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The Rad9-Rad1-Hus1 complex and Bif-1 regulate multiple mechanisms that affect sensitivity to DNA damage

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Title:
The Rad9-Rad1-Hus1 complex and Bif-1 regulate multiple mechanisms that affect sensitivity to DNA damage
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English
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Meyerkord, Cheryl L
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University of South Florida
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Apoptosis   ( mesh )
Autophagy   ( mesh )
Endocytosis   ( mesh )
Cell Cycle Proteins   ( mesh )
Adaptor Proteins, Signal Transducing   ( mesh )
Membrane Proteins   ( mesh )
Programmed cell death
Apoptosis
Autophagy
Endocytosis
BH3-only proteins
Dissertations, Academic -- Cancer Biology -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
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Includes bibliographical references.
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by Cheryl L. Meyerkord.
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Document formatted into pages; contains 149 pages.
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Includes vita.

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oclc - 642713755
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The Rad9-Rad1-Hus1 Complex and Bif-1 Regul ate Multiple Mechanisms that Affect Sensitivity to DNA Damage by Cheryl L. Meyerkord A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cancer Biology College of Graduate School University of South Florida Major Professor: Hong-Gang Wang, Ph.D. Srikumar Chellappan, Ph.D. W. Douglas Cress, Ph.D. Lori Hazlehurst, Ph.D. Date of Approval: February 9, 2009 Keywords: Programmed cell death, Apopt osis, Autophagy, Endocytosis, BH3-only proteins Copyright 2009, Cheryl L. Meyerkord

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Dedication I would like to dedicate this dissertation to my brave mother and everyone who has battled cancer, whether they won or lost. It is for them that we get up and go to work each day. They are the driving force behind our motivation to some day find a cure.

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Acknowledgments First and foremost, I express my deepes t gratitude to my mentor, Dr. Hong-Gang Wang, for all of his patience, help and guidan ce. I am fortunate to have had him as a mentor. I am very grateful for having an ex traordinary committee and would like to thank Dr. W. Douglas Cress, Dr. Srikumar Chellappan and Dr. Lori Hazlehurst for all of their support, direction and construc tive criticism. I am deeply hon ored to have Dr. Robert Weiss of Cornell University as the outside chairperson for my dissertation committee. I extend many thanks to all of the memb ers of the Wang laboratory, both past and present, who have contributed to my traini ng and provided a work environment that was conducive to my growth as a scientist. I w ould especially like to thank Dr. Yoshinori Takahashi for always being there for me dur ing the hard times and helping me keep things in perspective. He has not only taught me a lot about science, but also about life. I deeply appreciate the friends that I have made while at Moffitt, especially Daniele Gilkes, Amy Hazen and Rachel Havi land who have supported me throughout my Ph.D. I would especially like to thank them for treating me like family and always being there for me when I needed someone. I woul d also like to thank Patricia Massard and Cathy Gaffney for all of their help and support. Importantly, I would like to thank my fa mily who were always there to support me, no matter what struggle or obstacle they were deali ng with in their own lives. Without their love and support, I never would have made it this far.

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This work was funded by a predoctoral traineeship from the Department of Defense Breast Cancer Res earch Program (BC050563).

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Note to the Reader The original of this document contains colo r that is necessary fo r understanding the data. The original dissertation is on file with the USF library in Tampa, Florida.

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i Table of Contents List of Figures................................................................................................................ ....iv List of Abbreviations.......................................................................................................vii i Abstract....................................................................................................................... .........x Introduction................................................................................................................... .......1 Cancer......................................................................................................................1 The DNA Damage Response...................................................................................2 The Rad9-Rad1-Hus1 Complex...............................................................................5 Structure and Role in the DNA Damage Response.....................................6 Regulation of DNA Repair..........................................................................8 Regulation of Additiona l Cellular Processes...............................................9 Defects Resulting from Loss of a Functional 9-1-1 Complex...................10 Programmed Cell Death.........................................................................................11 Apoptotic Cell Death.................................................................................11 The extrinsic pathway....................................................................13 The intrinsic pathway.....................................................................14 DNA Damage-Induced Programmed Cell Death......................................16 Autophagic Cell Death...............................................................................17 Crosstalk between Apoptotic and Autophagic Cell Death........................18 Autophagy..............................................................................................................20 Mechanisms that Regulate Autophagy......................................................21 ATG genes.....................................................................................22 Bif-1...............................................................................................25 Deregulation of Autophagy in Cancer.......................................................26 Crosstalk between the Autopha gic and Endocytic Pathways................................28 Endocytosis and Vesicle Trafficking.....................................................................30 EGFR as a Model for Endocytic Trafficking and Degradation.................31 Deregulation of Endoc ytosis in Cancer.....................................................34 Summary................................................................................................................34 Loss of Hus1 Sensitizes Cells to EtoposideInduced Apoptosis by Regulating BH3-Only Proteins.............................................................................................................3 6 Abstract..................................................................................................................36 Results....................................................................................................................37 Loss of Hus1 Sensitizes Cells to Eto poside-Induced Apoptosis...............37

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ii Loss of Hus1 Enhances Bim and Puma Expression at Both the Protein and mRNA Level in Response to DNA Damage..........................39 Knockdown of Bim and Puma Conf ers Resistance to EtoposideInduced Apoptosis in Hus1 -Deficient Cells..............................................51 Loss of Hus1 Enhances the Binding of Rad9 to Bcl-2 to Potentiate the Apoptotic Response.............................................................................55 Discussion..............................................................................................................64 Materials and Methods...........................................................................................68 Reagents.....................................................................................................68 Cell Culture, Transfec tion and Infection...................................................68 Analysis of Cell Death and Apoptosis.......................................................69 Semi-Quantitative Reverse Transcription-PCR.........................................70 Chromatin Fractionation............................................................................70 Subcellular Fractionation and Coimmunoprecipitation.............................71 Immunofluorescence..................................................................................72 Loss of Hus1 Enhances DNA Damage-Induced Ca spase-Independent Cell Death and Autophagy.................................................................................................................. .73 Abstract..................................................................................................................73 Results....................................................................................................................74 Loss of Hus1 Enhances Caspase-Independent Cell Death in Response to DNA Damage........................................................................74 Loss of Hus1 Enhances DNA Damage-Induced Autophagy.....................76 Autophagy Plays a Cytoprotective Role in Response to DNA Damage......................................................................................................79 The BH3 Mimetic, ABT-737, Does Not Significantly Induce Autophagy..................................................................................................83 Discussion..............................................................................................................86 Materials and Methods...........................................................................................89 Reagents.....................................................................................................89 Cell Culture, Transfec tion and Infection...................................................89 Analysis of Cell Death and Apoptosis.......................................................90 Analysis of LC3 Localization....................................................................90 Analysis of LC3 Modification...................................................................91 Knockdown of Bif-1 Accelerates Endocytic Vesicle Trafficking and Enhances EGFR Degradation.............................................................................................................92 Abstract..................................................................................................................92 Results....................................................................................................................93 Knockdown of Bif-1Does Not Affect Internalization of Endocytic Cargo, but Accelerates EGF Co -Localization with Late Endosomes/Lysosomes..............................................................................93 Knockdown of Bif-1 Promotes EGFR Degradation..................................96 EGFR Degradation is Mediated through Both Proteasomal and Lysosomal Mechanisms...........................................................................100

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iii Discussion............................................................................................................103 Methods and Materials.........................................................................................107 Reagents...................................................................................................107 Cell Culture, Transfec tion and Infection.................................................107 Endocytosis of HRP.................................................................................108 EGFR Endocytosis and Degradation.......................................................108 Scientific Significance.....................................................................................................110 References Cited..............................................................................................................1 21 About the Author...................................................................................................End Page

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iv List of Figures Figure 1. Simplified schematic of the DNA damage response..........................................5 Figure 2. Comparison of the Rad9-Rad1-Hus1 complex and PCNA................................7 Figure 3. Overview of the extrinsi c and intrinsic apoptotic pathways............................13 Figure 4. Model of the mechan isms that regulate autophagy..........................................22 Figure 5. Convergence of the autophagic and endocytic pathways for lysosomal degradation.................................................................................................................... .....30 Figure 6. Endcoytic trafficking of the EGFR...................................................................32 Figure 7. Loss of Hus1 sensitizes cells to et oposide-induced cell death.........................37 Figure 8. Loss of Hus1 enhances the cleavage of caspase-3 and PARP..........................38 Figure 9. Loss of Hus1 sensitizes cells to et oposide-induced apoptosis..........................39 Figure 10. Expression of Bcl-2 family members in response to etoposide treatment...................................................................................................................... ......40 Figure 11. Loss of Hus1 results in upregulation of Bim and Puma expression in response to camptothecin treatment...................................................................................41 Figure 12. Loss of Hus1 results in upregulation of Bim and Puma expression in response to hydroxyurea treatment....................................................................................41 Figure 13. Restoration of Hus1 suppresses the upregulation of Bim and Puma in response to DNA damage..................................................................................................42 Figure 14. Induction of Bim and Puma e xpression in response to etoposide treatment is regulated at the transcriptional level..............................................................44 Figure 15. Induction of Bim expression in response to etoposide treatment is regulated at the mRNA level..............................................................................................45

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v Figure 16. Induction of Puma expression in response to etopos ide treatment is regulated at the mRNA level..............................................................................................46 Figure 17. Etoposide-induced upregulation of Bim expression occurs at the transcriptional level.......................................................................................................... ..46 Figure 18. Etoposide-induced upregulation of Puma expression occurs at the transcriptional level.......................................................................................................... ..47 Figure 19. Loss of p53 suppresses DNA damage-induced Puma expression....................48 Figure 20. Loss of p53 does not affect etoposid e-induced cell death................................49 Figure 21. Loss of p53 does not affect etoposide-induced apoptosis................................49 Figure 22. FoxO3a is not responsible fo r the upregulation of Bim and Puma expression in response to etoposide treatment...................................................................50 Figure 23. E2F1 is not responsible fo r the upregulation of Bim and Puma expression in response to etoposide treatment...................................................................50 Figure 24. Knockdown of Bim expressi on suppresses PARP cleavage in Hus1 deficient cells................................................................................................................ .....53 Figure 25. Knockdown of Bim expression conf ers resistance to etoposide-induced cell death in Hus1 -deficient cells.......................................................................................53 Figure 26. Knockdown of Bim and Puma expression suppresses etoposideinduced caspase-3 cleavage in Hus1 -deficient cells..........................................................54 Figure 27. Knockdown of Bim and Puma expression confers resistance to etoposide-induced apoptosis in Hus1 -deficient cells.........................................................55 Figure 28. Loss of Hus1 results in a defect in the binding of Rad9 to chromatin.............56 Figure 29. Rad9 is predominantly detect ed in the cytosolic fraction of Hus1 deficient cells................................................................................................................ .....57 Figure 30. Rad9 is predominantly located in the cytosol of Hus1 -deficient cells.............58 Figure 31. Binding of cytosolic Rad9 to Bc l-2 is increased up on treatment with etoposide and enhanced by loss of Hus1 ...........................................................................60 Figure 32. Etoposide-induced binding of Ra d9 to Bcl-2 is enhanced by loss of Hus1 ............................................................................................................................... ....61

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vi Figure 33. Knockdown of Rad9 further suppr esses caspase-3 ac tivation in shBim and shPuma expressing cells..............................................................................................62 Figure 34. Rad9 collaborates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-induced apoptosis..........................................................................................63 Figure 35. Rad9 collaborates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-induced cell death..........................................................................................63 Figure 36. Proposed model for the role of the Rad9-Rad1-Hus1 complex in the regulation of DNA damage-induced apoptosis..................................................................67 Figure 37. Camptothecin-induced cell deat h is moderately inhibited by Z-VADFMK............................................................................................................................ .......75 Figure 38. Camptothecin-induced caspase -3 activity is abrogated by Z-VADFMK............................................................................................................................ .......75 Figure 39. Loss of Hus1 enhances DNA damage-induced GFP-LC3 foci formation...................................................................................................................... ......78 Figure 40. DNA damage-induced LC3 modification is enhanced by loss of Hus1 ...........79 Figure 41. Knockdown of Atg7 or Bif1 suppresses LC3 modification............................81 Figure 42. Knockdown of Atg7 or Bif1 suppresses DNA damage-induced LC3 foci formation................................................................................................................. ....82 Figure 43. Inhibition of autophagy results in enhanced cell death in response to camptothecin treatment......................................................................................................83 Figure 44. ABT-737 does not significantly enhance GFP-LC3 foci formation................85 Figure 45. Knockdown of Bif-1 does not affect the internalizati on of a fluid phase marker......................................................................................................................... .......93 Figure 46. Knockdown of Bif-1 accelerates the co-localizatio n of EGF with LAMP-1-positive vesicles.................................................................................................95 Figure 47. Knockdown of Atg5 does not affect EGF localization....................................96 Figure 48. Knockdown of Bif-1 e nhances EGFR degradation..........................................97 Figure 49. Quantification of total EGFR levels and degradation......................................98

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vii Figure 50. Quantification of phosphorylated EGFR levels and degradation.....................99 Figure 51. Knockdown of Atg5 does not affect EGFR degradation................................100 Figure 52. The lysosomal and proteasomal pathways regulate EGFR degradation........101 Figure 53. The lysosomal and proteasomal pathways collaborate to regulate EGFR degradation...........................................................................................................102 Figure 54. Proposed model for the role of the Rad9-Rad1-Hus1 complex and Bif1 in the regulation of se nsitivity to DNA damage...........................................................120

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viii List of Abbreviations 9-1-1 complex: the complex composed of Rad9, Rad1 and Hus1 ATG: autophagy-related ATM: ataxia-telangiectasia mutated ATR: ATMand Rad3-related BAR: Bin-Amphiphysin-Rvs Bcl-2: B-cell lymphoma 2 BER: base excision repair BH: Bcl-2 homology domain Bif-1: Bax-interacting factor-1 Bim: Bcl-2 interacting mediator of cell death BRCA1: breast cancer gene 1 or breast a nd ovarian cancer sus ceptibility gene 1 BSA: bovine serum albumin Cdc25: cell division cycle 25 cdk: cyclin dependent kinase Chk: checkpoint kinase DAPI: 4, 6-diamidino-2-phenylindole DDR: DNA damage response DNA: deoxyribonucleic acid DMEM: Dulbecco's modified Eagle's medium DMSO: dimethylsulfoxide EGF: epidermal growth factor EGFR: epidermal grow th factor receptor GAPDH: glyceraldehyde3-phosphate dehydrogenase GFP: green fluorescent protein HEPES: N-2-hydroxyethylpiperazi ne-N'-2-ethanesulfonic acid HRP: horseradish peroxidase HRR: homologous recombination repair Hus1: hydroxyurea sensitive 1 HU: hydroxyurea IR: ionizing radiation LAMP-1: lysosome-associated membrane protein-1 LC3: microtubule-associated protein light chain 3 MDC1: mediator of DN A damage checkpoint 1 MEF: mouse embryonic fibroblast MOMP: mitochondrial outer membrane permeablization MRN: MRE11-Rad50-Nbs1 mRNA: messenger ribonucleic acid

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ix MVB: multivesicular body Nbs1: Nijmegen breakage syndrome NGF: nerve growth factor PARP: poly(ADP-ribose) polymerase PAS: phagophore assembly sites PCNA: proliferating cell nuclear antigen PE: phosphatidylethanolamine PBS: phosphate buffered saline PCR: polymerase chain reaction PI3K: phosphatidyli nositol 3-kinase PI3KC3: phosphatidylinosito l 3-kinase class III Puma: p53 upregulated m odulator of apoptosis Rad: radiation sensitive RIPA: radioimmunopreci pitation assay buffer RT-PCR: reverse transcription-polymerase chain reaction SDS-PAGE: sodium dodecyl sulfat e-polyacrylamide gel electrophoresis SH3: Src-homology 3 shRNA: short hairpin RNA siRNA: short interfering RNA Tor: target of rapamycin TrkA: tropomyosin-related kinase A TUNEL: Terminal deoxynucleotidyl transf erase-mediated dUTP nick end labeling UV: ultraviolet radiation UVRAG: ultraviolet irradiati on resistance-associated gene Vps: vacuolar protein sorting

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x The Rad9-Rad1-Hus1 Complex and Bif-1 Regul ate Multiple Mechanisms that Affect Sensitivity to DNA Damage Cheryl L. Meyerkord ABSTRACT The resistance of cancer cells to traditi onal chemotherapeutic agents is a major obstacle in the successful treatment of can cer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperac tivating survival signaling pathways. In an attempt to better understa nd the molecular mechanisms th at underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-11 complex plays an integral role in the response to DNA damage and regulate s many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigeni city. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1 sensitizes cells to DNA damaging agents by upregulating BH3-only prot ein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enha nces the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also pr ovide evidence that di sruption of the 9-1-1 complex sensitizes cells to caspase-indepe ndent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As

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xi autophagy has been implicated in caspase-inde pendent cell death, thes e data suggest that the enhanced autophagy observed in Hus1 -knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif1, did not suppress, but rather prom oted DNA damage-induced cell death in Hus1 deficient cells, suggesting that in apoptosiscompetent cells autophagy may be induced as a cytoprotective mechanism. The aberrant ac tivation of survival signals, such as enhanced EGFR signaling, is another mech anism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the colocalization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results sugge st that Bif-1 may enhance su rvival not only by inducing autophagy, but also by regulati ng EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

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1 Chapter One: Introduction Cancer Despite major advances in the treatment of cancer, this diseas e is still the second leading cause of death in the United States (Jemal et al. 2008). It is es timated that in 2008 over 1.4 million people were diagnosed with cancer and over 0.5 million people died from this disease. Cancer is caused by both internal factors, such as inherited mutations, and external factors, such as exposure to DNA damaging agents (American Cancer Society, 2008). Tumorigenesis is a multistep process that is characterized by the accumulation of genetic mutations that transf orm normal cells into malignant derivatives (Hanahan and Weinberg, 2000). These mutati ons can activate oncogenes and inactivate tumor suppressor genes resulting in genomic instability and driving tumor progression. Paradoxically, DNA-damaging agents are some of the most effective drugs used for the treatment cancer. In addition, the efficacy of DNA damage-based chemotherapy may be influenced by the ability of a ce ll to repair damaged DNA (Helleday et al. 2008). Therefore, deciphering the cel lular mechanisms that are activated in response to DNA damage may not only lead to a better understand ing of the causes of cancer, but also to better, more effective treatment strategies.

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2 The DNA Damage Response The genomes of eukaryotic cells are c onstantly being subjected to endogenous and exogenous genotoxic stresses. Damage re sulting from exposure to such stresses threatens cell survival and can lead to cancer, as well as other genetic diseases. In order to preserve genomic integrity and ensure that an accurate copy of the genome is passed on to subsequent generations, cells have evolve d a core surveillance machinery that senses damaged or abnormally structured DNA a nd coordinates cell cycle progression with DNA repair. In cases when damage is excessive or repair is unfa vorable, the cell death machinery is activated in order to elimin ate damaged cells (Melo and Toczyski, 2002; Niida and Nakanishi, 2006; Zhou and Elledge, 2000). In response to genotoxic stress, a comp lex network of interacting checkpoint signaling pathways act in concert to execu te an appropriate DNA damage response (DDR) (Harper and Elledge, 2007). In mamma lian cells, two relate d phosphatidylinositol 3-kinase-related serine /threonine kinases play a central role in the regu lation of the DDR (Abraham, 2001; Matsuoka et al. 2007). The ATM (ataxiatelangiectasia mutated)mediated pathway is activated in respons e to DNA damaging agents that induce doublestrand breaks (Lavin, 2008), whereas the ATR (ATMand Rad3-related)-dependent pathway responds to a broad spectrum of ge notoxic stresses including those that inhibit replication and induce singl e-strand DNA breaks or bulky DNA lesions (Cimprich and Cortez, 2008). Mutations in ATM lead to the autosomal recessive disorder ataxia-telangiectasia, which is characterized by immunodeficien cy, radiosensitivity, neurodegeneration and cancer predisposition (Boder, 1985). Individu als, mice and cells that lack ATM are

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3 viable, which indicates that ATM is not re quired for differentiation, normal cell cycle progression or other essentia l cellular functions (Shiloh and Kastan, 2001). Under normal conditions, ATM exists as an inactive hom odimer. In response to DNA damage, ATM undergoes a conformational change, which stim ulates its kinase act ivity. ATM is then autophosphorylated at serine 1981 leading to the dissociation of inactive homodimers to form active monomers (Bakkenist and Ka stan, 2003). DNA damage also induces the association of ATM with the MRE11-Rad50Nbs1 (MRN) complex, which acts as an adaptor for the recruitment of downstream signaling proteins and facilitates the full activation and proper local ization of ATM (Berkovich et al. 2007; van den Bosch et al. 2003). ATM-mediated phosphorylation of H2AX ( H2AX) recruits MDC1, which acts as a positive feedback loop to facilitate further ATM phosphor ylation of H2AX and the recruitment of additional ATM-MRN complexe s, thereby propagating the DDR (Stucki and Jackson, 2006). In contrast to ATM, ATR is required for viability (Brown and Baltimore, 2000); however, hypomorphic mutations of ATR ar e associated with Seckel syndrome (O'Driscoll et al. 2003). ATR is constitutively bound to ATRIP (ATR-interacting protein) even in the absence of DNA damage or replicative stress (Cortez et al. 2001). ATRIP binds to the single-stranded DNA-coa ting protein, RPA (rep lication protein A), which facilitates the recru itment of ATR to DNA and th e activation of downstream signaling (Zou and Elledge, 2003). However, recruitment of ATR to the site of DNA damage is not sufficient to activate ATR signa ling, several other protei ns must be present in order for ATR to execute all of its ce llular functions. Rad17, the Rad9-Rad1-Hus1 (91-1) complex, TopBP1 (topoisomerase-binding pr otein-1) and Claspin are all required for

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4 the full activation of ATR-mediated dow nstream signaling (Chini and Chen, 2003; Delacroix et al. 2007; Kumagai et al. 2006; Weiss et al. 2002; Zou et al. 2002). The loading of the 9-1-1 complex onto the DNA resu lts in the recruitment of TopBP1 through its interaction with Rad9 (Delacroix et al. 2007; Lee et al. 2007). TopBP1 then binds to the ATR-ATRIP complex and enhances the kinase activity of ATR (Kumagai et al. 2006; Mordes et al. 2008). Upon activation, ATM and ATR are res ponsible for relaying the DNA damage signal to downstream transducer and effector proteins (Chen et al. 2001; Niida and Nakanishi, 2006; Zou et al. 2002). ATM and ATR may regulate as many as 700 substrates in response to DNA damage (Matsuoka et al. 2007). While ATM and ATR share some substrate specificity, these kina ses have also been shown to selectively phosphorylate different substrates (Cimprich and Cortez, 2008; Kim et al. 1999; Zhou and Elledge, 2000). Two proteins that play a key role in conveying the DNA damage response are the checkpoint proteins, C hk2 and Chk1, which are phosphorylated by ATM and ATR, respectively. Along with ATM and ATR, Chk1 and Chk2 are responsible for transducing the DNA damage signal to downs tream effector proteins, such as p53, MDM2, BRCA1, E2F1, Cdc25A and Cdc25C (Bartek and Lukas, 2003; Kastan and Bartek, 2004). Through the phospho rylation of these, and many other proteins, ATM and ATR respond to DNA damage in order to regul ate cell cycle arrest/p rogression, facilitate DNA repair, regulate transcriptional events and induce apoptosis (Zhou and Elledge, 2000) (Figure 1).

