UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of tumor suppressor Inhibitor of Growth 1 in lesion bypass pathway and genomic stability Wong, Pak Cheung Ronald 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2011_fall_wong_pakcheungronald.pdf [ 2.81MB ]
Metadata
JSON: 24-1.0072283.json
JSON-LD: 24-1.0072283-ld.json
RDF/XML (Pretty): 24-1.0072283-rdf.xml
RDF/JSON: 24-1.0072283-rdf.json
Turtle: 24-1.0072283-turtle.txt
N-Triples: 24-1.0072283-rdf-ntriples.txt
Original Record: 24-1.0072283-source.json
Full Text
24-1.0072283-fulltext.txt
Citation
24-1.0072283.ris

Full Text

THE ROLE OF TUMOR SUPPRESSOR INHIBITOR OF GROWTH 1 IN LESION BYPASS PATHWAY AND GENOMIC STABILITY  by  Pak Cheung Ronald Wong  B.Sc., The Chinese University of Hong Kong, 2002 M. Phil., The Chinese University of Hong Kong, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Experimental Medicine)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    October 2011  © Pak Cheung Ronald Wong, 2011  ii Abstract The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. In this thesis, I demonstrated a novel tumor suppressor function of p33ING1 (ING1b) in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. For the first time, we have shown that chromatin remodeling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.  Previously, our group showed that ING1b is phosphorylated by the S phase checkpoint kinase Chk1 at S126 residue.  We further showed that ING1b cooperated with Chk1 in maintaining genomic stability. We found that ING1b interacted with Chk1 after UV through ING1b S126 residue. Furthermore, we found that ING1b S126 residue was required for PCNA monoubiquitination, Pol foci and therefore preventing chromosomal aberrations after UV. These data suggest that ING1b cooperates with Chk1 through the S126 residue in mediating PCNA monoubiquitination, lesion bypass and genomic stability.  We further explored the role of the E3 ligase Rad18, a key regulator for the lesion bypass pathway, in melanoma using melanoma tissue microarray. We found that Rad18 expression was significantly upregulated in melanoma. Strong Rad18 expression correlated  iii with worse 5-year patient survival in the sun-protected sites. Interestingly, we found an opposite role of Rad18 on patient survival in the sun-exposed sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown.  The work presented in this thesis leads to a better understanding of the role of ING1b and lesion bypass pathway in genomic stability and melanoma development. It has implications on designing new strategies for cancer therapy.    iv Preface 1. A version of chapter 3 has been published.  [Wong RP], Lin H, Khosravi S, Piche B, Jafarnejad SM, Chen DW, Li G. (2011) Tumor suppressor ING1b maintains genomic stability upon replication stress. Nucleic Acids Research. 2011 May 1;39(9):3632-42.  G.L. provided facilities, research materials, and contributed to experimental design and manuscript preparation. I designed and performed most of the experiments, and prepared the manuscript. H.L. performed Western blotting in Figure 3.11B, 3.12C and D, and Facs analysis in 3.11C; S.K. performed Western blotting in Figure 3.9H; B.P. performed Western blotting in Figure 3.12B; S.M.J. preformed Western blotting in Figure 3.9F; and D.W.C. prepared metaphase chromosome spread in Figure 3.8E. 2. A manuscript based on chapter 4 is under preparation.  [Wong RP], Jafarnejad SM, Cheng Y, Khosravi S, Tang P, Li G. ING1b Cooperates with Chk1 in regulating PCNA monoubiquitination and genomic stability.  G.L. provided facilities, research materials, and contributed to experimental design and manuscript preparation. I designed and performed most of the experiments, and prepared the manuscript. S.M.J. performed immunoprecipitation in Figure 4.1; Y.C. and P.T. performed confocal microscopy in Figure 4.3; and S.K. prepared metaphase chromosome spread in Figure 4.4. 3. A manuscript based on chapter 5 has been prepared and submitted.  [Wong RP], Aguissa-Touré  A, Wani  A, Khosravi  S, Martinka  M 1 , Martinka  M 2 , Li G. Elevated expression of Rad18 regulates melanoma cell proliferation.  v  G.L. provided facilities, research materials, and contributed to experimental design and manuscript preparation. I designed and performed most of the experiments, and prepared the manuscript. A.A. performed Western blotting in Figure 5.6 and 5.8; A.W. performed Western blotting in Figure 5.7; S.K. performed Western blotting in Figure 5.3A; M.M. 1  assisted with immunohistochemical staining; and M.M. 2  assisted with the scoring of the tissue microarray.  List of publications: 1. Wong RP, Lin H, Khosravi S, Piche B, Jafarnejad SM, Chen DW, Li G. (2011) Tumor suppressor ING1b maintains genomic stability upon replication stress. Nucleic Acids Res. 2011 May 1;39(9):3632-42.  With permission to reprint, this work is located in chapter 3. 2. Wong RP, Jafarnejad SM, Cheng Y, Khosravi S, Tang P, Li G. ING1b Cooperates with Chk1 in regulating PCNA monoubiquitination and genomic stability. Manuscript is under preparation. This work is located in chapter 4. 3. Wong RP, Aguissa-Touré A, Wani A, Khosravi S, Martinka M,  Martinka M, Li G. Elevated expression of Rad18 regulates melanoma cell proliferation. Manuscript has been prepared and submitted. This work is located in chapter 5.   vi Table of Contents  Abstract .............................................................................................................................. ii Preface ............................................................................................................................... iv Table of Contents .............................................................................................................. vi List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii Acknowledgements ...........................................................................................................xiv Dedication .........................................................................................................................xvi Chapter 1: General Introduction ........................................................................................1 1.1 Genomic Instability, Replication Stress and Cancer .......................................................... 2 1.1.1 Genomic instability as a feature of cancer ..................................................................... 2 1.1.2 Source of replication stress ........................................................................................... 3 1.1.3 Replication failure and cancer....................................................................................... 4 1.2 Lesion Bypass Pathway .................................................................................................... 5 1.2.1 Lesion bypass subpathways .......................................................................................... 5 1.2.2 PCNA ubiquitination and lesion bypass ........................................................................ 8 1.2.3 TLS for UV lesions .................................................................................................... 11 1.3 S Phase Checkpoint ........................................................................................................ 12 1.3.1 The replication checkpoint activation.......................................................................... 12 1.3.2 The role of ATR-Chk1 pathway in cell cycle arrest and replication fork stabilization .. 15 1.3.3 S phase checkpoint and lesion bypass pathway ........................................................... 16 1.4 Tumor Suppressor Inhibitor of Growth ........................................................................... 17 1.4.1 Functional domains of ING proteins ........................................................................... 18 1.4.2 Biological functions of ING proteins .......................................................................... 24 1.4.3 ING1 and chromatin remodeling................................................................................. 27 1.4.4 Chromatin remodeling, DNA replication and ING proteins ......................................... 28 1.4.5 Alterations of ING1 in human cancer.......................................................................... 29 1.5 Cutaneous Melanoma ..................................................................................................... 29  vii 1.5.1 Epidemiology of melanoma ........................................................................................ 29 1.5.2 Melanoma biology...................................................................................................... 30 1.5.3 Etiology of melanoma ................................................................................................ 31 1.5.4 UV radiation and melanoma ....................................................................................... 33 1.5.5 Genomic instability in melanoma ............................................................................... 34 1.5.6 Treatment for melanoma ............................................................................................ 35 1.6 Objective and Hypotheses............................................................................................... 36 Chapter 2: Material and Methods .................................................................................... 38 2.1 Cell Culture, Antibodies, Expression Plasmids, Chemicals, and UV Irradiation ............... 38 2.2 Expression Plasmid, siRNA Transfections and shRNA Construction............................... 39 2.3 Subcellular Fractionation ................................................................................................ 40 2.4 Histone Extraction, Western Blot and Immunoprecipitation ............................................ 40 2.5 Immunofluorescence ...................................................................................................... 41 2.6 Analysis of Metaphase Chromosome .............................................................................. 41 2.7 Replication Fork Progression by qPCR ........................................................................... 42 2.8 Cell Synchronization ...................................................................................................... 43 2.9 Sulforhodamine B Assay ................................................................................................ 43 2.10 Construction of TMA ..................................................................................................... 43 2.11 Immunohistochemistry of TMA ...................................................................................... 44 2.12 Evaluation of Immunostaining ........................................................................................ 44 2.13 Statistical Analyses ......................................................................................................... 45 Chapter 3: The Role of Tumor Suppressor ING1b in Genomic Stability upon Replication Stress .............................................................................................................. 46 3.1 Background and Rationale .............................................................................................. 46 3.2 Results ........................................................................................................................... 46 3.2.1 ING1b knockdown cells are more sensitive to UV at S phase...................................... 46 3.2.2 ING1b knockdown cells show defects in replication fork progression and enhanced genomic instability after UV ................................................................................................... 56 3.2.3 ING1b regulates PCNA monoubiquitination and TLS ................................................. 60 3.2.4 ING1b is required for Rad18-mediated PCNA-Ub ...................................................... 65 3.2.5 ING1b maintains histone H4 acetylation during S phase and is required for Rad18- mediated PCNA-Ub ................................................................................................................ 68 3.3 Discussion ...................................................................................................................... 72  viii Chapter 4: ING1b Cooperates with Chk1  in Genomic Stability .................................... 78 4.1 Background and Rationale .............................................................................................. 78 4.2 Results ........................................................................................................................... 79 4.2.1 ING1b S126 is required for interaction with Chk1. ..................................................... 79 4.2.2 ING1b cooperates with Chk1 in PCNA-Ub ................................................................. 81 4.2.3 ING1b S126 is required for Pol foci formation after UV irradiation .......................... 83 4.2.4 ING1b S126A cells showed enhanced genomic instability after UV ............................ 85 4.3 Discussion ...................................................................................................................... 87 Chapter 5: Elevated Expression of Rad18 Regulates Melanoma Cell Proliferation....... 90 5.1 Background and Rationale .............................................................................................. 90 5.2 Results ........................................................................................................................... 90 5.2.1 Rad18 is upregulated in melanoma ............................................................................. 90 5.2.2 Rad18 expression is correlated with melanoma patient survival .................................. 94 5.2.3 Rad18 is an independent prognostic factor for melanoma at the sun-protected sites ..... 98 5.2.4 Rad18 regulates melanoma cell proliferation ............................................................ 100 5.2.5 Rad18 regulates pAkt and cyclin D1 expression ....................................................... 104 5.3 Discussion .................................................................................................................... 108 Chapter 6: Conclusions ................................................................................................... 113 6.1 Summary of Findings ................................................................................................... 113 6.2 New Insights into the Mechanisms of Lesion Bypass .................................................... 115 6.3 The Role of Rad18 in Melanoma Development ............................................................. 119 6.4 Future Directions .......................................................................................................... 119 6.5 Targeting Cancer Susceptibility Pathways .................................................................... 121 Bibliography .................................................................................................................... 124   ix List of Tables  Table 5.1 Correlations of Rad18 staining with clinicopathological parameters of 146 cases of primary melanoma patients .................................................................................................. 93 Table 5.2 Univariate Cox regression analysis on overall and disease-specific 5-year survival of 70 cases of primary melanoma at sun-protected sites ....................................................... 97 Table 5.3 Multivariate Cox regression analysis on overall and disease-specific 5-year survival of 70 cases of primary melanoma at sun-protected sites. ......................................... 99   x List of Figures  Figure 1.1 Translesion DNA synthesis ...................................................................................7 Figure 1.2 The structural domains of the Y-family polymerases .......................................... 10 Figure 1.3 Replication checkpoint activation and function ................................................... 14 Figure 1.4 Functional domains in ING proteins ................................................................... 21 Figure 1.5 ING domains and functions ................................................................................ 23 Figure 3.1 Sequencing ING1b cDNA in HCT116 cells. ....................................................... 49 Figure 3.2 ING1b KD sensitizes cells to UV at S phase ....................................................... 50 Figure 3.3 ING1b knockdown increases UV sensitivity ....................................................... 51 Figure 3.4 Nucleotide excision repair efficiency in ING1b KD cells .................................... 52 Figure 3.5 Cell cycle analysis for ING1b KD cells .............................................................. 53 Figure 3.6 ING1b KD cells fail to recover from stalled replication ...................................... 54 Figure 3.7 ING1b knockdown cells showed increased stalled replication after UV .............. 55 Figure 3.8 ING1b KD cells show defects in replication fork progress and enhanced genomic instability after UV .............................................................................................................. 58 Figure 3.9  ING1b regulates PCNA-Ub and upon replication stress at S phase ..................... 62 Figure 3.10  ING1b is required for Polε foci formation after UV ......................................... 64 Figure 3.11 ING1b is required for Rad18-mediated PCNA-Ub during stalled replication ..... 66 Figure 3.12  ING1b maintains histone H4 acetylation during S phase and is required for Rad18-mediated PCNA-Ub ................................................................................................. 70 Figure 3.13  ING1b is not required for expression of ATP-dependent chromatin remodelling factors, SNF5 (BAF47) and BRG1 ...................................................................................... 76 Figure 4.1  ING1b S126 is required for interaction with Chk1 ............................................. 