderson,

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46 46 2003)], galectin-3 (Takenaka et al., 2004) FOXO (Nakamura et al., 2000), INI1/hSNPF5 (Craig et al., 2002) p27Kip1 (Min et al., 2004), p27WAF1 (Keeshan et al., 2003), and topo II (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b). Mislocalization of a protein can render it ineffective as a tumor suppressor or as a target for chem otherapy. However, it is possible that blocking nuclear export of any or all these proteins may induce tumor suppression, apoptosis, or in the case of topo II may reverse drug resistance to topo II inhibitors. This may be true in MM where the cells possess a CRM1mediated mechanism whereby topo II is exported from the nucleus and away from the DNA, rendering topo II inhibitors ineffective to produce cleavable complexes and DNA strand breaks. In previous reports, we have s hown that myeloma cells, under highdensity conditions, will export topo II into the cytoplasm both in vivo and in vitro (Engel et al., 2004; Turner et al., 2004; Valkov et al., 2000b). We found that nuclear export of topo II contributes to drug resistance (Engel et al., 2004) and the resistance was not due to differences in drug uptake, cell cycle or cellular topo II protein levels. In addition, topo II nuclear export has been shown to be CRM1-mediated and that topo II protein contains two functional nuclear export signals at amino acids 1017-1028 and 1054-1066 (Turner et al., 2004). Export by both signals was blocked by treatment of the cells with leptomycin B indicating that a CRM1-dependent pathw ay mediates export (Turner et al., 2004).

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47 47 In this study, we demonstrated that myeloma cells grown at high density are highly resistant to topo II directed chemotherapeutic drugs (figure 2.1A) and that drug resistance correlated with nuclear export of topo II (figures 2.1B, 2.2, and 2.4). Based on these data we proposed that blocking CRM1-mediated export of topo II may make myeloma cells more sensitive to topo II directed chemotherapy. To evaluate w hether the lack of topo II export would sensitize cells, we were able to knockdown CRM1 mRNA and protein expression in cells transfected with CRM1 specific siRNAs, and by using the CRM 1-inhibiting drug, ratjadone C. CRM1 inhibition by siRNA and ratjadone C in human myeloma cells was found to prevent nuclear export of topo II in plateau density cell cultures (figures 2.1B and 2.4). Depletion or i nhibition of CRM1 by siRNA or ratjadone C caused high-density myeloma cells to bec ome four-fold more sensitive to the topo II inhibitors, doxorubicin and etoposid e as measured by apoptosis (figures 2.3 and 2.5). Depletion of topo II protein by specific topo II siRNA knockdown reversed this synergistic e ffect, indicating that topo II was the targeted molecule for CRM1 synergistic activity (figure 2.6). In conclusion, maintaining topo II in the nucleus by inhibiting CRM1 greatly enhanced the cytotoxic effect of the topo II inhibitors, doxorubicin and etoposide in myeloma cells. Band depl etion assays indicated that more DNA/topo II complexes were stabilized in cells when CRM1 was inhibited because there was more topo II present in the cell nuclei (figure 2.8A).

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48 48 Increased cleavable complexes result ed in increased strand breaks (comet assay) (figure 2.8B) and subsequent apoptosis. These findings may have potential therapeutic value in the tr eatment of multiple myeloma.

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49 49 Chapter 3 ABCG2 expression, function an d promoter methylation in human multiple myeloma SUMMARY We investigated the role of t he breast cancer resistance protein (BCRP/ABCG2) in drug resistance in mult iple myeloma (MM). Human MM cell lines, and MM patient plasma cells isol ated from bone marrow, were evaluated for ABCG2 mRNA expression by quantit ative PCR, and ABCG2 protein by Western blot analysis, immunofluoresc ence microscopy and flow cytometry. ABCG2 function was determined by measur ing topotecan and doxorubicin efflux using flow cytometry, in the presenc e and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABC G2 promoter was determined using bisulfite sequencing. We found t hat ABCG2 expression in myeloma cell lines increased after exposure to topotec an and doxorubicin, and was greater in log-phase cells when compared to quiesc ent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated at least in part, by promoter methylation both in cell lines and in pati ent plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression.

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50 50 These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter me thylation, affected by cell density, upregulated in response to chemotherapy and may contribute to both intrinsic and acquired drug resistance. INTRODUCTION The development of drug resistanc e to chemotherapeutic agents remains one of the primary obstacles in cancer treatment. Membrane drug-efflux pumps such as P-glycoprotein (M DR1), multidrug resistance protein (MRP), and ABCG2 have been shown to produce resistance to several commonly used chemotherapeutic agents. Breast cancer resistance protein (BCRP) or ATP binding cassette protein G2 (ABCG2) is a 655 amino-acid polypeptide transporter that forms a homodimer (Doyle et al., 1998) and has been reported as a tetramer in plasma membranes (Xu et al., 2004). ABCG2 is a half-transporter, containing a single N-terminal ATP-binding casse tte and six transmembrane segments. ABCG2 was first described in dr ug resistant MCF-7/AdrVp cells (Doyle et al., 1998) and has been the subject of recent reviews (Abbott, 2003; Allen and Schinkel, 2002; Doyle and Ross, 2003) Like other members of the ATP binding cassette family of membrane transporte rs, such as MDR1 and MRP1, ABCG2 is expressed in a variety of malignancie s, where it may produce resistance to chemotherapeutic agents. Am ong cultured human cell lines that express high levels of ABCG2 are fibrosarcoma, ovar ian cancer, breast cancer, and myeloma

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51 51 cell lines (Allen et al., 1999). Hum an neoplasms frequently found to express ABCG2 protein include: adenocarcinomas ar ising from the digestive tract, the endometrium, and the lung; me lanoma; soft tissue sarcomas (Candeil et al., 2004; Diestra et al., 2002) and hematological malignancies such as acute myeloid leukemia (AML) (Litman et al ., 2000) and acute lymphoblastic leukemia (ALL) (Sauerbrey et al., 2002). Several studies have been performed to investigate potential correlations bet ween ABCG2 expression and clinical outcomes. Studies from pat ients with AML dem onstrated significantly increased expression of ABCG2 mRNA in the rel apsed/refractory samples compared to pre-treatment (van den Heuvel-Eibrink et al., 2002). AML patients with highlevels of ABCG2 expression had significantly shorter overall survival rates (Uggla et al., 2005) while decreased ABCG2 was f ound to be a prognostic factor in adult patients who achieved complete remission of AML (Benderra et al., 2004). The substrate specificity of ABCG2 includes the anti-neoplastic drugs primarily targeting topoisomerases, includi ng anthracyclines and camptothecins. Topoisomerase I and II inhibitors that are substrates of ABCG2 include topotecan, SN-38, CPT-11, mitox antrone, daunomycin, doxorubicin, and epirubicin (Doyle et al., 1998; Litman et al ., 2000). Topotecan in particular is an excellent substrate for ABCG2. In addition, flavopiridol resistance is mediated by ABCG2 (Honjo et al., 2001). Recently, se veral potent and specific inhibitors of ABCG2 have been developed, potentially opening the door to clinical applications of ABCG2 inhibition. Thes e inhibitors include the targeted agents