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5 DNA damage Replication inhibition Rad17 Rad1H u s 1R a d 9 Rad17 Rad1H u s 1R a d 9 Rad1H u s 1R a d 9 ATRIP ATR TopBP1 Claspin MRN MDC1 ATM H2AX Chk2 Chk1 MDM2 p53 E2F1 BRCA1 Cdc25 CdkCell cycle arrest ApoptosisTranscriptionDNA repair p21 Figure 1. Simplified schematic of the DNA damage response. The Rad9-Rad1-Hus1 Complex The members of the Rad (radiation sensitive) family are key regulators in sensing DNA damage and regulating checkpoint activation (Parrilla-Castellar et al. 2004). In fission yeast, certain members of the Rad family including Rad9, Rad1, Hus1, Rad17 and Rad3 (ATR) are essential for both the DNA damage and DNA replication checkpoints (Rhind and Russell, 1998). Eviden ce suggests that the functions of these proteins are

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6 conserved in mammals, which highlights the cr itical role for these proteins in the DDR (Parrilla-Castellar et al. 2004). Structure and Role in the DNA Damage Response Three members of the Rad family, Rad9, Rad1 and Hus1, form a heterotrimeric clamp that acts as a putative sensor for DNA damage (Burtelow et al. 2001; Hang and Lieberman, 2000; Roos-Mattjus et al. 2002; St. Onge et al. 1999; Volkmer and Karnitz, 1999). Biochemical and molecular modeling data suggest that the 9-1-1 complex bears structural similarity to the homotrimeric PC NA (proliferating cell nuc lear antigen) sliding clamp (Venclovas and Thelen, 2000), which is loaded onto DNA during replication, recombination and repair (Tsurimoto, 1999). Another member of the Rad family, Rad17, along with the four small subunits of the replication factor C complex (RFC), is responsible for loading the 9-1-1 complex onto DNA in response to various types of DNA damage (Bermudez et al. 2003; Lindsey-Boltz et al. 2001; Rauen et al. 2000) (Figure 2). Together with Rad17, the 9-1-1 co mplex is responsible for facilitating ATRmediated signaling pathways that are re quired for an appropriate response to DNA damage. While the 9-1-1 comp lex is required for full activ ation of the ATR-mediated DDR, it appears to be dispensable for activ ation of the ATM-mediated pathway (Weiss et al. 2002). However, it has been shown that ATM can phosphorylate Rad9 on serine 272. This event is required for checkpoint activation in res ponse to IR (Chen et al. 2001), suggesting that 9-1-1and AT M-mediated responses may not be completely exclusive. Thus, the 9-1-1 complex collaborates with ATR and ATM to activate downstream signaling pathways and ce ll cycle checkpoints.

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7 RFC1 3 4 5 2 PCNAP C N AP C N A PolymeraseRFC loading complex Sliding clamp Rad1H u s 1R a d 9 Rad17 3 4 5 2 2 Checkpoint sliding clamp Checkpoint loading complexDNA replication DNA damage response(Alkylation, UV, IR, Replication inhibitors) p p p RFC1 3 4 5 2 RFC1 3 4 5 2 PCNAP C N AP C N A PolymeraseRFC loading complex Sliding clamp Rad1H u s 1R a d 9 Rad17 3 4 5 2 2 Rad17 3 4 5 2 2 Checkpoint sliding clamp Checkpoint loading complexDNA replication DNA damage response(Alkylation, UV, IR, Replication inhibitors) p p p p p p Figure 2. Comparison of the Rad9 -Rad1-Hus1 complex and PCNA. Unlike the other members of the 9-11 complex, Rad9 possesses a carboxyterminal region that is constitutively phos phorylated and inducibly hyperphosphorylated in response to DNA damage (Chen et al. 2001; Roos-Mattjus et al. 2003; St Onge et al. 2001; St Onge et al., 2003). While this region is not re quired for interaction with Rad17, Rad1 or Hus1, it is required for Chk1 phos phorylation and downstream signaling (RoosMattjus et al. 2003). It has been suggested that th e C-terminus of Rad9, in which the phospho-tail is located, is responsible not on ly for translocation of the 9-1-1 complex to the nucleus (through a nuclear localization si gnal) (Hirai and Wang, 2002), but also for the recruitment of signaling pr oteins to DNA lesions, thereby facilitating the activation of downstream signaling pathways (Roos-Mattjus et al. 2003).

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8 Regulation of DNA Repair In addition to playing a central role in DDR signaling pathways, the 9-1-1 complex has also been shown to play a dire ct role in several DNA repair mechanisms. A role for the 9-1-1 complex in base excision re pair (BER) (especially long-patch BER) has been well described (Helt et al. 2005). It has been show n that the 9-1-1 complex regulates the early steps of BER by binding to and enhanc ing the activity of the DNA glycosyslase MutY homologue (MYH) (Chang and Lu, 2005; Shi et al. 2006) and apurinic/apyrimidini c endonuclease 1 (Gembka et al. 2007), which results in the removal of damaged bases. The 9-1-1 complex also interacts with DNA polymerase to augment its activity (Toueille et al. 2004). In addition, the 9-1-1 co mplex can also bind to flap endonuclease 1 (Friedrich-Heineken et al. 2005; Wang et al. 2004a) and DNA ligase I (Smirnova et al. 2005; Wang et al. 2006a), thereby stimulating the cleavage of flaps and the sealing of the final nick, respectively. Thus the 9-1-1 complex plays an integral role in the regulation of all of th e steps of BER. In addition to regulating BER, members of the 9-1-1 complex may also regulate homologous recombination repair (HRR), as knockdown of either Rad9 or Hus1 decrea ses the efficiency of HRR (Pandita et al. 2006; Wang et al. 2006b). Moreover, the 9-1-1 comple x may also regulate translesion synthesis in yeast by binding to tran slesion polymerases (Kai and Wang, 2003; Sabbioneda et al. 2005). Furthermore, Rad9 and Rad1 possess 3 to 5 exonuclease activity and therefore may f acilitate the processing of double-stranded DNA to singlestranded DNA (Bessho and Sancar, 2000; Parker et al. 1998). These resu lts suggest that the 9-1-1 complex may respond to DNA damage both by activating downstream signaling pathways and by directly medi ating various forms of DNA repair.

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9 Regulation of Additiona l Cellular Processes In addition to regulating the processes described above, members of the 9-1-1 complex have also been shown to play a ro le in the maintenance of telomeres (Francia et al. 2006; Nabetani et al. 2004; Pandita et al. 2006). Indeed, this ro le is evolutionarily conserved as progressive telomere shortening has been observed in Caenorhabditis elegans strains that lack Hus1 or MRT-2 (the orthologue of Rad1) (Ahmed and Hodgkin, 2000; Hofmann et al. 2002). Interestingly, Rad9 has been shown to function as a transcription factor that ca n transactivate p53 target gene s, including p21, which is a well known regulator of the cell cycle (Yin et al. 2004). Therefore, in addition to playing a role in checkpoint signaling pathways as a member of the 9-1-1 complex, Rad9 may also be able to directly affect cell cycle arrest at the G1 to S-phase transition through the transcriptional activation of p21. In addition to playing a ke y role in the regulation of DNA damage checkpoints, Rad9 also has been shown to induce apoptosis through its interaction with the an ti-apoptotic proteins, Bcl-2 and Bcl-xL (Ishii et al. 2005; Komatsu et al. 2000a; Komatsu et al. 2000b; Yoshida et al. 2002; Yoshida et al. 2003). Moreover, phosphorylation of Rad9 by either c-Abl (Yoshida et al. 2002) or protein kinase C (Yoshida et al. 2003) promotes the binding of Ra d9 to anti-apoptotic proteins thereby enhancing apoptosis. Furthermore, Rad9 can be cleaved by caspase-3, resulting in disruption of the 9-1-1 complex and releas e of the BH3 domain-containing fragment of Rad9 into the cytosol where it binds to BclxL to potentiate the apoptotic response (Lee et al. 2003).

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10 Defects Resulting from Loss of a Functional 9-1-1 Complex As described above, the 9-1-1 complex plays an integral role in the regulation of a multitude of cellular processes including the DDR, DNA repair and apoptosis. Therefore, disruption of the 9-1-1 complex affects ma ny downstream signaling processes. Impaired function of the 9-1-1 complex results in def ects in cell cycle arrest at both the S and G2/M checkpoints, an increase in chromosomal abnormalities and increased sensitivity to genotoxic stresses including topoisomerase poisons, ultraviolet radiation (UV), hydroxyurea (HU) and ioni zing radiation (IR) (Bao et al. 2004; Dang et al. 2005; Hopkins et al. 2004; Kinzel et al. 2002; Loegering et al. 2004; Pandita et al. 2006; Roos-Mattjus et al. 2003; Wang et al. 2004b; Wang et al. 2006b; Wang et al. 2003; Weiss et al. 2000; Weiss et al. 2003). Moreover, loss of Rad9 or Hus1 results in embryonic lethality, which is, at least in part, attributable to widesp read apoptosis during embryogenesis (Hopkins et al. 2004; Weiss et al. 2000). Although Hus1-/mouse embryonic fibroblast (MEF) cells have a cel lular proliferative defect, crossing of Hus1+/mice to a p21-/background results in Hus1-/-p21-/MEFs that are viable and can be grown in culture (Weiss et al. 2000). The defects described a bove emphasize the importance of the role of the 9-1-1 complex in the regul ation of the DDR and cell cycle checkpoints, maintenance of genomic integrity, proper em bryonic development and continued viability in culture. While loss of Hus1 has been shown to increase sensitivity to DNA damageinduced cell death, the molecular mechanisms by which this occurs have yet to be elucidated. In this study, we demonstr ate for the first time that loss of Hus1 sensitizes cells to etoposide treatment through the upre gulation of the BH3-only proteins, Bim and

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11 Puma. Furthermore, loss of Hus1 results in a defect in th e binding of Rad9 to chromatin and release of Rad9 into the cytosol, which in turn enhances the in teraction of Rad9 with Bcl-2 to amplify the apoptotic response. Programmed Cell Death The term programmed cell death can be defi ned as “a genetica lly controlled celldeath process that is turned on in response to external or internal signals” (Maiuri et al. 2007b). Programmed cell death has been shown to play an important role in development by regulating the formation and deletion of structures, control ling cell numbers and eliminating abnormal and damaged cells (Baehre cke, 2002). For years, the term apoptosis has been used interchangeably with pr ogrammed cell death (Chipuk and Green, 2005; Edinger and Thompson, 2004). However, ac cumulating evidence from more recent studies suggest that several forms of cell death are regulat ed or “programmed” (Edinger and Thompson, 2004; Kroemer et al. 2009). Programmed cell death now encompasses processes such as autophagic cell death and programmed necrosis, in addition to apoptosis (Degterev and Yuan, 2008; Edinger and Thompson, 2004; Kroemer et al. 2009; Lockshin and Zakeri, 2004; Okada and Mak, 2004). Apoptotic Cell Death Apoptosis is an evolutionarily conser ved form of programmed cell death that results in the self-destruction of a cell. This physiological “cell suicide” program is essential for development and plays an im portant role in th e regulation tissue homeostasis, as it allows fo r the elimination of damaged or redundant cells (Zimmermann

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12 et al. 2001). Disruption of the apoptotic pathway can lead to the development of numerous pathological conditions. Increased apoptosis is as sociated with diseases such as neurodegenerative disorders, myelodysplasti c syndromes, acquired immune deficiency syndrome and ischemic injury, while impaired apoptosis can lead to cancer and autoimmune disorders (Thompson, 1995). Apoptotic cell death is defined by certain morphological and biochemical features that are distinct from other forms of cell death and include membrane blebbing, chromatin condensation, nuclear fragmenta tion, loss of adhesion and cell shrinkage and the externalization of phosphatidylserine. The resulting apoptotic bodies are removed by phagocytes, thereby avoiding the ini tiation of an immune response (Kerr et al. 1972; Kroemer et al. 2009). Apoptosis is a tightly regulated process that eventually leads to the activation of cysteinyl aspartate-specific pr oteases, known as caspa ses (Nicholson, 1999). The apoptotic caspases can be separated into two functional groups: the initiator caspases (caspase-2, -8, -9 and -10) and the effector caspases (caspase-3, -6 and -7). Cleavage of procaspase zymogens results in their activa tion allowing them in turn to cleave hundreds of downstream proteins (Luthi and Martin, 200 7). The cleavage of these substrates results in the biochemical and morphological changes that are associated with apoptotic cell death (Taylor et al. 2008). Apoptosis is primarily mediated th rough two pathways: the extrinsic pathway, which is activated by ligati on of death receptors, and the intrinsic or stress-induced, mitochondrial pathway (Figure 3).

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13 Death receptor FADD Procaspase-8 Caspase-8 Death ligand DED DED DED DED DD DD DD DD DED DD DD DED DD DED DD DD DED DISCExtrinsic Apoptosis Caspase-3 Caspase-3 tBid Pro caspase-9 Apaf-1 Cytochrome c Pro caspase-9 Apaf-1 Cytochrome c Caspase-9 Caspase-9Apoptosome Intrinsic Bcl-2, Mcl-1 Bax, Bak Bim, Puma, tBidDeath stimuli IAPs Smac Omi Smac Omi Bad, Noxa Figure 3. Overview of the extrinsic and intrinsic apoptotic pathways. The extrinsic pathway The extrinsic pathway is activated by the binding of a ligand to its cognate death receptor. The tumor necrosis factor receptor (TNFR) family consists of more than 20 proteins including TNFR, Fas (CD95 or A po-1), the TNF-related apoptosis-inducing ligand (TRAIL), DR3, DR4 (TRAIL-R1) and DR5 (TRAIL-R2), among others (Ashkenazi, 2002). Members of the TNFR fa mily contain cysteine-rich extracellular domains and an intracellular death domain (DD). While the extracellular domain is important for receptor trimer ization (which requires the pre-ligand assembly domain) and

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14 provides ligand specificity, the intracellular death domain is critical for transmitting the death signal to downstream signaling pathways (Jin and El-Deiry, 2005). Ligand binding results in receptor activation, which recruits adaptor proteins, such as Fas-associated death domain (FADD) or TNF-associated death domain (TRADD). The DD of the receptor binds to the DD of FADD, which e xposes the death effect or domain (DED) of FADD. The DED of FADD in turn binds to the DED of procaspase -8 (and procaspase10) to form the death-inducing signaling co mplex (DISC). Aggregation of procaspase-8 within the DISC leads to autoproteolysis resulting in the activation of caspase-8 and subsequent cleavage and activation of effector caspases to elicit the apoptotic response (Ashkenazi, 2002; Jin and El-Deiry, 2005; Zimmermann et al. 2001). In addition to cleaving downstream effector caspases, caspase-8 has also been shown to cleave Bid; this truncated form of Bid (tBid) th en translocates to the mitoc hondria to activate the intrinsic pathway and amplify the apoptotic response (Li et al. 1998; Luo et al. 1998). The intrinsic pathway The members of the Bcl-2 family play a cen tral role in the re gulation of apoptosis induced through the intrinsi c pathway (Adams and Cory, 2007). The Bcl-2 family consists of both anti-apoptotic and pro-apopt otic members. Proteins such as Bcl-2, BclxL, Bcl-w and Mcl-1, are anti-apoptotic and thus prevent activation of apoptosis through the mitochondrial pathway. Most of these protei ns share structural similarity within all four of the conserved Bcl-2 homology (BH) domains. The pro-apoptotic Bcl-2 family members can be subdivided into the multidomain proteins, including Bax, Bak and Bok, and the BH3-only proteins, which include Bim, Puma, Bid, Bad, BNIP3 and Noxa,

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15 among others. The multi-domain proteins are st ructurally similar to the anti-apoptotic proteins, but lack the BH4 domain, and are essential for inducing apoptosis through the mitochondrial pathway. The members of the BH3-onl y family lack structural similarity to other Bcl-2 family members, except within their BH3 domain. The BH3-only proteins act as sensors for damage signals and induce apoptosis by neutralizi ng the anti-apoptotic proteins or by directly activating the multidomain, pro-apoptotic proteins of the Bcl-2 family to release apoptogenic factors fr om the mitochondria (G alonek and Hardwick, 2006; Strasser, 2005). Therefore, the members of the Bcl-2 family ultimately control the decision of whether a cell is to live or die, based on the relative ratio of antito proapoptotic proteins (Cory et al. 2003; Oltvai and Korsmeyer, 1994). As mentioned above, the BH3-only proteins act as sensors fo r various stress stimuli, including cytokine deprivation, hypoxia, oncogene activation and DNA damage, and are potent inducers of mitochondrial apopt osis (Willis and Adams, 2005). Therefore, these proteins must be regulated in order to prevent inappropriate activation of apoptosis and also to ensure that the apoptotic respons e is fully activated when necessary. It has been shown that BH3-only proteins can be regulated by a vari ety of mechanisms, including transcripti onal upregulation, post-translationa l modification, sequestration to cytoskeletal components and proteasomal degr adation (Puthalakath and Strasser, 2002). The regulation of the BH3-only pr oteins is tightly orchestrated and ensures that activation of Bax/Bak, and thus the intrinsic pa thway, occurs only when appropriate. Activation of the multi-domain proteins is required for mitochondrial outer membrane permeablization (MOMP), which results in the release of apoptogenic factors from the mitochondria (Green and Evan, 2002; Reed, 2003). MOMP results in the release

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16 of cytochrome c into the cytosol where it bi nds to Apaf-1. Apaf-1 can then oligomerize and recruit procaspase-9 to form the apoptos ome. The clustering of procaspase-9 in the apoptosome results in the cleavage of procas pase-9 to its active form and subsequent cleavage and activation of effector caspases. In addition to releas e of cytochrome c, MOMP also results in the release of other toxic pr oteins (Saelens et al. 2004), such as Smac/Diablo and Omi/Htr2A, which antagonize th e inhibitor of apoptosis (IAP) proteins. The binding of Smac/Diablo and Omi/HtrA2 to IAPs, such as XIAP and cIAP, abrogates their inhibitory effects on caspases (such as caspase-3 and -9), thus augmenting caspase activation and the apoptotic re sponse. AIF and endonuclease G ar e also released from the mitochondria and aid in DNA fr agmentation and chromatin condensation, respectively. Together, these mitochondrial prot eins act in concert to ensure that the apoptotic response is effectively executed to completion. DNA Damage-Induced Pr ogrammed Cell Death The intrinsic apoptotic pathway can be triggered by various intracellular and extracellular stresses incl uding those caused by exposure to DNA-damaging agents, such as the chemotherapeutic drugs, camptothecin and etoposide (Cory et al. 2003; Reed, 2003). In addition to activating apoptosis, camptothecin and etoposide have also been shown to activate other forms of program med cell death, such as autophagy (Abedin et al. 2007; Shimizu et al. 2004) (see below). Camptoth ecin and etopos ide are two commonly used chemotherapeutic agents th at target topoisomerases to induce DNA damage. Topoisomerases are a family of en zymes that regulate th e topology of DNA by inducing transient singlestrand (topoisomerase I enzymes) or double-strand

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17 (topoisomerase II enzymes) breaks in DNA to resolve torsional strains (Wang, 2002). Campothecin and etoposide target topoisomerase I and II, re spectively. The cytotoxicity of these drugs is a result of their ability to stabilize the covalent interaction between a topoisomerase and DNA, known as the cleav age complex (Montecucco and Biamonti, 2007; Pommier, 2006). Camptothecin and etopo side can both induce singleand doublestrand breaks. When the replication machinery encounters the camptothecintopoisomerase I-DNA complex, topoisomerase I re leases the cleaved strand resulting in a singleand double-strand break in the DNA (Kaufmann, 1998; Pommier, 2006). The molar ratio between topoisomerase II and et oposide determines whether a single-strand or double-strand break will be induced (Mont ecucco and Biamonti, 2007). Thus, both of these agents are able to elicit a DNA damage response that activates both ATMand ATR-mediated signaling to induce apoptosis via the intrinsic apoptotic pathway; however, the mechanisms by which these t opoisomerase poisons induce autophagy are not well understood. Autophagic Cell Death As mentioned above, mechanisms of pr ogrammed cell death, such as autophagy, play an essential role during development. Autophagy is an evolutionarily conserved process for the bulk degradation of subcellu lar constituents (Levine and Klionsky, 2004; Yoshimori, 2004). Evidence is accumulating that suggest that autophagy is involved in a wide variety of physiological proces ses and conditions, including aging, neurodegenerative diseases, infectious diseases and cancer (Kundu and Thompson, 2008; Levine and Kroemer, 2008; Mizushima et al. 2008) (for a detailed description of

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18 autophagy see below). Although autophagy is gene rally thought to pl ay a cytoprotective role, excess induction of autopha gy could result in the digesti on of essential proteins and organelles, thereby promoting the collapse of cellular functions and leading to cell death (Levine and Yuan, 2005; Tsujimoto and Shimizu, 2005). In addition, it has been suggested that while cells may preferentially die by activating the apoptotic machinery, cell death will be induced by any available route, including autophagy, if cells are exposed to harsh enough conditions (Lockshin and Zakeri, 2004). Autophagic cell death is morphologically and biochemically different from apoptotic cell death. Characteristics of au tophagic cell death include the absence of chromatin condensation, massive vacuoliz ation of the cytoplasm, accumulation of double-membraned vacuoles and little or no uptake by phagocytic cells (Kroemer et al. 2009). While the expression “aut ophagic cell death” implies that death is actually executed by autophagy, it is generally accepted th at the term simply describes cell death with autophagy (Kroemer et al. 2009; Levine and Yuan, 2005). Currently, the direct causative role of autophagy in cell death remains a key and controversial issue. Crosstalk between Apoptotic and Autophagic Cell Death Recent evidence suggests the functional relationship between the apoptotic and autophagic cell death pathways is quite complex (Maiuri et al. 2007b). Depending on the stimulus or cellular context, the interplay between autopha gy and apoptosis could occur through several mechanisms: autophagy could indu ce apoptosis or act in the later stages of apoptosis to aid in the elimination of apoptotic bodies, autophagy could delay or prevent apoptosis through th e clean-up of damaged mitoc hondria or the two processes

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19 may be mutually exclusive, acting as a back -up in case the other pa thway fails (Scarlatti et al. 2009). In addition, comm on cellular stresses can ac tivate various signaling pathways that elicit both the inductio n of autophagy and apoptosis (Maiuri et al. 2007b). As such, several proteins have been identif ied that regulate both the autophagic and apoptotic pathways. Beclin 1 was originally identified through a y east two-hybrid screen aimed at identifying novel Bcl-2 an d Bcl-xL binding partners (Liang et al. 1999). Recently, the BH3 domain of B eclin 1, which is required for its interaction with antiapoptotic Bcl-2 family members, has been desc ribed, thus Beclin 1 can be classified as a BH3-only protein (Maiuri et al. 2007a). Furthermore, other BH3-only proteins, specifically Bad and BNIP3, as well as the pharmacological BH3 mimetic, ABT-737, were shown to disrupt the interaction be tween Beclin 1 and Bcl-2/Bcl-xL thereby stimulating autophagy (Maiuri et al. 2007a). Atg5, a key compone nt of the ubiquitin-like conjugation system in autophagy (see below), can be cleaved by calpain resulting in the N-terminal fragment of Atg5 translocating to the mitochondria where it triggers MOMP (Yousefi et al. 2006). p53, a transcription factor th at is known to regulate DNA damageinduced apoptosis, was recently shown to induce DRAM (damage-regulated autophagy modulator). Knockdown of DRAM not only abrogated the inducti on of autophagy, but also inhibited the initia tion of apoptosis (Crighton et al. 2006). Other proteins, such as DAPk (death-associated protein kinase), act as regulators of cell death and can mediate processes that are involved in both the apoptotic and au tophagic pathways including membrane blebbing (a characteristic of apopt osis) and cytoplasmic vesicle formation (a characteristic of autophagy) (Inbal et al. 2002). In addition, activation of the DNA damage-responsive transcription factor, E2 F1 results in the upregulation of the