80 Figure 4.2 ING1b cooperates with Chk1 in PCNA-Ub ......................................................... 82 Figure 4.3  ING1b S126 is required for Pol foci formation after UV irradiation ................. 84 Figure 4.4 ING1b S126A cells showed enhanced genomic instability after UV ................... 86 Figure 5.1 Rad18 expression is reduced in melanoma .......................................................... 92 Figure 5.2 Correlation of Rad18 expression with melanoma patient survival ....................... 96 Figure 5.3 Rad18 enhances melanoma cell proliferation .................................................... 101 Figure 5.4 Rad18 knockdown inhibits colony formation of melanoma cells ....................... 102  xi Figure 5.5 Rad18 overexpression stimulated melanoma cell growth independent of its E3 ligase activity .................................................................................................................... 103 Figure 5.6 Rad18 knockdown does not induce replication stress in melanoma cells under non-stress condition ........................................................................................................... 106 Figure 5.7 Rad18 knockdown inhibited pAkt and cyclin D1 in cells. ................................. 107 Figure 5.8 Rad18 knockdown enhances DNA double stress breaks after UV ..................... 111 Figure 6.1 Model for ING1b in maintaining genomic stability through regulating lesion bypass ............................................................................................................................... 118 Figure 6.2 Targeting ING1 deficient tumor ........................................................................ 123   xii List of Abbreviations Abbreviation Definition   9-1-1 Rad9-Hus1-Rad1 6-4PPs Pyrimidine-(6-4)-pyrimidone photoproducts 8-oxo-G 7,8-dihydro-8-oxoguanine Akt Thymomo viral proto-oncogene ATM Ataxia telangiectasia mutated ATPIP ATR interacting protein ATR ATM- and Rad3-related BPDE Benzo(a)pyrene diolepoxide BRCA1 Breast cancer 1, early onset BRCA2 Breast cancer 2, early onset BRCT BRCA1 C-terminal BrdU Bromodeoxyuridine CBP CREB binding protein CDC25 Cell division control 25 CDK Cyclin dependent kinase CGH Comparative genomic hybridization CPDs Cyclobutane pyrimidine dimers DDT DNA tolerance pathway dNTPs Deoxynucleotide triphosphates DSBs DNA double strand breaks DUB Deubiquitinating enzyme ERK Extracellular regulated MAP kinase FA Fanconi anemia FHIT Fragile histidine triad GADD45 Growth arrest and DNA-damage-inducible HAT Histone acetylase HDAC Histone deacetylase HLTF Helicase-like transcription factor HR Homologous recombination HSP90 Heat shock protein 90 IFN-α Interferon α ING Inhibitor of growth INK4A Cylin-dependent kinase inhibitor 2A LID Lamin interaction domain LZL Leucine zipper-like MCM Minichromosome maintenance MDM2 Transformed mouse 3T3 cell double minute 2 MEFs Mouse embryonic fibroblast MEK MAP kinase kinase  xiii MITF Microphthalmia-associated transcription factor MMR Mismatch repair mTOR Mechanistic target of rapamycin (serine/threonine kinase) NBS Nijmegen breakage syndrome NER Nucleotide excision repair NLS Nuclear localization signal PBD Partial bromodomain PCAF p300/CPB-associated factor PCNA Proliferating cell nuclear antigen PCNA-Ub PCNA monoubiquitination PDGFR Platelet-derived growth factor receptor PHD Plant homeodomain PI3K Phosphoinositide-3-kinase PIKKs PI3K-related protein kinases PIP PCNA interacting peptide PLK1 Polo-like kinase 1 Pol Polymerase PRR Postreplicative repair PtdIns5P Phosphatidylinositide-5-phosphate PTEN Phosphatase and tensin homolog RFC Replication factor C RGP Radial growth phase ROS Reactive oxygen species RPA Replication protein A SAID SAP30-interacting domain SHPRH SNF2 histone linker PHD RING helicase ssDNA single stranded DNA TLS Translesion DNA synthesis TOPBP1 Topoiosmerase II binding protein 1 TRRAP Transformation/transcription domain-associated protein TS Template switching Ub Ubiquitin UBC13 Ubiquitin conjugating enzyme 13 UBM Ubiquitin-binding motif UBZ Ubiquitin-binding zinc finger USP1 Ubiquitin specific peptidase 1 UV Ultraviolet radiation VEGFR Vascular endothelial growth factor receptor VGP Vertical growth phase WNT wingless-type MMTV integration site family XP Xeroderma pigmentosum γH2AX H2AX (phopsho S139)  xiv Acknowledgements I would like to thank my supervisor Dr. Gang Li who has encouraged and supported me throughout my Ph.D. study. Dr. Li inspired me to develop critical thinking in research, to relentlessly strive for quality of my work and to be diligent at all time. He has shown me a path to be a successful scientist. I would like to express my gratitude to my supervisory committee members, Drs. Michael Cox, LeAnn Howe, Vincent Duronio and Paul Rennie for their support and constructive inputs in my project, and also for their kind advice for my career development.  I would like to thank Dr. Magdalena Martinka for assisting the evaluation of tissue microarray staining and her lectures on melanoma. I also thank Drs. Alan Lehmann, Kyungjae Myung, Junya Furukawa, Sarwat Jamil and Yemin Wang for generously providing reagents; Dr. Tim Lee and Paul Wighton for technical support in image analysis, and all the colleagues and volunteers in Li Lab and Jack Bell Research Centre for their friendly support and collaborations. I am honoured to be the recipient of studentship from Canadian Cancer Society Research Institute and trainee award from Canadian Institutes of Health Research-Skin Research Training Centre; University Graduate Fellowship and travel awards from Department of Dermatology and Skin Sciences and Canadian Cancer Society Research Institute. This work was generously supported by research grants from Canadian Institutes of Health Research and Canadian Dermatology Foundation. I would like to express my appreciation for Dr. Harvey Lui, Karen Ng and other staffs from the Department of Dermatology and Skin Sciences, and Dr. Vincent Duronio, Patrick  xv Carew and Cornelia Reichelsdorfer from the Experimental Medicine Program for providing the excellent training environment at UBC.   xvi Dedication I dedicate this thesis to my family, my mom and dad, my grandmother, and my dearest sisters, Tiffany, Sandy and Mable, for their unconditional love and enormous support throughout all these years.    1 Chapter 1: General Introduction Genomic instability plays a pivotal role in cancer development. It encompasses a wide range of alterations from point mutations to chromosomal rearrangement. The human genome is highly prone to genomic alterations during DNA replication (Aguilera & Gomez- Gonzalez, 2008). While unrepaired lesions on DNA template leads to replication stalling, prolonged stalling of replication will cause catastrophic events such as recombination and chromosomal rearrangement which eventually contribute to tumorigenesis (Aguilera & Gomez-Gonzalez, 2008; Kerzendorfer & O'Driscoll, 2009). Activation of S phase checkpoint prevents genotoxic events primarily by stopping cell cycle progression. S phase checkpoint proteins also directly stabilize stalled replication to prevent replication collapse (Andreassen et al, 2006; Branzei & Foiani, 2005; Friedel et al, 2009; Lambert & Carr, 2005). The lesion bypass pathway acts to resolve stalled forks to ensure genomic stability (Chang & Cimprich, 2009; Friedberg, 2005; Jansen et al, 2007). The tumor suppressor Inhibitor of Growth 1 (ING1) is found to be downregulated, mislocalized or mutated in various cancers (Bromidge & Lynas, 2002; Chen et al, 2003a; Chen et al, 2001; Ito et al, 2002; Krishnamurthy et al, 2001; Nouman et al, 2002b; Ohmori et al, 1999; Oki et al, 1999; Tokunaga et al, 2000; Toyama et al, 1999). Previously, our lab demonstrated that ING1b plays a role in response to ultraviolet radiation (UVR) (Cheung et al, 2000; Cheung et al, 2001). In this thesis, I continued this characterization of the tumor suppressor functions of ING1b. I discovered that ING1b was involved in regulation of the lesion bypass and connected the S phase checkpoint in such regulation. Moreover, I investigated the role of a key regulator of the lesion bypass, Rad18, in melanoma development using tissue microarray.  2 1.1 Genomic Instability, Replication Stress and Cancer 1.1.1 Genomic instability as a feature of cancer Hanahan and Weinberg proposed hallmarks of cancer to provide a logical framework to understand the complex properties of neoplastic diseases (Hanahan & Weinberg, 2000). Cancer cells often acquire these capabilities through a succession of genetic alterations. Mutations occur at extreme low rates in normal cells thanks to various genome surveillance and maintenance systems. In neoplastic cells, these systems are compromised, and thus, the accumulation of genetic or epigenetic alterations is accelerated, driving tumorigenesis (Negrini et al, 2010; Salk et al, 2010). Therefore, genomic instability is a common feature of cancer which is termed as an „enabling characteristic‟ of cancer by Hanahan and Wienberg in their recent follow-up to the classic “Hallmarks of cancer” article (Hanahan & Weinberg, 2011). With the technological advancement in genetic analysis with techniques like comparative genomic hybridization (CGH) and high throughput DNA sequencing, we come to appreciate that gains and losses of gene copy number and mutations are common events found across the cancer genome (Korkola & Gray, 2010; Stratton, 2011). Genomic instability refers to alterations in the genome ranging from point mutations to gene amplifications, deletions and chromosomal rearrangements. They are further subdivided into different classes according to the specific events. 1) Chromosome instability refers to changes in the number of chromosome leading to chromosome gain or loss (Draviam et al, 2004). It can be caused by the failure in mitotic chromosome transmission or the spindle mitotic checkpoint. 2) Micro- or minisatellite instability is marked by repetitive DNA expansion or contractions which can occur upon replication slippage, mismatch repair (MMR) or homologous recombination (HR). 3) Gross chromosomal rearrangement, such as  3 translocations, duplications, inversions and deletions, can result from errors in homologous recombination (HR) events, for instance unequal sister-chromatid exchange or HR between non-allelic repeated DNA fragments (Aguilera & Gomez-Gonzalez, 2008; Frohling & Dohner, 2008). Genomic alterations can also be acquired through epigenetic mechanisms such as DNA methylation and histone modifications (Berdasco & Esteller, 2010; Esteller, 2007; Jones & Baylin, 2007). Despite the broad spectrum of events leading to genomic instability, many of them arise from failure of replication due to replication stress. 1.1.2 Source of replication stress During DNA replication, the stable double stranded DNA structure is opened up to give rise to highly recombinogenic single stranded DNA (ssDNA). Any perturbation to the replication fork, defined as replication stress from now on, might lead to replication fork collapse, generating DNA breaks and recombination events (Hyrien, 2000). Replication stalling can occur at genomic regions with specific G-rich repeats or with long AT-rich repeats that can lead to formation of DNA secondary structures. These are known as the common fragile sites (Mirkin, 2007; Pichiorri et al, 2008). Gaps and breaks can arise at these sites following partial inhibition of DNA synthesis (Sutherland, 1977). Another factor leading to replication stress is the presence of DNA adducts on the DNA template during replication. The source of DNA damage could arise intrinsically, e.g. from generation of reactive oxygen species that give rise to oxidized DNA bases, or from spontaneous DNA depurination from hydrolysis (Lindahl, 1993); or extrinsically from environmental agents, e.g. from ultraviolet radiation (UVR) that generates pyrimidine dimers (Batista et al, 2009), or from alkylating agents that gives rise to alkylated bases (Sedgwick et al, 2007). Replicative polymerases Polε and Polδ duplicate DNA with high fidelity but are unable to  4 recognize DNA adducts on the template, consequently, resulting in stalled replication. Prolonged stalling of replication can lead to double-stranded breaks (DSBs) or single- stranded DNA gaps (Higgins et al, 1976; Sogo et al, 2002) which is a major source of genomic instability in cells. 1.1.3 Replication failure and cancer Replication failures can contribute to cancer development. This is first evidenced by the observation that common fragile sites are found at the breakpoints in rearranged chromosomes observed in malignant cells (Yunis & Soreng, 1984). For instance, the fragile histidine triad (FHIT) gene, considered as a multiple suppressor gene, maps to a region associated with deletions of translocation breakpoints in human lung cancer (Hibi et al, 1992), breast cancer (Negrini et al, 1996), oesophageal adenocarcinoma (Fang et al, 2001), pancreatic tumors (Shridhar et al, 1996) and renal cell carcinoma (Shridhar et al, 1997). Moreover, patients with various human genetic diseases carrying defects in pathways that protect replication failure are predisposed to cancer, for instance ATM, found to be mutated in ataxia telangiectasia, is a protein kinase activated by DSBs (Lavin, 1999); p53, a versatile DNA damage responsive gene, is found to be mutated in Li-Fraumeni syndrome (Bell et al, 1999); Nijmegen breakage syndrome (NBS) gene product is a substrate for ATM and part of the complex involved in HR for DSB repair  (Matsuura et al, 1998); defects in translesion DNA polymerase, which is required for recovering from UV-induced replication blockage, is found in Xeroderma Pigmentosum variant (XPV)  (Moriwaki & Kraemer, 2001); and Fanconi Anemia (FA) is an autosomal recessive cancer susceptible disease for which gene products in the FA pathway are involved in the DNA intrastrand crosslink repair (Haushalter  5 & Kadonaga, 2003). Those examples highlight the importance of faithful and complete replication to prevent genomic instability and cancer development. 1.2 Lesion Bypass Pathway To prevent tumorigenesis, cells evolved specialized mechanism to resolve replication blockage due to DNA adducts known as lesion bypass, also called post-replication repair (PRR) or DNA tolerance pathway (DDT). It is further subdivided into two subpathways, namely the translesion DNA synthesis (TLS) and template switching (TS) (Bienko et al, 2005; Ohmori et al, 2001; Stelter & Ulrich, 2003). 1.2.1  Lesion bypass subpathways In translesion DNA synthesis, the usage of replicative polymerases is switched to the Y-family polymerases upon replication blockage by DNA adducts (Bienko et al, 2005; Burgers et al, 2001; Ohmori et al, 2001) (Figure 1.1). The Y-family polymerases possess a more open active site when compared to the replicative polymerases and therefore are able to accommodate DNA adducts (Friedberg et al, 2000; Friedberg et al, 2005; Ohmori et al, 2001). Moreover, the translesion DNA polymerases are devoid of 3‟ to 5‟ proofreading exonuclease activity allowing them to replicate across lesions (Prakash et al, 2005). Therefore, these polymerases are also considered to be error-prone if they are not properly employed. In humans, the Y-family polymerases include Rev1, Polκ, Pol and Polι (Guo et al, 2009). Polκ is implicated in the bypass of alkylated bases such as those caused by methyl methanesulfonate (Lin et al, 2011); Polε is known to be required for replication across UV lesion with rather high accuracy (Friedberg et al, 2002; Masutani et al, 1999; Ohmori et al, 2001); Polι can incorporate nucleotides opposite an abasic site (Johnson et al, 2000) and N2- adducted guanine (Washington et al, 2004a); and Rev1 may resemble Polι for bypassing N2-  6 adducted guanine (Washington et al, 2004b).  While template switching is an error-free pathway in which replication blockage is resolved by switching of DNA template to the nascent daughter strand of the sister chromatid (Bruno et al, 2003; Chiu et al, 2006; Motegi et al, 2006), the mechanism for TS is less well understood.  7 Figure 1.1 Translesion DNA synthesis (A) The replicative Polδ is stalled at a lesion, exemplified by a CPD. RPA and Rad6/Rad18 are recruited to the template, downstream of the lesion, after unwinding by the MCM helicase. (B) Next, Rad6/Rad18 ubiquitinates PCNA. (C) This results in the exchange of Polδ for Pol at the primer terminus. (D) Pol synthesizes a few nucleotides opposite and beyond the CPD. (E) A second polymerase switch repositions Polδ at the primer terminus, leading to resumption of processive replication. Modified from Jansen et al, 2007, with permission to reprint.   8 1.2.2 PCNA ubiquitination and lesion bypass Proliferating cell nuclear antigen (PCNA) is a trimeric protein that forms a DNA sliding clamp and serves as a DNA polymerase processivity factor (Moldovan et al, 2007). During DNA replication, replication factor C (RFC) binds to the RNA primer-DNA template junction and load PCNA onto DNA (Bowman et al, 2004; Majka & Burgers, 2004). Upon PCNA loading, Polα is released and Polε is loaded to mediate the leading strand elongation (Kunkel, 2004); while on the lagging strand, the Okazaki fragment is synthesized by Polα and Polδ and subsequently ligated via DNA ligase I (Nick McElhinny et al, 2008). Upon replication blockage, ubiquitination of PCNA at K164 is essential for activation of both TLS and TS. TLS is believed to be activated by monoubiquitination of PCNA (PCNA-Ub) (Prakash et al, 2005) while TS is activated by K63-linked polyubiquitination of PCNA (Moraes et al, 2001). During replication stalling, the E3 ligase Rad18 is recruited to sites of replication, together with E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinates PCNA at K164 (Bailly et al, 1994; Haracska et al, 2001; Watanabe et al, 2004) (Figure 1.1); whereas polyubiquitination of PCNA requires the initial monoubiquitination on PCNA and is further catalyzed by another E2-E3 complex, the Mms2-Ubc13-Rad5 complex in yeast (Parker & Ulrich, 2009) and the Rad5 orthologues in human are found to be SHPRH and HLTF (Motegi et al, 2008; Unk et al, 2006).  