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52 52 gefitinib (Iressa) and imatinib me sylate (Gleevec) (Houghton et al., 2004) as well as the more specific inhibitors fu mitremorgin C (Rabindran et al., 2000), tryprostatin A (Woehlecke et al., 2003; Zhao et al., 2002), and GF120918 (Glaxo) (Maliepaard et al., 2001a). The normal tissue localization of ABCG2 is in hematological stem cells, placenta, bile canaliculi, colon, sma ll bowel, and brain microvessel endothelium (Maliepaard et al., 2001a). Given the specific tissue lo calizations, the role of ABCG2 in healthy tissues may be to pr otect an organism or tissue from potentially harmful toxins. ABCG2 expr ession has been associated with Akt signaling (Mogi et al., 2003) and its promoter contains an estrogen-response element (Ee et al., 2004). However, r egulation by the microenvironment or in direct response to chemotherapeutics has not been reported in multiple myeloma (MM). It has been shown that the ABCG2 promoter contains a potential CpG island, which may be regulated by methylat ion (Bailey-Dell et al., 2001). Another ABC family transporter, MDR1, has a promot er with a similar CpG island that has been shown to regulate gene expression via meth ylation of this si te (Baker et al., 2005; David et al., 2004; Fryxell et al., 1999; Kusaba et al., 1999). In the current study, we determined t hat ABCG2 is present and functional in human MM cells. Using quantitative PCR (QPCR), cytological staining, Western blot analyses, and functional efflux of chemotherapeutic drugs we found that ABCG2 may be involved in drug resistance. In vitro, we found that ABCG2 expression increased in response to exposure to the ABCG2 substrates,

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53 53 doxorubicin and topotecan. Cell density al so affected ABCG2 expression, as myeloma cells grown at log-phase densitie s had greater levels of ABCG2 than cells cultured at higher (pla teau) densities. Myeloma patients treated with a highdose chemotherapy (HDC) r egimen that included topotecan had an increase in ABCG2 mRNA and protein expression a fter treatment and at relapse when compared to pre-treatment samples. Expr ession of ABCG2 is regulated at least in part by promoter methyl ation both in cell lines and in plasma cells from patients. Demethylation of the promoter using 5-aza2’-deoxycytidine increased ABCG2 expression. Thus, ABCG2 may cont ribute to intrinsic drug resistance in human MM, and this may be augmented by exposure to chemotherapeutic agents that are substrates for ABCG2. MATERIALS AND METHODS Cell lines Human MM cell lines, RPMI-8226 (8226) and NCI-H929 (H929) were obtained from the American Type Cultur e Collection, Manassas, VA, USA. Mitoxantrone resistant-8226 (8226MR) cells were isolated by Dr. William Dalton at the H. Lee Moffitt Cancer Center, Tam pa, Florida (Hazlehurst et al., 1999). All cell lines were grown in RPMI-1640 m edia containing peni cillin/streptomycin (Gibco, Gaithersburg, MD, USA), and 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37C and 5% CO2.

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54 54 Clinical trial with high-dose melphalan and topotecan Human myeloma cells were obtained fr om patients enrolled in a phase I/II HDC protocol using melphalan, VP-16 phosphate and dose-escalated topotecan (MTV trial) followed by peripheral blood st em cell transplant. This protocol was approved by the University of South Fl orida Institutional Review Board, and signed informed consent was obtained from all patients prior to their participation in the study. Patients were infused fo r three consecutive days with melphalan (50 mg/m2/day IV over 30 min), followed immediately by topotecan (from 0 to 9 mg/m2/day IV over 30 min), follo wed by VP-16 phosphate (1200 mg/m2/day VP16 equivalents IV over 4 hour s) for two days. The dose escalation scheme for topotecan was: dose level (DL) 1, 0 mg/m2 total dose over 3 days; DL 2, 10 mg/m2 total dose; DL 3, 15 mg/m2 total dose, DL 4, 20 mg/m2 total dose; and DL 5, 27 mg/m2 total dose over three days. Bone marrow aspirates were taken before the start of HDC (pre -HDC), on the day after completion of three days of melphalan/topotecan infusion (before the first of two days of VP-16), and in patients who had relapsed from this HDC prot ocol. Plasma cells were isolated from bone marrow aspirates by Ficoll gradient separation followed by CD138 antibody/magnetic bead (Miltenyi Biotech, Auburn, CA, USA) purification according to the manufacturer's instruct ions. Percent purity of CD138 selected cells for all patient samples was rout inely between 80 and 99%. The analysis of patient plasma cells for ABCG2 mRNA expression was not an original endpoint

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55 55 of the MTV trial. An IR B approved amendment allowed us to analyze aliquots of residual bone marrow aspirate s for ABCG2 expression. Real-time quantitat ive PCR (QPCR) A quantitative primer/probe set was designed to evaluate and to quantify ABCG2 mRNA. Total RNA was extract ed from human myeloma cell lines and patient CD138 selected cells by using the guanidine isothiocyanate and phenol/chloroform method (Chomczynski and Sacchi, 1987) (Trizol, Gibco) with the addition of 20 g glycogen as a carrier for the RNA. Reverse transcription of RNA was performed using Omniscrip t reverse transcriptase (Qiagen, Germantown, MD, USA), according to the manufacturer's protocol. Primers and probes for real time PCR were designed using Primer Express software (Applied Biosystems, Fost er City, CA, USA). Each primer set consisted of standard PCR primers (Tm 58-60C) designed to span gene introns in order to exclude any possible genomic DNA contamination. Detection and quantitation of each gene was accomplis hed using an amplicon-specific fluorescent oligonucleot ide probe (Tm 68-70C), with a 5' reporter dye (carboxyfluorescein) and a downstream 3' quencher dye (carboxytetramethylrhodamine). The s equence of the prim ers used for ABCG2 detection were 5'-TTT CCA AGC GTT CAT TC A AAA A-3' (forwa rd primer), 5'TAC GAC TGT GAC AAT GAT CTG AGC-3' (reverse primer), and 5'-TTG CTG GGT AAT CCC CAG GCC TCT-3' (fl uorescent probe) (Integrated DNA