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20 expression of autophagic proteins, including LC3, Atg1 and Atg5 (Polager et al. 2008). Thus, numerous proteins play a role in both the apoptotic and autophagic pathways, which highlights the intricate crosst alk between these two pathways. In this report, we demonstrate that in hibition of apoptosis, through treatment with Z-VAD-FMK, results in the induction of caspa se-independent cell de ath in response to camptothecin treatment. Furthermore, disr uption of the 9-1-1 complex, through loss of Hus1 enhanced DNA damage-induced autophagy. Th ese results suggest that in response to genotoxic stresses, autophagy may be induced as a cell death mechanism. Surprisingly, inhibition of autophagy, through knockdown of Atg7 or Bif-1, enhanced cell death in response to camptothecin treatment, suggesti ng that the induction of autophagy observed in Hus1 -deficient cells is actually a cytoprotect ive mechanism. It is of interest to determine whether the inhibition of caspase activity in these autophagy-deficient cells would suppress cell death in response to DNA damage. Autophagy Autophagy is a tightly regulated process for the bulk degradation of cytoplasmic constituents (Yoshimori, 2004). This evolutiona rily conserved process plays a role in the maintenance of cellular homeostasis by r ecycling nutrients and removing damaged organelles, misfolded proteins and invasive microorgansisms. In addition, recent studies have shown that autophagy is involved in a variety of phys iological processes, including development, differentiation, tissue rem odeling and cell survival, whereas the deregulation of autophagy has been implicated in the pathogenesis of certain diseases, such as cancer, cardiomyopathy, muscular diseases and neurodegenerative disorders

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21 (Kundu and Thompson, 2008; Levine and Kli onsky, 2004; Levine and Kroemer, 2008). At least three distinct type s of autophagy have been desc ribed; chaperone-mediated autophagy, microautophagy and macroa utophagy (Cuervo, 2004; Klionsky et al. 2007). The studies included in this report focus on marcroautophagy, hereafter referred to as autophagy. Upon the initiation of autophagy, a portion of the cytoplasmic components is sequestered into cup-shaped membrane st ructures known as isolation membranes or phagophores (Levine and Klionsky, 2004). The isolation membrane elongates and the edges eventually fuse to form a double-me mbraned vesicle known as an autophagosome. The autophagosome matures when it fuses with endosomes and lysosomes to become an autolysosome, within which the enclosed components are degraded by lysosomal hydrolases. Autophagy occurs at basal levels in virtually all cells, but can be upregulated in response to environmental changes, such as starvation and exposure to DNA damaging agents (Crighton et al. 2006; Levine and Kroemer, 2008; Polager et al. 2008; Shimizu et al. 2004). Mechanisms that Regulate Autophagy As mentioned above, the process of autophagy involves multiple steps, including initiation, cargo selection and packaging, vesicle nucleation, ve sicle expansion and completion, retrieval, docking and fusion and ly sosomal degradation of vesicles and their contents (Levine and Klionsky, 2004). The initia tion of autophagy is mainly regulated by downstream signaling through the target of rapamycin (TOR) and the autophagy-related (Atg) proteins, including th e phosphatidylinosito l 3-kinase class III (PI3KC3)-Atg6

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22 (yeast homologue of mammalian Beclin 1) co mplex (Levine and Klionsky, 2004; Xie and Klionsky, 2007) (Figure 4) DegradationLysosome Atg Beclin 1 Class III PI3KDocking and fusion Autophagic vacuole formation Isolation membrane expansion TOR Atg12 Conjugation system LC3 Conjugation system LC3 Atg4 LC3 ATP+ Atg7 LC3 Atg7 LC3 Atg3 LC3 PE Atg12 ATP+ Atg7 Atg12 Atg7 Atg12 Atg10 Atg12 Atg5 Atg12 Atg5 Atg16 Bif-1 UVRAG DegradationLysosome Atg Beclin 1 Class III PI3KDocking and fusion Autophagic vacuole formation Isolation membrane expansion TOR Atg12 Conjugation system LC3 Conjugation system LC3 Atg4 LC3 ATP+ Atg7 LC3 Atg7 LC3 Atg3 LC3 PE LC3 Atg4 LC3 ATP+ Atg7 LC3 Atg7 LC3 Atg3 LC3 PE Atg12 ATP+ Atg7 Atg12 Atg7 Atg12 Atg10 Atg12 Atg5 Atg12 Atg5 Atg16 Bif-1 UVRAG Atg9 Atg9 Figure 4. Model of the mechanisms that regulate autophagy. ATG genes Genetic screening in yeast has lead to the discovery of at least 30 autophagyrelated genes, many of which have known orthologues in higher eukaryotes (Klionsky et al. 2003; Xie and Klionsky, 2007). The correspondi ng gene products of a subset of the ATG genes form what has been described as the core autophagy machinery. This machinery can be subdivided into three ma in functional groups: (1) the Atg9 cycling

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23 system, (2) the PI3KC3-Beclin 1 complex and (3) the ubiquitin-like (Ubl) protein system (Xie and Klionsky, 2007). Atg9 is one of the most well characterized molecules for the investigation of the biogenesis of autophagosomes and is the only known transmembrane Atg protein (Noda et al. 2000). In yeast, Atg9 has been shown to shuttle between periva cuolar sites, known as phagophore assembly sites (PAS), and pe ripheral sites which include mitochondria (Reggiori et al. 2005). In contrast, the mammalian or thologue of Atg9 localizes to the trans -Golgi network and late endosomes, but not to mitochondria (Yamada et al. 2005; Young et al. 2006). These results suggest that Atg9-c ontaining vesicles could be a source of membranes for the biogenesis or expans ion of autophagosomes by delivering donor membranes to the PAS. While the efficient transport of Atg9 to the PAS requires the Atg9 transport proteins Atg23 and Atg27, the retrieval of Atg9 from the PAS depends on the Atg1-Atg2-Atg18 complex (X ie and Klionsky, 2007). Vesicle nucleation is an ear ly step in autophagosome fo rmation and results in the formation of double-membraned structures. The class III phosphatidylinositol 3-kinase (PI3KC3), Vps34 (vacuolar protei n sorting 34), interacts with Beclin 1 to form a complex that is required for ve sicle nucleation (Volinia et al. 1995). This complex interacts with p150 (the mammalian homologue of yeast Vps15) a protein kinase that is thought to activate Vps34 and mediate the binding of the complex to membranes. The Vps34 complex most likely functions at the PAS by recruiting PtdIns (3)P-binding proteins. The process of autophagosome formation can be bl ocked by treatment with PI3K inhibitors, such as 3-MA and wortmannin (Mizushima et al. 2001), suggesting that Vps34 is an essential regulator of autophagosome formation.

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24 In addition to the Vsp34-Beclin 1 complex and Atg9, two ubiquitin-like conjugation systems, Atg12-At g5 and Atg8-phosphatidylethanolamine (PE), also play a role in the regulation of au tophagic vesicle formation (Ohsumi, 2001). The first system consists of Atg12 (the ubiqui tin-like protein), Atg7 (similar to an E1 ubiquitin-activating enzyme) and Atg10 (similar to an E2 ubi quitin-conjugating enzyme), which are responsible for transferri ng Atg12 to Atg5 (Mizushima et al. 1998; Shintani et al. 1999; Tanida et al. 1999). This complex then recruits Atg16, which can homooligomerize to mediate the formation of large protei n complexes containing Atg12, Atg5 and Atg16 (Mizushima et al. 2003; Mizushima et al. 1999). The second syst em is composed of Atg8 (the ubiquitin-like prot ein), Atg4 (which cleaves At g8 exposing a glycine residue that is then accessible for activation by At g7), Atg7 (the E1-like protein) and Atg3 (the E2-like enzyme), which are required for transferring Atg8 to PE (Ichimura et al. 2000; Kirisako et al. 2000). Unlike the Atg12-Atg5 system Atg8 conjugation to PE is reversible through Atg4-mediated cleavage of Atg8 (Kirisako et al. 2000). It has been shown that the Atg12-Atg5 conjugate is required for the stability and proper localization of Atg8 (Mizushima et al. 2001; Suzuki et al. 2001). While the Atg12-Atg5 complex was found to localize to forming autopha gosomes and dissociate before vesicle completion, the Atg8-PE conjugate is located on autophagosomes during their formation and after completion and is eventually de graded within the autolysosome (Kabeya et al. 2000; Kirisako et al. 1999; Mizushima et al. 2003; Mizushima et al. 2001). Accordingly, Atg8 is one of the best mark ers of autophagosomes and has been used extensively as an indicator for the ini tiation of the autophagic pathway and for autophagosome formation. The mammalia n homologue of Atg8, the microtubule-

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25 associated protein light chain 3 (LC3), is modi fied in a similar mechanism to that of Atg8 (Ichimura et al. 2000; Kabeya et al. 2000). Upon the induction of autophagy LC3 is cleaved by Atg4 to produce a cy tosolic form known as LC3-I. LC3-I is then conjugated to PE to form LC3-II, which is recru ited to the autophagosomal membrane. Thus examining the modification and localizati on of LC3 are well described methods to monitor the induction of autophagy (Mizushima and Yoshimori, 2007; Tasdemir et al. 2008). Bif-1 Bif-1, also known as Endophilin B1 or SH 3GLB1, was originally discovered as a Bax-interacting protein (Cuddeback et al. 2001; Pierrat et al. 2001). In addition to regulating apoptosis through its interaction with Bax, Bif-1 ha s also been found to play an integral role in the regulation of au tophagy. Bif-1 interacts with Beclin 1 through UVRAG (ultraviolet radiation resistance-associ ated gene) to promote the activation of PI3KC3/Vps34 and the formation of autophagosomes (Takahashi et al. 2007). Furthermore, Bif-1 has an intrinsic abili ty to induce membrane curvature (Farsad et al. 2001), suggesting that Bif-1 may collabor ate with the Beclin 1-UVRAG-PI3KC3 complex to provide the driving force for th e curvature of the isolation membrane. In response to nutrient starvation, Bif-1 accumulate s in foci in the cytosol where it colocalizes with LC3 and Atg5 (Takahashi et al. 2007). As mentioned above, LC3 is a well-known marker for autophagosomes and Atg5 has been shown to locate to phagophores throughout the elongation step of autophagosome formation, but is removed before the completion/sealing of the autophagosome (Kabeya et al. 2000; Klionsky et

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26 al. 2008; Mizushima et al. 2001). Taken together, these re sults implicate Bif-1 in the regulation of the early stages of autophagosome formation and suggest that Bif-1 may play a role in the biogenesis or expans ion of phagophores. Indeed, Bif-1 was found to localize to Atg9-positive vesicles (Takahashi et al. 2008). As the formation and trafficking of Atg9-positive ve sicles is essential for the biogenesis and expansion of autophagosomal membranes during the induction of autophagy (Noda et al. 2000; Young et al. 2006), these results provide further evid ence that Bif-1 is involved in these processes. Further studies ar e required to determine the pr ecise molecular mechanisms by which Bif-1 regulates autophagosome formation. Deregulation of Autophagy in Cancer Accumulating evidence suggests that autophagy may both enhance and inhibit tumor development and progression (Mizushi ma, 2005). In the early stages of tumor development, tumors are limited in growth by a lack of blood vessels, which provide necessary oxygen and nutrients. In this se tting, induction of au tophagy would provide cells with nutrients in a starvation setti ng and thus be cytoprotective (Degenhardt et al. 2006). Alternatively, autophagy could protect cells from undergoing apoptotic cell death induced by various chemothe rapeutic agents (Abedin et al. 2007; Amaravadi et al. 2007; Carew et al. 2007; Paglin et al. 2001). Conversely, autophagy may hinder tumor progression by removing damaged or malfunctio ning organelles, such as mitochondria, thereby limiting exposure to genotoxic subs tances, such as reactive oxygen species, which would result in enhanced genetic muta tions and favor tumorigenesis (Edinger and Thompson, 2003). Indeed, the concept that auto phagy may be beneficial during the early

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27 stages of tumorigenesis and detr imental during later stages is consistent with the findings that the rate of autophagy is decreased in ma lignant pancreatic adenocarcinoma cells as compared to premalignant cells (Toth et al. 2002). Recent studies have shown that genetic deregulation of autophagy regulatory proteins contributes to tumo rigenesis. While monoallelic deletions of Beclin 1 are frequently detected in breast, ovarian and prosta te cancers (Aita et al. 1999), monoallelic mutations in UVRAG occur at a high frequency in colon cancer cells (Ionov et al. 2004). Furthermore, homozygous deletion of Bif-1 has been confirmed in mantle cell lymphomas (Balakrishnan et al. 2006) and decreased Bif-1 expression has been described in gastric carcinomas, colorectal adenocarcinomas, urinary bladder and gallbladder cancers (Coppola et al. 2008a; Kim et al. 2008; Lee et al. 2006). A recent study demonstrated that Bif-1 expression is d ecreased in a significant portion of prostate cancers, although the majority of prostate can cer samples examined had high levels of Bif-1 (Coppola et al. 2008b). In addition, Beclin 1+/and Bif-1-/mice both have a significantly enhanced o ccurrence of spontaneous tumor development (Qu et al. 2003; Takahashi et al. 2007; Yue et al. 2003) and ectopic expre ssion of UVRAG suppresses tumorigenesis in nude mice (Liang et al. 2006). Moreover, it was shown that Atg4C and Atg5 also possess tumor suppres sive capabilities (Marino et al. 2007; Yousefi et al. 2006). In addition to the involvement of autophagy genes in the regulation of tumorigenesis, it has also been shown that tumor suppressor proteins, such as PTEN, p53 and DAPk, activate autophagy while oncogenes, such as Akt and Bcl-2, suppress autophagy (Botti et al. 2006). These studies highlight th e strong correlation that exists between proteins that regulate the induc tion of autophagy and tumor suppression and

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28 those that inhibit au tophagy and oncogenesis (Levine a nd Kroemer, 2008). While great strides have been made to determine th e connection between the deregulation of autophagy and cancer, further stud ies are needed to determin e the precise mechanisms by which autophagy functions in tumorigenesis and tumor suppression. Once the association between autophagy and cancer has been determ ined, it will allow for the manipulation of autophagy to enhance therapeutic treatments for cancer. Crosstalk between the Autophagic and Endocytic Pathways The autophagic and endocytic pathways represent branches of the lysosomal degradation system; these pathways are res ponsible for the degradation of cytoplasmic constituents and exogenous substances/macrom olecules, respectively. It has been shown in yeast that two distinct complexes form to regulate these processes; complex I (Vps15, Vps34, Atg14 and Atg6) and complex II (V ps15, Vps34, Vps38 and Atg6) that are involved in autophagy and vac uolar protein sorting/endocyt osis, respectively (Kihara et al. 2001). Complex I and II share three comm on members that are evolutionarily conserved: the adaptor protein, Vps15 (p150 in mammals), a class III PI3K, Vps34, and Atg6 (the yeast orthologue of mammalian Bec lin 1). Certain members of these complexes have been shown to play a role in both the autophagic and endocytic pathways, while others have been found to only be involved in the regulation of one process, but not the other. Mammalian Vps34 has been implicated not only in the regulation of autophagy, but also in endocytic trafficking and sorting of cell-surface receptors and the formation of internal vesicles in multivesicular endosom es (Backer, 2008). While Vps34 is required

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29 for the production of PtdIns(3)P for memb rane trafficking, part icularly at late endosomes/multivesicular bodies, it is dispensa ble for the internaliz ation of cell surface receptors and the uptake of fluid phase markers (Johnson et al. 2006). Another member of the mammalian Vps34 complex, UVRAG, has been shown to interact with the class C Vps complex, a key component of the endosom al fusion machinery (Peterson and Emr, 2001), to promote autophagosome matura tion by enhancing fusion with late endosomes/lysosomes (Liang et al. 2008). Similar to Vps34, UVRAG is not involved in the internalization of endocytic cargo, however it does acceler ate intracellular trafficking and degradation (Liang et al. 2008). In addition, the role of UVRAG in the class C Vps complex, which regulates endocytic vesicle tra fficking, was shown to be distinct from its role in the Vps34-Beclin 1-Bif-1 comp lex, which induces au tophagy by regulating autophagosome formation (Liang et al. 2008). Whereas Vps34 and UVRAG both play a role in the autophagic and endocytic pathwa ys, as mentioned above, Beclin 1 has only been shown to be required for autophagy a nd is expendable for endocytosis and vesicle trafficking (Zeng et al. 2006). In addition to various proteins being involved in the regulation of both the autophagic and endocytic pathways, these path ways have been shown to converge at the prelysosomal and lysosomal level for degradation (Gordon et al. 1992; Gordon and Seglen, 1988) (Figure 5). Indeed, it has been shown that autophagic vacuoles can directly fuse with vesicular and multivesicular bodies (MVB) (before fusing with lysosomes) to form what are known as amphisome s (Fader and Colombo, 2008; Liou et al. 1997). While much has been discovered about th e interplay between the autophagic and

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30 endocytic pathways, the molecular mechanisms that regulate vesicula r trafficking and the convergence between these two pathways are not fully understood. Lysosome Autophagosome Amphisome Autolysosome Degradation Endocytosis Endosome Lysosome Autophagosome Amphisome Autolysosome Degradation Endocytosis Endosome Figure 5. Convergence of the autophagic and endocytic pathways for lysosomal degradation. Endocytosis and Vesicle Trafficking The endocytic pathway functions in cellula r homeostasis through the regulation of internalization, transport, sorting and de gradation of macromol ecules (Fader and Colombo, 2008). In addition to a role for endocytosis in vesicle trafficking and degradation, evidence is accumulating that s uggest that endocytosed receptors may be able to activate specialized signaling complexe s that are not assembled at the cell surface

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31 (Sorkin and Von Zastrow, 2002). Indeed, adapto r and effector proteins have been found to localize to endosomes containing epider mal growth factor receptors (EGFR) (Di Guglielmo et al. 1994; Wiley, 2003) and EGFR can maintain signaling even after internalization and fusion with endosomes (Miaczynska et al. 2004). Therefore, it is possible that endocytic traffi cking could regulate signaling pathways that are distinct from those initiated at the cell surface (Vieira et al. 1996). Although evidence suggests that signaling through endocytosis may activ ate specialized signaling pathways, receptor endocytosis is generally considered to downregulate growth factor signaling through lysosomal degradation (Citri and Yarden, 2006). EGFR as a Model for Endocytic Trafficking and Degradation The EGFR was one of the first growth f actor receptors that was observed to be internalized following ligand binding (Gorden et al. 1978; Haigler et al. 1979). Based on extensive studies, which have investigated the mechanisms behind the internalization, sorting and degradation of this receptor, the EGFR is now a prototype for endocytic vesicle trafficking (Citri and Yarden, 2006; Sorkin and Von Zastrow, 2002). Internalization and sorting of the EGFR leads to the removal of activated receptors from the cell surface. Receptors can then be recycl ed back to the membrane; alternatively, the receptors can be degraded, thereby downre gulating EGFR-mediated proliferation and survival signaling (Katzmann et al. 2002) (Figure 6).

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32 EGFR EGF Activated EGFR Early endosome Multivesicular body Lysosome Fusion Activation of downstream signaling Recycled back to cell surface Degradation EGFR EGF Activated EGFR Early endosome Multivesicular body Lysosome Fusion Activation of downstream signaling Recycled back to cell surface Degradation Figure 6. Endcoytic trafficking of the EGFR. Ligand binding results in EGFR di merization and phosphorylation, which provides docking sites that ar e required for the recruitmen t of adaptor and effector proteins that are involved in the regula tion of the endocytic pathway (Grandal and Madshus, 2008). The E3 ubiquitin ligase, Cbl, is recruited to phosphorylated tyrosine 1045 of EGFR (Levkowitz et al. 1999). Cbl-mediated EGFR ubiquitination results in further recruitment of other signaling molecules and ubiquiti n-binding proteins, including Eps15 and CIN85 (Kirisits et al. 2007), which act as a scaffold for endophilins. Together these proteins are responsible for inducing the curvature of the plasma membrane and thus, regulate the formation of clathrin coated pits (Soubeyran et al. 2002). Dynamin mediates vesicle fission from the plasma memb rane, which releases the vesicle into the

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33 cytoplasm (McNiven et al. 2000). The clathrin then dissoci ates from the vesicle, which then fuses with a tubular-vesicular network located at the periphery of the cell. This fusion results in the delivery of the ligand-bound receptors to early endosomes (Katzmann et al. 2002; Sorkin and Von Zastrow, 2002). The EGFR is then sorted through various intracellular trafficking even ts, which depend on Cbl-mediated ubiquitin signals (Levkowitz et al. 1998). PI3KC3 is responsible for membrane invagination and thus the formation of internal vesicles to form MVBs (Futter et al. 2001). The ligandbound receptors then accumulate in the lim iting (outer) and lumenal membranes of MVBs. The limiting membrane then fuses with lysosomal membranes resulting in the delivery of lumenal contents to the hydrolytic interior of th e lysosome where the contents are then degraded (Futter et al. 1996). In contrast, proteins that remain on the limiting membrane of MVBs avoid degradation and are subsequently recycled back to the plasma membrane or transported to othe r sites within the cell (Katzmann et al. 2002). Thus, the integrity of the endocytic pathway can be monitored by tracking the fate of activated EGFR. In this study, we have found that knoc kdown of Bif-1 does not affect the uptake of a fluid phase marker, horse radish peroxi dase, or the internalization of epidermal growth factor (EGF). Interestingly, knockdown of Bif-1 accelerated th e co-localization of internalized EGF with late endosomes/lysoso mes. Furthermore, EGFR degradation was enhanced by loss of Bif-1. These results in dicate a novel role fo r Bif-1 in vesicle trafficking within the endocytic pathway. Fu rther studies are need ed to determine the molecular mechanisms by which loss of Bif-1 accelerates vesicle trafficking and receptor degradation.