All Y-family TLS DNA polymerases contain one or two ubiquitin-binding domains, either the ubiquitin-binding motif (UBM) in Polι and Rev1 or the ubiquitin-binding zinc finger (UBZ) in Pol and Polκ (Guo et al, 2009) through which they interact with the ubiquitinated PCNA. In addition, Polι, Pol and Polκ contain the PCNA interacting peptide (PIP) box (Guo et al, 2009; Prakash et al, 2005); while Rev1 interacts with PCNA via its N-  9 terminal BRCT domain (Guo et al, 2006) as well as with other TLS polymerase (Guo et al, 2003) (Figure 1.2). The TLS polymerases are found to be very dynamic which enter and leave replication foci rapidly (Sabbioneda et al, 2009).  The TLS polymerases are only recruited through multiple domains on them to carry out lesion bypass at the sites of stalled replication when the TLS substrate is present. This serves as a safeguard mechanism to ensure that this potentially mutagenic pathway will not be casually deployed. Many factors have been shown to be involved in the regulation of PCNA ubiquitination. The key event is recruitment of Rad18 to the sites of stalled replication by the single stranded DNA (ssDNA) binding protein, replication protein A (RPA). This event is essential for Rad18 E3 ligase to monoubiquitinate PCNA in vivo (Davies et al, 2008). Recently, Cimprich and colleagues showed that HLTF and SHPRH are involved in determining which TLS polymerases are used in a damage specific manner (Lin et al, 2011). They demonstrated that HLTF is responsible for facilitating TLS by Pol across UV lesions and SHPRH facilitates TLS by Polκ across the MMS-induced lesions to prevent mutagenesis (Lin et al, 2011). Besides Rad18 and Rad5 (yeast) or SHPRH/HLTF (human), another E3 ligase, CRL4 Cdt2  was found to be responsible for PCNA monoubiquitination possibly under non-stress conditions (Terai et al, 2010). While ubiquitination of PCNA is mediated by E3 ligases, the ubiquitin specific peptidase 1 (USP1), a deubiquitinating enzyme (DUB), was found to be able to remove ubiquitin from PCNA and inactivate the lesion bypass (Huang et al, 2006). Despite identification of various factors involved in PCNA ubiquitination, the regulation of the pathway remains elusive.  10 Figure 1.2 The structural domains of the Y-family polymerases Protein size is represented proportionately. BRCT BRCA1 C terminus-like domain, UBM ubiquitin binding motif, UBZ ubiquitin binding zinc finger motif, PAD polymerase associated domain, NLS nuclear localization signal, PIP PCNA interaction peptide. Adopted from Guo et al, 2009, with permission to reprint.   11 1.2.3 TLS for UV lesions Ultraviolet radiation is the major environmental risk factor for melanoma development (Miller & Mihm, 2006). This is exemplified by the fact that patients with xeroderma pigmentosum (XP), who carry defects in the nucleotide excision repair that repairs for UV lesions, are predisposed to skin cancer development. In a subgroup of XP, the XP variant (XPV), those patients carry mutations in the Polh gene which encodes for Pol, demonstrate sun-light sensitivity and are prone to skin cancer development (Cleaver, 1972; Moriwaki & Kraemer, 2001). Additionally, cells derived from patients with XPV which are deficient in Polε exhibit a prolonged S phase arrest and enhanced H2AX phosphorylation following UV exposure, suggesting that the DSBs may arise from replication fork collapse (Limoli et al, 2002; Wang et al, 2007). Polε is recruited to the sites of replication and colocalizes with PCNA and Rad18 in foci upon UV irradiation (Acharya et al, 2008; Kannouche et al, 2004; Watanabe et al, 2004). These observations suggest that TLS plays a crucial role in genomic stability and prevent cancer development. UV inflicts two types of DNA lesions, cyclobutane pyrimidine dimmers (CPDs) and 6-4 photoproducts (6-4PPs). CPDs are responsible for the majority of UV-induced mutations in mammalian cells because they are formed much more frequently than 6-4PPs (Armstrong & Kunz, 1990; Bourre et al, 1987; Brash, 1997; Brash & Haseltine, 1982; Canella & Seidman, 2000), and are removed much less efficiently than 6-4PPs by the nucleotide excision repair (NER) (Mitchell et al, 1985). In contrast, 6-4PPs impose a large structural distortion in DNA and are more readily detected and removed by NER (Kim & Choi, 1995; Kim et al, 1995). Pol is proficient in accurately replicating across TT, TC and CC CPDs. This is supported by the observation that inactivation of Pol in human and yeast leads to an increase in C to T  12 transitions, which is a result from misincorporation of dNTPs when other TLS polymerase(s) is utilized (Stary et al, 2003; Yu et al, 2001). It was found that the elevated mutation frequency in XPV cells is due to the activity of another Y-family polymerase, Polι, in incorporating G or T opposite the 3‟ nucleotide of the CPDs (Tissier et al, 2000). In spite of the importance of TLS pathway in preventing photocarcinogenesis, the status of the components in this pathway during skin cancer development is not known. 1.3 S Phase Checkpoint 1.3.1 The replication checkpoint activation S phase checkpoint kinases that sense DNA damage and replication failure play a prominent role as an anti-cancer barrier (Gorgoulis et al, 2005; Syljuasen et al, 2005). Blockage of polymerase function due to replication stress generates ssDNA resulting from the uncoupling of MCM helicases and polymerases (Lupardus et al, 2002). ssDNA is coated by the RPA where two complexes, the ataxia-telangiectasia mutated and RAD3-related (ATR) kinase, a member of the PI3K-like kinases (PIKKs) and ATR-interacting protein (ATRIP) complex (ATR-ATRIP), and RAD9-RAD1-HUS1 (9-1-1) complex, are recruited to the site of stalled replication independently (Melo et al, 2001; Zou & Elledge, 2003). 9-1-1 further recruits topoisomerase-binding protein-1 (TOPBP1) that, together with other components, contributes to ATR activation (Delacroix et al, 2007; Lee et al, 2007). Activation of ATR leads to global checkpoint response by phosphorylating numerous targets. One important target is the downstream effector kinase Chk1. ATR phosphorylates Chk1 on S317 and S345 which activates Chk1 (Guo et al, 2000; Liu et al, 2000; Lopez-Girona et al, 2001; Walworth & Bernards, 1996). Claspin, a checkpoint mediator, is recruited at the replication fork and is required for Chk1 activation (Kumagai & Dunphy, 2000). In addition  13 to claspin, another replication-fork associated complex, timeless and timeless-interacting protein, also contributes to the activation of Chk1 by ATR (Errico et al, 2007; Unsal-Kacmaz et al, 2007) (Figure 1.3).  14 Figure 1.3 Replication checkpoint activation and function When a polymerase encounters a lesion that prevents its progression, the MCM helicase and the polymerase on the opposing DNA strand will continue to unwind and synthesize DNA. This functional uncoupling between the stalled polymerase and helicase leads to accumulation of ssDNA on the stalled strand, which results the recruitment of ATR-ATRIP complex as well as claspin, 9-1-1 which is necessary for the activation of Chk1. Activation of this pathway promotes genomic stability by downregulating further origin firing, facilitating replication restart, stabilizing replication forks, and blocking cell cycle progression. Adopted from Paulsen and Cimprich, 2007, with permission to reprint.   15 1.3.2 The role of ATR-Chk1 pathway in cell cycle arrest and replication fork stabilization ATR is essential for survival of proliferating cells (Brown & Baltimore, 2000) suggesting that it is required for cells to cope with intrinsic cellular stress. Abrogation of ATR and its effector kinase Chk1 leads to increased genomic instability (Casper et al, 2002; Syljuasen et al, 2005) during S phase indicating that the ATR-Chk1 pathway is essential for maintenance of genomic stability during DNA replication. Indeed, in agreement with this notion, checkpoint mutants were shown to have high rates of gross chromosomal rearrangements (Myung et al, 2001). The ATR-Chk1 plays an important role in cell cycle arrest during replication stress. Cell division control 25 (CDC25) phosphatases are the key regulators of the S phase progression. Upon activation of ATR-Chk1, Chk1 phosphorylates CDC25A which promotes CDC25A ubiquitination and degradation, leading to reduced cyclin-dependent kinase 2 (CDK2) activities (Busino et al, 2003; Jin et al, 2003; Melixetian et al, 2009). Furthermore, Chk1 also phosphorylates CDC25C which increases binding of CDC25C to 14-3-3 proteins which sequester and inhibit CDC25C function and prevents activation of the CDK1-cyclinB kinase and mitotic entry (Peng et al, 1997; Sanchez et al, 1997). Activation of the ATR-Chk1 pathway also inhibits the late origin firing. This prevents the replication forks from encountering DNA lesions before they are repaired. It has been shown that ATR-Chk1 pathway inhibits the CDC45-chromatin binding that is required for the initiation of the replication origins (Liu et al, 2006b). S phase checkpoint proteins have direct function in fork stabilization as well to protect the integrity of the existing stalled replication forks (Lopes et al, 2001; Tercero & Diffley, 2001). This is supported by the observation that the replisome components, such as  16 Polε and PCNA, dissociate from stalled forks in the absence of ATR signaling (Dimitrova & Gilbert, 2000; Trenz et al, 2006). ATR has been shown to phosphorylate a number of substrates at stalled forks such as the replication factor C complex, RPA1 and RPA2, the minichromosome maintenance (MCM)2-7 complex, MCM10, and several polymerases (Bao et al, 2001; Brush et al, 1996; Cortez et al, 2004; Liu et al, 2006a; Matsuoka et al, 2007; Oakley et al, 2001; Wang et al, 2001; Yoo et al, 2004). MCM2 phosphorylation by ATR promotes checkpoint recovery and resumption of replication through recruitment of the Polo- like kinase-1 (PLK1) (Trenz et al, 2008). However, the functional role of other phosphorylation substrates at stalled forks is not clear. Stabilization of stalled replication forks by S phase checkpoint is also thought to occur through stabilization of the replisome with the fork (Cobb et al, 2003; Lucca et al, 2004). These events might be mediated by the physical association of checkpoint proteins at the stalled fork. For instance, the components of the ATR-Chk1 signaling pathway, TOPBP1, BRCA1, Claspin and Chk1 itself, are required for preventing fork collapse at common fragile sites (Arlt et al, 2006). However, the mechanism of how the S phase checkpoint proteins maintain stalled forks stability remains to be elucidated. 1.3.3 S phase checkpoint and lesion bypass pathway Although both the S phase checkpoint and lesion bypass pathway has been implicated in resolution of, and recovery from, stalled replication, there are limited studies addressing whether the S phase checkpoint regulates lesion bypass. A recent study showed that ATR regulates TLS. ATR was found to phosphorylation Pol at S601 which is required for survival and lesion bypass following UV irradiation (Gohler et al, 2011). Mec1, the yeast homologue of ATR in human, was found to be required for TLS through Rev1  17 phosphorylation (Pages et al, 2009; Sabbioneda et al, 2007). Moreover, TLS polymerases, Polκ and Polδ in yeast, were shown to interact with the 9-1-1 checkpoint clamp and recruited to the sites of DNA damage (Sabbioneda et al, 2005). However, there are inconsistent findings for the role of checkpoint proteins, ATR and Chk1, in PCNA monoubiquitination. Studies using fission yeast and Xenopus extracts suggest that ATR is not required for UV-induced PCNA-Ub (Chang et al, 2006; Frampton et al, 2006). On the other hand, ATR and Chk1 are found to be required for B[a]P Di-hydrodiol Epoxide (BPDE)-induced PCNA-Ub (Bi et al, 2006). It has recently been shown that Chk1 regulates PCNA-Ub through stabilization of Claspin and interacting protein Timeless which is independent of its kinase activity and ATR function (Doyon et al, 2006). Although these data support the idea that S phase checkpoint is involved in regulation of PCNA-Ub and lesion bypass, the mechanism is yet to be defined. 1.4 Tumor Suppressor Inhibitor of Growth The Inhibitor of Growth (ING) family proteins are defined as type II tumor suppressors which include five genes in mammals, ING1 to ING5. These proteins are highly conserved among species with common and distinct functional domains in each family member (Soliman & Riabowol, 2007). The founding member ING1 gene was identified in 1996 by Riabowol and colleagues using subtractive hybridization between cDNAs from normal mammary cell line and seven breast cancer cell lines followed by an in vitro screening for cDNAs that were capable of promoting neoplastic transformation (Garkavtsev et al, 1996).  Since the initial discovery of ING1, ING2, ING3, ING4 and ING5 were discovered by sequence homology. These proteins carry diverse biological functions.  18 1.4.1 Functional domains of ING proteins ING proteins are well-conserved in eukaryotic proteomes (He et al, 2005). All ING genes with the exception of ING3 are found near chromosome ends and the function and expression of ING5 could be affected by telomere erosion (He et al, 2005). p33ING2 shares 60% homology with p33ING1b and encodes a 33-kDa protein (Shimada et al, 1998). Compared to ING1b, ING2 contains a leucine zipper domain (LZL) which is thought to mediate hydrophobic protein-protein interaction (Feng et al, 2002). p29ING4 and p28ING5 are highly homologous with 73% identity (Shiseki et al, 2003). There are four isoforms in ING1, namely ING1a, b, c and d, with identical C-terminus (Campos et al, 2004a). All these isoforms share a common exon (exon 2), which contains the plant homeodomain (PHD). ING1b is the most widely expressed isoform (Jager et al, 1999; Nouman et al, 2002c). The expression of ING1c is lower than ING1b and there is little ING1a expression in all tissues examined (Saito et al, 2000). It has been shown that ING proteins carry out diverse biological functions through common and specific domains (Figure 1.4). The PHD domain is present in all ING proteins at their C termini (Soliman & Riabowol, 2007). The PHD zinc finger comprises of a signature C4HC3 sequence which binds two zinc ions in a cross braced topology (Aasland et al, 1995; Bienz, 2006; Pascual et al, 2000). The PHD domain is shown to interact with the nuclear phosphatidylinositol-5-phosphate (PtdIns5P) which is required for loading of ING proteins onto the chromatin directed through the p38 stress-activated protein kinase upon stress (Jones et al, 2006). PHD has also been shown to interact with methylated histone H3K4 which marks the actively transcribed genes. The ING2 mediates active gene repression through the PHD domain (Martin et al, 2006; Palacios et al, 2006; Pena et al, 2006; Shi et al, 2006). The lamin interaction domain (LID) was found in all ING proteins  19 which is required for interaction with lamin A. It is responsible for stabilizing ING1 and targets ING1 to the nucleus (Han et al, 2008). It is not known if this function is the same for other ING proteins. All ING proteins contain a nuclear localization signal (NLS) and are therefore nuclear proteins. The nuclear localization of these proteins is important for their tumor suppressor functions as evidenced by the fact that nuclear ING1 expression is lost in several cancers (Gong et al, 2005). The NLS of ING1 contain two copies of a putative nucleolar translocation signal which interacts with the proteins karyopherin-α and -β for nuclear import (Russell et al, 2008). The nucleolar translocation of ING1 after UV was reported and was found to be required for ING1-induced apoptosis (Scott et al, 2001a). The LZL domain is present in all ING proteins except ING1. This domain contains four to five conserved leucine or isoleucine residues spanning every seven amino acids (forming hydrophobic patch). Therefore, the LZL domain often interacts with other proteins with LZL to form heterodimer or with itself to form homodimer through hydrophobic interaction (Chen et al, 2003b; Kobe & Kajava, 2001). LZL in ING2 was shown to be important for its association with p53 and for the proper functioning of ING2 in apoptosis and NER (Wang et al, 2006b). However, it is not known if LZL in other ING proteins may carry similar or unique functions. Since LZL is responsible for dimer formation, it has been proposed that ING proteins may form homo- or hetero-dimer(s) among themselves. ING proteins also contain specific functional domains. For instance, both ING1b and ING2 contain specific domains to interact with PCNA. ING1b carries a PIP box (Scott et al, 2001b) while ING2 utilizes another distinct domain for PCNA interaction (Larrieu et al, 2009). ING1b interaction with PCNA is increased upon UV through its PIP box and such interaction is required for UV-induced apoptosis (Scott et al, 2001b) while ING2 interaction  20 with PCNA is essential for normal DNA replication (Larrieu et al, 2009). A SAP30- interacting domain (SAID) is reported to be present in ING1b and potentially in ING2 (Kuzmichev et al, 2002; Loewith et al, 2001). This interaction is thought to bridge ING1 and ING2 to SAP30-containing HADC1/2 complexes. Another unique domain in ING1b is the partial bromodomain (PBD). The PBD binds SAP30 of mSin3A-HDAC1 which might tether ING1b with the mSin3A-HDAC1 complex (Kuzmichev et al, 2002) (Figure 1.5).  21 Figure 1.4 Functional domains in ING proteins (A) ING1 protein domains and their interacting partners. (i) The PCNA-interacting protein motif of ING1b binds specifically to PCNA. (ii) The region that shows sequence homology to bromodomains called a partial bromodomain (PBD) was identified by bioinformatics analysis, binds Sap30 of the mSin3–HDAC1 complex. (iii) The novel conserved region (NCR) identified by bioinformatics analyses. (iv) The nuclear localisation signal (NLS) is conserved in most ING proteins and targets them to the nucleus. (v) The ING1 proteins contain a 14-3-3 recognition motif to which 14-3-3 binds when ING1 is phosphorylated on serine 199. (vi) The PHD finger found in the ING proteins is the most highly conserved region in the ING family in all species examined, from yeast to humans. (vii) The poly basic region (PBR) of ING1 and ING2 is juxtaposed with the PHD finger and is both necessary and sufficient to mediate interaction with bioactive phosphoinositides and activate them. (B) Representation of the known ING proteins and their structural features. Numbers indicate the boundaries of the sequence motifs and domains in ING1b are drawn approximately to scale. In this diagram, PIP represents the PCNA-interacting protein motif, PBD is a sequence with partial homology to bromodomains, LZL is a leucine-zipper-like region, the NCR is a novel conserved region of currently unknown function, NLS represents the nuclear localisation sequence, the NTS in p28ING4 represents a truncated NLS that retains a nucleolar translocation sequence but is not effective in localising proteins to the nucleus, PHD represents the highly conserved plant homeodomain finger and PBR is a polybasic region. Modified from Soliman and Riabowol, 2007, with the permission to reprint.  