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56 56 Technologies). Two microliters of c DNA were assayed per well, and the QPCR performed as previously described (Turner et al., 2004). The ABCG2 expression data were found to be log normally di stributed. Consequently, the geometric means were used to average both withi n and between patient data. Changes due to treatment were assessed by taking t he logarithm of the ratio of the data compared to baselines level. Statisti cal significance was assessed using the Wilcoxon signed rank test. P-values below 0.05 were considered to be statistically significant. Western blot for cell lines and patient myeloma samples Human 8226 and H929 myeloma cells we re harvested by centrifugation, washed with cold PBS, and lysed by sonica tion in 2% SDS buffe r. Protein from 2x105 cells per lane was separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham) usi ng a Biorad Mini-Transblot Apparatus (Biorad, Hercules, CA, USA). Membranes were blocked for one hour at ambient temperature in a blocking buffer contai ning 0.1 M Tris-HCl, 0.9% NaCl, 0.5% Tween-20 (TBST) and 5% non-fat dry milk. ABCG2 was identified by incubation in a 1:1000 dilution of BXP-21 antibody (Kamiya, Seattle, WA, USA) in blocking buffer overnight at 4C. Membranes we re washed three times for 10 minutes in TBST, and incubated for one hour with a goat anti-mouse IgG antibody linked to horseradish peroxidase (Sigma-Aldrich, S t. Louis, MO, USA) in blocking buffer at a 1:2000 dilution. Antibody bindin g was visualized by Enhanced

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57 57 Chemiluminesence (Amersham, GE Health care, USA) on autoradiography film (Kodak, USA). Protein loading on gels was assessed by Coomassie blue staining of the Western blots. Blots were incubated at room temperature in a shaker apparatus with 250 mg/L Coomassie blue in 50% methanol and 10% glacial acetic acid. Blots were then destained for 2 hours in a solution containing 50% methanol and 10% glacial acetic acid. Protein staini ng was compared visually to ensure equal loading in each lane, and unless other wise noted was equivalent in each immunoblot. Flow cytometry/ABCG2 functional assay ABCG2 expression was assayed by flow cytometry using an antibody that specifically recognizes only membr ane bound ABCG2 epitopes (Bcrp1-PE, Chemicon/Millipore, Billeric a, MA, USA). Human myeloma patient cells and myeloma cell lines were fixed with 4% paraformaldehyde for 10 minutes and washed in phosphate buffered sali ne (PBS). Approximately 105 cells were labeled with 10 l of Bcrp1-PE antibody in 190 l of 1% bovine serum albumin (BSA) in PBS at 37C for 30 minutes. Labeled cells were washed in PBS and assayed by flow cytometry on a FACS can (Becton Dickenson, Franklin Lakes, NJ, USA). ABCG2 function was assayed as the efflux of the ABCG2 substrates doxorubicin and topotecan or as the effl ux of Hoechst 33342 (Sigma) (Kawabata et al., 2003). ABCG2 function was a ssayed in myeloma patient bone marrow

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58 58 aspirates obtained before and after exposure to 1 M topotecan (aspirates obtained prior to HDC on the MTV protocol), and in H929, 8226 and 8226MR cell lines. The plasma cells were isolat ed from bone marrow aspirates (frozen in liquid nitrogen) using CD138 magnet ic bead-antibody conjugates (Miltenyi Biotec) after separation by a Ficoll gradi ent. Topotecan is very fluorescent and accumulates in cells that do not expre ss ABCG2. Myeloma cell lines and CD138 purified patient samples were inc ubated with 40 M t opotecan or 1 M doxorubicin for 20 minutes at 37C, was hed twice in ice-cold PBS, and analyzed by flow cytometry for topotecan and doxorubicin fluorescence. 14C-Mitoxantrone uptake was also used to assess ABCG2 function. Cells were incubated for two hours with 14C-mitoxantrone with and without a large molar excess of unlabeled mitoxantrone. Radioactivity was measured by liquid scintillation counting (Perkin-Elmer, We llesley, MA, USA). Controls used were identical cell samples wit hout drug or co-incubated with the specific ABCG2 inhibitor tryprostatin A (Woehlecke et al ., 2003; Zhao et al., 2002). Tryprostatin A was synthesized by Dr. Chunchun Zhang and Dr. James M. Cook, at the University of Wisconsin-Milwaukee. To determine if decreased drug uptake was due to ABCG2 activity, both patient samples and cell lines were co-incubated with the specific ABCG2 inhibitor tryprostat in A. This drug blocks the drug efflux function of ABCG2, resulting in incr eased fluorescence due to intracellular topotecan or doxorubicin accumulation. ABCG2 function was expressed as the

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59 59 change in relative fluorescence in t opotecan treated versus untreated control cells. Immunofluorescent microscopy and quantitative measurement of ABCG2 Patient plasma cell samples and myeloma cell lines (1x105 cells) were plated on double cytoslides (Shandon) by cyto-centrifugation at 500 rpm, and fixed and stained with anti-ABCG2 (BXP-21, Kamiya Biomedical Labs) according to the protocol in Engel et al (Engel et al., 2004). Slides were washed with PBS, air-dried and the nuclei stained with 4',6-d iamidino-phenylindole dihydrochloride hydrate (DAPI) (Vector laboratories, Bur lingame, CA, USA). Cellular membrane ABCG2 staining was performed directly on paraformaldehyde fixed cells using the membrane specific antibody, Bcrp1-FI TC (Chemicon). Immunofluorescence was observed by a Leitz Orthoplan microscope with a CCD camera. Cell density and low dose drug treatment The model used to assess possible microenvironmental effects involved incubating cells at highand low-density cu lture conditions, assuming that highdensity conditions mimic the in vivo bone marrow environment. We have shown previously that myeloma cells grown at different densities exhibit specific characteristics, including drug resistance to topoisomerase I and II inhibitors that depends on the nuclear to cytoplasmic traf ficking of topoisomerases (Engel et al., 2004; Turner et al., 2004; Valkov et al ., 2000a). Myeloma cell lines (8226, H929,