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34 Deregulation of Endocytosis in Cancer Deregulation of EGFR signaling occurs in nearly 50% of all human tumors (Rodemann et al. 2007). In addition to overexpression and gain -of-function mutations, the evasion of downregulati on by endocytic/lysosomal degrad ation is another mechanism that enhances EGFR signaling and drives tumorigenesis (Grandal and Madshus, 2008; Kirisits et al. 2007; Yarden and Sliwkowski, 2001). Defective downregulation of EGFR can prolong signaling and thus positively regula te survival and proliferation. Indeed, it has been shown that preventing the do wnregulation of EGFR facilitates cell transformation (Levkowitz et al. 1998). Furthermore, EGFR mutations that impair ubiquitination and thereby stab ilize the EGFR have been de scribed in cancer patients. These mutations have been shown to protec t cells from apoptosis and promote growth (Grandal and Madshus, 2008). Ther efore, elucidating the mech anisms that regulate the endocytic trafficking and degradation of EGFR could lead to more lucrative therapeutic approaches for the tr eatment of cancer. Summary As I mentioned above, a functional 9-1-1 complex is required for the maintenance of genomic stability and thus prevents the accumulation of mutations that could lead to cancer. Additionally, cells w ith a disrupted 9-1-1 complex are hypersensitive to genotoxic stress-induced apoptosis. However, the molecular mechanism by which loss of a functional 9-1-1 complex activates the apoptot ic pathway had yet to be determined. Our data indicate that loss of Hus1 sensitizes cells to etoposid e-induced apoptosis through the upregulation of Bim and Puma. Furthermore, loss of Hus1 enhances the interaction of

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35 Rad9 with Bcl-2 to potentiate th e apoptotic response. Interest ingly, our data suggest that disruption the 9-1-1 complex not only sensitiz es cells to caspasedependent cell death, but also to caspase-independent cell deat h in response to DNA damage. Moreover, the results presented in this study indicate that loss of Hus1 enhances DNA damage-induced autophagy. Since excessive induc tion of autophagy could resu lt in cell death, autophagy may be the mechanism underlying ca spase-independent cell death in Hus1 -deficient cells. However, inhibition of autophagy, by knoc kdown of Atg7 or Bif-1, enhanced the cytotoxicity of camptothecin, suggesting that autophagy is be ing induced as a cytoprotective mechanism rather than a pro-death mechanism in response to DNA damage. Finally, our results describe a nove l role for Bif-1 in endocytic vesicle trafficking and receptor degradati on. It is therefore likely that Bif-1 promotes survival not only through its regulation of autophagy, but also by affecting EGFR signaling through its regulation of the endocytic pathway. Impor tantly, the results de scribed here better define the mechanisms that are regulated by the 9-1-1 complex and Bif-1 that affect sensitivity to DNA damage.

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36 Chapter Two: Loss of Hus1 Sensitizes Cells to Etoposide-Induced Apoptosis by Regulating BH3-Only Proteins1 Abstract The Rad9-Rad1-Hus1 (9-1-1) cell cycle chec kpoint complex plays an integral role in the DNA damage response. Cells with a de fective 9-1-1 complex have been shown to be sensitive to apoptosis induced by certai n types of genotoxic stress. However, the mechanism linking the loss of a functional 9-11 complex to the cell death machinery has yet to be determined. Here, we report that etoposide treatment dramatically upregulates the expression of the BH3-only proteins, Bim and Puma, in Hus1 -deficient cells. Inhibition of either Bim or Puma expression in Hus1 -knockout cells confers significant resistance to etoposide-induced apoptosis, while knockdown of both proteins results in further resistance, suggesting that B im and Puma cooperate in sensitizing Hus1 -deficient cells to etoposide treatment. Moreover, we found that Rad9 collabo rates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-i nduced apoptosis. In response to DNA damage, Rad9 localizes to chromatin in Hus1 -wild-type cells, whereas in Hus1 deficient cells Rad9 is predominantly located in the cytoplasm where it binds to Bcl-2. Taken together, these resu lts suggest that loss of Hus1 sensitizes cells to etoposideinduced apoptosis, not only by inducing Bim and Puma expression, but also by releasing Rad9 into the cytosol to augm ent mitochondrial apoptosis. 1 Meyerkord CL, Takahashi Y, Araya R, Takada N, Weiss RS, Wang HG (2008). Loss of Hus1 sensitizes cells to etoposide-induced apoptosis by regulating BH3-only proteins. Oncogene 27: 7248-59.

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37 Results Loss of Hus1 Sensitizes Cells to Etoposide-Induced Apoptosis Knockout of Hus1 results in cell cycle checkpoint defects and enhanced cell death in response to DNA damage induced by hydroxyur ea (HU) and ultraviolet (UV) radiation (Weiss et al. 2000; Weiss et al., 2003; Weiss et al., 2002). In this study, we examine the sensitivity of Hus1-deficient cells to etoposide, one of the most potent drugs used for cancer therapy (Montecucco and Biamonti, 2007) In order to determine whether loss of Hus1 would sensitize cells to etoposide-induced cell death, Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with increasing doses of etoposide for 24 h. Measurement of cell death by trypan blue exclusion assay revealed that knockout of Hus1 greatly enhanced the dose-dependent susceptibility of MEFs to etoposide (Figure 7a). Consistently, the hypersensitivity of Hus1-deficient cells to etoposide-induc ed cell death also occurred in a time-dependent manner (Figure 7b). Figure 7. Loss of Hus1 sensitizes cells to etoposide-induced cell death. ( a) Hus1+/+ p21-/and Hus1-/-p21-/MEFs were treated with increa sing doses of etoposide for 24 h. Viability was determined by trypan blue exclusion assay (mean s.d.; n=3). ( b ) Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for varying time points and subjected to trypan blue exclusion assay (mean s.d.; n=3). 0 10 20 30 40 50 6001.563.616.2512.525 Etoposide ( g/ml) 24 hPecent Cell Death Hus1+/+p21-/Hus1-/-p21-/0 10 20 30 40 50 60 0122448Hours etoposide (6.25 g/ml)Percent Cell Death Hus1+/+p21-/Hus1-/-p21-/a b

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38 To determine whether the increase in cell death observed in Hus1 -deficient cells is due to the enhanced inducti on of apoptosis, activation of cas pase-3, as well as cleavage of its downstream substrate, poly(ADP-ribos e) polymerase (PARP), were examined by immunoblot analysis. Hus1-/-p21-/cells exhibited a robus t induction of caspase-3 processing, which correlated with PARP cleavage, upon 24 h treatment with 12.5 g/ml etoposide that was further enhanced at a higher dose (Figur e 8). In contrast, Hus1+/+p21-/cells showed only slight activation of caspa se-3 and minimal PARP cleavage even upon treatment with the highest dose of etoposid e (Figure 8). Induction of apoptosis in response to etoposide treatment was furthe r analyzed by examination of nuclear morphology for chromatin condensation and nucl ear fragmentation. As shown in Figures 9a and b, Hus1deficient cells were almost thr ee times more sensitive to etoposideinduced apoptosis. Taken together, these results suggest that MEFs that lack Hus1 are not only sensitive to hydroxyurea and UV radiation, as previously described, but that these cells are also sensitive to DNA damage indu ced by topoisomerase II poisons, such as the chemotherapeutic drug, etoposide. Figure 8. Loss of Hus1 enhances the cleavage of caspase-3 and PARP. Hus1+/+p21-/and Hus1-/-p21-/MEF cells were treated with vary ing doses of etoposide for 24 h. Total cell lysate was normalized for protein cont ent and subjected to SDS-PAGE/immunoblot analysis. Tubulin0 1.56 3.12 6.25 12.5 25 0 1.56 3.12 6.25 12.5 25 Hus1+/+p21-/-Etoposide ( g/ml) Active Caspase-3 PARPHus1-/-p21-/-

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39 Figure 9. Loss of Hus1 sensitizes cells to etoposide-induced apoptosis. (a) Hus1+/+ p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for 0 or 48 h. Apoptosis was determined by examination of nuclear morphology. Arrows indicate apoptotic nuclei. (b) Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for the times indicate d. The percent of apoptotic cells was quantified based on nuclear morphology (mean s.d.; n=3). Loss of Hus1 Enhances Bim and Puma Expression at Both the Protein and mRNA Level in Response to DNA Damage Since our results indicate that loss of Hus1 sensitizes cells to etoposide-induced apoptosis, the expression levels of members of the Bcl-2 family were examined (Figure 10). In response to etoposide treatment, the expression levels of anti-apoptotic Bcl-2-like proteins and pro-apoptotic multi-domain proteins remained relatively stable in both Hus1+/+p21-/and Hus1-/-p21-/MEFs. Notably, the basal level of Bcl-xL was higher in Hus1-/-p21-/cells compared to Hus1+/+p21-/cells, presumably to neutralize elevated Bax expression in cells lacking Hus1 (Weiss et al. 2000). Interestingly, the expression of the BH3-only proteins, Bim and Puma, was indu ced following etoposide treatment. Whereas a b 0h 48h Hus1+/+p21-/-Hus1-/-p21-/0 10 20 30 40 50 60 012243648Hours Etoposide (6.25 g/ml)Percent Apoptotic Hus1+/+p21-/Hus1-/-p21-/-

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40 the expression of these proteins was only slightly induced and peaked at 24 h in Hus1 wild-type cells, the upre gulation of all three is oforms of Bim, as well as Puma, was much more dramatic and persisted to later time points in Hus1 -deficient cells. Similar results were observed after treatment with other DNA damaging agents including camptothecin, an inhibitor of topoisomerase I, and hydroxyurea, an inhibito r of DNA replication (Figures 11 and 12, respectively). Mcl-1 Bcl-xL Bax Bak Bim L Puma Tubulin 03122436480312243648 6 Etoposide (h) Hus1+/+p21/-Hus1/-p21/-Bim S Bim EL 6 Bcl-2 Figure 10. Expression of Bcl-2 family memb ers in response to etoposide treatment. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for the indicated time points. Total cell lysate was prepared and analyzed by SDSPAGE/immunoblot using the in dicated antibodies.

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41 Bim EL Bim L Bim S Puma Tubulin CPT (h) 0361224364803612243648 Hus1+/+p21-/-Hus1-/-p21-/Figure 11. Loss of Hus1 results in upregulation of Bim and Puma expression in response to camptothecin treatment. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 500 nM camptothecin (CPT) for the times indicated. Total cell lysate was prepared and analyzed by SDS-PAGE/immunoblot using the indicated antibodies. 0361224364803612243648 Hus1+/+p21-/-Hus1-/-p21-/-Bim EL Puma Tubulin HU (h) Figure 12. Loss of Hus1 results in upregulation of Bim and Puma expression in response to hydroxyurea treatment. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 50 M hydroxyurea (HU) for the times indicate d. Total cell lysate was prepared and analyzed by SDS-PAGE/immunoblot us ing the indicated antibodies.

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42 To confirm that the etoposide-induced upregulation of Bim and Puma expression is a direct result of loss of Hus1 we examined whether rest oration of Hus1 expression would suppress the induction of these BH3-only proteins in response to etoposide treatment. To this end, Hus1-/-p21-/MEFs that were infected with retrovirus to express either Hus1 ( Hus1-/-p21-/Hus1 ) or control GFP ( Hus1-/-p21-/GFP ) (Weiss et al. 2002) were treated with etoposide fo r varying time points and the expression of Bim and Puma was examined. Expression of Hus1, but not control GFP, significantly reduced etoposideinduced expression of Bim and Puma in Hus1 -deficient MEFs (Figur e 13). These results indicate that upre gulation of Bim and Puma in response to etoposide-induced DNA damage is indeed a direct result of loss of Hus1 Figure 13. Restoration of Hus1 suppresses the upregulatio n of Bim and Puma in response to DNA damage. Hus1-/-p21-/MEFs stably expressing Hus1 or GFP were treated with 6.25 g/ml etoposide for varying time points. Total cell lysate was prepared and analyzed by SDS-PAGE/immunoblot using the indicated antibodies. Bim EL Bim L Bim S Puma Tubulin Etoposide (h) 0361224364803612243648 Hus1GFP GFP Hus1-/-p21-/-

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43 The activity of BH3-only pr oteins can be regulated by various mechanisms including transcripti onal upregulation, post-transla tional modification, proteasomal degradation and sequestration to cytoskelet al components (Puthalakath and Strasser, 2002; Willis and Adams, 2005). To examine whether the induction of Bim and Puma expression observed in Hus1 -deficient cells is regulated at the transcriptional level after exposure to etoposide, Hus1-/-p21-/cells were treated with et oposide in the presence of a transcriptional inhibitor, actinomycin D, a translational inhibitor, cycloheximide, or control DMSO. As shown in Figure 14, trea tment with either actinomycin D or cycloheximide abrogated the expression of Bim, as well as Puma, even in the presence of etoposide. In contrast, the expression of these BH3-only proteins was significantly induced in response to etoposide treatment in the control DMSO-treated cells (Figure 14), indicating that upregulation of both Bim and Puma, in re sponse to etoposide-induced DNA damage, occurs at the transcriptional level. Indeed, semi-quantitative RT-PCR analyses revealed that the expression of Bim and Puma mRNAs are increased in response to etoposide treatment (Figures 15-18). The levels of Bi m and Puma mRNAs continued to increase until approximately 24 to 36 h and then decreased at later time points, presumably due to induction of cell deat h (Figures 15 and 16). Notably, a greater upregulation of Bim, especially Bim S, th e most potent isoform, and Puma mRNAs was observed in Hus1 -deficient cells, as compared to Hus1 -wild-type cells. Consistently, when cells were treated with a higher dose of etoposide for a shorter time course, a clear induction of Bim and Puma mRNAs occurred in a time-dependent manner, with more dramatic increases seen in Hus1 -deficient cells (Figures 17 and 18). Notably, the increase in Bim and Puma protein levels (Figure 10) in Hus1 -deficeint cells after etoposide

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44 treatment is greater than the increase in their mRNA levels (Figures 15 and 16), suggesting that etoposide-mediated upregulatio n of Bim and Puma is regulated through both transcriptional and post-tr anscriptional mechanisms. -actin Bim EL Puma Bim L Bim S Hus1-/-p21-/-Cycloheximide+ +ActinomycinD + +Etoposide+++ Figure 14. Induction of Bim and Puma ex pression in response to etoposide treatment is regulated at the transcriptional level. Hus1-/-p21-/MEFs were treated with control DMSO (-), 1 g/ml actinomycin D or 5 g/ml cycloheximide alone or in combination with 3.125 g/ml etoposide for 24 h. Total cell lysate was prepared and the expression of Bim and Puma was an alyzed by SDS-PAGE/immunoblot.

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45 Figure 15. Induction of Bim expression in response to etoposide treatment is regulated at the mRNA level. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for 0, 3, 6, 12, 24, 36 or 48 h. (a) Semi-quantitative RT-PCR was used to examine the mRNA level of Bim. (b, c, d) Quantification of the levels of (b) Bim EL, (c) Bim L and (d) Bim S. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBim S 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBim S Hus1+/+p21-/Hus1-/-p21-/0.0 0.5 1.0 1.5 2.0 2.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBimL 0.0 0.5 1.0 1.5 2.0 2.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBimL Hus1+/+p21-/Hus1-/-p21-/0.0 0.5 1.0 1.5 2.0 2.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBim EL 0.0 0.5 1.0 1.5 2.0 2.5 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionBim EL Hus1+/+p21-/Hus1-/-p21-/a b c d Bim EL Bim L Bim S GAPDH 0361224364803612243648 Etoposide (h) Hus1+/+p21-/-Hus1-/-p21-/-

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46 Figure 16. Induction of Puma expression in response to etoposide treatment is regulated at the mRNA level. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for 0, 3, 6, 12, 24, 36 or 48 h. (a) Semi-quantitative RT-PCR was used to examine the mRNA level of Puma. (b) Quantification of Puma expression. 01234 Hus1+/+p21-/-Hus1-/-p21-/-Etoposide (h) Bim EL Bim L Bim S GAPDH 01234 Figure 17. Etoposide-induced upregulation of Bim expression occurs at the transcriptional level. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 25 g/ml etoposide for 0, 1, 2, 3 or 4 h. Semi-quantitative RT-PCR was used to examine the mRNA level of Bim. a b Puma GAPDH 0361224364803612243648 Etoposide (h) Hus1+/+p21-/-Hus1-/-p21-/0.0 2.0 4.0 6.0 8.0 10.0 12.0 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionPuma 0.0 2.0 4.0 6.0 8.0 10.0 12.0 03612243648 Hours Etoposide (6.25 g/ml)Fold InductionPuma Hus1+/+p21-/Hus1-/-p21-/-

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47 Hus1+/+p21-/-Hus1-/-p21-/0123401234 Etoposide (h) Puma GAPDH Figure 18. Etoposide-induced upregulation of Puma expression occurs at the transcriptional level. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 25 g/ml etoposide for 0, 1, 2, 3 or 4 h. Semi-quantit ative RT-PCR was used to examine the mRNA level of Puma. Since we found that Bim and Puma are regulated, at least in part, at the transcriptional level, we investigated which transcription factors are responsible for the upregulation of these BH3-only proteins in response to DNA damage. As it has been shown that p53 can transcact ivate both Bim and Puma in response to DNA damage (Burns and El-Deiry, 2003; Nakano and Vousden, 2001), we first examined the possibility that p53 is respons ible for the etoposide-induced upregulation of these BH3only proteins. To this end, we examined the effect of loss of p53 on DNA damageinduced Bim and Puma expression by treating Hus1-/p53-/and Hus1-/p21-/MEFs with etoposide for varying time points. As shown in Figure 19, loss of p53 resulted in a slight inhibition of Bim expression and moderate inhibition of Puma expression. However, knockout of p53 did not affect etoposide-induced ce ll death or apoptos is, regardless of Hus1 status (Figures 20 and 21, re spectively). Taken t ogether, these results suggest that p53 is involved in inducing Puma expression and to a lesser extent Bim expression, but that other factors are also responsible for th e induction of Bim and Puma in response to

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48 etoposide treatment. FoxO3a and E2F1 are also candidates for transc ription factors that may regulate Bim and Puma expression, as both have been shown to upregulate the expression of these BH3-only proteins in response to DNA damage (Dijkers et al. 2000; Hershko and Ginsberg, 2004; Sunters et al. 2003; Yang et al. 2006). However, knockdown of either FoxO3a or E2F1 in Hus1 -deficient cells did not inhibit etoposideinduced upregulation of Bim or Puma expres sion (Figure 22 and 23, respectively). These results indicate that FoxO3a and E2F 1, along with p53, are not essential for the upregulation of Bim and Puma in re sponse to etoposide treatment in Hus1 -deficient cells. Therefore, further studies are needed to identify the transcription factors that are responsible for etoposide-induced Bim a nd Puma expression in cells lacking Hus1 p53 Puma Bim EL Bim L Bim S 0361224364803612243648 Etoposide Treatment (h) Hus1-/-p53-/-Hus1-/-p21-/-actin Figure 19. Loss of p53 suppresses DNA damage-induced Puma expression. Hus1-/-p53-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for the times indicated. Total cell lysate was prepared a nd the expression of p53, Bim and Puma were examined by SDS-PAGE/immunoblot analysis.

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49 0 10 20 30 40 50 60 70 80 90 100Percent Cell DeathHus1-/-p53-/-Hus1+/-p53-/-Hus1+/+p21-/-Hus1-/-p21-/0 10 20 30 40 50 60 70 80 90 100Percent Cell DeathHus1-/-p53-/-Hus1+/-p53-/-Hus1+/+p21-/-Hus1-/-p21-/Figure 20. Loss of p53 does not affect etoposide-induced cell death. Hus1+/-p53-/-, Hus1-/-p53-/-, Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide or control DMSO for 48 h. Cell death was m easured by trypan blue exclusion assay. The data shown represent the per cent cell death of etoposide-treated cells minus the percent cell death of control DMSO-treat ed cells (mean s.d.; n=3). 0 10 20 30 40 50 60 70 80 90 100Percent ApoptosisHus1-/-p53-/-Hus1+/-p53-/-Hus1+/+p21-/-Hus1-/-p21-/Figure 21. Loss of p53 does not affect etoposide-induced apoptosis. Hus1+/-p53-/-, Hus1-/-p53-/-, Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for 48 h. The cells were harvested and prepar ed for TUNEL staining and analysis by flow cytometry. The data shown represent the pe rcent apoptosis of etoposide-treated cells minus the percent apoptosis of control DMSO-treated cells (mean s.d.; n=3).

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50 E2F1 Bim EL Bim L Bim S Puma -actin 061224061224 Etoposide Treatment (h) ScrambledshE2F1 Hus1-/-p21-/-FoxO3a Puma Bim EL 0361224364803612243648 Etoposide Treatment (h) shScrambledshFoxO3a Tubulin Hus1-/-p21-/Figure 22. FoxO3a is not responsible fo r the upregulation of Bim and Puma expression in response to etoposide treatment. Hus1-/-p21-/MEFs stably expressing shRNA targeting FoxO3a or a control scrambled shRNA were treated with 6.25 g/ml etoposide for the times indicated. Total cell ly sate was prepared and the expression of FoxO3a, Bim and Puma were examined by SDS-PAGE/immunoblot analysis. Figure 23. E2F1 is not responsible for the upregulation of Bim and Puma expression in response to etoposide treatment. Hus1-/-p21-/MEFs stably expressing shRNA targeting E2F1 or a control scra mbled shRNA were treated with 6.25 g/ml etoposide for the indicated times. Total cell lysate was pr epared and the expression of E2F1, Bim and Puma were examined by SDS-PAGE/immunoblot analysis.

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51 Knockdown of Bim and Puma Confers Resist ance to Etoposide-Induc ed Apoptosis in Hus1-Deficient Cells Our results clearly show that loss of Hus1 not only results in the upregulation of Bim and Puma expression, but also promotes caspase-3 activation and cell death induced by etoposide treatment. Since BH3-only proteins play a key role in the initiation of apoptosis (Huang and Strasser, 2000; Puthalak ath and Strasser, 2002; Willis and Adams, 2005), we examined whether the upregulati on of Bim and Puma is responsible for sensitizing Hus1 -deficient cells to etoposide treatmen t. As a dramatic induction of all three isoforms of Bim was observed in re sponse to etoposide treatment, we first investigated whether inhibition of Bim e xpression would suppress DNA damage-induced cell death in Hus1-/-p21-/MEFs. Transfection of siRNA sp ecific for Bim abrogated its expression, even upon treatment with et oposide (Figure 24). Knockdown of Bim expression resulted in a decrease in PARP cl eavage (Figure 24) and partial resistance to DNA damage-induced cell death (Figure 25), as compared to siGFP or mock transfected cells. These results suggest that upregulat ion of Bim expression contributes to the sensitivity of Hus1 -deficient cells to et oposide-induced apoptosis. We next examined whether the upregulation of Puma expression is also involved in sensitizing Hus1 deficient cells to etoposide treatment. To this end, a lentiviral delivery system was used to transduce Hus1-/-p21-/MEFs with shRNA targeting Puma or Bim, or a control scrambled shRNA. Whereas etoposide treatment resu lted in the induction of Bim and Puma expression in control shScrambled expressing cells, the upregulation of these proteins was suppressed by their respectiv e shRNA (Figure 26). Consiste nt with the siBim results shown in Figure 24, expression of shBim suppressed etoposide-induced apoptosis

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52 (Figures 26 and 27). Moreover, knockdown of Puma expressi on significantly suppressed etoposide-induced caspase-3 ac tivation and apoptosis, as co mpared to control cells (Figures 26 and 27). Since knockdown of eith er Bim or Puma al one only partially suppressed etoposide-induced cell death, we ne xt examined whether Bim and Puma act redundantly or synergistically to induce apoptosis in respons e to etoposide treatment. To this end, shBim expressing Hus1-/-p21-/MEFs were infected with lentivirus expressing shRNA targeting Puma, which resulted in efficient knockdown of Puma expression (Figure 26). Importantly, knockdown of both Bim and Puma resulted in further inhibition of caspase-3 processing and apoptosis, when compared to Hus1-/-p21-/cells expressing shBim or shPuma alone (Figur es 26 and 27). Taken together, these results indicate that Bim and Puma cooperate in sensitizing Hus1 -deficient cells to etoposide-induced apoptosis.