22   23 Figure 1.5 ING domains and functions The PIP box binds to PCNA and promotes ING1-mediated apoptosis. The PBD and NCR/LID binds to SAP30, HDAC and HAT, and regulates their activities. The LZL domain of ING2 is required for DNA repair and apoptosis. The NLS is responsible for the nuclear localization of ING proteins. The PBR domain binds to PtdInsPs and PHD domain binds to histone mark which play a role in regulation of gene transcription. Adopted from Aguissa- Touré et al., 2010, with permission of reprint.   24 1.4.2 Biological functions of ING proteins ING proteins have been shown to confer tumor suppressor functions through regulating various biological processes. For instance, ING1, ING2 and ING3 are all shown to induce apoptosis when expressed ectopically (Cheung & Li, 2002; Chin et al, 2005; Nagashima et al, 2003). Both ING1 and ING2 are involved in regulation of cellular senescence (Garkavtsev et al, 1996; Ludwig et al, 2011; Menendez et al, 2009; Pedeux et al, 2005) and DNA repair (Kuo et al, 2007; Wang et al, 2006a). While ING4 was found to inhibit cell migration and angiogenesis (Li & Li, 2010; Li et al, 2008; Serra-Pages et al, 1995; Unoki et al, 2006). Because of the ubiquitous expression of p33ING1 (ING1b) in various tissue and the its well known function as a tumor suppressor, herein, I am going to focus on introducing the known biological functions of ING1b which is the subject of this thesis.  ING1b was initially shown to regulate cell cycle progression. When ING1 was first identified, Riabowol and colleagues observed that an increase in cells arrested in G0/G1 when they overexpressed ING1 in human diploid fibroblasts and conversely, suppression of ING1 results in entry of cells into S phase and therefore it was named as the “inhibitor of growth” (Garkavtsev et al, 1996). Later, it was shown that ING1b is involved in G2/M transition through regulating cyclin B1 expression (Takahashi et al, 2002; Tsang et al, 2003). ING1 expression was also found to be inversely correlated with cyclin E expression (Ohgi et al, 2002). Moreover, it has also been shown that ING1 expression changes throughout the cell cycle. ING1 expression is reduced from G0 to G1, then increases in late G1, peaking during S phase and subsequently decreases in G2 (Garkavtsev & Riabowol, 1997). Therefore, it indicates that ING1 is involved in the cell cycle regulation.  25  ING1 plays an important role in cellular stress response. Ectopic expression of ING1b sensitizes cells to doxorubicin, etoposide and taxol (Scott et al, 2001a; Takahashi et al, 2002; Tsang et al, 2003; Vieyra et al, 2002). ING1b is also required for UV-induced apoptosis in melanoma cells (Cheung & Li, 2002). A previous study showed that ING1 induces apoptosis in an isoform-specific manner. ING1b, but not p47ING1 (ING1a), promotes UV- and hydrogen peroxide-induced apoptosis (Vieyra et al, 2002). Intriguingly, downregulation of ING1b sensitized p53-deficient glioblastoma cells to cisplatin-induced apoptosis (Tallen et al, 2008). Previously, we and others showed that ING1b knockdown sensitized S-phase arrested melanoma cells to UV (Kuo et al, 2007) and MEFs from ing-/- mice exhibited increased sensitivity to UV (Kichina et al, 2006). However, the mechanism for UV hypersensitivity in ING1 deficient cells is unclear. In this thesis, we addressed the mechanism for the replication stress sensitivity in ING1b depleted cells.  ING1b has also been linked to the regulation of NER following UV exposure. Overexpression of ING1b increases repair efficiency of the UV damaged reporter plasmids (Cheung et al, 2001) and ING1b knockdown leads to reduced removal of endogenous CPDs (Kuo et al, 2007). ING1b was also found to interact with a DNA damage responsive gene, GADD45 (Cheung et al, 2001). ING1b is also required for recruitment of XPA, a lesion recognition factor, and HDAC inhibitor treatment rescues NER in ING1b deficient cells suggesting that ING1b facilitates accessibility of the NER machinery through chromatin remodeling (Kuo et al, 2007).  All ING proteins except ING3 have been shown to interact with p53 and the ING- induced cell cycle arrest and apoptosis is compromised in p53 deficient cells suggesting the function of ING on cell growth is p53-dependent (Berardi et al, 2004; Campos et al, 2004a;  26 Soliman & Riabowol, 2007). Expression of antisense ING1 inhibited expression of p21WAF1 in cells with functional p53 (Garkavtsev et al, 1998), suggesting that p53 also requires ING1 for its functions. Cooperation of ING1 and p53 is further supported by the observation that only co-expression of ING1 and p53 was able to enhance apoptosis in glioma and esophageal carcinoma cell lines (Shimada et al, 2002; Shinoura et al, 1999). It was also observed that ING1 triggers senescence in p53-dependent manner (Abad et al, 2011). Other studies suggest that ING1 regulates p53 through MDM2. It has been proposed that ING1b competes with MDM2 with p53 binding and increases p53 stability and activity (Leung et al, 2002). ARF was shown to interact with ING1b and ARF is required for ING1b- induced cell cycle arrest (Gonzalez et al, 2006). However, it should be noted that many of these functions were characterized with ectopic expression of ING1b. It is not clear if such cooperation of ING1b and p53 also occurs at physiological level. There also appears to be p53-independent functions of ING1. It has been shown that ING1b regulates cyclin B1 expression in p53-independent manner since ectopic expression of ING1b in p53-null Saos-2 cells downregulated cyclin B1 (Takahashi et al, 2002). ING1b also regulates HSP70 expression at transcriptional level in a p53-independent manner (Feng et al, 2006). There are also p53-independent functions of ING1b on cell cycle regulation. It has been shown that ectopic expression of ING1b induced G2/M arrest upon doxorubicin treatment in p53- deficient H1299 lung carcinoma cells (Tsang et al, 2003). Moreover, ING1 inhibits B cells regardless of p53 status (Coles et al, 2007; Coles et al, 2008). Therefore, more studies are needed to further elucidate the p53 dependency of ING1 functions.  ING1 has been shown to play a role in tumorigenesis. Knockdown of ING1 promotes the ability of anchorage independent growth in soft agar and increases foci formation  27 suggesting a role for ING1 in preventing cell transformation (Garkavtsev et al, 1998; Garkavtsev et al, 1996; Takahashi et al, 2002). This notion is further supported by in vivo experiments in mice. Knockout of p37ING1b, the potential human ING1b orthologues, in mice leads to enhanced aggressive diffuse large B-cell lymphomas and spontaneous follicular B-cell lymphomas (Coles et al, 2007; Coles et al, 2008). These observations confirm the role of ING1 as tumor suppressor. 1.4.3 ING1 and chromatin remodeling ING proteins are found to be in stable complex with various chromatin remodeling proteins. Endogenous ING1b was found to be associated with a complex that contains mSin3, SAP30, histone deacetylase 1 (HDAC1), RbAp48 and additional components of the mSin3A transcriptional co-repressor complex (Skowyra et al, 2001) and purified ING1b complex were found to deacetylate core histones in vitro. ING1b and ING2 have also been shown to recruit SIRT1 to the Sin3/HDAC complex (Binda et al, 2008). Overexpression of ING1b induces hyperacetylation of histone H3 and H4 (Vieyra et al, 2002); conversely, our group showed that ING1b knockdown reduces histone H4 acetylation (Kuo et al, 2007). Moreover, ING1b was found to co-immunoprecipitate with HATs such as p300, CBP, PCAF and TRRAP; whereas ING1a was found to associate with HDAC1 (Vieyra et al, 2002). Therefore, there appears to be conflicting results in identification of ING1b complex since both HATs and HDACs were found. It might imply that ING1b could associate with different members at different conditions or different ING1b complexes are present. So far, ING1b complex was identified in non-stress conditions. It is conceivable that ING1b partners could be changed upon stress. This is evidenced by the fact that ING1b associations with PCNA and p15 PAF  are induced after UV (Scott et al, 2001b; van Bueren et al, 2007).  28 Other ING proteins were found to be in various chromatin remodeling complexes. ING2, similar to ING1, is found in a complex with HDAC1 (Doyon et al, 2006); ING3 has been shown to stably associate with the Tip60/NuA4 HAT complex (Doyon et al, 2004); while ING4 and ING5 were found to interact with the HBO1 HAT and HBO1 or the MOZ/MORF HAT, respectively (Doyon et al, 2006). 1.4.4 Chromatin remodeling, DNA replication and ING proteins Since the chromatin structure is a barrier for DNA replication, it has to be remodeled before and after this process (Falbo & Shen, 2006). Various chromatin remodeling complexes are required to ensure concerted removal of histone prior to replication fork and histone disposition after DNA replication (Dhalluin et al, 1999; Groth et al, 2007a; Groth et al, 2007b). Chromatin remodeling is also implicated in DNA replication under stress and preservation of genomic stability. The ATP-dependent chromatin remodeling enzyme Ino80 is found to bind to stalled replication forks, possibly promoting replication recovery by mobilizing histones ahead of the fork to mediate polymerase resumption (Shimada et al, 2008). Although chromatin remodeling has been implicated in various processes involving DNA repair, transcription and replication (Hanawalt, 2007), little is known about its role in DNA replication under stress and the functional interplay between S phase checkpoint and chromatin remodeling.  Several members of ING family proteins have been implicated in regulation of DNA replication. ING2 has been shown to be required for normal replication progression. ING2 is found to associate with PCNA and is required for loading of PCNA onto chromatin (Larrieu et al, 2009). ING4- and ING5-associated HAT, HBO1, was found to be required for normal progression through S phase. Knockdown of HBO1 causes accumulation of cell population  29 in S phase (Doyon et al, 2006). The ING5 HAT complexes were found to interact with MCM helicases which are essential for DNA replication (Doyon et al, 2006). However, it is not known if ING proteins are also involved in DNA replication under stress. 1.4.5 Alterations of ING1 in human cancer ING1 expression is often found to be downregulated or lost in many human cancers, including lymphoid malignancies (Ohmori et al, 1999), bladder cancer (Sanchez-Carbayo et al, 2003), gastric cancer (Graham et al, 1999), breast cancer (Tokunaga et al, 2000), and non- small cell lung carcinoma (Kameyama et al, 2003). ING1b was also found to be mislocalized to the cytoplasm in glioma (Vieyra et al, 2003). Intriguingly, ING1 expression has also been shown to be upegulated in basal cell carcinoma (Chen et al, 2003a), and melanoma (Campos et al, 2002) The reason for such upregulation is not known. But ING1 mutation was found in 20% of primary melanoma (Campos et al, 2004b). Mutants of p53 are also found to be upregulated in cancer. More investigation will be needed to understand if ING1 mutation correlates with its expression level. Since nuclear localization of ING1 is critical for its function, a nuclear to cytoplasmic shift of ING1b his been reported in certain cancers such as melanoma, oral squamous cell carcinoma and brain tumors (Nouman et al, 2002a; Vieyra et al, 2003; Zhang et al, 2008) 1.5 Cutaneous Melanoma 1.5.1 Epidemiology of melanoma Although melanoma only accounts for 4% of all dermatologic cancers, it is responsible for 80% of death from skin cancer. Only 14% of patients with metastatic melanoma survive for five years (Miller & Mihm, 2006). Although there is a trend of reduction in cancer incidence in many forms of cancer, the incidence of melanoma has  30 doubled in the last decade (Dauda & Shehu, 2005; Thompson et al, 2005). In Canada, melanoma is the seventh most common cancer in men and the eighth most common cancer in women (Canadian Cancer Society: Canadian Cancer Statistics 2010).  The strongest risk factors for melanoma are a family history of melanoma, multiple benign or atypical nevi, and a previous melanoma. Immunosuppression, sun sensitivity and exposure to ultraviolet radiation are additional risk factors. Melanoma is the most common among light-skin population who have 10-fold higher risk of developing melanoma than Black, Asian or Hispanic populations (Ries et al, 2000). Therefore, both genetic predisposition and environmental factors contribute to the genesis of the disease. 1.5.2 Melanoma biology Cutaneous melanocytes originate from neural-crest progenitors that migrate to the skin during embryonic development. Melanocytes from the skin reside in the basal cell layer of the epidermis and in the hair follicles and their homeostasis is regulated by epidermal keratinocytes (Slominski et al, 2004). The ratio of melanocytes to keratinocytes varies in different regions of the body, ranging from 1:4 to 1:10 (Fitzpatrick & Szabo, 1959). Keratinocytes produce factors that stimulate the production of melanin pigments from melanocytes which affect the skin color. The key role of melanin is to protect DNA from UV damage. Consequently, individuals with pigmentary disorder such as vitiligo and albinism, for which their melanocytes are nonfunctional, are hypersensitive to UV radiation (Boissy & Nordlund, 1997).  When mutations in growth regulatory genes, including those involved in growth signaling and cell adhesion, occur in normal melanocytes, they can escape the tight regulation by keratinocytes (Haass et al, 2004). As a result, melanocytes can proliferate and  31 spread that lead to the formation of nevus or common mole. Melanocyte proliferation can be restricted to the epidermis (junctional nevus), the dermis (dermal nevus) or overlapping components of both (compound nevus) which are generally benign. But with additional genetic alterations, nevus cells can progress to the radial growth phase (RGP) melanoma, where the cells are able to proliferate intraepidermally. RGP cells can enter the vertical growth phase (VGP) with nodules and nests of cells invading the dermis. The VGP cells obtain invasive ability and metastaic potential. Not all melanoma pass through each individual phase. They can also progress directly to metastatic malignant melanoma (Clark et al, 1984). 1.5.3 Etiology of melanoma Melanoma is a complex genetic disease for which its development could be influenced by the genetic alterations de novo, from heredity or from environments. It encompasses a progressive change in various cell signaling pathways which eventually leads to the manifestation of the histological features in different stages of melanoma development.  The Ras/Raf/MEK/ERK pathway regulates cell proliferation and is required for the strong and sustained proliferation of melanoma cells which is hyperactivated in up to 90% of human melanomas (Cohen et al, 2002). In melanoma, ERK can be activated by the production of autocrine growth factors (Satyamoorthy et al, 2003). NRAS mutations occur between 15% and 30% of melanomas which also account for the activation of ERK pathway. The most commonly mutated component of this pathway is BRAF. BRAF is mutated in 50% to 70% of melanomas and the most common mutation is the V600E substitution (Davies et al, 2002). Intriguingly, similar frequency of BRAF mutations is found in benign nevi compared to primary and metastatic melanomas (Pollock et al, 2003). This is due to the fact  32 that activation of BRAF leads to senescence through induction of p16 INK4a  (Michaloglou et al, 2005; Wajapeyee et al, 2008). Therefore, activation of the ERK pathway is not sufficient to drive malignant transformation of melanocytes.  Inactivation of the CDKN2A gene occurs in 20% to 40% cases of familial melanoma 6 which encodes for two tumor suppressor proteins p16 INK4A  and p19 ARF  (Kamb et al, 1994; Nobori et al, 1994).  INK4A is an inhibitor for CDK4 which suppresses cell proliferation through blocking the cell cycle at the G1/S checkpoint in cells with damaged DNA or activated oncogenes (Sharpless & Chin, 2003). CDK4 and cyclin D1, a downstream target of INK4A, are also found to be mutated in some melanomas (Sauter et al, 2002; Sotillo et al, 2001; Zuo et al, 1996).  It suggests that mutations in CDKN2A potentially abrogate the oncogene-induced senescence and drive melanomagenesis. However, another study showed that BRAF V600E -induced senescence in nevus melanocytes is in part INK4A-independent, suggesting presence of other melanoma suppressor(s) (Michaloglou et al, 2005).  The phosphoinositide-3-kinase (PI3K)-Akt signaling cascade plays an essential role in melanoma proliferation (Dai et al, 2005; Madhunapantula & Robertson, 2009). Akt is activated by phosphorylations at Thr308 and Ser473. Akt phosphorylates a number of substrates which result in sustained cell proliferation and cell cycle progression, invasion, angiogenesis and reduced apoptosis (Chudnovsky et al, 2005; Hsu et al, 2002; Smalley & Herlyn, 2005). The mechanism of activation of Akt in melanoma could be explained in part by the findings that mutation of  PI3K, the upstream activator of Akt, is found in 3% of metastatic melanoma (Omholt et al, 2006); loss of function of PTEN, the negative regulator of Akt, in 5-20% of late stage melanoma (Wu et al, 2003); and the overexpression of Akt itself in up to 60% of melanoma (Stahl et al, 2004). However, expression level of Akt does  33 not necessarily correlate with its activity (Stahl et al, 2003). Therefore, there may be other mechanism regulating Akt activation in melanoma. Other important pathways implicated in melanoma development include the melanocortin and MITF differentiation pathways and the WNT, β-Catenin, and Cadherin cell adhesion and signaling  (Miller & Mihm, 2006). 