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60 60 8226MR) grown at 2x105 cells/ml media were defined as low-density (log-phase), and cells grown at 2x106 cells/ml were defined as high-density (plateau-phase). Cell lines were placed at log and plateau density conditions and grown for 24 hours at 37C in 5% CO2. Cells were harvested and assayed for ABCG2 expression by flow cytometry, immunos taining, mRNA analysis (QPCR), and Western blot as described above. In addi tion, logand plateau-phase cells were further incubated in the presence of 1 M topotecan or 0.1 M doxorubicin for 20 hours at 37C in a 5% CO2 incubator and harvested for the determination of ABCG2 expression. Bisulfite sequencing and dem ethylation by 5-aza-2'deoxycytidine of the ABCG2 promoter in patient samples and myeloma cell lines Genomic DNA from patient biopsies or cell lines was extracted using the DNeasy Tissue Kit (Qiagen). Two micrograms of DNA were subjected to bisulfite conversion according to methods published in Warnecke et al (Warnecke et al., 2002). Primers were designed to clone t he bisulfite-converted CpG island-rich portions of the human ABCG2 promoter using standard PCR conditions (ABCG2 forward, GGA TAA TAT TAG GTA AGG TTG AGT AA, ABCG2 reverse, TCA AAA TAA CTC CCT CCA AAC AAA AC). ABCG2 low-expressing H929 cells, whic h had highly methylated promoter CpG islands, were treated with the dem ethylating agent, 5-aza-2'-deoxycytidine, to determine if ABCG2 promoter dem ethylation allowed increased ABCG2

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61 61 expression. Cells were incubated for 72 hours in media containing 5-aza-2'deoxycytidine (Sigma) at a concentrat ion of 100 nM and harvested to detect ABCG2 expression by flow cytometry, immunostaining, QPCR, and Western analysis. Methylation-specific quantitative PCR Genomic DNA samples that were extr acted from patient myeloma cells for bisulfite sequencing were further anal yzed to determine the percentage of methylated alleles. Primers were desi gned to anneal specifically to methylated and non-methylated CpG dinucleotides in a region of the ABCG2 promoter. This area of the ABCG2 promoter was previously found to be methylated by bisulfite sequencing (data not shown). The prim ers used had the following sequences: 5'TGA TTG GGT AAT TTG TGC GTT AGC G-3', methylat ed forward primer; 5'TGA TTG GGT AAT TTG TGT GTT AGT GTT-3', un-meth ylated forward primer, and 5'-AAA TAA ACC AAA ATA ATT AAC TAC3', reverse primer that was used for both PCR reactions. The PCR reaction was performed in a 96-well optical reaction plate. The reacti on mixture consisted of 2 l of bisulfite DNA, 0.2 M each primer and 23 l of SYBR green P CR mix (Biorad) according to the manufacturer’s protocol. QPCR was performed in an ABI 5700 sequence detection system. For each sample, the ex act number of alleles that were methylated and non-methylated were assa yed, and the data ex pressed as the percentage of methylated alleles.

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62 62 RESULTS Quantitative PCR of ABCG2 Table 3.1 and Figure 3.1 show the re lative expression of ABCG2 mRNA in several human cancer cell lines. High levels of expression were found in mitoxantrone resistant 8226MR ce lls (Hazlehurst et al., 1999) and MCF-7/mitox cells (Taylor et al., 1991) while par ental 8226 cells (827.4 RU) and H929 cells (56.1 RU) had intermediate and low levels of expression, respectively. ABCG2 mRNA copy numbers were normalized to housekeeping gene GAPDH copy numbers and expressed as relative units (RU). Plasma cells obtained from patients prior to high-dose chemotherapy also had intermediate levels of expression, with a geometric me an of 118.4 RU (Table 3.2). Patient bone marrow aspirates obtained prior to HDC, after three days of melphalan alone (DL 1) or after three da ys of melphalan and topotecan (DL 2-5), or at relapse from HDC, were analyz ed for ABCG2 mRNA expression (Table 3.2). These were unused aliquots of bone marrow aspirates from MTV trial patients (Sullivan et al., 2001; Valkov et al., 2000a) for which we obtained IRB approval for ABCG2 analysis. All possible residual samples from this trial were analyzed; 42 paired

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63 63 Table 3.1. ABCG2 mRNA expre ssion determined by QPCR in human cancer cell lines. Results giv en as mean (SD). PBMC indicates peripheral blood mononuclear cells. *S ee Warnecke et al (Warnecke et al., 2002). †See Knutsen et al(Knutsen et al., 2000). samples from 31 patients (paired either pre-HDC and during HDC, or pre-HDC and relapse). Ten patients had samples from all three time points. The frozen samples were thawed, selected using CD138 immunomagnetic beads and analyzed by QPCR. Patients that had received three days of melphalan followed by two days of VP-16 (Table 3.2; 6 patients, DL 1) had no significant change in ABCG2 expression compared with pr e-HDC plasma cells (P = 0.56), nor when relapse values were compared with pre-HDC Cell/Tissue Type ABCG2 mRNA (copies per cell normalized to GAPDH) Normal PBMC 1.1 (1.0) 8226MR myeloma* 4402.7 (195.5) 8226 myeloma 827.4 (30.6) H929 myeloma 56.1 (3.1) CCRF leukemia 0.0 HL-60 leukemia 0.0 KG1A leukemia 0.6 (0.1) MCF-7 breast cancer 37.9 (1.1) MCF-7/Mitox† 5040.4 (589.3) MDA 231 breast cancer 16.9 (0.2) MDA 361 breast cancer 0.4 (0.1) A375 melanoma 13.2 (0.4) SK5 melanoma 19.7 (2.8) SK28 melanoma 9.2 (0.3) CRL 1974 melanoma 29.0 (1.8)