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53 0 5 10 15 20 25 30 35Percent Cell DeathMock DMSO siGFPsiBim Mock Etoposide 0 5 10 15 20 25 30 35Percent Cell DeathMock DMSO siGFPsiBim Mock Etoposide Figure 24. Knockdown of Bim expressi on suppresses PARP cleavage in Hus1 deficient cells. Hus1-/-p21-/MEFs were mock transfected or transiently transfected with siRNA targeting GFP or Bim. Thirty-six hours after transfection, cells were treated with control DMSO or 6.25 g/ml etoposide for 30 h. Whole ce ll lysate was subjected to SDSPAGE/immunoblot analysis with antibodies to PARP (full length PARP is shown), Bim, Puma and Tubulin. The expression of Bim in DMSO-treated mock transfected cells represents the basal le vel of Bim expression. Figure 25. Knockdown of Bim expression co nfers resistance to etoposide-induced cell death in Hus1 -deficient cells. Hus1-/-p21-/MEFs were mock transfected or transiently transfected with siRNA targe ting GFP or Bim. Thirty-six hours after transfection, cells were treat ed with control DMSO or 6.25 g/ml etoposide for 30 h. Viability was determined by trypan blue excl usion assay (mean s.d.; n=2). The cell death of DMSO-treated mock transfected cells represents the basal le vel of cell death. Etoposide Mock DMSO Mock siGFP siBimPARP Bim EL TubulinHus1-/-p21-/-

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54 Bim EL Bim L Bim S Puma Tubulin++++Etoposide shBim shScramshPuma shBim/ shPuma Active Caspase-3 Hus1-/-p21-/Figure 26. Knockdown of Bim and Puma ex pression suppresses etoposide-induced caspase-3 cleavage in Hus1 -deficient cells. Hus1-/-p21-/MEFs were infected with lentivirus expressing shR NA targeting Bim, Puma, Bi m and Puma, or a control scrambled shRNA (shScram). After selection on puromycin, cells were treated with 12.5 g/ml etoposide or control DMSO for 16 h. Knockdown of Bim and Puma was confirmed by SDS-PAGE/immunoblot analysis.

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55 0 20 40 60 80 100Relative Apoptosis (%) shScram shBim shPuma shBim/shPuma Figure 27. Knockdown of Bim and Puma exp ression confers resistance to etoposideinduced apoptosis in Hus1 -deficient cells. Hus1-/-p21-/MEFs stably expressing shRNA targeting Bim, Puma, Bim and Puma, or a control scrambled shRNA (shScram) were treated with 12.5 g/ml etoposide or control DMSO fo r 16 h. Induction of apoptosis was measured by caspase-3 activity assay. The cas pase-3 activity of control DMSO-treated cells was subtracted from the amount of caspase-3 activity observed in the etoposide treated cells. The data are represented as pe rcent relative apoptosis as normalized to the control infected cells (mean s.d.; n=3). Loss of Hus1 Enhances the Binding of Rad9 to Bcl-2 to Potentiate the Apoptotic Response It has been shown that DNA damage promotes the binding of the 9-1-1 checkpoint complex to chromatin to initiate the DNA damage response and facilitate the activation of downstream pr oteins (Parrilla-Castellar et al. 2004; Zhou and Elledge, 2000). Consistently, Rad9 binding to chromatin was enhanced in Hus1 -wild-type cells in a time-dependent manner after etoposide trea tment (Figure 28). In contrast, chromatin bound Rad9 was barely detectable in Hus1 -deficient cells, although a slight increase was noticeable after etoposide treatment. These resu lts are consistent with previous findings

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56 which show that hydroxyureaand UV-indu ced binding of Rad9 to the chromatin is decreased in Hus1-/cells (Zou et al. 2002). Taken together, thes e results indi cate that loss of Hus1 results in a defect in the binding of Rad9 to chromatin in response to DNA damage. Chromatin Fraction Soluble Fraction Rad9 Rad9 RPA RPA 00 22 88EtoposideHus1+/+p21-/-Hus1-/-p21-/Chromatin Fraction Soluble Fraction Rad9 Rad9 RPA RPA 00 22 88EtoposideHus1+/+p21-/-Hus1-/-p21-/Figure 28. Loss of Hus1 results in a defect in the bi nding of Rad9 to chromatin. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 12.5 g/ml etoposide for 0, 2 or 8 h and subjected to subcellu lar fractionation. The resulting chromatin bound and soluble fractions were analyzed by SDS-PAGE/immunoblot using antibodies specific for Rad9 and RPA as a control. Previous evidence from our laboratory and others demonstrate that Rad9 can interact with Bcl-2 or BclxL through a BH3-like domain within its N-terminus to promote apoptosis following DNA damage (Ishii et al. 2005; Komatsu et al. 2000a; Komatsu et al. 2000b; Lee et al. 2003; Yoshida et al. 2002; Yoshida et al. 2003). These results indicate that Ra d9 not only has functions in th e nucleus as a member of a DNA damage checkpoint complex, but also in the cytosol as an inducer of apoptosis. Therefore, the effect of loss of Hus1 on the intracellular localization of Rad9 was

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57 examined. Consistent with previous studies (Burtelow et al. 2000), Rad9 was detected in both the nuclear and cytosolic fractions of Hus1+/+p21-/cells when analyzed by subcellular fractionation (Fi gure 29). Moreover, etoposide treatment resulted in Rad9 accumulation and hyperphosphorylation in the nucleus of Hus1+/+p21-/cells (Figure 29). In contrast, Rad9 was primarily dete cted in the cytosolic fraction of Hus1 -deficient cells and remained hypophosphorylated even upon DNA damage (Figure 29). These results suggest that Rad9 chromatin bi nding and hyperphosphorylation are Hus1 -dependent. Furthermore, immunofluorescent analysis re vealed that Rad9 formed punctate nuclear foci in Hus1 -wild-type cells after etoposide treatment, whereas the Rad9 signal accumulated in perinuclear foci upon treatment w ith etoposide in Hus1 -deficient cells (Figure 30). Tubulin Rad9 PARP + + + + + + Etoposide CytNucWCL CytNucWCL Hus1+/+p21-/-Hus1-/-p21-/Figure 29. Rad9 is predominantly detect ed in the cytosolic fraction of Hus1 -deficient cells. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 12.5 g/ml etoposide or control DMSO for 12 h and subjected to subc ellular fractionation. The resulting cytosolic (Cyt) and nuclear (Nuc) fracti ons, along with whole cell lysa te (WCL), were analyzed by SDS-PAGE/immunoblot.

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58 Etoposide Rad9 DAPI Merged Hus1+/+p21 -/Hus1-/-p21 -/+ + Etoposide Rad9 DAPI Merged Hus1+/+p21 -/Hus1-/-p21 -/+ + Figure 30. Rad9 is predominantly located in the cytosol of Hus1 -deficient cells. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 12.5 g/ml of etoposide or control DMSO for 12 h. Localization of Rad9 was an alyzed using fluorescence microscopy. In order to determine whether Rad9 binds to Bcl-2 family members during apoptosis, the interaction between Rad9 and Bcl-2 was examined in Hus1+/+p21-/and Hus1-/-p21-/MEFs in response to etoposide treatm ent. Since the majority of Rad9 was detected in the cytosolic fraction of Hus1 -deficient cells, co immunoprecipitation of cytosolic Rad9 with Bcl-2 was performed. While etoposide treatment enhanced Rad9 interaction with Bcl-2, a signi ficant amount of cytosolic Rad9 was bound to Bcl-2 even in the absence of DNA damage (Figure 31). Thus, it is possible that subcellular

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59 fractionation using a hypotonic buffer may alte r the conformation or localization of Rad9 and Bcl-2, which affects their interaction. Indeed, it has been shown that Rad9 can leak from the nucleus during subcellular fractio nation even in the absence of DNA damage (Burtelow et al. 2000). In order to confirm the in teraction of Rad9 with Bcl-2, the coimmunoprecipitation was repeated using whol e cell lysates. As shown in Figure 32, a minimal amount of Rad9 was bound to Bcl-2 in the absence of DNA damage, regardless of Hus1 status. Treatment with etoposide resulted in an induction of Rad9 binding to Bcl2 that was much greater in Hus1 -deficient cells as compared to Hus1 -wild-type cells. These results suggest that, in response to DNA damage, Rad9 may also contribute to the enhanced sensitivity of Hus1 -deficient cells through its in teraction with anti-apoptotic Bcl-2 family members.

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60 Figure 31. Binding of cytosolic Rad9 to Bcl-2 is increased upon treatment with etoposide and enhanced by loss of Hus1 Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 12.5 g/ml etoposide or control DMSO fo r 12 h. The cytosolic fractions were subjected to immunoprecipitation in the presence or absence of anti-Bcl-2 monoclonal antibody. The resulting immunoc omplexes were analyzed by SDSPAGE/immunoblot. The amount of Rad9 in th e immunocomplexes was quantified and normalized to cytosolic Rad9. The levels of Bcl-2 bound Rad9 are listed relative to those of untreated Hus1-/-p21-/cells, which was set as 1.0. ++ IB: anti-Rad9 Protein G IP: anti-Bcl-2 Cytosolic Fraction Rad9 -actin Etoposide Hus1+/+p21-/Hus1-/-p21-/0.670.771.001.59

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61 ++ IB: anti-Rad9 IP: anti-Flag IP: anti-Bcl-2 WCL Rad9 -actin Etoposide Hus1+/+p21-/Hus1-/-p21-/0.40 2.181.006.94 ++ IB: anti-Rad9 IP: anti-Flag IP: anti-Bcl-2 WCL Rad9 -actin Etoposide Hus1+/+p21-/Hus1-/-p21-/0.40 2.181.006.94 Figure 32. Etoposide-induced binding of Ra d9 to Bcl-2 is enhanced by loss of Hus1 Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 12.5 g/ml etoposide or control DMSO for 12 h. Whole cell lysate (WCL) wa s subjected to immunoprecipitation with anti-Bcl-2 or control anti-Flag monoclona l antibodies. The resulting immunocomplexes and WCL were analyzed by SDS-PAGE/i mmunoblot. The amount of Rad9 in the immunocomplexes was quantified and normali zed to total Rad9. The levels of Bcl-2 bound Rad9 are listed relative to those of untreated Hus1-/-p21-/cells, which was set as 1.0. We therefore examined whether Rad9 cooperates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-induced apoptosis. Hus1-/-p21-/MEFs stably expressing shBim and shPuma were infected with le ntivirus expressing shRNA targeting Rad9. Indeed, knockdown of Rad9 resulted in furt her inhibition of caspa se-3 activation, as compared to the shBim and shPuma expressi ng cells (Figures 33 a nd 34). Consistently, suppression of Rad9 expression in the shBi m and shPuma expressing cells conferred further resistance to etoposide-induced cell death (Figure 35). Ta ken together, these results indicate that Rad9 acts in colla boration with Bim and Puma to sensitize Hus1 deficient cells to etopos ide-induced apoptosis.

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62 Tubulin Active Caspase-3 Puma Bim EL Bim L Bim S Rad9 +Etoposide shBim/ shPuma/ shRad9 +shBim/ shPuma shScram + Hus1-/-p21-/Figure 33. Knockdown of Rad9 further supp resses caspase-3 activation in shBim and shPuma expressing cells. Hus1-/-p21-/MEFs stably expre ssing shBim and shPuma were infected with lentivirus expressing sh RNA targeting Rad9. Cells were treated with 12.5 g/ml etoposide or control DMSO for 16 h. Knockdown of Rad9, as well as Bim and Puma, was confirmed by SDS-PAGE/immunoblot analysis.

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63 Figure 34. Rad9 collaborates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-induced apoptosis. Hus1-/-p21-/MEFs stably expressing shBim and shPuma were infected with lentivirus expressing sh RNA targeting Rad9. Cells were treated with 12.5 g/ml etoposide or control DMSO for 16 h. Induction of apoptosis was measured using a caspase-3 activity assay. Figure 35. Rad9 collaborates with Bim and Puma to sensitize Hus1 -deficient cells to etoposide-induced cell death. Hus1-/-p21-/MEFs stably expressing control shRNA, shBim and shPuma, or shBim and shPuma plus shRad9 were treated with 12.5 g/ml etoposide for 0, 24 or 48 h. Cell death was de termined by trypan blue exclusion assay (mean s.d.; n=3). 0 10 20 30 40 50 60 70 80 90 02448Hours Etoposide (12.5 g/ml)Percent Cell Death shScrambled shBim/shPuma shBim/shPuma/shRad9 0 20 40 60 80 100Relative Apoptosis (%)shScram shBim/ shPuma shBim/ shPuma/ shRad9 0 20 40 60 80 100Relative Apoptosis (%)shScram shBim/ shPuma shBim/ shPuma/ shRad9

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64 Discussion In this study, we have de monstrated that loss of Hus1 results in the upregulation of the BH3-only proteins, Bim and Puma, whic h is partially responsible for sensitizing Hus1 -deficient cells to etoposide-induced apopt osis. In addition, we found that in the absence of Hus1 Rad9 functions as a BH3-only prot ein and cooperates with Bim and Puma to promote apoptosis in response to etoposide treatment. There are currently two models for the activation of apoptosis by the Bcl-2 family members: the direct model and the hierarchy model (Galonek and Hardwic k, 2006). The direct model proposes that BH3-only proteins have varying levels of poten cy due to their ability to bind various Bcl2-like family members (Certo et al. 2006; Chen et al. 2005; Willis et al. 2007). Thus, Bim, Puma and tBid are the most potent as they can bind all of th e anti-apoptotic Bcl-2 family members, whereas Noxa and Bad are le ss potent as they can only bind to a subset of the Bcl-2-like proteins. In the hierarc hy model, on the other hand, Bim and Puma, as well as tBid, are more potent as they act dow nstream of the other BH3-only proteins and the Bcl-2-like proteins and can bind directly to the multi-domain pro-apoptotic proteins, resulting in their activation a nd the induction of apoptosis (Kim et al. 2006; Kuwana et al. 2005; Letai et al. 2002). This model suggests that loss of both Bim and Puma would result in complete inhibition of apoptosi s mediated through the intrinsic pathway. Our results show that knockdown of both Bim and Puma diminishes the hypersensitivity of Hus1 -deficient cells to etoposide-induced apopt osis, indicating that both Bim and Puma indeed play a central role in the activa tion of this programmed cell death pathway. Interestingly, suppression of Rad9 expr ession in the Bim and Puma double-knockdown cells resulted in further inhibition of DNA da mage-induced apoptosis. Therefore, our data

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65 argue in favor of the direct model and suggest that it is the ratio of the BH3-only proteins to the Bcl-2-like proteins that regulates apoptosis. Our results demonstrate that Bim a nd Puma mRNA expression is induced by etoposide treatment. Among known transcription factors, p53, FoxO3a and E2F1 were the most likely candidates for regulators of Bim and Puma expre ssion in response to etoposide treatment, as these transcription f actors have been shown to upregulate both Bim and Puma in response to DNA damage (Burns and El-Deiry, 2003; Dijkers et al. 2000; Hershko and Ginsberg, 2004; Nakano and Vousden, 2001; Sunters et al. 2003; Yang et al. 2006). However, our results suggest that these transcripti on factors are not required for the upregulati on of Bim and Puma in Hus1 -deficient cells in response to DNA damage. While we cannot rule out the possi bility that loss of p53, FoxO3a or E2F1 results in compensation by othe r transcription factors, our data indicate that these transcription factors are not e ssential for the upregulation of Bim and Puma in response to etoposide treatment in Hus1 -deficient cells. Several other tr anscription factors have been shown to regulate BH3-only expression (S hibue and Taniguchi, 2006). It has been reported that Myc plays a role in th e enhanced expression of Bim (Egle et al. 2004). Additionally, JNK and its downstream pathwa y, c-Jun/AP-1, have been shown to be involved in the transcripti onal upregulation of Bim (Jin et al. 2006; Putcha et al. 2003; Whitfield et al. 2001). Furthermore, JNK was reporte d to phosphorylate p73 at several residues and this phosphorylation was require d for p73-mediated induction of Puma in response to DNA damage (Jones et al. 2007). It is of interest to determine whether these proteins are responsible for etoposide-induced Bim and Puma expression in cells lacking Hus1

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66 Rad9 has been shown to play a role in multiple cellular processes including: regulation of cell cycle checkpoints, transcript ional activation of p53 targets, initiation of DNA repair and when DNA repair is unfavor able, induction of apoptosis (Lieberman, 2006). It has been suggested that the primary fu nction of Rad9 is to act as a sensor in the DNA damage response pathway to promote surv ival by initiating cell cycle arrest and facilitating DNA repair (Brandt et al. 2006; Loegering et al. 2004). Thus, it is not surprising that loss of Rad9 sens itizes cells to DNA damage as these cells lack the ability to activate appropriate cell cycle checkpoints and DNA repair On the other hand, Rad9 may function as a pro-apoptotic factor in cells with unrep airable, excessively damaged DNA or a disrupted 9-1-1 complex, such as through loss of Hus1 Hus1 -deficient cells have a defective cell cycle checkpoint, thus making them more sensitive to DNA damage. In the absence of Hus1 Rad9 was found to be mostly located in the cytosol, where it formed perinuclear foci and associated w ith Bcl-2 in response to DNA damage. These results suggest that loss of Hus1 results in an abrogation of the nuclear functions of Rad9 and an augmentation of its pro-apoptotic functions. Taken together, the results presented here indicate that the 9-1-1 complex plays a critical role in the suppre ssion of etoposide-induced apopt osis by regulating the induction of the BH3-only proteins, Bim and Puma. Loss of Hus1 results in enhanced upregulation of these BH3-only proteins that initiate mitochondrial apoptosis in response to DNA damage. Moreover, disruption of the 9-1-1 complex, through loss of Hus1, switches Rad9 from functioning as a mediator of cell cycle checkpoints and DNA repair to an inducer of apoptosis. Thus, the 9-1-1 complex may act as a checkpoint sensor to decide whether a cell should survive or undergo apoptosis in response to DNA damage (Figure 36).

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67 Functional 9-1-1 complexDisrupted 9-1-1 complexDNA damage Bim and Puma expression Bim and Puma expression ApoptosisApoptosis Nucleus Cytoplasm Rad9 Rad9 binding to Bcl-2 family members Ra d 1Hus1Rad9R ad 1Rad9 Functional 9-1-1 complexDisrupted 9-1-1 complexDNA damage Bim and Puma expression Bim and Puma expression ApoptosisApoptosis Nucleus Cytoplasm Rad9 Rad9 binding to Bcl-2 family members Ra d 1Hus1Rad9R ad 1Rad9 Figure 36. Proposed model for the role of the Rad9-Rad1-Hus1 complex in the regulation of DNA damage-induced apoptosis. Cells with a functional 9-1-1 complex can suppress Bim and Puma expression in response to DNA damage, which results in resistance to apoptosis. In cells with a disrupted 9-1-1 complex, through loss of Hus1 exposure to DNA damaging agents results in the upregulation of Bim and Puma expression, which activates the mito chondrial apoptotic pathway. Loss of Hus1 also results in the cytoplasmic localization of Ra d9 and enhances the inte raction of Rad9 with Bcl-2 to potentiate th e apoptotic response.

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68 Materials and Methods Reagents Etoposide, camptothecin, hydroxyurea, protea se inhibitor cocktail, phosphatase inhibitor cocktails I and II, cycloheximid e and actinomycin D were purchased from Sigma (St. Louis, MO). Puromycin was purch ased from Calbiochem (San Diego, CA). Antibodies were purchased from the followi ng commercial sources : anti-tubulin, antiactin, anti-Flag and FITC-conjugated goat an ti-rabbit secondary antibody from Sigma, anti-Bim and anti-Puma from Calbiochem, anti-Mcl-1 from Rockland Immunochemicals (Gilbertsville, PA), anti-PARP and anti-cleav ed caspase-3 from Cell Signaling (Danvers, MA), anti-Bcl-2 from BD Pharmingen (S an Diego, CA), anti-RPA from Oncogene (Cambridge, MA), anti-GFP from Clontech (Mountain View, CA), anti-Bak and antiFoxO3a from Upstate (Lake Placid, NY), an ti-Bax and goat anti-rabbit IgG-horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Rad9 and antiBcl-xL polyclonal rabbit antisera have been previously described (Komatsu et al. 2000b). Protein G agarose beads, trypan blue and culture medium were purchased from Invitrogen (Carlsbad, CA). The Nucleofector machine, as well as all Nucleofector solutions, was purchased from Amaxa Biosystems (Gaithersburg, MD). Cell Culture, Transfection and Infection Hus1+/+p21 -/and Hus1-/-p21-/cells (Weiss et al. 2000), Hus1-/-p21-/GFP and Hus1-/p21-/Hus1 cells (Weiss et al. 2002), as well as Hus1+/-p53-/and Hus1-/-p53-/-cells (Weiss et al. 2002) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine seru m, 1.0 mM L-glutamine, 0.1 mM MEM non-

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69 essential amino acids, 100 g/ml streptomycin and 100 U/ml penicillin. The 21nucleotide siRNA duplexes targeting Bim a nd GFP were synthesized and purified by Dharmacon (Lafayette, CO). The siRNA se quence targeting mous e Bim mRNA was 5’AAUCAUGUACAAUCUCUUCAU-3’ The siRNA specific for GFP has previously been described (Hirai and Wang, 2002). Hus1-/-p21-/MEFs were tran sfected with 10 g of siRNA per 1 x 106 cells using the Nucleofector syst em. After transfection, cells were allowed to recover for 36 h. The medium, c ontaining any cells which may have died due to transfection, was removed and replaced wi th treatment medium containing control DMSO or etoposide. The pLKO.1-based lentiviral shRNAs targeting Bim (TRCN0000009692), Puma (TRCN0000009710) and Rad9 (TRCN0000012638) were purchased from Open Biosystems (Huntsville, AL). The pLKO.1-ba sed scrambled control shRNA vector was purchased from Sigma (St. Louis, MO). R ecombinant lentivirus was produced by cotransfecting the appropriate shRNA plas mid with the ViraPower Packaging Mix (Invitrogen) into 293FT cells. The resulting supernatant containing shRNA-expressing lentivirus was used to transduce Hus1-/-p21-/MEFs according to the manufacturer’s protocol. Analysis of Cell Death and Apoptosis Cell death was assessed by trypan blue ex clusion assay. Apoptosis was scored by the presence of nuclear chromatin condens ation and DNA fragmentation and evaluated by fluorescence microscopy. Briefly, cells were harvested, fixed in 4% paraformaldehyde for 10 min at room temperature and washed w ith PBS. Cell nuclei we re stained with 0.5

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70 g/ml bis-benzimide trihydrochloride (Hoech st 33258, Molecular Probes, Eugene, OR). At least 200 cells were counted for each sa mple and percent apoptosis was calculated [(apoptotic nuclei) / (all nucle i) x 100]. The induction of ap optosis was analyzed using a Caspase-3 Assay Kit (Sigma), an In Situ Cell Death Detectio n (TUNEL) kit (Roche Applied Science, Indianapolis, IN) and by examination of caspase-3 processing and PARP cleavage by SDS-PAGE/immunoblot analysis. Semi-Quantitative Reverse Transcription-PCR Semi-quantitative reverse transcripti on-PCR was performed using the Qiagen (Valencia, CA) OneStep RT-PCR syst em according to the manufacturer’s recommendations. The primers for Bim and GAPDH have been previously described (Wong et al. 2005). The primers for Puma ar e 5’-GTGATCCGGACACGAAGACT-3’ and 5’-GACTCTAAGTGCTGCTGGGC-3’. Fo r quantification, Bim and Puma mRNA levels were normali zed to GAPDH mRNA. Chromatin Fractionation Chromatin fractionation was carried out as described previously (Mendez and Stillman, 2000). Briefly, cells were washed twice with PB S and resuspended in buffer A (10 mM HEPES, pH 7, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT) containing protease inhibito rs. Triton X-100 was added to a final concentration of 0.1% and the cells were in cubated for 5 min on ice. The nuclei were collected by low-speed centrifugation (4 min, 1,500 x g, 4 C). The supernatant was clarified by high-speed centri fugation (15 min, 12,500 x g, 4 C) to remove insoluble

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71 aggregates and the resulting lysate was de signated the soluble fraction. The nuclei were washed once in buffer A and then lysed in buffer B (3 mM EDTA 0.2 mM EGTA, 1 mM DTT) with protease inhibitors for 10 min on ice. Insolubl e chromatin was collected by centrifugation at 2,000 x g for 4 mi n at 4 C and washed once in buffer B. The final pellet was resuspended in 2X Laemmli buffer, bo iled for 10 min and used as the chromatin fraction. Subcellular Fractionation and Coimmunoprecipitation Subcellular fractionation was performe d as previously described (Wang et al. 1996). Briefly, cells were washed with PBS then resuspended in hypotonic lysis buffer (5 mM Tris HCl, pH 7.5, 5 mM NaCl, 1.5 mM MgCl2, 0. 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, 10 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin and 10 g/ml pepstatin A). After incubation on ice for 30 min, cells were homogenized using a Dounce homogenizer. Samples were s pun down at 510 x g for 5 min at 4 C. The supernatant (cytosolic fraction) was cleared by centrifugation at 720 x g for 5 min at 4 C, while the pellet (nuclear fraction) was washed with hypotonic lysis buffer, centrifuged at 720 x g for 5 min at 4 C, then lysed in radioimmunoprecipitation assay buffer. Coimmunoprecipitation of Rad9 with Bc l-2 was performed as previously described with minor modifications (Yoshida et al. 2002; Yoshida et al. 2003). Briefly, whole cell lysate was prepared in 1% NP -40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, 10 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin and 10 g/ml pepstatin A). One milligram

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72 of lysate was precleared by incubation with protein G agarose beads for 1 h at 4 C. The precleared lysate was then incubated with anti-Bcl-2 (BD Pharmingen), or anti-Flag monoclonal antibody as negative control, overnight at 4 C. The immunocomplexes were then incubated with protein G agarose beads for 1.5 h at 4 C. After extensive washing in lysis buffer, the resulting immunocomplexes were subjected to SDS-PAGE/immunoblot analysis with anti-Rad9 polyclonal antibody. For coimmunoprecipitati on analysis of the cytosolic fraction, subcellular fractionation was performed as described above, then NP40, SDS and NaCl were added to a final concentration of 1%, 0.1% and 150 mM, respectively. Coimmunoprecipitation wa s performed as described above. Immunofluorescence Hus1+/+p21 -/and Hus1-/-p21-/MEF cells were grown on gelatin-coated glass coverslips. After treatment with etoposide, ce lls were washed once with PBS and fixed in 4% paraformaldehyde for 20 min at 4 C. Cells were then washed three times with PBS and permeabilized in 0.5% Triton X-100 plus 1% normal goat serum (NGS) for 30 min at room temperature. After four washes with PB S, the cells were blocked in 5% milk/ 3% BSA/ 1% NGS for several hours at 4 C. The cells were then incubated in primary antibody in blocking solution overnight at 4 C. After several washes with PBS, cells were incubated in blocking solution for 30 min at 37 C, then in FITC-conjugated goat anti-rabbit secondary antibody for one hour at 37 C. The cells were washed several times with PBS before the addition of mounting media containing DAPI (4’, 6’diamidino-2-phenylindole; Vector Laborator ies, Burlingame, CA). The fluorescent images were analyzed using an automated Zeiss Axiovert fluorescence microscope.