1.5.4 UV radiation and melanoma The link of UV radiation with melanoma development is first supported by the fact that the risk of melanoma in fair-skinned people is higher than the people with darker complexions. Fair-skinned individuals have lower melanin production in their skin and therefore more susceptible to UV damage (Marrett et al, 1992). This is also supported by the epidemiologic evidence that patients with XP, a family of diseases characterized by grossly deficient repair of DNA photolesions, have greatly increased risk of melanoma (Kraemer et al, 1987; Lambert et al, 1995). Melanoma can also be induced in vivo in certain animals by exposure to UV radiation (Jhappan et al, 2003; Kusewitt et al, 1991; Setlow et al, 1989). UV radiation can be subdivided into UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm) wavebands. UVC contribution to the development of skin cancers is negligible since it is prevented from reaching the surface of the earth by the atmospheric ozone layer. The UVB is the most effective wavelength for inducing DNA photoproducts in the basal layer of epidermis. Therefore, UVB induces typical signature mutation, C to T transitions, which is believed to be caused by the mis-incorporation of A opposite C during replication (Runger & Kappes, 2008).  Recently, it has become appreciated that UVA also contribute to the mutagenesis process because over 20% of UVA is able to reach the basal germinative layers while that of UVB is less than 10% (Bruls et al, 1984). The basal cell  34 layers are where melanocytes are situated and presumably more susceptible to UVA radiation. UVA causes generation of reactive oxygen species (ROS) which reacts predominantly with guanine and generates the 7,8-dihydro-8-oxyguanine (8-oxoG). These lesion is known to cause G to T transversions (Cheng et al, 1992). In fact, it was shown that there are more UVA fingerprint mutations in human squamous tumors compared to that of UVB (Agar et al, 2004). Although epidemiological studies indicate the involvement of UV in melanoma development, the mechanism of the photocarcinogenesis is not clear. Only very low frequency of the UVB signature mutations, C to T, are found in melanoma suppressor p16 INK4A  (Peris et al, 1999). C to T mutations have been described in PTEN of melanomas (Wang et al, 2010). A high frequency of T:A to A:T mutations is found in BRAF in malignant melanomas and melanocytic nevi (Pollock et al, 2003). Although these mutations are found predominantly in melanomas with intermittently sun-exposed areas or chronically UV-exposed areas, this type of mutation is not located in the di-pyrimidine sites nor is it associated with any of the common type of UV-induced lesions. It has been proposed that error-prone translesion DNA synthesis of a nearby pyrimidine dimer could contribute to these BRAF mutations (Thomas et al, 2006). It has also been proposed that UV-induced genomic instability could contribute to melanoma development (Dahle & Kvam, 2003), however, the underlying mechanism is not clear. 1.5.5 Genomic instability in melanoma Genomic instability has been observed in melanoma. Curtin et al. reported that there are distinct sets of genomic alterations in four different groups of melanoma analyzed with various degrees of genome gains and losses, changes in intrachromosomal copy number, and  35 focal amplifications (Curtin et al, 2005). CGH analysis revealed that melanoma differs from melanocytic nevi by the presence of frequent chromosomal aberrations (Bauer & Bastian, 2006).  It is proposed that replication stress is a feature of cancer and precancerous lesions that leads to the constitutive activation of the DNA damage response pathway (Bartkova et al, 2010; Bartkova et al, 2005; Halazonetis et al, 2008). In fact, recent studies showed that there is an elevation of γH2AX, which is a very sensitive marker for DNA damage, in melanoma and melanocytic lesions (Warters et al, 2005; Wasco et al, 2008). However, the source for replication stress in melanoma cells is not known. Moreover, the contribution of the DNA damage response pathway to genomic stability during melanoma development remains elusive. 1.5.6 Treatment for melanoma Surgical removal is by far the most effective treatment for melanoma if it can be detected early. However, metastatic melanoma is refractory to conventional chemotherapy and there is no effective treatment available. Therefore, there are different novel therapeutic strategies being proposed. Dacarbazine historically was the standard treatment for melanoma and over the past decade it has been replaced with temozolomide, an oral formulation of dacarbazine (Middleton et al, 2000). However, the result for patient survival is disappointing with 5-year survival rates for treated patients ranging from 3 to 14% (Barth et al, 1995). Interferon-α (IFN-α) has been used for melanoma treatment for decades. Recently, it was shown that IFN has immunomodulatory effect in which infiltration of immune cells in the tumor was observed (Moschos et al, 2006). This suggests that immunotherapy is  36 potentially an effective treatment for melanoma. Strategies like stimulating T cell responses have been under clinical trial and show some better response rates compared to traditional chemotherapy (Wolchok & Saenger, 2007). With the advancement in the knowledge on melanoma genetics, new therapeutic strategies are being developed to target the altered signaling pathways in melanoma. For instance, inhibitors targeting VEGFR, PDGFR, Raf, MEK, CDK4/6, mTor, Akt, Ras, Hsp90 and PI3K have been proposed and applied in clinical trial for melanoma treatment (Gray- Schopfer et al, 2007).  The efficacy of these therapies is yet to be proven. Meanwhile, more studies are warranted on the etiology of melanoma to identify novel therapeutic targets. 1.6 Objective and Hypotheses ING1b has been shown to be tumor suppressor and it plays an important role in UV stress response. Previously studies showed that ING1b deficient cells are hypersensitive to UV (Kichina et al, 2006; Kuo et al, 2007). However, the reason for such sensitivity is not clear. In this thesis, we sought to understand the physiological role of ING1b in UV stress response. Since ING1b was found to be abrogated in cancer, we used small interfering RNA to knock down ING1b expression and studied the biological responses of these cells to UV. We found that ING1b played an important role in resolving stalled replication due to UV. We hypothesized that ING1b was involved in the regulation of the lesion bypass pathway through PCNA monoubiquitinaton. We further investigated if ING1b regulates PCNA monoubiquitination and genomic stability through chromatin remodeling. Previously our group identified that ING1b is phosphorylated at S126 residue by Chk1, the key effector kinase in the S phase checkpoint (Garate et al, 2007). Since Chk1 has also been implicated in the regulation of PCNA monoubiquitination and genomic stability, we hypothesized that ING1b cooperated with Chk1 in regulation of PCNA  37 monoubiquitination. We further investigated if ING1b S126 is required for TLS and maintaining genomic stability after UV.  Since the lesion bypass pathway plays an important role in genomic stability, we hypothesized that the expression of the E3 ligase Rad18, a key regulator for lesion bypass, was altered in melanoma. We took advantage of the melanoma tissue microarray (TMA) and studied the expression of the Rad18 in melanocytic lesions. We discovered the dual role of Rad18 on patient survival in the sun-exposed and sun-protected area. We also found that Rad18 was involved in regulation of melanoma proliferation. Therefore, we further investigated the mechanism by which Rad18 affected melanoma cell proliferation.   38  Chapter 2: Material and Methods 2.1 Cell Culture, Antibodies, Expression Plasmids, Chemicals, and UV Irradiation Melanoma cell lines, RPM-MC, SK-mel-3 and MMRU, HCT116 and HEK293 cells were cultured in Dulbecco‟s Modified Eagle Media (DMEM) (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS. Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen) in 5% CO2 humidified atmosphere at 37 o C. Immortalized melanocytes, a gift from Dr. Meenhard Herlyn (The Wistar Institute, PA, USA), were cultured in melanocyte growth media supplemented with 5 µg/ml bovine pituitary extract, 1 ng/ml basic fibroblast growth factor, 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, 10 ng/ml phorbol myristate acetate and 4% FBS (PromoCell, Heidelberg, Germany). Anti-actin and rabbit anti-Flag antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-BrdU from BD Biosciences; anti-53BP1, anti-γH2AX (Ser 139) and anti-PCNA from Millipore (Billerica, MA, USA); anti-AcH4K5/8/12/16, anti-AcH3K9/14 and anti-H4 from Millipore; anti-ubiquitin, BRG1, cyclin D1, ING1b, Lamin B1, and pATM (Ser 1981) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Rad18 and anti- RPA from Abnova (Walnut, CA, USA); anti-SNF5 from Abcam (Cambridge, MA, USA); anti-CPD from Cosmo Bio (Tokyo, Japan); anti-Akt, anti-pChk1 (Ser345) and anti-pAkt (Ser473) from Cell Signaling (Danvers, MA); and mouse anti-Flag from Applied Biological Materials (Richmond, BC, Canada). BrdU was purchased from Sigma-Alrich. Ultraviolet irradiation was performed by removal of medium, washing with PBS and exposure to controlled dose of UVC (254 nm) light using a crosslinker (UltraLum, Claremont, CA, USA).  39 2.2 Expression Plasmid, siRNA Transfections and shRNA Construction Expression plasmids were transfected into HCT116 cells by Effectene Transfection Reagent (Qiagen, Mississauga, ON, Canada) according to the manufacturer‟s instruction. UV irradiation and assays were performed at 24 h after transfection. siRNAs were synthesized by Qiagen. Two ING1b siRNAs were used with target sequences as follows: siRNA-1: 5‟- acccacgtactgtctgtgcaa-3‟ and siRNA-2: 5‟-ttggtacacgtgtaacaagaa-3‟; the target sequence for Rad18 siRNA is 5‟-atggttgttgcccgaggttaa-3‟; and the target sequence for control is: 5‟- aattctccgaacgtgtcacgt-3‟. siRNA was transfected to cells by siLenFect Lipid reagent (Bio- Rad, Mississauga, ON, Canada) according to manufacturer‟s instruction. Assays were performed 48 h after transfection. Short-hairpin RNA targeting ING1b sequence 5‟-aaccatgttgagtcctgccaa-3‟ was constructed using HuSH-29 shRNA system (Origene, Rockville, MD, USA) according to the manufacturer‟s instruction. In brief, shRNA cassette was generated by annealing top and bottom strands of the oligos containing shRNA target sequence and was cloned into BamH1 and HindIII cloning sites on the pRS vector with U6 promoter. pRS vector and ING1b- shRNA were transfected into the retroviral packaging cell line, Phoenix Ampho cells (Orbigen, San Diego, CA, USA). Retroviral particles were collected 3 days after transfection and concentrated by ultracentrifugation. pRS vector and ING1b shRNA retroviral particles were used to infect HEK293. Infected cells were selected with 1 µg/ml puromycin. Stable knockdown was confirmed by Western blot. Flag-Polε is a gift from Dr. A.R. Lehmann and p3Flag14-HsRad18 is a gift from Dr. K. Myung. pcDNA Rad18 wild type and C28F mutant expression plasmids was a gift from Dr. A. Lehmann (University of Sussex, UK).  40 2.3 Subcellular Fractionation Fractionation of chromatin bound and unbound fractions were described previously (Kannouche et al, 2004; Kuo et al, 2007). Briefly, cytoplamic and nucleoplasmic proteins were isolated by Cytoskeletal Buffer (CSK) (100 mM NaCl, 300 mM sucrose, 3 mM MgClR2R, 10 mM PIPES [pH 6.8], 1 mM EGTA, 0.2% Triton X-100) with protease inhibitors for 15 min on ice. After centrifugation at 900 × g for 5 min at 4 o C, chromatin bound proteins in the pellet were resuspended in modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 0.1% NP-40, 0.25% sodium dodecyl sulfate) and sonicated. 2.4 Histone Extraction, Western Blot and Immunoprecipitation Cells were lysed in Cell Lysis Buffer (10 mM Tris-HCl [pH 7.5], 1 mM MgClR2R, and 0.5% NP-40) for 10 min on ice. Nuclei were pelleted by centrifugation at 1,000 × g for 5 min at 4 o C. Nuclei were resuspended in Extraction Buffer (0.5 M HCl, 10% glycerol and 0.1 M β-mercaptoethanol) for 1 h on ice. After centrifugation at 10,000 × g, the acid-soluble fraction was taken and neutralized by 2 M NaOH. Proteins were quantified and prepared for Western blotting as described (Kuo et al, 2007) using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) equipped with Odyssey 2.1 software. For pAkt and pChk1, all washings were done in 1×TBS/T, whereas all others were done in 1×PBS/T. For immunoprecipitation, cells were resuspended in modified RIPA buffer followed by incubation with 2 μg of specific antibody at 4oC overnight. Immunocomplex was pulled down by 50 μl protein G, washed thrice with modified RIPA for 5 min followed by WB. For immunoprecipitation of Polε, cells were incubated with 1% formaldehyde 10 min at room temperature for crosslinking followed by stopping the reaction with 0.125 M glycine prior to  41 lysis with Cell Lysis Buffer. The nuclei were resuspended in modified RIPA followed by immunoprecipitation with specific antibody. 2.5 Immunofluorescence Cells were fixed with 1% paraformaldehyde in 1PBS for 10 min at room temperature and permeabilised with methanol/acetone (1:1) at -20 o C for 10 min. For BrdU staining, the slides were treated with 2N HCl for 10 min at room temperature followed by neutralization with sodium borate [pH 10.5] for 5 min. The slides were incubated with specific primary antibodies followed by incubation with Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit secondary antibodies (Invitrogen). The slides were mounted with Vectorshield mounting media with DAPI (Vector Laboratories, CA, USA). The images were obtained by a laser scanning confocal microscope, LSM 780, equipped with the ZEN software, under the 10× eyepiece and 63× oil immersion lens (Carl Zeiss, ON, Canada).  10- 15 optical sections each with 0.4 μm distance in the z-direction were obtained for each image. The images were further processed into 2D by maximum intensity projection provided from the ZEN software. The weighted colocalization coefficient of BrdU staining with Rad18 staining was calculated using the colocalization module equipped in the ZEN software under the same threshold values for all images. The number of Rad18 foci was quantified with the Image J software. 2.6 Analysis of Metaphase Chromosome Cells were arrested at G1 by serum starvation, irradiated with or without UV and released in the presence of 0.1 µg/ml colcemid for 18 h. Cells were trypsinized and resuspended with 0.5 ml of medium. 10 ml of 0.075 M KCl was added slowly and gently. Cells were incubated at 37 o C for 15 min. 3-5 drops of fixative (3:1 methanol/acetic acid) were added to stop the reaction and the cells were centrifuged at 1,200 × g for 8 min.  42 Supernatant was removed, leaving 0.5 ml to resuspend the pellet. 10 ml of fixative was added gently and slowly. Cells were pelleted, resuspended and fixed in the same way twice. The pellet was finally resuspended in 0.5 ml of fresh fixative. 30 µl of the chromosome preparation was dropped on a pre-chilled microslide and let spread by gravity. Chromosomes were stained with a 1:20 Giemsa stain (Sigma-Aldrich) for 20 min, washed with water and mounted with Cytoseal 60 mounting medium (Fisher Scientific, Ottawa, ON, Canada). The slides were observed using an AxioPlan microscope (Zeiss) with a 100× oil immersion lens and 10× eyepiece. An image was obtained using a QImaging Ketiga Ex camera and was analysed with Northern Eclipse software. 2.7 Replication Fork Progression by qPCR Cells were trypsinized and resuspended in lysis buffer (5 mM Tris-HCl, 100 mM EDTA pH 8.0, 1% SDS and 80 µg/ml proteinase K) and incubated at 55 o C for 2 h. Genomic DNA was isolated by phenol/choloroform extraction. 250 ng of genomic DNA was used for qPCR analysis of DNA replication using SYBR PCR Master Mix with the 7900 HT Fast Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Primers used were as follows: Ori, 5‟-ccagaatccgatcatgcacc -3‟ (forward), 5‟-tccgtttttgcaggttgtgc-3‟ (reverse); 3.5 kb distal to the Ori, 5‟-ctgggtgtcagatcccagtt-3‟ (forward), 5‟ atggtccccaggatacacaa-3‟ (reverse); and β-globin, 5‟- caacttcatccacgttcacc-3‟ (forward), 5‟- acacaactgtgttcactagc-3‟ (reverse). qPCR conditions: dissociation, 95 o C, 30s; and annealing/extension, 60 o C, 1 min.  43 2.8 Cell Synchronization For synchronization at G1, cells were starved in serum free medium for 24 h. For cell synchronization at the G1-S boundary, cells were treated with 2 mM thymidine for 17 h and released into fresh medium for 8 h followed by a second thymidine block or treatment with 1 µg/ml aphidicolin for 14 h. 2.9  Sulforhodamine B Assay Cells were fixed in 10% trichloroacetic acid for 1 h at 4 o C, rinsed and dry, followed by staining with 0.057% sulforhodamine B for 15 min at room temperature (Li et al, 1998). Stained plates were washed with 1% acetic acid thrice and let dry. SRB dye from the stained cells was dissolved in 10 mM Tris-HCl (pH 10.5). Absorbance at 550 nm was measured. 2.10 Construction of TMA TMA construction was previously described (Dai et al, 2005). Briefly, formalin- fixed, paraffin-embedded tissues from 66 cases of dysplastic nevi, 180 cases of primary melanoma and 53 cases of metastatic melanoma were collected for the study. All specimens were obtained from the 1990 to 2004 archives of the Department of Pathology, Vancouver General Hospital. The use of human skin tissues in this study was approved by the Clinical Research Ethics Board of the University of British Columbia and was performed in accordance to the Declaration of Helsinki guidelines. The most representative tumor area was carefully selected and marked on the hematoxylin and eosin-stained slide. The TMA was assembled using a tissue-array instrument (Beecher Instruments, Silver Spring, MD). Duplicate 0.6-mm-thick tissue cores were taken for each biopsy specimen. Multiple 4-µm sections were cut with a Leica microtome (Leica Microsystems Inc, Bannockburn, IL) and transferred to adhesive-coated slides. One section from each TMA was routinely stained with  44 hematoxylin and eosin. The remaining sections were stored at room temperature for immunohistochemical staining. 