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64 64 ABCG2 mRNA expression/cell 2 Patient Dose Level MTV1 Pre-HDC During HDC Relapse from HDC 1 1 143.5 421.3 282.5 2 1 7.4 12.3 29.6 3 1 87.9 29.8 ND 4 1 140.8 67.5 38.9 5 1 20.6 49.0 ND 6 1 2.4 24.5 0.1 7 1 37.6 ND 40.4 8 2 8.6 18.7 12.4 9 2 8.7 111.8 62.3 10 3 33.2 86.4 422.6 11 3 30.0 35.6 38.9 12 3 253.2 154.7 ND 13 3 9.5 ND 44.1 14 3 10.2 ND 67.7 15 4 193.8 122.3 1234.8 16 4 882.6 1133.4 ND 17 4 443.9 1908.0 ND 18 4 202.8 1791.1 ND 19 4 813.6 1595.6 ND 20 4 446.0 852.5 ND 21 4 45.9 14.3 ND 22 4 342.2 164.0 ND 23 4 1604.1 1877.3 ND 24 4 558.1 316.2 ND 25 4 42.9 97.4 ND 26 4 176.3 ND 354.1 27 4 100.2 ND 140.6 28 5 44.2 183.1 ND 29 5 78.6 146.6 ND 30 5 53.9 31.6 103.0 31 5 24.7 2893.8 238.2 Pre-HDC versus During HDC3 Dose Level 1 (n = 6) 29.3 48.2 p = 0.564 Dose Levels 2-5 (n = 20) 118.4 232.5 p = 0.033 Pre-HDC versus Relapsed from HDC Dose Level 1 (n = 5) 26.6 p= 1.00 17.6 Dose Levels 2-5 (n = 11) 31.7 p= 0.001 117.1 Table 3.2. ABCG2 mRNA expression determined by QPCR in CD138 selected human plasma cells from bone marrow aspirates obtained from patients with multiple myeloma prior to and during highdose chemotherapy, and at the time of relapse. The QPCR was repeated twice for each patient, and the value is the geometric mean of those two observations (see methods). 1Patients on dose level 1 received three days of melphalan followed by two days of VP-16 phosphate. Those on dose levels 2-5 rece ived three days of melphalan followed immediately by dose-escalated topotecan each day for three days, followed by two days of VP-16 phosphate. 2The expression of ABCG2 is normalized to that

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65 65 of GAPDH. 3The analyses presented are for paired samples only, that is, pre + during and pre + relapse. 4p-values are from a Wilcoxon signed rank test. Abbreviations: MTV, melphalan + topotecan + VP-16 phosphate; HDC, highdose chemotherapy; ND, not done either because of insufficient CD138 cells isolated or because the patient has not relapsed from HDC. (5 patients; P = 1.00). In contrast, those patients that rece ived melphalan and topotecan (Table 2; 20 patients, DL 2-5) had a significant in crease in ABCG2 expression compared to pre-HDC samples (P = 0.033). In addition, patient samples from relapse in DL 25 (Table 2) also had a statistically significant increase in ABCG2 when relapse samples were compared with pre-HDC valu es (11 patients; P = 0.001). A statistical analysis comparing ABCG2 mRNA levels and patient clinical outcome (best response to high-dose chemother apy) failed to find any statistically significant prognostic value of mRNA leve ls in this limited number of samples. ABCG2 protein expression determined by Western analysis, flow cytometry and immunofluorescence The protein expression of ABCG2 in human myeloma cell lines was assayed by Western blot, immunofluore scence staining, and flow cytometry, and found to correlate well with the QPCR data (Figure 3.1). Mitoxantrone-resistant 8226 (8226MR) cells expressed very high le vels of ABCG2, while parental 8226 cells and H929 cells expressed intermediat e and low levels, respectively. Flow cytometric analysis of human myelom a cell lines for ABCG2 membrane expression also showed high levels of expression.

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66 66 Figure 3.1. ABCG2 Expression a nd function in myeloma cell lines (A) Flow cytometric analysis of MM cell lines for ABCG2 membrane expression was performed. Mitoxant rone resistant 8226MR cells express more ABCG2 than wild-type 8226 cells, and H929 cells express ve ry little, as shown by the shift in fluorescence. (B) Immunostaining of myeloma cell lines using an anti-ABCG2 FITC (Chemicon) labeled antibody (ABC G2 is green and DAPI is blue). (C) Western blot of protein (25 g/lane) extracted from myeloma cell lines for ABCG2. (D) Functional analysis of ABC G2 using topotecan as a substrate and the ABCG2 specific inhibitor tryprostat in A. Topotecan, a very good ABCG2 substrate and a naturally fluorescent mole cule, is effluxed in high (8226MR) and moderate (8226) ABCG2 expressers, but is accumulated by H929 cells (which do not express ABCG2). Tryprostatin A (T rypA) blocks the efflux of topotecan, demonstrating that topotecan efflux is ABCG2 dependent. 120 80 40 0 120 80 40 0 120 80 40 0 Isotype Anti-ABCG2 PE No drug Topotecan Topotecan/trypA mRNA= 4402.7 mRNA= 827.4 mRNA= 56.1 A C D H929 8226 8226 8226MR H929 B Counts FL2 ABCG2 FL2 ABCG2 120 80 40 0 120 80 40 0 120 80 40 0

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67 67 8226MR expressed more ABCG2 than wild-type 8226, and H929 expressed very little as shown by the shift in fluorescence (Figure 3.1A). Immunostaining of myeloma cell lines us ing an anti-ABCG2 Bc rp1-FITC labeled antibody (Chemicon) demonstrated high leve ls of ABCG2 expression in 8226MR and 8226 cells, but not in H929 cells (Figur e 3.1B). A Western analysis of protein extracted from myeloma cell lines for ABC G2 also followed the same pattern of protein expression (Figure 3.1C). ABCG2 functional assay: topotecan efflux The purpose of the functional assa y was to evaluate ABCG2 mediated drug efflux in myeloma cell lines. T opotecan, an exceptional ABCG2 substrate and a naturally fluorescent molecule, was effluxed in high (8226MR) and moderate (8226) ABCG2 expressers, but wa s not effluxed by H929 cells (low expressing cells). To show that efflux was specific to ABCG2 and not other transporters, the inhibitor tryprostatin A was used as a control (Figure 3.1D). Tryprostatin A efficiently blocked the efflux of topotecan in 8226 and 8226MR cells, demonstrating that in these ce ll lines topotecan efflux was ABCG2 dependent (Figure 3.1D) (Woehlecke et al., 2003; Zhao et al., 2002). ABCG2 function was expressed as the change in relative fluorescence in topotecan treated versus untreated control cells.

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68 68 Figure 3.2. Functional assay in patient myeloma cells. Patient samples with high ABCG2 and low ABCG2 mRNA (as measured by QPCR), were assayed for ABCG2 function. The high ABCG2 expre sser effluxed topotecan more efficiently than the lower expresser. Efflux wa s shown to be ABCG2 specific by the addition of tryprostatin A.