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73 Chapter Three: Loss of Hus1 Enhances DNA Damage-Induced Caspase-Independent Cell Death and Autophagy Abstract The Rad9-Rad1-Hus1 (9-1-1) complex plays a central role in the decision of whether a cell should survive or undergo cell death in response to DNA damage. Although it has been shown that disruption of the 9-1-1 complex sensitizes cells to certain genotoxic stresses, the precise mechan isms that are responsible for enhanced cell death are not fully understood. We have rece ntly described the mechanism behind the sensitivity of Hus1 -deficient cells to etoposide-induced apoptosis. Here, we provide evidence that loss of Hus1 also sensitizes cells to caspase-independent cell death. Treatment with the pan-caspase inhibito r, Z-VAD-FMK, only moderately inhibited camptothecin-induced cell death, suggesting that Hus1 -knockout cells die through a caspase-independent mechanism in response to camptothecin-induced DNA damage. Moreover, we found that loss of Hus1 enhances LC3 foci formation and modification, indicating that disruption of the 9-11 complex enhances DNA damage-induced autophagy. Interestingly, inhibition of autophagy, by knockdown of Atg7 or Bif-1, does not suppress, but rather promotes, camptoth ecin-induced cell death. Taken together, these results suggest that the 9-1-1 complex plays a key role in the regul ation of both caspasedependent and caspase-indepe ndent cell death in respons e to DNA damage and that autophagy is induced as a survival mech anism when this complex is disrupted.

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74 Results Loss of Hus1 Enhances Caspase-Independent Cell Death in Respons e to DNA Damage As mentioned above, we have found th at disruption of a functional 9-1-1 complex, through loss of Hus1 sensitizes cells to DNA damage-induced cell death (Meyerkord et al. 2008). Treatment of Hus1 -deficient cells with DNA damaging agents promotes the upregulation of the BH3-only proteins, Bim and Puma and enhances the interaction of Rad9 with Bcl-2 thereby inducing the activatio n of caspase-3 and apoptosis (Meyerkord et al. 2008). In order to determin e if apoptosis is the only mechanism that sensitizes Hus1 -deficient cells to DNA damage-induced cell death, Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with camptothecin in the presence or absence of the pan-caspase inhibitor, Z-VADFMK. Consistent with a previous report (Wang et al. 2004b), knockout of Hus1 greatly enhanced the su sceptibility of MEFs to camptothecin-induced cell death (Figure 37) Moreover, the induction of caspase-3 activity observed in Hus1 -deficient cells was greater than that in Hus1 -wild-type cells (Figure 38). Whereas caspase-3 was ac tivated in a time-dependent manner in Hus1 -wildtype cells, caspase-3 activity incr eased until the 24 h time point in Hus1 -deficient cells, after which, it decreased presumably due to cell death. Importantly, treatment with ZVAD-FMK completely inhibited the inducti on of caspase-3 activity, regardless of Hus1 status (Figure 38). Interestingly, the DNA damage-induced cell death observed in Hus1 deficient MEFs was only slightly suppre ssed by addition of Z-VAD-FMK (Figure 37), suggesting that an alternat e cell death mechanism is ac tivated when apoptosis is inhibited.

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75 0 10 20 30 40 50 60 70 DMSOZ-VAD-FMKDMSOZ-VAD-FMK 02 44 8 CPT Treatment (h)Percent Cell Death Hus1+/+p21-/Hus1-/-p21-/0 10 20 30 40 50 60 70 DMSOZ-VAD-FMKDMSOZ-VAD-FMK 02 44 8 CPT Treatment (h)Percent Cell Death Hus1+/+p21-/Hus1-/-p21-/Figure 37. Camptothecin-induced cell deat h is moderately inhibited by Z-VADFMK. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 1 M camptothecin (CPT) for 24 or 48 h in the presence or absence of 50 M Z-VAD-FMK. At the indicated times, cells were harvested and cell death was dete rmined by trypan blue exclusion assay (mean s.d.; n=3). 0 50 100 150 200 250 300 350 400 450 01224364812243648 DMSO Z-VAD-FMK CPT Treatment (h)Caspase-3 Activity ( FU/ g protein/h) Hus1+/+p21-/Hus1-/-p21-/0 50 100 150 200 250 300 350 400 450 01224364812243648 DMSO Z-VAD-FMK CPT Treatment (h)Caspase-3 Activity ( FU/ g protein/h) Hus1+/+p21-/Hus1-/-p21-/Figure 38. Camptothecin-induced caspase-3 activity is abrogated by Z-VAD-FMK. Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 1 M camptothecin (CPT) for the times indicated in the presence or absence of 50 M Z-VAD-FMK. At the indicated times, cells were harvested and the induction of apoptosis was measured by caspase-3 activity assay (mean s.d.; n=3).

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76 Loss of Hus1 Enhances DN A Damage-Induced Autophagy It has been shown that cells that are re sistant to apoptosis initiated through the mitochondrial pathway, such as those that lack both Bax and Bak (Wei et al. 2001; Zong et al. 2001), undergo caspase-ind ependent, but autophagy-dependent cell death in response to DNA damage (Shimizu et al. 2004). Autophagy is an evolutionarily conserved intracellular proce ss for the bulk degradation of cytoplasmic components that is initiated in response to environmen tal changes (Levine and Klionsky, 2004; Yoshimori, 2004). Although autophagy is genera lly thought to play a cytoprotective role by recycling nutrients under starvation conditions and prev enting the accumulation of damaged organelles, excessive autophagy c ould result in the overconsumption of functional proteins and organe lles, thus leading to cell death (Levine and Yuan, 2005; Tsujimoto and Shimizu, 2005). In order to ex amine whether autophagy is involved in the enhanced caspase-independe nt cell death observed in Hus1 -deficient cells, we established Hus1+/+p21-/and Hus1-/-p21-/MEFs that stably expresse d GFP-LC3, a well characterized marker for autophagy that is used to visualize autophagosomes (Kabeya et al. 2000). In control DMSO-treated cells, GFP-LC3 was mo stly located diffusely throughout the cell; although, the basal level of GFP-LC3 foci formation was higher in Hus1 -deficient cells (Figure 39). Treatment with rapamycin, whic h inhibits mTOR to induce autophagy (Noda and Ohsumi, 1998), resulted in the fo rmation of GFP-LC 3 foci in both Hus1 -wild-type and Hus1 -deficient MEFs, indicating that both of these cell lines are capable of inducing autophagy. Interestingly, in re sponse to treatment with eith er etoposide or camptothecin, GFP-LC3 foci formation was greatly induced in Hus1 -deficient cells, while relatively few foci were observed in Hus1 -wild-type cells (Figure 39). These results suggest that

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77 autophagy is induced in response to DNA da mage and that the level of DNA damageinduced autophagy is enhanced by loss of Hus1 In order to confirm these results, we next examined the effect of Hus1 -deficiency on LC3 modificatio n. During the induction of autophagy, LC3 is processed from a cytosolic form, LC3-I, to the phosphatidylethanolamine (PE)-conjugated, membrane-bound form, LC3-II (Kabeya et al. 2000; Klionsky et al. 2008). Consistent with the resu lts from the analysis of GFPLC3 foci formation, an accumulation of LC3-II was observed in response to etoposide treatment (Figure 40), indicati ng that autophagy is indeed being induced in response to DNA damage. Moreover, the modification of LC3 was much greater in Hus1 -deficient cells, as compared to Hus1 -wild-type cells (Figure 40). Ta ken together, these results indicate that loss of a func tional 9-1-1 complex promotes the induction of DNA damageinduced autophagy.

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78 Hus1+/+p21-/-Hus1-/-p21-/-DMSO Rapamycin Etoposide Camptothecin Hus1+/+p21-/-Hus1-/-p21-/Hus1+/+p21-/-Hus1-/-p21-/-DMSO Rapamycin Etoposide Camptothecin Figure 39. Loss of Hus1 enhances DNA damage-induced GFP-LC3 foci formation. Hus1+/+p21-/and Hus1-/-p21-/MEFs stably expressing GFPLC3 were treated with 12.5 g/ml etoposide or 500 nM camptothecin fo r 12 h or 500 nM rapamycin for 3 h. The localization of GFPLC3 was examined by fluorescence microscopy.

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79 LC3-I Tubulin+/++/++/++/+ -/--/--/--/-Hus1 0h12h24h48h Etoposide Treatment (6.25 g/ml)LC3-II Figure 40. DNA damage-induced LC3 modifi cation is enhanced by loss of Hus1 Hus1+/+p21-/and Hus1-/-p21-/MEFs were treated with 6.25 g/ml etoposide for the times indicated. Total cell lysate was prepared a nd the modification of LC3 was examined by SDS-PAGE/immunoblot analysis. Autophagy Plays a Cytoprotective Role in Response to DNA Damage As loss of Hus1 resulted in enhanced autopha gy in response to DNA damage, we next investigated whether autophag y is responsible for sensitizing Hus1 -deficient cells to caspase-independent cell deat h induced by camptothecin treatment. To this end, Hus1+/+p21 -/and Hus1-/-p21-/MEFs were infected with lentivirus expressing shRNA targeting Atg7, Bif-1 or a control scramble d shRNA. Both Atg7 and Bif-1 are required for autophagosome formation and are thus ke y regulators of the induction of autophagy. While Atg7 acts as the E1-like enzyme th at mediates both of the ubiquitin-like conjugation systems (Tanida et al. 1999), Bif-1 binds to UVRAG to regulate the activation of PI3KC3 (Takahashi et al. 2007). As shown in Figur e 41, the expression of Atg7 and Bif-1 was efficiently knocked down in cells stably expres sing shAtg7 or shBif-

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80 1, respectively. Consistent with etoposide treatment (Figure 40), LC3 modification was enhanced in shScrambled-expressing cells after camptothecin treatment (Figure 41). Similar to a previous study (Komatsu et al. 2005), loss of Atg7 expression completely abolished the induction of autophagy, as dete rmined by modification of LC3-I to LC3-II (Figure 41). Moreover, knockdown of Bif1 significantly suppressed LC3 modification (Figure 41), as previously reported (Takahashi et al. 2007). Consistently in response to camptothecin treatment a greater numb er of LC3 foci were observed in Hus1 -deficient cells expressing shScrambled (Figure 42). No tably, LC3 foci formation was suppressed by knockdown of either Atg7 or Bif-1 (Figure 42), suggesting that the initiation of autophagy is indeed being inhibited in these ce lls. To determine whether the induction of autophagy results in cell death in respons e to DNA damage, we treated the shBif-1, shAtg7 and control shScrambled-expressing cel ls with camptothecin and examined cell death as determined by trypan blue exclus ion. Surprisingly, inhi bition of autophagy, by suppression of either Atg7 or Bif-1, significantly enhanced cell death when compared to shScrambled-expressing control cells (Figure 43 ). These results suggest that in response to treatment with DNA damaging agents, autoph agy is induced to promote cell survival, rather than cell death.

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81 Atg7 Bif-1 LC3-I LC3-II ++++++CPT shScramshAtg7shBif-1 shScramshAtg7shBif-1 Hus1+/+p21-/-Hus1-/-p21-/-actin Figure 41. Knockdown of Atg7 or Bi f-1 suppresses LC3 modification. Hus1+/+p21-/and Hus1-/-p21-/MEFs stably expressing shAtg7, sh Bif-1 or a control scrambled shRNA (shScram) were treated with treated with 1 M camptothecin (CPT) for 36 h. Total cell lysate was prepared and LC3 modifica tion was examined by SDS-PAGE/immunoblot analysis.

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82 DMSODMSO CPTCPT Hus1+/+p21-/-Hus1-/-p21-/-shScram shAtg7 shBif-1 DMSODMSO CPTCPT Hus1+/+p21-/-Hus1-/-p21-/-shScram shAtg7 shBif-1 Figure 42. Knockdown of Atg7 or Bif-1 s uppresses DNA damage-induced LC3 foci formation. Hus1+/+ p21-/and Hus1-/-p21-/MEFs stably expressing shAtg7, shBif-1 or a control scrambled shRNA (shScram) were tr eated with 500 nM camptothecin (CPT) for 12 h. Immunocytochemistry was used to examine endogenous LC3 localization.

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83 0 10 20 30 40 50 60 70 024480244802448 shScram shBif-1 shAtg7 CPT Treatment (h)Percent Cell Death ** ** P 0.01 Hus1+/+p21-/Hus1-/-p21-/0 10 20 30 40 50 60 70 024480244802448 shScram shBif-1 shAtg7 CPT Treatment (h)Percent Cell Death ** ** P 0.01 Hus1+/+p21-/Hus1-/-p21-/Figure 43. Inhibition of autophagy results in enhanced cell death in response to camptothecin treatment. Hus1+/+p21-/and Hus1-/-p21-/MEFs stably expressing shAtg7, shBif-1 or a control scrambled shRN A (shScram) were treated with 1 M camptothecin (CPT) for the times indicated. Cell death wa s determined by trypan blue exclusion assay (mean s.d.; n=3). The BH3 Mimetic, ABT-737, Does Not Significantly Induce Autophagy As described above, BH3-only proteins can regulate autophagy by binding to Bcl2 thereby releasing Beclin 1 to induce autopha gosome formation and t hus the initi ation of autophagy (Liang et al. 1999; Maiuri et al. 2007a). We have shown that the expression of the BH3-only proteins, Bim and Puma, are dramatically upregulated in Hus1-deficient cells in response to camptothecin treatment (Meyerkord et al. 2008). In order to determine whether the upregulation of th ese BH3-only proteins is the underlying mechanism behind the enha nced autophagy seen in Hus1-deficient cells, we treated Hus1+/+p21-/and Hus1-/-p21-/MEFs stably expressing GFP-LC3 with the pharmacological BH3-mimetic, ABT-737. ABT737 binds to Bcl-2 and Bcl-xL and

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84 mimics BH3-only protein binding (Oltersdorf et al. 2005), thus releasing Beclin 1 to induce autophagy (Maiuri et al. 2007a). Therefore, if th e excess binding of BH3-only proteins to Bcl-2-like prot eins is the underlying mechan ism of enhanced autophagy induction in Hus1 -deficient cells, ABT-737 should prom ote GFP-LC3 foci formation in camptothecin-treated Hus1 -wild-type cells, such that the GFP-LC3 foci formation in these cells would be similar to that of Hus1 -deficient cells trea ted with camptothecin alone. However, co-treatment of ABT-737 a nd camptothecin resulted in only a slight induction of autophagy in Hus1 -wild-type cells, indicating th at upregulation of BH3-only proteins is not the main mechanism responsible for DNA damage-induced autophagy in these cells (Figure 44).

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85 Hus1+/+p21-/-Hus1-/-p21-/-DMSOCPT +ABT-737 CPT Figure 44. ABT-737 does not significantl y enhance GFP-LC3 foci formation. Hus1+/+p21-/and Hus1-/-p21-/MEFs stably expressing GFPLC3 were treated with 500 nM camptothecin (CPT) in the presence or absence of 10 M ABT-737 for 12 h. (a) The localization of GFPLC3 was examined by fluorescence microscopy. (b) The number of GFP-LC3 dots per Hus1+/+ p21-/cell was determined by analyzing images from a fluorescent microscope. a b 0 50 100 150 200 250 CPTCPT + ABT-737P = 0.32Number of Autophagosomes (GFP-LC3 dots)/cell 0 50 100 150 200 250 CPTCPT + ABT-737P = 0.32Number of Autophagosomes (GFP-LC3 dots)/cell

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86 Discussion In the present study, we have demonstr ated that the 9-1-1 complex plays an integral role in the regulation of autoph agy and cell death indu ced by treatment with DNA damaging agents. Co-treatment of ZVAD-FMK with camptothecin resulted in only a moderate inhibition of cell death, regardless of Hus1 status. These results suggest that DNA damage not only promotes apoptos is, as mentioned previously (Meyerkord et al. 2008), but also induces a caspase-independ ent form of cell death when caspase activity is inhibited. Nota bly, a greater induction of cell death was observed in camptothecin-treated Hus1 -deficient MEFs as compared to Hus1 -wild-type MEFs, even in the presence of Z-VAD-FMK, indicating that the 9-1-1 complex may play a role in the suppression of both caspase-dependent and caspase-independent cell death induced by DNA damage. In addition to the role of the 9-1-1 co mplex in the suppression of DNA damageinduced cell death, we have also found that the 9-1-1 complex is responsible for the suppression of autophagy. DNA damage-indu ced autophagosome formation was enhanced when the 9-1-1 complex was disrupted through loss of Hus1 Notably, loss of Hus1 enhanced the level of autophagosome fo rmation, even in the absence of DNA damage. Interestingly, it has been shown that Hus1 -deficient MEFs have an increased frequency of spontaneous chromosomal a bnormalities and increas ed expression of DNA damage-responsive genes (Levitt et al. 2007; Weiss et al. 2000; Zhu and Weiss, 2007). Therefore, one possible explana tion for the enhanced autopha gy observed in these cells is to dispose of mitochondria that have b een damaged through activation of the DDR. Indeed, it has been shown that DNA damage-i nduced autophagy is involved in the early

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87 removal of damaged mitochondria (Abedin et al. 2007). Consistent with previous reports (Abedin et al. 2007; Kanzawa et al. 2004; Katayama et al. 2007; Paglin et al. 2001), we found that autophagy can be induced after exposure to DNA damaging agents. Furthermore, while both Hus1 -wild-type and Hus1 -deficient cells are capable of inducing autophagy, DNA damage-induced autophagy was enhanced in cells lacking Hus1 It has also been shown that inhibition of DNA-PK, whic h plays a major role in the repair of IRinduced double-strand DNA breaks, sensitizes cells to IR thro ugh the induction of autophagy (Daido et al. 2005). Taken together, these results suggest that loss of either DNA repair machinery or a functional DDR can sensitize cells to DNA damage-induced autophagy. Alternatively, loss of Hus1 could affect the induction of autophagy by disrupting signaling upstream of Akt and mT OR. Akt phosphorylation of mTOR results in the suppression of autophagy induction (Kondo et al. 2005). In addition, it has been reported that knockdown of ATR prevents Akt activation in response to genotoxic stress (Caporali et al. 2008). Moreover, Hus1 has been s hown to be required for signaling downstream of ATR (Weiss et al. 2002), suggesting that loss of Hus1 may also disrupt signaling pathways that are required for the suppression of autophagy. Furthermore, inhibition of mTOR, by treatment with rapamy cin, induced similar levels of autophagy in both Hus1 -wild-type and Hus1 -deficient cells. Taken together these results indicate that signaling downstream of Akt and mTOR is important for the suppression of autophagy and that disruption of this signaling, by loss of Hus1 could promote autophagy. While the precise mechanism linking the 9-1-1 complex to the autophagic machinery has yet to be determined, our results sugge st that this may occur indepe ndent of the upregulation of

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88 BH3-only proteins in Hus1 -deficient cells, which convers ely was shown to be required for DNA damage-induced apoptosis (Meyerkord et al. 2008). Evidence from various studies suggests th at autophagy plays a dual role in cell survival and cell death (Baehrecke, 2005; Ts ujimoto and Shimizu, 2005). Indeed, it has been shown that autophagy is induced in Bax / Bak-/cells after treatment with etoposide (Shimizu et al. 2004) and in L929 fibroblast ce lls treated with Z-VAD-FMK (Yu et al. 2004). Furthermore, the induction of autophagy ob served in these cells is required for cell death (Shimizu et al. 2004; Yu et al. 2004). In contrast, we found that inhibition of autophagy, through knockdown of Atg7 or Bif-1, re sulted in enhanced cell death in response to treatment with camptothecin, s uggesting that autophagy is induced as a cytoprotective mechanism in these cells in response to DNA damage. Therefore, it is possible that autophagy may function as an al ternate cell death mechanism only when the apoptotic pathway is inhibi ted, such as through loss of Bax / Bak or by treatment with ZVAD-FMK. However, if the apoptotic machinery is intact, such as in shAtg7and shBif1-expressing Hus1-/-p21-/cells, DNA damage-induced autophagy may play a prosurvival role. Accumulating evid ence now suggests that this ma y indeed be the case, that autophagy may act primarily as a survival me chanism by which the cell rids itself of potentially harmful constituents in order to maintain cellular homeostasis (Kroemer and Levine, 2008; Levine and Kroemer, 2009). Further studies, in which Hus1-/-p21-/cells expressing shAtg7 or shBif-1 ar e treated with camptothecin in the presence or absence of Z-VAD-FMK, will be required to determin e whether DNA damage-induced autophagy acts as a cell death or survival mech anism in apoptosis-impaired cells.