2.11 Immunohistochemistry of TMA TMA slides were incubated at 55°C for 30 min followed by three 5-minite washes in xylene. Tissues were rehydrated by serial 5-min washes in 100%, 95% and 80% ethanol followed by distilled water. Antigen was retrieved by heating the tissues at 95°C for 30 minutes in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 20 min. Blocking was performed with universal blocking serum (DAKO Diagnostics, Mississauga, Ontario, Canada) for 30 min. The samples were incubated with anti-Rad18 antibody (1:300; Abnova, Taipei, Taiwan) at 4°C overnight followed by biotin-labeled secondary antibody and streptavidin-peroxidase (DAKO Diagnostics) for 30 min each at room temperature. The samples were developed using 3,3‟- diaminobenzidine substrate (Vector Laboratories) and counterstained with hematoxylin. The slides were dehydrated with standard procedure and sealed with coverslips. Negative controls were preformed by omitting the Rad18 antibody during primary antibody incubation. 2.12 Evaluation of Immunostaining The Rad18 staining in TMAs was evaluated blinded by two independent observers (including one dermatopathologist) simultaneously to reach a consensus score for each core. Overall intensity of Rad18 staining was scored as 0, 1+, 2+ and 3+, while percentage of stained cells was categorized into 1 (1-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%).  In case of discrepancy between duplicate cores, the average score from the two tissue cores was taken as the final score. The level of Rad18 staining was estimated by immunoreactive score (IRS) (Remmele & Stegner, 1987) which is calculated by multiplying the scores of staining  45 intensity and the percentage of positive cells. Rad18 staining was defined as negative (0), weak (1-4), moderate (6-8), and strong (9-12). 2.13 Statistical Analyses The χ2 test was used to analyze the difference in Rad18 staining during various stages of melanoma progression and also the association of Rad18 staining with the clinicopathological parameters in primary melanoma patients, including age, sex, tumor thickness, ulceration, histological subtype, and location. The Kaplan-Meier method and log- rank test were used to evaluate the correlation of Rad18 staining with the overall and disease- specific survival of melanoma patients. Univariate and multivariate Cox regression was employed to evaluate the relative risk and their 95% confidential intervals (CIs) of melanoma patients with different Rad18 expression. All statistical analyses were performed using SPSS version 11.5 (SPSS Inc, Chicago, IL) software.  46 Chapter 3: The Role of Tumor Suppressor ING1b in Genomic Stability upon Replication Stress 3.1 Background and Rationale Unrepaired DNA lesions, such as photolesions generated by UV radiation, stall replication forks progression because replicative DNA polymerases are unable to recognize modified DNA bases (Kunkel, 2004). The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions (Hoege et al, 2002; Lehmann, 2006; Stelter & Ulrich, 2003). This process is important for preventing genomic instability and cancer development (Limoli et al, 2002; Moriwaki & Kraemer, 2001; Wang et al, 2007). Previously, it was shown that cells deficient in tumor suppressor ING1b are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we sought to investigate the physiological role of ING1b upon UV irradiation. We explored the role of ING1b in resolution of stalled replication due to UV photolesions. We hypothesized that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Since ING1b is a component of the chromatin remodeling complex(es) containing HATs and HDACs (Skowyra et al, 2001; Vieyra et al, 2002), we studied whether the function of ING1b in regulation of the lesion bypass is through histone acetylation. 3.2 Results 3.2.1 ING1b knockdown cells are more sensitive to UV at S phase We investigated the physiological role of ING1b in UV response in HCT116 cells, which retain normal DNA damage checkpoint (Cahill et al, 1998; Lengauer et al, 1997). It expresses wild type ING1b (Figure 3.1) and p53 for which many functions of ING proteins are dependent (Aguissa-Toure et al, 2010; Li et al, 2009). We knocked down ING1b  47 expression with 70% knockdown (KD) efficiency at 48 h after siRNA transfection (Figure 3.2A). We found that ING1b KD sensitized cells to UV (Figure 3.2B). We generated stable ING1b KD cells by shRNA in HEK293 cells and found that ING1b shRNA cells were more sensitive to UV (Figure 3.3), which is consistent with the hypersensitivity of ING1b-deficient cells to UV (Kichina et al, 2006; Kuo et al, 2007). Since we showed previously that ING1b is required for NER (Kuo et al, 2007), we analyzed the persistence of UV-induced DNA lesions in control and ING1b KD HCT116 cells (Figure 3.4). Consistent with our previous finding (Kuo et al, 2007), repair of UV lesions is retarded in ING1b KD cells. However, we found that the extent of DNA lesions was comparable in control and ING1b KD cells up to 12 h after UV, suggesting that the sensitization of ING1b KD cells to UV at this time is probably not due to the difference in NER efficiency in these cells. We further analyzed the effect of ING1b KD on progression of cells from G1 to G2/M in a single cell cycle. Without UVR, ING1b KD cells progressed from G1 to G2/M approximately 4 h faster than the control cells (Figure 3.5A and C). This is consistent with the role of ING1b in cell proliferation (Coles et al, 2007; Garate et al, 2007). On the other hand, we observed that there was a significant increase in the sub-G1 cell population in ING1b KD cells after UVR indicating that more cells were undergoing cell death (Figure 3.5A and B). We observed a significant increase in cells accumulated at S phase in ING1b KD cells 12 and 14 h after UV (Figure 3.6A and D); therefore we examined if increased cell death in ING1b KD cells after UV irradiation is due to the inability of these cells to complete S phase. We pulsed the cells with BrdU to label cells undergoing DNA replication and quantified the percentage of cells progressed to G2/M 12 h after UV. We found that the  48 percentage of BrdU labeled cells that had progressed to G2/M was significantly reduced in ING1b KD cells compared to control (Figure 3.6A and B). Meanwhile, ING1b knockdown did not affect S phase progression in non-irradiated cells (Figure 3.6C and D). These data indicate that ING1b KD cells contain defects in S phase recovery after UV but not in non- stress conditions. We further pulsed the cells with BrdU at different time points after UV and observed that ING1b KD reduced BrdU incorporation in S phase as indicated by a ‘flattened’ pattern in the BrdU positive population indicating that more extensive stalled replication occurred in the ING1b KD cells (Figure 3.7). These results suggest that cells lacking ING1b expression are defective in recovering from UV-induced stalled replication and thus fail to progress to G2/M, becoming apoptotic.  49 Figure 3.1 Sequencing ING1b cDNA in HCT116 cells. The mRNA was isolated from HCT116 cells, reverse transcribed and sent out for sequencing. The sequence was aligned against the cDNA sequence obtained from NCBI gene bank. *, matching DNA bases; HCT116, sequencing result from HCT116 cDNA; ING1b, ING1b cDNA sequence from NCBI gene bank.   50 Figure 3.2 ING1b KD sensitizes cells to UV at S phase (A) HCT116 cells were transfected with control or ING1b siRNA and harvested for Western blot (WB) at various time points. (B) ING1b KD sensitizes cells to UV. HCT116 cells were transfected with control or ING1b siRNA, irradiated with 10 J/m 2  UVC, and harvested at indicated times after UV for PI staining followed by FACS analysis. Data was shown as mean ± SEM from three independent experiments. (*P<0.05).   51 Figure 3.3 ING1b knockdown increases UV sensitivity (A) HCT116 cells transfected with control and ING1b siRNA were subjected to UVC with indicated dose for 48 h and sulrhodamine B cell proliferation assay. Data was presented as means ± SEM from three independent experiments. (B) Stable ING1b knockdown in HEK293 cells. pRS vector control and ING1b shRNA cells were harvested and subjected for Western blot (WB) with ING1b and -actin antibodies. (C) ING1b stable knockdown cells are more sensitive to UV. pRS vector and ING1b shRNA knockdown HEK293 cells were subjected to UVC with indicated dose and sulforhodamine B cell proliferation assay was performed 48 h after irradiation. Data was presented as means ± SEM from three independent experiments (**P<0.01).   52 Figure 3.4 Nucleotide excision repair efficiency in ING1b KD cells (A) HCT116 cells transfected with control and ING1b siRNA were irradiated with 10 J/m 2  UVC and harvested at various time points after UV.  Genomic DNA was isolated for slot- western analysis of UV-induced photolesions with antibody against cyclobutane pyrimidine dimers (CPDs). (B) Quantification of the amount of CPD remaining. The experiment is repeated once and the representative data is shown.   53 Figure 3.5 Cell cycle analysis for ING1b KD cells (A) HCT116 cells were transfected with control or ING1b siRNA for 48 h and arrested at G1 by serum starvation. Cells were irradiated with or without 10 J/m 2  UVC and released in the presence of 50 ng/ml nocodazole. Cells were harvested for cell cycle analysis at various time points. Percentage of each cell cycle phase was quantified and data was presented as means ± SEM. Student‟s t test was performed between each of the control and ING1b KD group (*P<0.05; **P<0.01). (B) Sub G1 phase; (C) G1 phase; (D) S phase; (E) G2/M phase.   54 Figure 3.6 ING1b KD cells fail to recover from stalled replication (A) Control or ING1b KD HCT116 cells were pulsed with 20 μM BrdU, irradiated with 10 J/m 2  UVC and chased for 12 h. Cells were stained with anti-BrdU antibody and PI, and analyzed by FACS. (B) Quantification of BrdU-labeled cells in G2/M phase from three independent experiments (*P<0.05). (C) HCT116 cells were transfected with control or ING1b siRNA for 48 h, pulsed with 20 µM BrdU for 1 h and chased. Cells were harvested for PI and BrdU staining followed by FACS analysis. (D) Percentage BrdU-labelled cells progressed to G2/M was quantified. Error bars represent means ± SEM. from three independent experiments.   55 Figure 3.7 ING1b knockdown cells showed increased stalled replication after UV (A) HCT116 cells were transfected with control or ING1b siRNA for 48 h and irradiated with 10 J/m 2  UVC. One hour before the indicated time point, cells were pulsed with 20 µM BrdU and harvested for BrdU incorporation analysis by FACS. Cell population with unperturbed replication was gated according to untreated cells (replicative). Cells with stalled replication were gated to the population with reduced BrdU incorporation (stalled). Cells treated with hydroxyurea (HU) were shown as positive control. (B) Quantification of stalled replication. Percentage stalled replication was calculated by the number of stalled cells over total number of cells in S phase. Data was presented as means ± SEM from three independent experiments (**P<0.01, Student‟s t-test).   56 3.2.2 ING1b knockdown cells show defects in replication fork progression and enhanced genomic instability after UV To further demonstrate the importance of ING1b for replication fork progression after UV, we analyzed the amplification of genomic DNA at a known replication origin in the lamin B2 gene (Giacca et al, 1994) by qPCR. We isolated genomic DNA from cells released from the G1-S boundary and analyzed DNA replication at the origin (Ori) and a region 3.5 kb distal to the origin at different time points after UV (Figure 3.8A). The amount of replicated DNA was significantly reduced in ING1b KD cells compared to control cells at 60 min (compare 1 vs 2 and 3 vs 4, Figure 3.8B). This was even more prominently at 240 min after UV (compare 5 vs 6 and 7 vs 8, Figure 3.8B), indicating that the ability of the replication fork to progress from the origin to 3.5 kb distal is impaired in cells lacking ING1b expression after UV. The effect of ING1b KD is less prominent during non-stress conditions (Figure 3.8C). We speculated that ING1b prevents the stalled replication forks from collapsing. It is believed that UV-induced DSBs are caused by collapse of replication forks arrested at the UV lesions (Limoli et al, 2002). Therefore, we analyzed the levels of H2AX phospohorylation at Ser139 (γH2AX) and phosphorylation of ATM at Ser-1981 which occur upon DSBs. We detected an increased level of γH2AX and pATM in ING1b KD cells when compared to control (Figure 3.8D), indicating that DSBs are more extensive in ING1b KD cells after UV. We further observed an increased number of aberrant structures in metaphase chromosomes in ING1b KD cells, a 4-fold increase in chromatid breaks and a 3-fold increase in chromosomal fusions, which appear to be chromatid exchange, compared to control cells after UV (Figure 3.8E and F). We observed a very low level of spontaneous chromosome  57 aberrations in control and ING1b KD cells (Figure 3.8G). Together, these data suggest that ING1b plays an important role in the response to UV-induced replication stress, preserving the genomic stability.  58 Figure 3.8 ING1b KD cells show defects in replication fork progress and enhanced genomic instability after UV (A) Schematic diagram for the replication origin at lamin B2 gene. Primers designed for the origin (Ori) and 3.5 kb distal of the origin (3.5 kb) are indicated. (B) Stalled replication at lamin B2 replication origin in ING1b KD cells after UV. Control and ING1b KD HCT116 cells were arrested at G1-S boundary, irradiated with 10 J/m 2  UVC and released for 60 and 240 min. Genomic DNA was isolated and analyzed by qPCR. All samples were normalized with β-globin gene, a region replicated at late S phase. Relative replicated DNA was calculated by amount of DNA in cells after release over amount of DNA in G1 arrested cells. Data was presented as mean ± SEM from three independent experiments (*P<0.05). (C) ING1b KD HCT116 cells were synchronized at G1-S boundary as in described in (B). Cells were washed and released in fresh media for indicated times. Genomic DNA was isolated followed by qPCR analysis for the amplification of a genomic region 3.5 kb distal to the replication start site at lamin B2 gene. (D) Enhanced and prolonged γH2AX and pATM after UV. Control and ING1b KD HCT116 cells were irradiated with 10 J/m 2  UVC, WCE and histones are extracted for WB. (E) Representative images of metaphase chromosome. Control and ING1b KD HCT116 cells were arrested at G1 by serum starvation, irradiated with 10 J/m 2  UVC and released in the presence of 0.1 µg/ml colcemid for 18 h. Metaphase chromosome was prepared on slides and stained with Giemsa stain. Arrow indicates chromosome aberrations. (F) Quantification of chromosomal aberrations. Percentage of cells containing chromatid breaks or chromosome fusions was counted. Thirty cells were counted on each slide and the experiment was done in triplicate (**P<0.01). (G) Low levels of spontaneous chromatid breaks and fusions in control and ING1b KD cells. HCT116 cells  59 transfected with control and ING1b siRNA were arrested at G1 by serum starvation and released in the presence of 0.1 µg/ml colcemid for 18 h. Metaphase chromosome was prepared and stained with Giemsa stain. One hundred cells were analyzed for the presence of chromosome aberrations.   60 3.2.3  ING1b regulates PCNA monoubiquitination and TLS We sought to elucidate the mechanism by which ING1b regulates replication fork stability upon during replication stress. As PCNA-Ub is essential for the lesion bypass pathway with respect to overcoming replication blockage, we investigated if ING1b regulates PCNA-Ub. We detected the monoubiquitinated form of PCNA by Western blotting which was induced by UV (Figure 3.9A). ING1b KD decreased PCNA-Ub by 40% and 70% at 6 and 24 h after UV respectively in HCT116 cells (Figure 3.9B), which indicates that PCNA-Ub is inhibited rather than delayed in ING1b KD cells. We also observed a reduction of PCNA-Ub in normal human fibroblasts (Figure 3.9C) and HEK293 cells (Figure 3.9D) upon ING1b depletion. To confirm that inhibition of PCNA-Ub by ING1b KD was not due to the off-target effect, we treated cells with another ING1b siRNA and found that it also reduced PCNA-Ub after UV (Figure 3.9E). Furthermore, inhibition of PCNA-Ub was also observed when cells were irradiated with a much lower dose of UV (1 J/m 2 ) (Figure 3.9F). Since PCNA-Ub occurs predominantly at S phase (Davies et al, 2008), we asked if ING1b affects PCNA-Ub at S phase. We synchronized cells at G1-S boundary and released them into S phase (Figure 3.8G, bottom panel) followed by UV irradiation. We observed a reduction of PCNA-Ub in ING1b KD cells after UV compared to the control (Figure 3.9G). PCNA-Ub is induced by various genotoxic agents, such as cisplatin, hydroxyurea and methyl methanesulfonate, all of which stall replication forks and lead to appearance of ssDNA (Hoege et al, 2002; Kannouche et al, 2004; Stelter & Ulrich, 2003). We therefore treated control and ING1b KD cells with these agents and found that ING1b KD inhibited PCNA-Ub induced by these agents, while PCNA-Ub was not detected in cells treated with etoposide and camptothecin which induce DSBs rather than stalling of replication (Figure 3.9H). It was  61 shown previously that translesion DNA polymerase Pol preferentially interacts with the monoubiquitinated form of PCNA (Kannouche et al, 2004). We asked if ING1b is required for Pol foci formation after UV. We found that Pol foci formation significantly increased after UVR but was dramatically reduced in ING1b KD cells (Figure 3.10A and B). In addition, Polη interaction with monoubiquitinated PCNA (Figure 3.10C) and Polη binding to chromatin (Figure 3.10D) after UV were reduced in ING1b KD cells. Our data suggest that ING1b regulates PCNA-Ub in S phase during replication fork stalling and facilitates Polη- dependent lesion bypass to avoid catastrophic consequence from replication fork collapse.  