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69 69 This analysis showed that ABCG2 protein and mRNA levels correlated well with function (Figure 3.1A, 3.1B, 3.1C, 3.1D). Similar results were found in patient bone marrow samples, where a high ABCG2 expressing sample was found to efflux topotecan more efficiently than a low expresser (Figure 3.2). In addition, doxorubicin was e ffluxed more efficiently by high ABCG2 expressing 8226MR than 8226 parental cells (Figure 3. 3). ABCG2 efflux of doxorubicin was inhibited by the addition of the ABCG2 i nhibitor tryprostatin A (Figure 3.3). 14C-Mitoxantrone uptake was also used to assay ABCG2 function, as we have previously described(Harker et al ., 1995). Human myeloma cells were incubated for 2 hours with 14C-mitoxantrone with and wi thout a large molar excess of unlabeled mitoxantrone. 14C-Mitoxantrone uptake corroborated the findings of topotecan uptake; high ABC G2 expressing 8226MR cells effluxed labeled drug more efficiently than low expressing 8226 parental cells. 8226MR cells had an equilibrium cellular radioacti vity of 5,37465.6 cpm/mg cellular protein, while parental 8226 cells had 13, 187102.9 cpm/mg cellular protein. Thus, the ABCG2 expressing 8226MR cells are able to efflux mitoxantrone more efficiently.

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70 70 Figure 3.3. ABCG2 expression increases in response to doxorubicin exposure. ABCG2 expression was assayed by fl ow cytometry in 8226 and 8226MR MM cells after exposure to 1 M doxorubi cin for 20 minutes. Higher ABCG2 expressing cells were able to efflux doxorubicin more than parental 8226 cells. The ABCG2 specific inhibitor, tryprostat in A decreased efflux in the 8226MR cell line but not the 8226 parental cell line, indicating that doxorubicin efflux was mediated by ABCG2. Myeloma cells treat ed with low dose doxorubicin, 0.1 M 8226MR and 1.0 M 8226 cells exhibit an increase in protein expression as determined by Western analysis (inset of each graph). Equal amounts of protein (25 g), was assayed. Both 8226 and 8226 MR cells demonstrated a 1.7-fold increase in ABCG2 protein after low-dose doxorubicin treatment. Control Control Doxorubicin Doxorubicin/TrypA Doxorubicin Doxorubicin/TrypA 8226 8226MR Ctrl Dox Ctrl Dox ABCG2 ABCG2

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71 71 Figure 3.4. ABCG2 expression is elevated in log phase myeloma cells (A) Flow cytometric data using an ABCG2 antibody (Chemicon) demonstrate that ABCG2 expressing 8226 cells have increased ABCG2 at log-phase density compared with log-phase H929 cells. (B-C) T he FACScan data are confirmed by immunostaining for ABCG2 (B), and by QPCR and Western analyses (C). Densitometry analysis of the immunoblot shows a 4:1 ratio of log:plateau ABCG2 in 8226 cells, and a 2:1 ratio of log:plat eau 8226MR ABCG2. Note, 8226 and 8226MR Western blots were exposed for different time intervals and do not reflect relative protein levels.

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72 72 Effect of the microenvironment and t opoisomerase inhibitors on ABCG2 expression We also examined the expression of ABCG2 as a function of cell density (Figure 3.4), and found that log-phas e (low density) 8226 and 8226MR cells express significantly more ABCG2 than plateau-phase (high density) cells, as shown by flow cytometry (Figure 3.4A), immunofluorescence microscopy (Figur e 3.4B), QPCR (Figure 3.4C), and Western blot analysis (Figure 3.4C). Changes in cell density failed to induce ABCG2 protein expression in H929 cells (Figure 3.4A). 8226MR and 8226 cells were also found to increase the expression of ABCG2 in response to low dose topotec an exposure (Figure 3.5). This was shown by immunofluorescence microsc opy (Figure 3.5A-B), Western blot analysis (Figure 3.5C inset), and protein expression measured as pixel intensity from immunofluorescence (Figure 3.5C). In addition, ABCG2 protein expression was measured by flow cytometry using the membrane epitope-specific antibody Bcrp1-PE (Chemicon), and showed an increa se in ABCG2 protein in log-phase 8226MR and 8226 cells after exposure to lo w dose topotecan (1 M) for 20 hours (Figure 3.5D). The low ABCG2 expr essing myeloma cell line H929 had no ABCG2 antibody binding (Figure 3.5D).

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73 73 Figure 3.5. ABCG2 expression incr eases in response to topotecan chemotherapy. (A-B) Multiple myelom a 8226MR, 8226, and H929 cells treated with low dose topotecan (B) exhibit an in crease in membrane ABCG2 over the no drug control (A), as shown by imm unostaining with ABCG2 antibody (Bcrp1 FITC). (C) Protein expression of ABCG2 was quantified as pixel intensity (from immunofluorescence microscopy), and also assessed by Western analysis (inset of each graph). (D) ABCG2 expression was measured by flow cytometry and showed an increase in log-phase 8226MR a nd 8226 cells after exposure to low dose topotecan (1 M) for 24 hours. T he ABCG2 non-expressing cell line (H929) shows no increase in ABCG2 antibody binding

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74 74 Figure 3.6. ABCG2 increases in pat ient plasma cells after high-dose chemotherapy and at relapse (A-B) A patient bone marrow aspirate taken before (A) and during (B) HDC with melphalan and topotecan exhibited an increase in immunofluoresc ence of ABCG2 (green). (C) This same patient demonstrated an increase in ABCG2 ex pression by immunofluorescence at relapse as well. (D) ABCG2 protein expression pre-HDC and at relapse by Western blot in four patients on the MT V study. Laser densitometry analysis of the immunoblots shows a 1. 54and 1.94-fold increase in ABCG2 during HDC for patients A and B, respectively, and a 3.68and 1.34-fold increase in ABCG2 at relapse for patients C and D, respectively. In all cases this is relative to the preHDC ABCG2 protein expression.

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75 75 8226 parental and 8226MR cells treat ed with low dose topotecan exhibit an increase in membrane ABCG2 compared to the no drug control, as shown by immunostaining with ABCG2 antibody (MXB-21) (Figure 3.5A-B). 8226MR and 8226 parental ce ll cultures treated with low dose doxorubicin for 20 hours also demonstrated an increase in ABCG2 expression by Western blot analysis (insets figure 3.3).As was seen at the mRNA level (Table 3.2), patient bone marrow aspirates taken before, during, and after HDC with melphalan and topotecan showed changes in ABCG2 expression (Figure 3.6). A limited number of patient sa mples from the MTV trial were available for these Western and immunofluorescence analyses. The same patient sample from dose level 5 showed increased ABCG2 expression after three days of exposure in vivo to topotecan (Figure 3.6B), as well as at relapse (Figure 3.6C). Four different patient samples analyzed by i mmunoblotting (from dose levels 4 and 5 of the MTV trial) also demonstrated an in crease in ABCG2 protein expression after three days of topotecan or at rel apse from high-dose chemotherapy (Figure 3.6D). ABCG2 promoter methylation The methylation status of a prev iously described CpG island in the ABCG2 promoter was exami ned via bisulfite sequencing. Figure 3.7 shows the CpG dinucleotides that were methylated in four cell lines tested. The promoter