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89 Materials and Methods Reagents Etoposide, camptothecin, digitonin, proteas e inhibitor cocktail and phosphatase inhibitor cocktails I and II were purchased from Sigma (St. Louis, MO). Z-VAD-FMK and rapamycin were purchased from Alexis Biochemicals (San Diego, CA). Puromycin was purchased from Calbiochem (San Diego, CA). Trypan blue and culture medium were purchased from Invitrogen (Carlsbad, CA ). ABT-737 was a kind gift from Abbott Laboratories. Antibodies were purchased fr om the following comme rcial sources: antitubulin, anti-actin and FITC-conjugated goat anti-rabbit secondary antibody from Sigma, anti-LC3 from Novus Biologicals (L ittleton, CO) and MBL International (Nakaku Nagoya, Japan), anti-Bif-1 from GeneTex (San Antonio, TX), bovine anti-goat and bovine anti-mouse IgG-horseradi sh peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA) and goat anti-rabbit IgG-horseradis h peroxidase from Amer sham Biossciences (Piscataway, NJ). The anti-Atg7 antibody was a generous gift from Dr. Isei Tanida (Tanida et al. 1999). Cell Culture, Transfection and Infection Hus1+/+p21 -/and Hus1-/-p21-/MEFs (Weiss et al. 2000) were maintained in Dulbecco’s modified Eagle’s medium supplem ented with 10% fetal bovine serum, 1.0 mM L-glutamine, 0.1 mM MEM non-essential amino acids, 100 g/ml streptomycin and 100 U/ml penicillin. To generate the G FP-LC3 expression vector, cDNA encoding a GFP-LC3 fusion protein wa s subcloned into the Bgl IIEco R I site of the pK1-IRES-puro

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90 vector. Amphotrophic 293T cells were tr ansfected with the GFP-LC3 vector. The resulting recombinant retrovi rus was used to infect Hus1+/+p21 -/and Hus1-/-p21-/cells. The pLKO.1-based lentiviral shRN As targeting Atg7 (TRCN0000007586) and Bif-1 (TRCN0000093178) were purchased from Op en Biosystems (Huntsville, AL). The pLKO.1-based scrambled control shRNA v ector was purchased from Sigma. The appropriate shRNA plasmid, along with the ViraPower Packaging Mix (Invitrogen), was co-transfected into 293FT cells to produce recombinant lentivir us. The supernatant containing the shRNA-expressing le ntivirus was used to infect Hus1+/+p21 -/and Hus1-/-p21-/cells MEFs according to the manufacturer’s protocol. Analysis of Cell Death and Apoptosis Cell death was assessed by trypan blue exclusion assay. The induction of apoptosis was analyzed using a Caspas e-3 Assay Kit (Sigma) according to the manufacturer’s protocol. Analysis of LC3 Localization For analysis of GFP-LC3 localization, Hus1+/+p21 -/and Hus1-/-p21-/MEF cells were grown on gelatin-coated chamber slides After treatment, cells were washed once with PBS, then fixed in 3.7% formaldehyde for 7 min at room temperature. The cells were washed three times with PBS before being mounted with media containing DAPI (4’, 6’-diamidino-2-phenylindole; Vector Laboratories, Bur lingame, CA). The fluorescent images were obtained using an automated Zeiss Axiovert fluorescence microscope.

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91 To examine the localization of endogenous LC3, cells were washed once with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were then washed three times with PBS and permeabilized in 100 g/ml digitonin for 15 min at room temperature. After three washes with PBS, the cells were blocked in 3% BSA for one hour at room temperature. Cells were th en incubated in primary antibody in blocking solution overnight at 4 C. After three washes with PBS, cells were incubated in 3% BSA blocking solution for 30 min, then in FITC-conjugated goat anti-rabbit secondary antibody for three hours at room temperature. The cells were wash ed three times with PBS before the addition of mounting media containing DAPI. The fluorescent images were analyzed using an automated Zei ss Axiovert fluorescence microscope. Analysis of LC3 Modification Modification of LC3 was analyzed by SDS-PAGE/immunoblot. After treatment, cells were harvested, washed once in ice-cold PBS, then lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-H Cl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate and 5 mM EDTA pH 8.0) containing protease and phosphatase inhibitor cocktails. Tota l cell lysate was subjected to SDSPAGE/immunoblot analysis with anti-LC3 antibody.

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92 Chapter Four: Knockdown of Bif-1 Acceler ates Endocytic Vesicle Trafficking and Enhances EGFR Degradation Abstract The Endophiln proteins are well known regulators of intracellular membrane dynamics. While the members of the Endophilin A subfamily regulate the formation of endocytic vesicles at the plasma membrane members of the Endophilin B subfamily are involved in regulating the membrane dynamics of organelles, such as the Golgi complex, mitochondria and autophagosomes. While th e mechanisms by which Bif-1/Endophilin B1 regulates membrane dynamics are well studied, th e role of Bif-1 in endocytic trafficking is not well defined. In this study, we repor t that knockdown of Bi f-1 expression does not affect the uptake of a fluid phase marker, horse radish peroxidase, or the internalization of EGF. However, loss of Bif-1 results in th e premature localizati on of EGF to late endosomes/lysosomes. Moreover, knockdown of Bif-1 accelerates the degradation of EGFR in response to EGF stimulation. We f ound that EGFR degrad ation is regulated by both the lysosomal and proteasomal pathways in Bif-1-knockdown cells. Taken together, these results identify Bif-1 as a novel regul ator of endocytic ve sicle trafficking and receptor degradation.

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93 0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350 0.400 020406080100Time (min)HRP Uptake (units/ g protein) WT K/DResults Knockdown of Bif-1Does Not Affect Internal ization of Endocytic Cargo, but Accelerates EGF Co-Localization with Late Endosomes/Lysosomes It has been shown that Bif-1 can in teract with Beclin 1 through UVRAG to promote the activation of PI3KC3/Vps34 and the induction of autophagy (Takahashi et al. 2007). The activation of Vps34 plays an esse ntial role not only in the regulation of autophagy, but also in vesicle transport, including endocytic trafficking (Backer, 2008). As loss of Bif-1 significantly reduces the ac tivity of Vps34, we inve stigated whether Bif1 also plays a role the regulat ion of endocytic trafficking. To determine whether loss of Bif-1 would affect the integrity of the early endocytic pathway, we first examined the effect of knockdown of Bi f-1 expression on the uptake of a fluid phase marker, horseradish peroxidase (HRP). As shown in Figure 45, knockdow n of Bif-1 did not affect the kinetics of HRP uptake, suggesting that Bif1 is not involved in the regulation of the early endocytic pathway a nd HRP internalization. Figure 45. Knockdown of Bif-1 does not affe ct the internalization of a fluid phase marker. Wild-type (WT) and Bif-1-knockdown (K /D) HeLa cells were incubated in uptake media for the times indicated. HRP ac tivity was measured using a 1-Step Turbo TMB-ELISA kit. Enzyme activity was normalized to protein concentration (mean s.d.; n =3).

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94 In order to directly investigate the role of Bif-1 in endocytic trafficking, we examined EGF internalization and endocytic tr ansport to lysosomes. In response to serum starvation, the EGFR accumulates on the cell surface. After stimula tion with EGF, the EGFR is rapidly activated by phosphorylation of the C-terminal cytoplasmic domain. The EGF-EGFR complex is then internalized, sorted into multivesicular bodies (MVBs), delivered to lysosomes and degraded, whic h efficiently downregulates EGFR signaling pathways (Katzmann et al. 2002). Therefore, disruptions in the endocytic pathway can be identified by changes in the localizati on of the EGF-EGFR complex. As shown in Figure 46, similar amounts of fluorescently c onjugated EGF were initially bound to the plasma membranes of Bif-1-knockdown cells and control wild-type HeLa cells, indicating that EGF-receptor binding was not affected by lo ss of Bif-1. Fifteen minutes after stimulation the EGF signal formed small foci that were located throughout wild-type cells. In contrast, the EGF signal accumulate d in large aggregates and partially colocalized with LAMP-1, a ma rker of late endosomes/lysosomes, in Bif-1-knockdown cells after 15 min of EGF stimulation. No tably, the difference in EGF localization between Bif-1-knockdown cells and control w ild-type cells was more pronounced at 30 min after stimulation. In wild-t ype cells the EGF signal formed larger foci and began to co-localize with LAMP-1, whereas the EG F signal was decreased in Bif-1-knockdown cells, suggesting that the degradation of EGF is accelerated by loss of Bif-1. To determine if the observed effect of loss of Bi f-1 on the endocytic system was due to the inhibition of autophagy, through knockdown of Bif-1, we established HeLa cells that stably expressed shRNA targeting Atg5 or a control scrambled shRNA. Atg5 plays a role in one of the ubiquitin-like conjugation syst ems and is thus required for autophagosome

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95 formation and the induction of autophagy (Mizushima et al. 2001). Importantly, knockdown of Atg5 did not result in an appreciable difference in the localization of EGF or co-localization with LAMP-1 at any of the times examined (Figure 47), indicating that inhibition of autophagy does not affect the endo cytic trafficking and degradation of EGF. Taken together, these results describe a nove l role for Bif-1 in the regulation of EGF trafficking to late endosomes/lysosomes th rough an autophagy-independent mechanism. Figure 46. Knockdown of Bif-1 accelerates th e co-localization of EGF with LAMP-1positive vesicles. The localization of Alexa Fluor 488-EGF (green) in wild-type (WT) and Bif-1-knockdown (K/D) HeLa cells was de tected by confocal microscopy. At the times indicated, the cells were fixed, pe rmeabilized, stained for LAMP-1 (red) and mounted in media containing DAPI (blue). All images were taken at the same exposure setting for the 488-EGF signal. 0 min 15 min 30 min WT K/D

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96 Figure 47. Knockdown of Atg5 does not affect EGF localization. The localization of Alexa Fluor 488-EGF (green) in control shSc rambled (shScram) or shAtg5-expressing HeLa cells was monitored by confocal micros copy. At the times indicated, the cells were fixed, permeabilized, stained for LAMP-1 (re d) and mounted in media containing DAPI (blue). All images were taken at the same exposure setting for the 488-EGF signal. Knockdown of Bif-1 Promotes EGFR Degradation Since loss of Bif-1 accelerated th e co-localization of EGF to late endosomes/lysosomes, we next investigated th e effect of loss of Bif-1 on the degradation of EGFR. To this end, we examined the expr ession of total and phosphorylated EGFR in response to stimulation with EGF. Indeed, the degradation of EGFR was enhanced by knockdown of Bif-1 (Figures 48 and 49). Moreover the rate of degradation of activated EFGR was markedly accelerated in Bi f-1-knockdown cells (Figures 48 and 50), suggesting that upon EGF stimulation, EGFR-m ediated signaling is downregulated more shAtg5 shScram 0 min 15 min 30 min

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97 rapidly in cells lacking Bif-1. Interestingly, the expression of EGFR before stimulation with EGF was lower in Bif-1-knockdown cells as compared to control wild-type cells, suggesting that loss of Bif-1 also affects th e stability or turnov er of EGFR. Notably, knockdown of Atg5 did not affect the degrad ation of total or phosphorylated EGFR (Figure 51), which is consistent with the EG F localization data (Fi gure 47). In support of our results, a recent study has shown that loss of Bif-1 resulted in the premature targeting of the TrkA receptor to late endosomes and ly sosomes, which resulted in the accelerated the degradation of TrkA (Wan et al. 2008). Taken together, these results suggest that Bif-1 not only plays a role in autophagy, as previously de scribed, but that Bif-1 also regulates endocytic vesicle traffick ing and the degradation of EGFR. Figure 48. Knockdown of Bif-1 enhances EGFR degradation. Wild-type (WT) and Bif-1-knockdown (K/D) HeLa cells were serumstarved overnight. Cells were incubated with 100 ng/ml EGF for the times indicated, th en washed and harves ted. Total cell lysates were subjected to SDS-PAGE/i mmunoblot analysis with anti bodies specific for total and phosphorylated EGFR (Tyr 1068). Total EGFR EGF Stimulation (h) Tubulin WTK/D00.5123400.51234 pEGFR(Tyr 1068)

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98 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 00.51234EGF Stimulation (h)EGF R WT K/D***P < 0.05 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 00.51234EGF Stimulation (h)EGF R WT K/D***P < 0.05 0 20 40 60 80 100 120 140 00.51234EGF Stimulation (h)EGFR (% of time 0 ) WT K/D Figure 49. Quantification of total EGFR levels and degradation. Cells were treated and lysate prepared as in Figure 48. Data from three independent experiments were analyzed (mean s.d.). (a) Quan tification of total EGFR leve ls. The levels of EGFR are listed relative to that of unstimulated wild-t ype cells, which was set as 1. (b) Quantification of the percen t EGFR relative to time 0 h. a b

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99 0.0 0.5 1.0 1.5 2.0 2.5 0.51234EGF Stimulation (h)pEGFR/total EGFR WT K/D Figure 50. Quantification of phosphoryla ted EGFR levels and degradation. Cells were treated and lysate prepared as in Figure 48. Data from three independent experiments were analyzed (mean s.d.). (a) Quantification of the ratio of phosphoEGFR to total EGFR. (b) Quantification of the percent phospho-EGFR relative to time 0.5 h. a b 0 20 40 60 80 100 120 0.51234EGF Stimulation (h)pEGFR (% of time 0.5) WT K/D*P < 0.05* 0 20 40 60 80 100 120 0.51234EGF Stimulation (h)pEGFR (% of time 0.5) WT K/D*P < 0.05* *

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100 Figure 51. Knockdown of Atg5 does not affect EGFR degradation. HeLa cells stably expressing shAtg5 or a control scrambled sh RNA were cultured in serum-free DMEM overnight. Cells were incubated with 100 ng/ml EGF for the times indicated, then washed and harvested. Total cell lysate s were subjected to SDS-PA GE/immunoblot analysis with antibodies specific for total and phosphorylated EGFR (Tyr 1068). EGFR Degradation is Mediated through Both Proteasomal and Lysosomal Mechanisms The degradation of EGFR has been show n to be regulated by both the lysosomal and proteasomal pathways (Ettenberg et al. 2001; Levkowitz et al. 1999; Levkowitz et al. 1998; Longva et al. 2002). However, the precise mechanisms that regulate EGFR degradation are not fully unders tood. In order to investigat e the mechanism by which the degradation of the EGFR is enhanced by knockdown of Bif-1, we treated Bif-1knockdown and wild-type cells with bafilomycin A1 or MG132, which are well known inhibitors of the lysosomal and proteasoma l pathways, respectively (Lee and Goldberg, 1998; Yoshimori et al. 1991). As shown in Figure 52, treat ment with either bafilomycin A1 or MG132 inhibited the degradation of EG FR. In contrast to a previous study (Alwan et al. 2003), our results indicated that treatment w ith bafilomycin A1 does not pEGFR (Tyr 1068) Total EGFR Atg5 Tubulin 0 0.5 1 2 400.5124 EFG Stimulation (h) shScrambled shAtg5

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101 completely inhibit EGFR degradation. Inte restingly, the EGFR was still substantially degraded in Bif-1-knockdown cells, despite treatment with bafilomycin A1 or MG132, suggesting that the lysosomal and prot easomal pathways may be functionally compensating for one another. To examine this possibility, Bif-1-wild-type and knockdown cells were treated with either bafilomycin A1 or MG132 alone or in combination and the degradation of EGFR wa s examined. Co-treatment of bafilomycin A1 and MG132 further suppressed the degrada tion of EGFR as compared to treatment with either inhibitor alone (F igure 53). These results suggest that the lysosomal and the proteasomal pathways collaborate to regulate the degradation of EG FR after stimulation with EGF. SFM SFM Baf MG132 Baf MG132 Baf MG132 Baf MG132 0.5 h EGF0.5 h EGF 3 h EGF3 h EGF WT K/DpEGFR(Tyr1068) Total EGFR Tubulin Figure 52. The lysosomal and proteasomal pathways regulate EGFR degradation. Wild-type (WT) and Bif-1-knockdown (K/D) He La cells were serum-starved overnight, then treated with 100 ng/ml EGF either alon e or in combination with either 250 nM bafilomycin A1 or 10 M MG132 for 0, 0.5 or 3 h. The degradation and phosphorylation of EGFR was examined by SDS-PAGE/immunoblot analysis with anti bodies specific for total and phosphorylated EGFR (Tyr1068).

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102 SFM SFM Baf Baf/MG132 Baf Baf/MG132 Baf Baf/MG132 Baf Baf/MG132 0.5 h EGF0.5 h EGF 3 h EGF3 h EGF WT K/DpEGFR(Tyr1068) EGFR Tubulin SFM SFM MG132 Baf/MG132 MG132 Baf/MG132 MG132 Baf/MG132 MG132 Baf/MG132 0.5 h EGF0.5 h EGF 3 h EGF3 h EGF WT K/DpEGFR(Tyr1068) EGFR Tubulin Figure 53. The lysosomal and proteasomal pa thways collaborate to regulate EGFR degradation. Wild-type (WT) and Bif-1-knockdow n (K/D) HeLa cells were serumstarved overnight then stimulated with 100 ng/ ml EGF in the presence of either 250 nM bafilomycin A1 (Baf) or 10 M MG132 or a combination of bafilomycin A1 and MG132 for 0, 0.5 or 3 h. The levels of phosphorylated and total EGFR were analyzed by SDSPAGE/immunoblot analysis. (a) Cells were tr eated with bafilomycin A1 alone or in combination with MG132. (b) Cells were treat ed with MG132 alone or in combination with bafilomycin A1. a b

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103 Discussion In this study, we have de scribed a novel role for Bi f-1 in the regulation of endocytic trafficking and receptor degradation. Our observations indicate that Bif-1 plays a critical role in the regulation of the later stages of the endocytic pathway with little affect on internalization. Examination of th e levels of both activated and total EGFR, by SDS-PAGE/immunoblot analysis, revealed that knockdown of Bif-1 enhanced the degradation of EGFR, especia lly the activated form. Furt hermore, knockdown of Bif-1 lead to the premature targeting of EGF to LAMP-1-positive foci, suggesting that the accelerated degradation of th e EGF-EGFR complex could be the result of precocious localization to late endosomes/lysosomes. Degradation of the EGFR appears to be mediated by both lysosomal and proteasomal mechanisms, as co-treatment with bafilomycin A1 and MG132 resulted in furt her suppression of EGFR degradation than treatment with either inhib itor alone. Taken together, our results suggest that although loss of Bif-1 accelerates intracellular traffi cking and receptor degradation, it does not affect internalization of endoc ytic cargo. However, further st udies are needed to rule out the possibility that Bif-1 may pl ay a role in vesicular traffi cking through the early steps of the endocytic pathway. In support of our findings, a recent repor t described a role for Bif-1 in the regulation of the endocytic tr afficking of nerve growth f actor (NGF)-tropomyosin-related kinase A (TrkA) (Wan et al. 2008). Consistent with our re sults, Ip and colleagues found that knockdown of Bif-1 did not affect the internalization of a fluid phase marker; however, they demonstrated that Bif-1 may regulate the size of early endosomes. In addition, knockdown of Bif-1 resulted in the premature trafficking of internalized NGF-

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104 TrkA to lysosomes. Furthermore, loss of Bi f-1 enhanced the degradation of TrkA and diminished signaling downstream of NGF-TrkA. Taken together, these studies indicate that Bif-1 plays a critical ro le in the regulation of endocyt ic vesicle trafficking and the degradation of inte rnalized receptors. Our laboratory has demonstrated that Bi f-1 can interact w ith Beclin 1 through UVRAG to promote the activation of PI3KC3/Vps34 (Takahashi et al. 2007). As mentioned above, the activation of Vps34 plays an critical role not only in the induction of autophagy, but also in the regulation of vesicle transport, including endocytic trafficking (Backer, 2008). It has recently been shown that knockdown of Vps34 reduced the rate of EGFR degradation and disrupt ed the invagination of late endosomes preventing the formation of MVBs (Johnson et al. 2006). As Bif-1 has been shown to promote the activity of Vps34, it would be a ssumed that loss of Bif-1 should therefore result in a decrease in endocytic vesicle tr afficking and a reduction in EGFR degradation. However, our results suggest that knockdown of Bif-1 doe s not suppress, but rather accelerates, endocytic traffick ing and EGFR degradation. One possible explanation for this discrepancy could be that Bif-1 suppr esses these processes by binding to UVRAG and interrupting the formati on of the UVRAG-C-Vps complex as described below. UVRAG is a Beclin 1-binding protein that regulates the activity of PI3KC3/Vsp34 and thus the formation of autophagosomes (Liang et al. 2006; Liang et al. 2007). In addition, a recent report has shown that UVRAG also regulates the maturation of autophagosomes and the traffi cking of endocytic vesicles through its interaction with the class C Vps complex (Liang et al. 2008). In stark contrast to our results, which suggest that cells expre ssing wild-type Bif-1 exhibit delayed EGFR

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105 degradation as compared to knockdown cells UVRAG expression was shown to enhance EFGR localization to endosomes, resulting in the accelerated degradation of EGFR (Liang et al. 2008). In addition, it was shown that the role of UVRAG in C-Vpsmediated autophagosome/endosome maturation is independent of the role of UVRAG in Beclin 1-mediated autophagosome formation and maturation (Liang et al. 2008). Furthermore, Beclin 1 has been shown to ex clusively regulate the autophagic pathway, not endocytic trafficking (Zeng et al. 2006). Taken together, thes e results suggest that targeting of UVRAG to the Beclin 1-Bi f-1 complex could sequester UVRAG from interacting with C-Vps, thereby augmenti ng its role in the autophagic pathway and abrogating its role in endocytic trafficking. Indeed, Bif-1 has been shown to interact with the proline rich domain of UVRAG, which is located next to th e phospholipid-interacting C2 domain (Takahashi et al. 2007). The C2 domain of UVRAG, along with the Cterminus, corresponds to the region that is required for the intera ction of UVRAG with CVps (Liang et al. 2008). Therefore, it is possible that binding of Bif-1 changes the conformation of UVRAG which could decrease its affinity for C-Vps and would thus decrease the maturation and fusion capabilitie s of UVRAG. Further studies are necessary to determine whether the expression of a Bi f-1 mutant that cannot bind to UVRAG would affect the localization of UVRAG, its interaction partners and its ability to regulate endosomal maturation and fusion. In addition, Bif-1 could also regulate UVRAG localization by tethering UVRAG to autophagosomes, rather than endosomes. Bif-1 contains an NBAR domain, which is required for membrane binding, driving memb rane curvature and perhaps regulating subcellular localization (Masuda et al. 2006). It is therefore po ssible that Bif-1 may bind

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106 to UVRAG and regulate its localization thereby targeting UVRAG to autophagosomes, not endosomes. Therefore, in the absence of Bif-1, UVRAG would be released from autophagosomes, allowing it to localize to endosomes and regulate the endocytic pathway. While our results demonstrate that Bif-1 plays an integral role in the later stages of endocytic trafficking, further studies are needed to determine the molecular mechanisms by which loss of Bif-1 accelerat es EGF trafficking and enhances the degradation of EGFR.