62 Figure 3.9  ING1b regulates PCNA-Ub and upon replication stress at S phase (A) UV induces PCNA monoubiquitination. HCT116 cells were irradiated with UVC at 10 J/m 2 , and harvested 4 h after irradiation for subcellular fractionation followed by immunoprecipitation with PCNA antibody, and WB with anti-ubiquitin and anti-PCNA antibodies. 10% of lysates were used as input control. (B) ING1b KD reduces PCNA-Ub after UV. Control and ING1b KD HCT116 cells were irradiated with 10 J/m 2  UVC for indicated times. Chromatin fraction was isolated for WB. (C) Primary human fibroblasts were transfected with control or ING1b siRNA for 48 h, irradiated with 10 J/m 2  UVC for 4 h. Chromatin bound proteins were subjected to WB. (D) PCNA monoubiquitination in HEK293 ING1b shRNA cells. Control and ING1b shRNA cells were irradiated with various doses of UVC, harvested 4 h after irradiation and chromatin bound proteins were subjected to WB. (E) HCT116 cells were transfected with control or ING1b siNRA (sequence 2) as described (Kuo et al, 2007) for 48 h, irradiated with 10 J/m 2  UVC for 4 h and harvested for chromatin bound proteins and WB. Quantification of band intensity was done by the Image J software. (F) ING1b is required for PCNA monoubiquitination and histone H4 acetylation at high and low dose UV. HCT116 cells transfected with control and ING1b siRNA were irradiated with 1 and 10 J/m 2  UVC for 6 h and harvested for chromatin bound proteins followed by WB analysis. (G) ING1b is required for efficient PCNA-Ub at S phase. Control and ING1b KD HCT116 cells were synchronized at G1-S boundary as previously, released for 3 h into S phase and irradiated with 10 J/m 2  UVC. Chromatin fraction was isolated for WB. DNA content was analyzed by FACS. (H) ING1b is required for PCNA-Ub upon replication stress. Control and ING1b KD HCT116 cells were treated with 100 µM CP, for 1 h and replaced  63 with fresh media for 5 h, 10 mM HU for 6 h; and 250 µM MMS, 0.25 µM CPT or 20 µM EP for 4 h. Chromatin fraction was isolated for WB.   64 Figure 3.10  ING1b is required for Polη foci formation after UV (A) HCT116 cells transfected with Flag-tagged Polε were irradiated with 10 J/m2 UVC for 6 h. Cells were fixed and observed under confocal fluorescence microscope. (B) Quantification of number of cells displaying Polε foci. Fifty cells were counted on each slide and the experiment was done in triplicate (**P<0.01). (C) Polε interaction with PCNA-Ub is reduced in ING1b KD cells. Control and ING1b KD HCT116 cells transfected with Flag- Polε were irradiated with 10 J/m2 UVC for 6 h. IP for nuclear Flag-Polε were performed as described in supplementary experimental methods. Relative interaction of Polε with PCNA- Ub was calculated by quantifying the band intensity of PCNA-Ub over that of Polε in the IP fraction (PCNA-Ub:Polε). (D) Polε binding to chromatin is reduced in ING1b KD cells. Control and ING1b KD HCT116 cells were transfected with Flag-tagged Polε for 24 h. Cells were subjected to 10 J/m 2 UVC for indicated time and harvested for subcellular fractionation followed by WB.   65 3.2.4  ING1b is required for Rad18-mediated PCNA-Ub We further examined if ING1b is required for PCNA-Ub mediated by the Rad18 E3 ligase. Ectopic expression of Rad18 enhanced PCNA-Ub in control cells in a time-dependent manner after UV. Rad18-enhanced PCNA-Ub was abrogated in ING1b KD cells at 1 h and more dramatically at 6 h (Figure 3.11A). Moreover, we observed a corresponding reduction of Rad18 binding to chromatin in ING1b KD cells when compared to the control while the total Rad18 expression did not change significantly (Figure 3.11A). We then knocked down the expression of ING1b and Rad18 alone or in combination and found that co-knockdown of ING1b and Rad18 resulted in a further reduction in PCNA-Ub and sensitized cells to UV compared to ING1b and Rad18 KD alone (Figure 3.11B). We further asked how ING1b regulates Rad18 functions. Rad18 is known to colocalize with sites of replication (Watanabe et al, 2004). We labeled sites of replication by pulsing cells with BrdU prior to UV and performed immunofluorescent staining for Rad18 and BrdU. We found that Rad18 formed punctate foci which colocalized with BrdU in control cells after UV (Figure 3.11D-F). Rad18 foci formation was abrogated in ING1b KD cells while BrdU foci could still be observed in these cells (Figure 3.11D-F). Furthermore, UV-induced interaction between Rad18 with PCNA in control cells while this association was dramatically reduced in ING1b KD cells (Figure 3.11G). These data suggest that ING1b is required for the engagement of Rad18 on chromatin at the sites of replication during replication blockage to mediate PCNA-Ub.  66 Figure 3.11 ING1b is required for Rad18-mediated PCNA-Ub during stalled replication (A) ING1b is required for Rad18-mediated PCNA-Ub. Control and ING1b KD HCT116 cells were transfected with either vector control or Flag-Rad18 plasmid. Cells were irradiated with 10 J/m 2  UVC and whole cell extract (WCE) or chromatin bound proteins (Chr) were analyzed by WB. Relative binding of Rad18 to chromatin is calculated as Chr:WCE. (B) ING1b cooperates with Rad18 on PCNA-Ub. HCT116 cells transfected with ING1b and Rad18 siRNAs alone or in combination were irradiated with 10 J/m 2  UVC for 6 h. Chromatin fraction was isolated for WB. (C) The effect of ING1b and Rad18 knockdown on cell survival after UV irradiation. HCT116 cells transfected with ING1b and Rad18 siRNAs alone or in combination were irradiated with 10 J/m 2  UVC. Cells were harvested for PI staining at indicated time followed by FACS analysis. Data was presented as means ± SEM from three independent experiments. Student‟s t test was performed between the control and knockdown groups at each time point (*P<0.05; *P<0.01). (D) ING1b is required for Rad18 foci formation at S phase after UV. Control and ING1b KD HCT116 cells were transfected with Flag-Rad18 plasmid. Cells were pulsed with 20 µM BrdU and irradiated with 10 J/m 2  UVC for 1 h. Immunofluorescent staining was performed using anti-BrdU and Flag antibodies. White box indicates the portion of nucleus displayed on the magnified panel. (E) Number of Rad18 foci was quantified by Image J. (F) Weighted colocalization coefficient of BrdU staining with Rad18 staining was analyzed with ZEN software. Thirty cells were counted on each slide and the experiments were done in triplicate (**P<0.01). (G) ING1b is required for Rad18-PCNA interaction. Control and ING1b KD HCT116 cells were transfected with Flag-Rad18, irradiated with or without 10 J/m 2  UVC. Immunoprecipitation  67 was performed using anti-Flag antibody. Relative Rad18-PCNA interaction is calculated as PCNA:Rad18.   68 3.2.5  ING1b maintains histone H4 acetylation during S phase and is required for Rad18-mediated PCNA-Ub We further studied the mechanism by which ING1b regulates PCNA-Ub. We previously showed that ING1b is required for histone H4 acetylation (AcH4) during the repair of UV-damaged DNA (Kuo et al, 2007). We asked whether ING1b affects histone H3 and H4 acetylation at S phase. We synchronized cells at the G1-S boundary, released them into S phase and irradiated the cells with UV. We then examined the acetylation level of H3 and H4 and found that both AcH3 and AcH4 levels increased moderately after UV in control cells. We observed a dramatic decrease in AcH4 level in ING1b KD cells when compared to control cells whereas only a slight reduction was seen for AcH3 (Figure 3.12A). We postulated that ING1b-regulated AcH4 might be important in PCNA-Ub. We first studied the effect of histone hyperacetylation induced by trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. TSA treatment enhanced AcH4 and PCNA-Ub after UV (Figure 3.12B). Furthermore, TSA treatment at various doses restored AcH4 and PCNA-Ub in ING1b KD cells (Figure 3.12C), indicating that histone hyperacetylation bypasses the requirement of ING1b in PCNA-Ub. Moreover, we found that treatment of TSA and NaBu, HDAC class I and II inhibitors, but not treatment with nicotinamide, a NAD-dependent HDAC inhibitor,restored PCNA-Ub in ING1b KD cells (Figure 3.12D). We asked whether ING1b regulates Rad18 through histone acetylation. Rad18 binding to chromatin was reduced in ING1b KD cells (Figure 3.11A and 3.12E). TSA treatment at various doses restored Rad18 binding to chromatin while the overall Rad18 expression did not change significantly (Figure 3.12E). We determined whether restoration of AcH4 in ING1b KD cells would rescue genomic instability and cell survival. Since TSA  69 treatment was shown to induce apoptosis in HCT116 cells upon prolonged treatment (Mattera et al, 2009; Sayan et al, 2007), we performed the experiment at a reduced dose of TSA (5 ng/ml) which did not cause significant apoptosis, while AcH4 was restored in ING1b KD cells at this dose (Figure 3.12F). We observed that restoration of AcH4 by TSA alleviated UV-induced γH2AX and UV-induced apoptosis in ING1b KD cells (Figure 3.12F). This suggests that ING1b regulates PCNA-Ub and genomic stability through maintaining H4 acetylation during S phase. These data suggest that ING1b is required for stabilization of the stalled replication fork through maintenance of histone H4 acetylation to mediate PCNA-Ub and lesion bypass, maintaining genomic stability.  70 Figure 3.12  ING1b maintains histone H4 acetylation during S phase and is required for Rad18-mediated PCNA-Ub (A) ING1b maintains AcH4 at S phase. Control and ING1b KD HCT116 cells were synchronized at G1-S boundary as previously and irradiated with 10 J/m 2  UVC. (B) TSA treatment enhances PCNA-Ub after UV. HCT116 cells pretreated with 50 ng/ml TSA for 1 h were irradiated with 10 J/m 2  UVC and incubated with TSA for 6 h. (C) TSA treatment restores PCNA-Ub in ING1b KD cells. Control and ING1b KD HCT116 cells were treated with various doses of TSA and irradiated with 10 J/m 2  UVC for 6 h. (D) TSA and sodium butyrate (NaBu) but not nicotinamide (Nico) restores PCNA-Ub in ING1b KD cells. Control and ING1b KD HCT116 cells were treated with 50 ng/ml TSA, 2.5 mM NaBu or 5 mM Nico and irradiated with 10 J/m 2  UVC for 6 h. (E) TSA treatment restores Rad18 binding to chromatin after UV. Control and ING1b KD HCT116 cells were treated with various doses of TSA for 1 h and irradiated with 10 J/m 2  UVC for 6 h. WCE and chromatin fraction were isolated for WB. Relative binding of Rad18 to chromatin is calculated as Chr:WCE. (F) TSA treatment alleviates genomic instability in ING1b depleted cells. HCT116 control and ING1b KD cells were treated with or without 5 ng/ml TSA, irradiated with 10 J/m 2  UVC for 24 h. Cells were harvested for WB and FACS analysis. Data was presented as mean ± SEM from three independent experiments (**P<0.01).  71   72 3.3 Discussion In this study, we characterized the physiological role of ING1b in the UV response. Although it was previously shown that ING1b deficient cells were more sensitive to UV irradiation (Kichina et al, 2006; Kuo et al, 2007), the mechanism by which ING1b status affects UV sensitivity is unknown. Consistent with previous findings, we observed that ING1b depleted cells were more sensitive to UV irradiation (Figure 3.2B). ING1b KD cells showed defects in recovering from UV-induced stalled replication and eventually became apoptotic (Figure 3.5 and 3.6). Furthermore, we observed that replication fork progression was inhibited in ING1b KD cells after UVR (Figure 3.8B). As a result, there was an increased formation of DSBs and elevated chromosomal aberrations in ING1b KD cells (Figure 3.8D-F). We conclude that ING1b is required for overcoming blocked replication forks due to the presence of UV lesions during DNA replication. Therefore, in the absence of ING1b, stalled replication remains unresolved and prolonged replication blockage leads to catastrophic events such as chromatid breaks and chromosomal fusions. It has also been shown that ING1b overexpression increases stress-induced apoptosis including UV in a p53-dependent manner (Cheung & Li, 2002; Garkavtsev et al, 1998). We reasoned that ING1b may play a separate role in apoptosis and S phase recovery. At 10 J/m 2  UVC, cells were allowed to progress throughout the cell cycle (Figure 3.5A) and therefore mechanisms involved in resolving stalled replication due to UV lesions would be essential for cell survival. In fact, we found that ING1b is required for monoubiquitination of PCNA which is central in regulating the lesion bypass mechanism to recover from stalled replication (Hoege et al, 2002; Stelter & Ulrich, 2003). Replication could be stalled even by the presence of very low levels of DNA lesions. We observed that PCNA-Ub and AcH4 are reduced in  73 ING1b KD cells irradiated with a sub-lethal dose of UV (1 J/m 2 ) (Figure 3.9F), suggesting a specialized role of ING1b in S phase recovery. In fact, our observation in ING1b knockdown cells is comparable to cells deficient in Rad18 E3 ligase, which is essential for PCNA-Ub. Rad18 deficient cells were found to be hypersensitive to DNA damaging agents and displayed enhanced genomic instability (Tateishi et al, 2003; Tateishi et al, 2000). Although we found that ING1b is required for Rad18-dependent PCNA-Ub, we also observed that co- knockdown of ING1b and Rad18 further reduced PCNA-Ub and sensitized cells to UV compared to ING1b and Rad18 knockdown alone (Figure 3.11B and C). This can be explained by the fact that Rad18 is not the only E3 ligase known to monoubiquitinate PCNA. Recently, another E3 ligase, CRL4(Cdt2), was found to monoubiquitinate PCNA in a Rad18- independent manner (Terai et al, 2010). Moreover, PCNA-Ub was found to be negatively regulated by the deubiquitinating enzyme, ubiquitin specific protease 1 (USP1) which removes ubiquitin from PCNA (Huang et al, 2006). ING1b might regulate PCNA-Ub and cell survival upon replication stress through these molecules. We observed that ING1b is required for the translesion DNA polymerase, Pol, to form foci which are required for TLS (Garg & Burgers, 2005; Kannouche et al, 2004) and for the interaction of Pol specifically with the monoubiquitinated form of PCNA. The ability of Pol to interact with PCNA through the Ub-binding zinc finger (UBZ) and the newly identified PCNA interacting domains are all required for full activation of TLS (Bienko et al, 2010). We postulate that ING1b is required for efficient PCNA-Ub in initiating TLS to bypass the UV lesions at stalled forks. We further demonstrated that ING1b is required for histone H4 acetylation at S phase (Figure 3.12A) and that TSA treatment which induces histone hyperacetylation enhances  74 PCNA-Ub (Figure 3.12B). During replication, new histone H4 is acetylated at K5 and K12 prior to assembly into chromatin and deacetylation occurs within 30-60 min. Inhibition of deacetylation may preserve AcH4 after chromatin assembly. This may explain why treatment with HDAC inhibitors bypasses the requirement of ING1b in PCNA-Ub (Figure 3.12C and D). Our results also indicate that reduction of AcH4 in ING1b KD cells during non-stress conditions does not lead to a significant retardation in normal S phase progression (Figure 3.6C and D), suggesting that reduction of AcH4 is not due to inhibition of DNA replication. Our observation in part corresponds with work previously done in yeast. Choy et al observed that cells deficient in Yng2, the yeast homologue of ING proteins, are highly sensitive to replication stress, including UV, hydroxyurea and MMS. Moreover, yng2 mutants showed a reduction in histone H4 acetylation and delayed S phase progression upon MMS treatment. In addition, TSA treatment alleviates the S phase delay in yng2 mutants after MMS treatment (Choy & Kron, 2002) which concurs with our observation that restoration of AcH4 in ING1b depleted cells rescues UV-induced DNA DSBs and apoptosis (Figure 3.12F). It indicates that the regulation of histone H4 acetylation and recovery from replication stress is conserved in eukaryotes. Other members of ING family proteins have also been implicated in regulating DNA replication to preserve genomic stability (Larrieu & Pedeux, 2009). The ING5 complex containing HBO1 is required for normal DNA replication (Doyon et al, 2006). Recently, ING2 was also shown to be required for normal DNA replication and is associated with PCNA (Larrieu et al, 2009). In the same study, the authors observed that ING1b is not involved in normal DNA replication which coincides with our observation (Larrieu et al, 2009). Our study suggests that ING1 plays a unique role in DNA replication upon stress.  75 Why is histone acetylation required for PCNA-Ub? Chromatin is a barrier for DNA replication and repair. It has to be remodeled before these processes are possible (Groth et al, 2007b; Peterson & Cote, 2004). We previously showed that ING1b regulates histone H4 acetylation and chromatin relaxation to provide accessibility for XPA in nucleotide excision repair (Kuo et al, 2007). It is possible that ING1b remodels the chromatin structure through H4 acetylation to provide accessibility for factors involved in lesion bypass. This is supported by the observation that Rad18 and Pol binding to chromatin was reduced in ING1b KD cells (Figure 3.10D and 3.11A) and TSA treatment restores Rad18 binding to chromatin in ING1b KD cells (Figure 3.12E). In fact, chromatin remodeling enzymes are found to be important for DNA replication upon stress. For instance, the ATP-dependent chromatin remodeling enzyme Ino80 is found to associate directly with replication forks and is required for fork stability and restart upon stress (Papamichos-Chronakis & Peterson, 2008; Shimada et al, 2008). More recently, Ino80 was also found to be required for PCNA- Ub (Falbo et al, 2009). Moreover, it has been shown that alterations in chromatin structure affect Polε dynamics in TLS (Sabbioneda et al, 2008). In our case, ING1b depletion did not affect expression of the ATP-dependent chromatin remodeling factors BRG1 and SNF5 (Figure 3.13) which suggests a more direct and distinct role of ING1b in regulation of lesion bypass. Therefore, it is conceivable that ING1b may affect replication fork stability in S phase recovery.  