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76 76 Figure 3.7. ABCG2 promoter methylation. (A) Cells were harvested at plateauphase and the DNA extracted and assay ed by bisulfite DNA sequencing analysis. The figure shows the methylati on status of the putative CpG island of the ABCG2 promoter in four cell lines. Filled circles represent methylated groups and the open circles demethylated CpG. (B) H929/5aza are cells treated with 100 nM 5-aza-2'-deoxycytidine for 72 hour s and the ABCG2 promoter assayed by bisulfite sequencing. 5-Aza-2'-deo xycytidine was able to augment ABCG2 transcription in low ABCG2 expressing H9 29 cells but had no effect in moderate expressing 8226 cells. (C, D) CD-138 selected MM patient samples were assayed for ABCG2 promoter methylation after bisulfite conversion using realtime quantitative PCR. The percentage of alleles that were methylated inversely correlated with ABCG2 mRNA expression.

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77 77 region of ABCG2 over-expressing cells, 8226MR, was completely unmethylated, whereas the H929 cells, which express very little ABCG2, had 13 methylated CpG dinucleotide groups (Figure 3.7A). We found that protein, mRNA, and topotecan efflux function in live cells correlated with the methylation of CpG dinucleotides in the ABCG2 promoter (Figure 3.1). Th erefore it is likely that ABCG2 expression is controlled in part by methylation of its promoter. We also tried to increase ABCG2 expre ssion by treating low-expressing H929 cells with 100 nM 5-aza-2'-deoxycytidi ne. This agent has been shown to increase gene expression by inhibiting DNA-methyltransferase I, thereby decreasing epigenetic methylation of DNA. After treatment of cells for 72 hours, we found that ABCG2 mRNA and protein in creased approximately six-fold in low expressing H929 cells, whereas m oderately expressing 8226 cells were unaffected (Figure 3.7B). Bisulfite sequenc ing of 5-aza-2’-deo xycytidine treated H929 DNA showed that the ABCG2 pr omoter CpG island was fully demethylated (Figure 3.7A). Methylation-specific quantitative PCR of patient myeloma cells The percentage of ABCG2 promoter alleles that were methylated was assayed using SYBR green based real-t ime quantitative PCR. These eight patient samples were all obtained preHDC from the MTV tria l. The data are expressed in Figures 3.7C and 3.7D as the percentage of alleles methylated for

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78 78 each patient sample compared to ABCG2 mRNA levels assayed by fluorescent probe based real-time quant itative PCR (as described previously in the methods). Figure 3.7C and 3.7D show that ABCG2 m RNA expression correlates inversely with the percentage of alleles methylated. High levels of methylated alleles resulted in a decrease in ABCG2 mRNA transcripts, whereas patient samples with low levels of methylat ed alleles had much higher amounts of ABCG2 mRNA. These data, along with cell culture methylation data, indicate that promoter methylation may contribute to the control of ABCG2 expression in human myeloma cells. DISCUSSION We have found that ABCG2 is pres ent and functional in human myeloma cell lines and patient plasma cells. Using QPCR, protein assays, and functional efflux of chemotherapeutic drugs, we have found that ABCG2 is potentially involved in drug resistance to spec ific agents in human myeloma cells. The principal findings of our in vitro experiments are that (i) human multiple myeloma cells lines have a wide range of ABCG2 expression and function, (ii) low density 8226 cells expr ess more ABCG2 than high-density cells, (iii) human myeloma cell lines with moderat e or high baseline levels of ABCG2 expression further increase this ex pression upon exposure to the ABCG2 substrates topotecan and doxorubicin, and (iv) methyl ation of the CpG island of the ABCG2 promoter correlates in versely with ABCG2 expression. In vivo we

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79 79 found that myeloma patients treated with topotecan had an increase in ABCG2 mRNA expression, both after three days of topotecan exposure an d at the time of relapse. In addition, we found that a CpG island in the ABCG2 promoter is heavily methylated in cells that do not ex press ABCG2. This promoter region was completely demethylated in cells that expressed high to intermediate levels of ABCG2. Therefore, expr ession of ABCG2 was regulat ed, at least in part, by promoter methylation, bot h in cell lines and in patient plasma cells. The ability to up-regulate ABCG2 in response to chemotherapy could confer a selective survival advantage to malignant plasma cells. Plasma cells are derived from hematologi cal stem cells that have demonstrated an intrinsic ability to produce ABCG2, and therefore, myeloma cells may come by this ability naturally (Kim et al., 2002; Scharenber g et al., 2002; Zhou et al., 2001b). However, clonal selection may play a par t in the further development of ABCG2 expression in myeloma. Rapidly gr owing log-phase myeloma cells also increased ABCG2 expression in vitro Increased drug resistance conferred to rapidly growing cells could possibly produc e an additive result with the unlimited replicative potential of c ancer cells, one of the “ha llmarks of cancer" (Hanahan and Weinberg, 2000). ABCG2 may contribut e to intrinsic drug resistance in myeloma, and its effect is likely in creased by exposure to chemotherapeutic drugs that are substr ates for ABCG2. In general, we observed the same result s in the limited number of patient plasma cells available from the HDC MT V trial. Patient cells exposed to

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80 80 topotecan in vivo had increased ABCG2 expression, as did plasma cells from relapse bone marrow aspirates. In additi on, patient plasma cells with increased CpG island methylation of the ABCG2 promoter had decreased ABCG2 mRNA expression. In studies performed in another hematological malignancy, AML, patients demonstrated significantly increas ed expression of ABCG2 mRNA in the relapsed/refractory samples compared to pre-treatment (van den Heuvel-Eibrink et al., 2002). AML patients with highlevels of ABCG2 expression had significantly shorter overall survival rates (Uggla et al., 2005) and decreased ABCG2 expression predicted a complete remission of AML in adult patients (Benderra et al., 2004). In a recent study, Raaijmakers et al (Raaijmakers et al., 2005), isolated plasma cells from normal bone marro w donors and from ten patients with myeloma prior to treatment with VAD chemotherapy using flow cytometry and an anti-CD38 antibody. They observed that ABCG2 expression was relatively high in both normal and malignant plasma cells, but that ABCG2 mediated efflux of mitoxantrone was significantly impaired in the malignant plasma cells. These results differ from ours, however, our func tional assay was limited to only a high and a low expressor of ABCG2 (Figure 3.2) was not compared to drug efflux in normal plasma cells, and the plasma cells were isolated using an anti-CD138 antibody and immunomagnetic beads from patients previously treated with chemotherapy. Thus, the two studies ma y be comparing different populations of plasma cells.