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107 Methods and Materials Reagents Horseradish peroxidase was from purchase d from Sigma (St. Louis, MO). The 1 step-Turbo TMB-ELISA kit was purchased from Pierce (Rockford, IL). Bafilomycin A1 was purchased from Wako Chemicals USA (Richmond, VA). MG132 and puromycin were purchased from Calbiochem (San Di ego, CA). Epidermal growth factor, Alexa Fluor 488 conjugated EGF, culture medium a nd penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA). Anti bodies were purchased from the following commercial sources: anti-tubulin from Sigma, anti-LAMP-1 from BD Pharmingen (San Diego, CA), total EGFR and phospho-EGFR fr om Cell Signaling (Da nvers, MA), antiAtg5 from MBL Internationa l (Naka-ku Nagoya, Japan), Al exa Fluor 594 chicken antimouse IgG from Invitrogen, bovine anti-mous e IgG-horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA) and goa t anti-rabbit IgG-horseradish peroxidase from Amersham Biosciences (Piscataway, NJ). Cell Culture, Transfection and Infection Wild-type and Bif-1-knockdown HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemen ted with 5% fetal bovine serum, 1.0 mM L-glutamine, 100 g/ml streptomycin and 100 U/ ml penicillin (Takahashi et al. 2005). The pLKO.1-based lentiviral shRN A targeting shAtg5 (TRCN0000151963) was purchased from Open Biosystems. The pLKO .1-based scrambled control shRNA vector was purchased from Sigma. Recombinant lent ivirus was produced by co-transfecting the appropriate shRNA plasmid w ith the ViraPower Packaging Mix (Invitrogen) into 293FT

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108 cells. The resulting supernatan t containing shRNA-expressing lentivirus was used to infect wild-type HeLa cells according to the manufacturer’s protocol. Endocytosis of HRP Analysis of enodcytosis by HRP uptake wa s performed as previously described (Johnson et al. 2006; Zeng et al. 2006). Briefly, Bif-1-knoc kdown and control wild-type HeLa cells were washed once in DMEM, th en incubated with 2 mg/ml HRP in DMEM containing 1% BSA for the indicated times. Cells were washed three times in ice-cold PBS containing 1% BSA and once with PBS. Cells were then scraped into PBS and collected by centrifugatio n at 400 x g for 4 min at 4C. Th e pellet was washed once with PBS, then lysed in PBS containing 0.5% Triton X-100 and prot ease inhibitors. HRP activity was measured using the 1-Step Turbo TMB-ELISA kit according to the manufacturer’s protocol. Enzyme activity was normalized to protein concentration. EGFR Endocytosis and Degradation To monitor endocytosis, the internaliza tion of EGF coupled to Alexa Flour 488 was examined as previously described (Liang et al. 2008). Briefly, Bif-1-knockdown and control wild-type HeLa cells were seeded on 2-well chamber slides. The following day, the cells were washed in DMEM, then cultu red in serum-free DMEM overnight in order to allow the EGFR to accumulate at the cell surface. Cells we re washed once in ice-cold PBS and then incubated in uptake medium (DMEM, 2% BSA and 20 mM HEPES, pH 7.5) containing 5 g/ml Alexa Fluor 488-EGF for one hour on ice. Unbound ligand was removed by washing the cells three times in ice-cold PBS. At the indicated times, the

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109 cells were fixed in 4% paraformaldehyde fo r 10 min at room temperature. Cells were then washed three times with PBS and permeabilized in 100 g/ml digitonin for 15 min at room temperature. After three washes with PBS, the cells were blocked for one hour at room temperature in 3% BSA. Cells were th en incubated in primary antibody in blocking solution overnight at 4 C. After three washes with PBS, cells were incubated in 3% BSA blocking solution for 30 min, then in FI TC-conjugated goat anti-rabbit secondary antibody for three hours at room temperature. The cells were wash ed three times with PBS before being mounted with media containing DAPI (4’, 6’-diamidino-2phenylindole; Vector Laborator ies, Burlingame, CA). Th e fluorescent images were obtained using a Leica confocal microscope. To monitor the degradation of the EG FR, Bif-1-knockdown and control wild-type HeLa cells were serum-starved overnight. R eceptor internalization and degradation were stimulated by incubating the cells in 100 ng/ml EGF (Invitrogen) in DMEM containing 20 mM HEPES and 0.2% bovine serum albumin at 37 C. At the indicated times, cells were washed once with ice-cold PBS, then co llected in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 2 g/ml aprotinin, 2 g/ml leupeptin, 100 g/ml phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 20 mM p -nitrophenyl phosphate, 1% Triton X-100) as prev iously described (Ren et al. 2004). Total cell lysates were subjected to SDS-PAGE/immunobl ot analysis with antibodies specific for total and phosphorylated EGFR (Tyr 1068). The levels of total and phosphorylated EGFR, as well as degradation, from three independent expe riments were quantified by densitometry.

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110 Scientific Significance Genomic DNA is constantly being subj ected to genotoxic stresses. DNA damage triggers the activation of complex, highl y coordinated DNA damage response (DDR) pathways, which can initiate cell cycle arrest and promote DNA repair or if DNA repair is unfavorable, activate the a poptotic machinery to induce ce ll death (Zhou and Elledge, 2000). Thus the DDR acts as a safeguard to pr otect genomic integrity and to prevent the accumulation of mutations which could lead to cancer, as well as other genetic diseases. A vast amount of data has accumulated that provides insight into the regulation of the DDR. However, research is st ill being conducted to elucidat e the precise mechanisms by which DNA damage is sensed and translated in to a signal that result s in the decision of whether to arrest the cell cycle or under go cell death. Further i nvestigation into the mechanisms that regulate the DDR are impe rative, as a better unde rstanding of these pathways will allow more educated approaches for novel treatment strategies that will hopefully contribute to the cure of cancer and possibly other diseases. At the forefront of the DDR is the Rad9-Rad1-Hus1 complex (9-1-1). This complex is a key mediator of the DDR and regulates many downstream signaling pathways that are not only involved in cell cy cle arrest and DNA repair, but also in the induction of cell death. It is well accepted that the 9-1-1 complex confers resistance to a variety of genotoxic stresses, including several traditional chemotherapeutic agents. Accordingly, disruption of the 9-1-1 comple x has been shown to sensitize cells to DNA

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111 damaging agents (Hopkins et al. 2004; Kinzel et al. 2002; Loegering et al. 2004; Wang et al. 2004b; Wang et al. 2006b; Weiss et al. 2000). Importantly, th e data presented in this report describe for the first time the mechanism by which loss of a functional 9-1-1 complex sensitizes cells to etoposide-induced apoptosis. We have shown that etoposide treatment dramatically upregulates the e xpression of the pro-apoptotic, BH3-only proteins, Bim and Puma, in Hus1 -deficient cells. The upregul ation of these proteins is responsible for sensitizing Hus1 -knockout cells to etoposid e-induced apoptosis, as knockdown of either Bim or Puma confer s resistance to etoposide treatment. Interestingly, knockdown of both Bim and Puma results in further resistance, indicating that these BH3-only proteins collaborate in sensitizing Hus1 -deficient cells to apoptosis induced by genotoxic stress. Furthermore, loss of Hus1 results in a defect in the binding of Rad9 to chromatin and release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptot ic response. However, future studies are necessary to determine the precise mechan isms linking the loss of a functional 9-1-1 complex to the upregulation of Bim and Pu ma. Taken together, our results clearly demonstrate a role for the 9-1-1 cell cycl e checkpoint complex in the suppression of genotoxic stress-induced apoptosis and suggest that this comp lex may play a pivotal role in determining whether a cell should survive or undergo apoptosis. Importantly, the results from our studies also reveal a role for the 9-1-1 complex in the regulation of caspase -independent cell death. Our da ta indicate that loss of a functional 9-1-1 complex sensitizes cells to camptothecin treatment, which is only moderately suppressed by co-treatment with the pan-caspase inhibitor, Z-VAD-FMK. These results indicate that loss of a functi onal 9-1-1 complex not only sensitizes cells to

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112 apoptosis, as described above, but also to caspase-independent cell death when the apoptotic machinery is inhibited. In addition, we found that loss of Hus1 results in enhanced autophagy in response to tr eatment with the DNA damaging agents, camptothecin and etoposide, suggesting that a functional 9-1-1 complex is required for suppression of DNA damage-induced autophag y. However, the mechanism by which loss of a functional 9-1-1 complex activates th e autophagic machinery has yet to be discovered. It has previously been shown that BH3-only proteins as well as the pharmacological BH3 mimic, ABT-737, can di srupt the interaction between Bcl-2 and Beclin 1, which releases Beclin 1 to induce apoptosis. Ther efore, it is possible that the upregulation of BH3-only proteins observed in Hus1 -deficienct cells in response to genotoxic stress could be responsible for e nhancing autophagy in these cells. However, this is unlikely as we found that co-treatment of Hus1 -wild-type cells with ABT-737 and camptothecin did not significantly en hance DNA damage-induce autophagy. These results suggest that the m echanism by which loss of Hus1 induces autophagy occurs independent of the upregulation of BH3-only protein expression. As loss of Hus1 both enhanced caspase-independen t cell death and the initiation of autophagy in response to DNA damage, we predicted that the induction of autophagy was responsible for the caspase-i ndependent cell death observed in Hus1 -knockout cells. Interestingly, inhibition of autophagy, th rough knockdown of Atg7 or Bif-1, did not suppress, but rather enhanced DNA damage-induced cell death in Hus1 -deficient cells. These results indicate that the induction of autophagy observed in Hus1 -knockout cells may play a cytoprotective role Interestingly, damaged mito chondria, which could release apopotogenic factors and activate the caspase cascade, have been shown to be removed

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113 by autophagy (Abedin et al. 2007; Mijaljica et al. 2007). Therefore, it is not difficult to imagine that inhibition of the clearan ce of damaged mitochondria by suppressing autophagy could amplify the apoptotic response. It is of interest to determine whether prevention of caspase activation (e.g. by tr eatment with Z-VAD-FMK) could suppress cell death when the inducti on of autophagy is inhibited. Taken together, our results highlight a role for the 9-1-1 complex in th e regulation of various cell death mechanisms and suggest that targeting th e 9-1-1 complex may be an e ffective treatment strategy for cancer, even in cells with impa ired apoptosis (see below). While an immense amount of research has focused on investigating the role of the 9-1-1 complex in the DDR, much less is know n about the role of the 9-1-1 complex in cancer progression. Interestingly, overexpressi on of members of the 9-1-1 complex has been observed in various types of cancer including breast cancer, non-small cell lung carcinoma and prostate cancer (Cheng et al. 2005; de la Torre et al. 2008; Maniwa et al. 2005; Zhu et al. 2008). Moreover, high Hus1 expr ession in ovarian tumors was found to correlate with poor prognosis and advanced stage (de la Torre et al. 2008). In addition, aberrantly high levels of Rad9 mR NA in breast cancer ce lls were associated with tumor size and local recurrence, sugges ting that Rad9 overexpression may play a role in tumor proliferatio n and local invasion (Cheng et al. 2005). Consistently, Rad9 protein abundance has been shown to strongl y correlate with advanced stage prostate cancer and knockdown of Rad9 led to decrea sed tumorigenicity in nude mice (Zhu and Lieberman 2008). The concept that overexpre ssion of Rad9 enhances tumorigenesis may seem contradictory given the well-describ ed role of Rad9 in apoptosis (Ishii et al. 2005; Komatsu et al. 2000a; Komatsu et al. 2000b; Yoshida et al. 2002; Yoshida et al. 2003).

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114 However, as Rad9 was found to accumulate in the nucleus of non-small cell lung carcinoma cells (Maniwa et al. 2005), it is not unreasonable to assume that overexpression of members of the 9-1-1 complex could result in enhanced complex formation and retention of the complex in the nucleus. Enforced nuclear localization would augment the nuclear functions of Rad9, such as activation of cell cycle checkpoints and initiation of DN A repair, and abrogate the cy tosolic functions of Rad9 in activating the apoptotic machiner y. It is reasonable to assume that rapidly dividing cancer cells may benefit from, or even require, a dditional DNA repair mechanisms (e.g. elevated expression of DDR proteins, such as Rad9 and Hus1) in order to offset the high level of DNA replication and the corresponding DNA damage that can occur during this process. Alternatively, the increase in DNA repair mechanisms could provide resistance by rapidly and efficiently repa iring DNA damage incurred by exposure to chemotherapeutic agents. Thus, overexpression of members of the 9-1-1 complex could provide cancer cells with a survival advantage and resist ance to chemotherapy thereby enhancing tumorigenicity. As described above, evidence is accumula ting that suggests that overexpression of members of the 9-1-1 complex promotes tumo rigenesis. Conversel y, disruption of the 91-1 complex sensitizes cells to commonly used DNA-damaging anticancer agents. In addition, the 9-1-1 complex plays an apical role in the DDR and a direct role in several DNA repair pathways. Therefore, the 9-1-1 complex may be a rational target for novel treatment strategies that would inhibit mu ltiple pathways involved in survival and resistance to genotoxic stress. Indeed, it ha s been shown that k nockdown of Hus1 in H1299 non-small cell lung carcinoma cells enhanced the cytotoxicity of cisplatin (Kinzel

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115 et al. 2002). However, inhibition of the multitude of cellular functions that are regulated by the 9-1-1 complex could also result in undes irable effects. Ideally, the agent used to disrupt the 9-1-1 complex would suppress th e pro-survival functions of the 9-1-1 complex (e.g. DNA repair) while avoiding unwan ted results from i nhibition of desired functions (e.g. apoptosis). Our re sults indicate that knockout of Hus1 not only disrupts the binding of Rad9 to chromatin (producing desired effects), but also upregulates BH3only protein expression and enhances the bi nding of Rad9 to Bcl-2 to augment the apoptotic response (avoiding undesirable effect s). Taken together, these results suggest that Hus1 may be an optimal target for di sruption of the 9-1-1 complex. Alternatively, agents could be designed that would promote the transloca tion of Rad9 from the nucleus to the cytosol, thereby attenuating the nuclear functions of Rad9, such as in the regulation of DNA repair, and potentia ting the cytosolic function of Rad9 as an inducer of apoptosis. However, the precise mechanisms th at regulate the translocation of Rad9 have yet to be determined, but may involve pos t-translational modifications, such as phosphorylation of Rad9 by c-Ab l or protein kinase C or cleavage of Rad9 by caspase3. In addition, agents could be designed th at would inhibit the interaction between members of the 9-1-1 complex. Ho wever, the effect of using this method to disrupt the 91-1 complex (without decreasing the expression of any of the members) on sensitivity to DNA damaging agents has yet to be examin ed. Therefore, additional studies are necessary to explore this po ssibility, as this strategy ma y also be therapeutically beneficial. Our results indicate that targeting the 91-1 complex would sensitize cells to both caspase-dependent and caspase-independent cell death in response to treatment with

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116 DNA damaging agents. Therefore, disruption of the 9-1-1 complex could efficiently sensitize cells with impaired apoptotic m achinery to commonly used chemotherapeutic agents. One of the hallmarks of cancer is th e ability to evade a poptosis (Hanahan and Weinberg, 2000). This resistance to apoptotic cell death is an important aspect not only of tumorigenesis, but also the development of resistance to anticancer drugs (Green and Evan, 2002; Hanahan and Weinbe rg, 2000). As such, a large area of research focuses its efforts on the restoration of apoptosis in cancer cells (Reed, 2006; Wang and El-Deiry, 2008). Importantly, a growing body of evidence indicates that autophagy may act as a cell death mechanism in cells with impaired apoptotic machinery (Shimizu et al. 2004; Yu et al. 2004). Therefore, rather than trying to restore apoptotic machinery, a more lucrative approach may be to disrupt the 9-1-1 complex and activate autophagy in these cells in order to manipulate the cell’s abil ity to induce alternate forms of cell death. Indeed, several commonly used chemotherape utic agents have been shown to induce autophagy (Kondo et al. 2005) and could potentially be used in combination with disruption of the 9-1-1 complex to sensitize cells that are resistant to apoptosis. However, this strategy may only be feasible in apoptosis -deficient cells, as our results and those of other laboratories suggest that autophagy can promote survival in cells that are apoptosiscompetent (Kroemer and Levine, 2008). In apoptosis-competent cells, an alternate approach could be to inhibit autophagy and di srupt the 9-1-1 complex in order to enhance apoptotic cell death in response to chemot herapy. While recent studies have provided insight into the interplay between the aut ophagic and apoptotic pathways and how this may affect sensitivity to chemotherapy, future studies are needed to further define the crosstalk between these two cell death m echanisms. Once this knowledge is obtained,

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117 controlling the balance between apoptotic and autophagic cell death could lead to enhanced tumor cell killing and thus provi de more efficient cancer treatments. Another strategy for enhancing the cytotoxi city of anticancer agents would be to inhibit certain functions of Bi f-1, in combination with disruption of the 9-1-1 complex. In this report, we demonstrate that kno ckdown of Bif-1 significantly enhances camptothecin-induced cell death in Hus1 -deficient cells. However, our results indicate that knockdown of Bif-1 expression suppre sses, but does not completely block the induction of autophagy. These results suggest that while Bif-1-mediated autophagy may protect cells from DNA damage-induced cell death, other functions of Bif-1 may also play a role in the suppression of cell death. In this study, we describe a novel role for Bif1 in the regulation of endocytic vesicle trafficking and receptor degradation. While knockdown of Bif-1 did not affect the internalizati on of either a fluid phase marker or EGF, it accelerated the co-loc alization of EGF with late endosomes/lysosomes and enhanced the degradation of activated EGFR. As knockdown Bif-1 enhances the degradation of EGFR, it can be assumed that inhibition of Bif-1 function could result in the rapid attenuation of EGFR signaling. Ther efore, suppression of Bif-1 function in cancer cells with hyperactivated EGFR si gnaling may be a potentially beneficial therapeutic strategy. Ho wever, as loss of Bif-1 has been shown to significantly enhance spontaneous tumorigenesis in mice (Takahashi et al. 2007), care should be taken when designing therapeutics that target Bif-1. In addition to the newly described role of Bif-1 in endocytic vesicle trafficki ng, our laboratory has also show n that Bif-1 is involved in the regulation of Bax-mediated apoptos is and the induction of autophagy (Cuddeback et al. 2001; Takahashi et al. 2007; Takahashi et al. 2005). Therefore, in order to properly

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118 target Bif-1 for anticancer therapy, the specific functions of Bif-1 that are responsible for its tumor suppressive capabil ities and the functions that are required for regulating sensitivity to DNA damage-induced cell deat h must first be defined. This knowledge could then be used for the rational design of anticancer agents that would specifically target the pro-survival functions of Bif-1 while leaving the anti-tumor functions intact. Given that both autophagy and EGFR signaling have been shown to promote survival, the role of Bif-1 in these processes may be the most likely candidates for conferring resistance to DNA damage-induced cell death. Interestingly, it has been suggested that in addition to the various gain-of-function mutations that occur in the EGFR, deregulation or ineffi cient EGFR degradation may also play a significant role in the abe rrant activation of EG FR signaling thereby enhancing tumor development (Gra ndal and Madshus, 2008; Kirisits et al. 2007). As EGFR signaling has been shown to pers ist even within endosomes (Miaczynska et al. 2004; Sorkin and Von Zastrow, 2002), enhan ced EGFR degradation, by inhibition of Bif1 function, could abrogate growth factor-m ediated survival signaling through EGFRmediated pathways. In addition, it has been s hown that EGFR signaling can be induced in response to chemotherapy and exposure to ioni zing radiation, even in the absence of ligand binding (Rodemann et al. 2007). EGFR that is activ ated in such a manner has been shown to confer resistance to D NA damaging chemotherapeutic agents by activating various mechanisms that e fficiently repair damaged DNA, thereby counteracting the cytoto xic effects of the chemothera py. Therefore, targeting Bif-1 functions in endocytic vesicle trafficking may have similar effects to current therapeutic approaches that inhibit EGFR signaling. Indee d, it has been shown that inhibiting EGFR

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119 signaling in combination with chemothera py or radiotherapy results in increased sensitivity both in preclinical and clinical studies (Nyati et al. 2006). The results presented in this report sugge st that inhibiting Bif-1 function could potentially target two survival pathways: autophagy and EGFR signa ling. In addition, our results indicate that the combination of inhi bition of Bif-1 and di sruption of the 9-1-1 complex, through loss of Hus1 enhances the cytotoxicity of camptothecin. These studies provide evidence supporting the concept that targeting Bif-1 and Hus1 would inhibit several survival signaling pa thways and thus may sensiti ze otherwise resistant cancer cells to commonly used chemotherapeutic agents. In response to genotoxic stress, a comple x network of signaling pathways act in concert to activate the DNA damage response. It is therefore important to determine the molecular mechanisms that regulate of these signaling pathways in order to improve the efficacy of cancer treatments. Importantly, the crosstalk between the autophagic and apoptotic pathways must also be deciphered in order to optimize ther apeutic benefits. In addition to manipulating the in terplay between these pathways, other factors such as those that regulate EGFR endocytic traffick ing and degradation, may also prove to be lucrative targets for sensiti zing cancer cells to chemothe rapy. Significantly, the results described in this report offer insight into so me of the mechanisms that both sensitize and provide resistance to DNA damaging agents (Fi gure 54). Further resear ch will lead to a better understanding of the molecular mechan isms are regulated by the 9-1-1 complex and Bif-1 that affect sensitivity to DNA da mage and will provide valuable knowledge that can be used for the rational design of novel chemotherapeutic strategies that will offer more effective treatments for cancer.

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120 Loss of Hus1 Functional 911 complex Functional 9-1-1 complex Bim and Puma Rad9-Bcl-2 Apoptosis DNA damage Bif-1 Endocytic vesicle trafficking Sensitivity to genotoxic stresses Autophagy Autophagy Autophagic cell death EGFR degradation Figure 54. Proposed model for the role of the Rad9-Rad1-Hus1 complex and Bif-1 in the regulation of sensitivity to DNA damage. A functional 9-1-1 complex confers resistance to genotoxic stresses by suppressing apoptosis. Loss of Hus1 sensitizes cells to DNA damage-induced apoptosis through the upre gulation of the BH3-only proteins, Bim and Puma. Moreover, loss of Hus1 enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. Loss of Hus1 also enhances DNA damage-induced autophagy, which promotes survival in apopt osis-competent cells. Inhibiting autophagy in apoptosis-competent cells may enhan ce DNA damage-induced apoptosis. Conversely, inducing autophagy in cells that are apoptosis-impaired may in crease the cytotoxicity of DNA-damaging chemotherapeutic agents. In addition, Bif-1 promotes survival in Hus1deficient cells, perhaps thr ough its regulation of autophagy and/or endocytic vesicle trafficking (EGFR signaling). Notably, loss of Hus1 sensitizes cells to both caspasedependent and caspase-independent ce ll death in response to DNA damage.

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About the Author Cheryl Meyerkord attended Wittenberg University, where she earned a Bachelor of Science Degree in Biology in May 2003. Af ter graduation, she joined the Cancer Biology Ph.D. program at the H. Lee Moffitt Ca ncer Center and Research Institute. She conducted her doctoral research under the mentorship of Dr. Hong-Gang Wang. Cheryl received funding for her graduate research from the Department of Defense Breast Cancer Research Program. During her time in the Cancer Biology Program, Cheryl contributed to several studies, which led to the publication of three papers. The focus of her research was on investiga ting various mechanisms that are regulated by the Rad9Rad1-Hus1 complex and Bif-1 that a ffect sensitivity to DNA damage.


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ABSTRACT: The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.
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Programmed cell death
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Autophagy
Endocytosis
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Cell Cycle Proteins.
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