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81 81 In previous studies, ABCG2 over-expression has been observed in drug resistant cell lines (Allen et al., 1999; Candeil et al., 2004; Honjo et al., 2002; Knutsen et al., 2000; Litman et al., 2000; Miyake et al., 1999; Robey et al., 2001; Ross et al., 1999). This over-expression of ABCG2, in the majority of cases, has been attributed to heavy amplification of the gene locus (Allen et al., 1999; Knutsen et al., 2000; Miyake et al., 1999). Also, significant increases in function have been found to occur due to specific mutations, but without a concurrent increase in gene transcription or translati on (Honjo et al., 2001; Honjo et al., 2002). Analysis of a putative ABCG2 pr omoter region presents a TATA-less promoter with several putative transcripti on factor binding sites. In addition, the promoter has an estrogen response elem ent, all of which may contribute to increased gene expression levels (BaileyDell et al., 2001; Ee et al., 2004). It has been reported that the ABCG2 promot er contains a potential CpG island, which may regulate expression by methylation (Bailey-Dell et al., 2001). The MDR1 promoter has a simila r CpG island. In a recent publication, it was found that hypermethylation of CpG dinucleot ides in the MDR1 promoter region strongly contributed to differences in gene expression in related cell lines (David et al., 2004). In our study, we examined the methylation status of the ABCG2 promoter region in cell lines that differ ed in their respective ABCG2 expression. We found that the promoter of very low level expressing cells was almost completely methylated, whereas high and medium ABCG2 expressers were either completely or almost completely unmethylated. Analysis of the ABCG2

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82 82 promoter via bisulfite s equencing showed that methylation occurred precisely at the putative CpG island as described by Ba iley-Dell, et al (Bailey-Dell et al., 2001). In addition, when low ABCG2 produc ing H929 cells were exposed to the de-methylating agent, 5-aza-2'-deoxycytidine, the cells were induced to express ABCG2 mRNA and protein (Figure 3.7B). Methylation was also shown to be important in human myeloma patient samples. The percentage of methylated alleles inversely correlated with ABCG2 mRNA expression (Figure 3.7C and 3.7D). Therefore, our data suggest that pr omoter methylation contributes to gene expression of ABCG2. In summary, our data suggest ABCG2 ma y be involved in the resistance of human myeloma cells, both in vitro and in vivo to chemotherapeutic agents that are substrates of ABCG2. Do xorubicin, VP-16 an d topotecan are all substrates of ABCG2. Doxorubicin is commonly used in the treatment of myeloma (vincristine + adriamycin + decadron, or VAD regimen). In this study we found that doxorubicin is actively effluxed by ABCG2 in vitro Single agent topotecan has been shown to have activity in relapsed and refractory multiple myeloma patients in a SWOG trial (Kraut et al., 1998). The overall response rate in these highly pre-treated patients was 16%. We have recently combined melphalan and VP-16 phosphate wit h dose-escalated topotecan in a phase I/II high-dose chemotherapy trial in high risk my eloma (Harker et al., 1995; Valkov et al., 2000a). and found this to be an active and tolerable regimen. Future trials that incorporate ABCG2 transport inhibi tors, such as GF120918, may increase

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83 83 the efficacy of topoisomerase I inhibitors in this di sease (Jonker et al., 2000; Maliepaard et al., 2001b).

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84 84 DISSERTATION SUMMARY Multiple myeloma is an incurable malignancy that kills approximately 17,000 individuals yearly in the United Stat es. In this dissertation we have shown that myeloma is intrinsically resistant to commonly used topoisomerase inhibitors by microenvironmental factors that induce nuclear export of topo II In addition, during disease progression myeloma acquire s additional multi-drug resistance by overexpression of the molecular transpor ter ABCG2. Topoisomerases are critical for cell division, especially in rapidly divi ding cells such as are found in cancer. Topoisomerases are an excelle nt drug target to treat c ancer, however, in order for topoisomerase drugs to be effective, the enzyme must be in direct contact with the DNA. Myeloma is intrinsically resistant to topoisomerase drugs via a mechanism whereby the drug target, topo II is exported. We found th at this mechanism is present at cell densities similar to thos e found in the bone marrow. High-density cells were found to be greater than 10-fo ld more drug resistant than low-density cells. Nuclear export could be blocked using a CRM1 inhibitor ratjadone C, CRM1 specific siRNA or a casein kinase II specific inhibitor. Blocking nuclear export was found to sensitize high-dens ity cells to topoisomerase drugs. Sensitization to topoisomerase inhi bitors was correlated with increased topoisomerase/DNA complexes and incr eased DNA strand breaks. This method

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85 85 of sensitizing human myeloma cells sugges ts a new therapeutic approach to this disease. We also examined acquired drug re sistance mediated by the molecular transporter ABCG2 in multiple myeloma. We found that ABCG2 expression in myeloma cell lines increased in respons e to treatment wit h topotecan or doxorubicin. In patient studies we found t hat after treatment with topotecan, and at relapse, patients had an increase in ABCG2 mRNA and protein expression. Increased protein expression correlated wit h decreased drug uptake in functional assays. We found that expression of ABC G2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, af fected by cell density, upregulated in response to chemotherapy, and may contribute to acquired drug resistance. The potential exists to utilize this effect with drug combinations which include a ABCG2 inhibitory agent.

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About the Author Joel Turner received a B.S. degree in Human Biology from the University of Wisconsin in 1983 and a M.S. in Mo lecular and Cellular Biology from the University of South Florida in 2004. He officially entered the Ph.D. program at the University of South Florida College of Arts and Sciences Biology program in the spring of 2005 and was admitted to c andidacy for doctoral degree August 9, 2007. Mr. Turner started worki ng as a laboratory technician at the age of 17 as a college freshman and since has continued in medical research for 28 years. Mr. Turner has been a lab technician and coordinat or at the H. Moff itt Cancer Center and Research Institute for eleven years. He has accumulated 40 peer-reviewed scientific publications and is a full-member of the American Association for Cancer Research. In addition, he has taught undergraduat e biology, anatomy and physiology during evening classes as an adjunct faculty at both Hillsborough Community College and the USF College of Public Health for the past four years.