76 Figure 3.13  ING1b is not required for expression of ATP-dependent chromatin remodelling factors, SNF5 (BAF47) and BRG1 HCT116 cells transfected with control and ING1b siRNA were irradiated with 10 J/m 2  UVC and harvested at various times for WB.   77  In conclusion, our data provide evidence for the physiological role of ING1b in the recovery from replication blockage through regulation of PCNA-Ub and histone H4 acetylation. To our knowledge, this is the first study linking histone acetylation to the PCNA- Ub pathway. Our study provides a novel insight into the regulation of lesion bypass mechanism and implicates the role of ING1b in genomic stability and tumor suppression.  Since we previously showed that ING1b is a downstream target of the checkpoint kinase Chk1 (Garate et al, 2007), in the next chapter we further investigated the role of ING1b in connecting the S phase checkpoint with the lesion bypass pathway and maintenance of genomic stability.  78 Chapter 4: ING1b Cooperates with Chk1  in Genomic Stability 4.1 Background and Rationale The ATR-Chk1 S phase checkpoint pathway is known to play an important role in maintaining genomic stability upon replication stress (Bartek et al, 2004; Cimprich & Cortez, 2008). S phase checkpoint proteins have direct function in fork stabilization to protect the integrity of the existing stalled replication forks (Lopes et al, 2001; Tercero & Diffley, 2001). Stabilization of stalled replication forks by S phase checkpoint is thought to occur through stabilization of the replisome with the fork (Cobb et al, 2003; Lucca et al, 2004) and through restraining the activity of recombination enzymes at stalled fork (Boddy et al, 2003; Kai et al, 2005). These events might be mediated by either the physical association of checkpoint proteins at the stalled fork, or through the kinase activity of the checkpoint kinases. Failure to stabilize replication forks can result in the replication fork collapse and ultimately in genomic instability (Casper et al, 2002; Cha & Kleckner, 2002). However, the mechanism of how the checkpoint proteins stabilize replication forks remains elusive. Chk1 is an effector kinase for the ATR-Chk1 checkpoint pathway. Previous studies suggest that ATR is not required for UV-induced PCNA monoubiquitination (Chang et al, 2006; Frampton et al, 2006). However, another study showed that ATR is required for PCNA ubiquitination induced by the carcinogen B[a]P di-hydrodiol epoxide (BPDE) (Bi et al, 2006). Therefore, the exact role of the ATR-Chk1 pathway in PCNA monoubiquitination and genomic stability is not clear.  Chk1 has been shown to be important for maintaining genomic stability during DNA replication (Syljuasen et al, 2005; Zachos et al, 2005). Recently, Chk1 was found to regulate PCNA monoubiquitination through stabilization of claspin which is independent of Chk1 kinase activity (Yang et al, 2008).  79 Our group demonstrated previously that ING1b is phosphorylated by Chk1 upon UV stress at S126 residue of ING1b (Garate et al, 2007). ING1b S126 phosphorylation peaks at S phase after UV irradiation and such phosphorylation stabilizes ING1b and prevents it from degradation through the proteosome (Garate et al, 2007; Garate et al, 2008). It suggests that ING1b is downstream of the S phase checkpoint. We recently showed that ING1b regulates genomic stability through PCNA monoubiqiutination (Wong et al, 2011). It is not clear if the S phase checkpoint regulates such function of ING1b during replication stress. Therefore, we hypothesize that ING1b cooperates with Chk1 in regulation of PCNA monoubiquitination and genomic stability upon replication stress. 4.2 Results 4.2.1 ING1b S126 is required for interaction with Chk1. To investigate if ING1b and Chk1 cooperate to regulate UV stress response, we first asked if ING1b interacts with Chk1 after UV. We performed immunoprecipitation and found that Chk1 co-precipitated with ING1b. Such interaction increased upon UV treatment (Figure 4.1A). Since we showed that Chk1 phosphorylates ING1b at S126, we examined if ING1b S126 is required for ING1b-Chk1 interaction. We expressed control vector, wild type (WT) or the phospho-defective mutant (S126A) of ING1b and performed co-immunoprecipitation. We found that the association of ING1b with Chk1 was significantly reduced in ING1b S126A mutant compared to WT (Figure 4.1B). We further investigated if the kinase activity of Chk1 is required for such interaction. We pulled down the wild type (WT) and kinase dead (KD) Chk1 and surprisingly, we found that ING1b was co-precipitated with both Chk1 WT and KD (Figure 4.1C). These data suggest that Chk1-ING1b interaction requires ING1b S126 residue while the kinase activity of Chk1 is dispensable.  80 Figure 4.1  ING1b S126 is required for interaction with Chk1 (A) Chk1-ING1b interaction increased upon UV. HCT116 cells were treated with or without 10 J/m 2  UVC for 1 h. Nuclear proteins were extracted for immunoprecipitation using anti- Chk1 antibody. Immunoprecipitate and 10% of input lysate were subjected to WB. (B) ING1b S126 is required for interaction with Chk1. HCT116 cells were transfected with vector control, Flag-ING1b or Flag-ING1b-S126A for 24 h. Nuclear protein was extracted for immunoprecipitation with anti-Flag antibody and WB. (C) Kinase activity is dispensable for Chk1-ING1b interaction. HCT116 cells were transfected with Flag-Chk1 (WT) or kinase dead Flag-Chk1 (KD). Nuclear protein was extracted for immunoprecipitation using anti- Flag antibody followed by WB. The experiment was repeated for three times. Representative images were shown.   81 4.2.2 ING1b cooperates with Chk1 in PCNA-Ub Since both ING1b and Chk1 were found to regulate PCNA monoubiquitination, we asked if they cooperate in this function. We performed knockdown of ING1b and Chk1 alone or in combination. We found that PCNA-Ub was reduced in ING1b and Chk1 knockdown alone. PCNA-Ub was reduced in ING1b and Chk1 co-knockdown to a similar level as the ING1b and Chk1 single knockdown (Figure 4.2A), suggesting that ING1b and Chk1 are epistatic in the regulation of PCNA-Ub. Moreover, ING1b knockdown did not affect Chk1 phosphorylation after UV irradiation (Figure 4.2B), supporting  our previous finding that ING1b is downstream of Chk1. Since ING1b S126 was required for interaction with Chk1, we examined if ING1b S126 is required for PCNA-Ub. HCT116 cells were sequentially transfected with siRNA targeting 5‟UTR of ING1b, and empty vector, vectors expressing WT or S126A ING1b. We found that PCNA-Ub was reduced in ING1b knockdown cells after UV as previously reported (Figure 4.2C) (Wong et al, 2011). We found that reconstitution of WT ING1b expression partially restored PCNA-Ub in ING1b knockdown cells. Partial rescue may be due to the transfection efficiency that not all the knockdown cells expressed the exogenous ING1b. More importantly, we found that reconstitution of ING1b expression with S126A mutant failed to restore PCNA-Ub in ING1b knockdown cells (Figure 4.2C). We conclude that ING1b S126A is required for efficient PCNA-Ub.  82 Figure 4.2 ING1b cooperates with Chk1 in PCNA-Ub (A) Chk1 and ING1b are epistatic in regulation of PCNA-Ub. HCT116 cells were transfected with siRNA against ING1b and Chk1 alone or in combination for 48 h. Cells were irradiated with 10 J/m 2  UVC for 1 h. Chromatin fraction was isolated for WB. (B) ING1b knockdown does not affect Chk1 activation. HCT116 cells were transfected with control or ING1b siRNA for 48 h. Cells were irradiated with 10 J/m 2  UVC and harvested at indicated times. Whole cell extract was isolated for WB. (C) ING1b S126 is required for PCNA-Ub. HCT116 cells were transfected with control siRNA or siRNA targeting 5‟UTR of ING1b. 24 h after transfection, cells were transfected again with plasmid expressing ING1b WT or S126A mutant for another 24 h. Cells were then irradiated with 10 J/m 2  for 4 h. Chromatin bound proteins were subjected for WB. The experiment was repeated for three times. Representative images were shown.   83 4.2.3 ING1b S126 is required for Pol foci formation after UV irradiation PCNA monoubiquitination is required for recruiting translesion DNA polymerase, Pol, after UV irradiation to bypass the UV photolesions (Kannouche et al, 2004). We asked if ING1b S126 is required for Pol foci formation after UV irradiation. We performed immunofluorescent staining for Flag-Pol after UV irradiation in cells reconstituted with WT or S126A ING1b. We found that Pol foci formation was significantly increased in ING1b WT cells 4 h after UV whereas Pol foci formation was abrogated after UV in cells reconstituted with ING1b S126A (Figure 4.3A and B). These data suggest that ING1b S126 is required for Pol-dependent lesion bypass.  84 Figure 4.3  ING1b S126 is required for Pol foci formation after UV irradiation (A) HCT116 cells were transfected with siRNA targeting ING1b. 24 h after siRNA transfection, cells were co-transfected with Flag-Pol and the siRNA resistant ING1b WT or ING1b S126A mutant. Cells were synchronized at G1/S boundary by thymidine block followed by aphidicolin block and were irradiated with or without 10 J/m 2  UVC for 4 h. Cells were fixed followed by immunofluorescent staining for ING1b and Flag-Pol. Cells positive for both ING1b and Flag-Pol were chosen for the analysis. Representative images for DAPI and Flag-Pol were presented. (B) Quantification of Pol foci. Images of thirty cells were taken from each group. The number of foci was quantified using Image J software under the same threshold for all images. Percentage cells with over 20 Pol was calculated. *P<0.05, χ2 test.   85 4.2.4 ING1b S126A cells showed enhanced genomic instability after UV Since we showed that depletion of ING1b causes enhanced chromosomal aberrations and genomic instability (Wong et al, 2011), we investigated if ING1b S126A would result in the same effect as ING1b knockdown. We reconstituted ING1b expression with WT or S126A ING1b in cells and prepared metaphase chromosomes with or without UV. We found that chromatid breaks and fusions were significantly increased in cells reconstituted with S126A mutant compared to the control (χ2 test) (Figure 4.4A and B); whereas we did not find significant chromosome aberrations in the non-irradiated cells (Figure 4.4C). This indicates that the function of ING1b in maintaining genomic stability is dependent on S126 residue.  86 Figure 4.4 ING1b S126A cells showed enhanced genomic instability after UV HCT116 cells were sequentially transfected with ING1b siRNA and siRNA resistant ING1b WT or S126A mutant as previously. Cells were synchronized at G1/S boundary by thymidine block followed by aphidicolin block. Cells were irradiated with 10 J/m 2  UVC followed by treatment with 0.1 µg/ml colcemid for 20 h. Metaphase chromosomes were spread on slides and analyzed. (B) Quantification of chromosome aberrations. Images of fifty random metaphase spreads were taken from each group. The percentage cells with chromatid breaks and chromosomal fusions were calculated. *P<0.05, χ2 test.    87 4.3 Discussion The ATR-Chk1 S phase checkpoint has been shown to regulate genomic stability upon replication stress in several ways, including arresting the cell cycle, preventing firing of late replication origins, stabilizing replication forks and promoting DNA repair and restart of DNA replication (Matsuoka et al, 2007; Mu et al, 2007; Stokes et al, 2007). Among these functions, the mechanism by which ATR-Chk1 stabilizes stalled replication is poorly understood. It was found that proteins involved in checkpoint signaling such as TOPBP1, BRCA1, claspin and Chk1, are required for preventing fork collapse at common fragile sites (Arlt et al, 2006). PCNA monoubiquitination plays an important role in resolving stalled replication to promote genomic stability (Chang & Cimprich, 2009). It was shown that Chk1 regulates PCNA-Ub through regulating the stability of claspin which in turn is required for Rad18 binding to chromatin (Yang et al, 2008). Previously, we showed that ING1b is required for maintaining genomic stability upon replication stress. We also observed that PCNA-Ub and Rad18 binding to chromatin were both reduced in ING1b knockdown cells (Wong et al, 2011). Therefore, the impact of ING1b and Chk1 on the Rad18-regulated PCNA monoubiquitination pathway is similar, suggesting that they might cooperate in such regulation. This is supported by our observation that the effect of ING1b and Chk1 knockdown on PCNA-Ub was indeed epistatic (Figure 4.2A). Moreover, we showed that PCNA-Ub was reduced in cells expressing ING1b S126A mutant at which Chk1 phosphorylates ING1b (Figure 4.2C) (Garate et al, 2007) and ING1b knockdown did not affect Chk1 activation through phosphorylation (Figure 4.2B). Our results suggest that ING1b is downstream of Chk1 in the regulation of PCNA-Ub.  88  One of the essential functions of PCNA-Ub is to recruit the translesion DNA polymerase, Pol, after UV irradiation to the sites of stalled replication to bypass the UV photolesions (Kannouche & Lehmann, 2006; Watanabe et al, 2004). Although Chk1 has been shown to regulate PCNA-Ub, it is not known if Chk1 regulates Pol recruitment to stalled forks. We showed that Pol foci formation was significantly reduced in ING1b S126A cells (Figure 4.3), suggesting that the Chk1-ING1b pathway is involved in the regulation of the Pol-dependent translesion DNA synthesis.  Prolonged stalling of replication can result in more serious consequence such as replication collapse, generation of DNA DSBs and resulting in gross chromosomal rearrangement (Branzei & Foiani, 2010). The requirement of ING1b S126 in PCNA-Ub and Pol foci formation indicates that the Chk1-ING1b pathway is required for resolution of stalled replication and prevents formation of chromosomal abnormalities. In fact, our observation agrees with this hypothesis. We observed a significant increase in chromatid breaks and chromosome fusions in cells expressing ING1b S126A mutant following UV (Figure 4.4A and B).  The mechanism by which the ATR/Chk1 checkpoint regulates PCNA-Ub and translesion DNA synthesis remains unclear. There seems to be conflicting results regarding the role of ATR and Chk1 as checkpoint kinases in this pathway. ATR was shown to be required for PCNA-Ub induced by BPDE (Bi et al, 2006) but not by UV (Chang et al, 2006; Frampton et al, 2006). Recently, ATR was shown to phosphorylate Pol at S601 which is required for translesion DNA synthesis and cell survival after UV (Gohler et al, 2011). Moreover, the kinase activity of Chk1 was found to be important for fork stabilization in the absence of Pol (Despras et al, 2010). A separate study reported that Chk1 kinase activity is  89 dispensable for PCNA-Ub. They found that kinase dead Chk1 can restore PCNA-Ub in Chk1 knockdown cells same as the wild type (Yang et al, 2008). In this study, we found that ING1b S126, which is phosphorylated by Chk1 upon stress, was required for PCNA-Ub, lesion bypass and genomic stability. This suggests that ING1b S126 phosphorylation by Chk1 might be required for such function. However, we also noticed that ING1b S126 was also required for the physical interaction between Chk1 and ING1b (Figure 4.1B). Moreover, the kinase activity of Chk1 is not required for this interaction since the kinase dead Chk1 was still able to co-precipitated with ING1b (Figure 4.1C). Therefore, it is conceivable that it is the Chk1-ING1b complex that is required for PCNA-Ub rather than phosphorylation of ING1b itself. Therefore, more studies are warranted in dissecting the mode of cooperation of Chk1 and ING1b in the lesion bypass mechanism. Our study provides a novel link between the S phase checkpoint and tumor suppressor ING1b in maintaining genomic stability upon replication stress.  The lesion bypass pathway plays an important role in maintaining genomic stability upon replication stress, in the next chapter, we examined the involvement of the E3 ligase Rad18, the key regulator of the lesion bypass pathway, in melanoma development using tissue microarray.  90 Chapter 5: Elevated Expression of Rad18 Regulates Melanoma Cell Proliferation 5.1 Background and Rationale To investigate the role of the lesion bypass pathway in human cancer development, we evaluated the expression of Rad18 in different stages of melanocytic lesions and the prognostic significance of Rad18 expression on melanoma patient survival. Despite the close association of Rad18 and lesion bypass with genomic stability, there is limited information on the status of Rad18 in human cancers. To our knowledge, there were three studies looking into the polymorphism and mutation of Rad18 in colorectal cancer and non-small cell lung cancer (Kanzaki et al, 2007; Kanzaki et al, 2008; Nakamura et al, 2009). However, the expression level of Rad18 in human cancer is not known. Since Rad18 is the E3 ligase for PCNA monoubiquitination, we hypothesized that Rad18 might play a role in melanoma development. Surprisingly, we found that Rad18 was involved in the regulation of melanoma cell proliferation. We further investigated the mechanism by which Rad18 affected melanoma cell proliferation. 5.2 Results 5.2.1 Rad18 is upregulated in melanoma  Forty-two cases of dysplastic nevi, 146 cases of primary melanoma and 49 cases of metastatic melanoma were evaluated for Rad18 staining by immunohistochemistry. Rad18 was found to be predominantly expressed in the nucleus (Figure 5.1A and B). This is consistent with the previous report on the subcellular localization of Rad18 in the cells (Miyase et al, 2005; Tateishi et al, 2000). We found that the number of cases with strong Rad18 staining was significantly increased from dysplastic nevi (5%) to primary melanoma (19%) (P=0.030, χ2) and metastatic melanoma (27%) (P=0.005, χ2) (Figure 5.1C), while  91 there is no significant difference between primary melanoma and metastatic melanoma, suggesting that Rad18 might play a role in melanoma transformation. We further evaluated the correlation of Rad18 staining with clinicopathological parameters in 146 cases of primary melanoma. While there is no significant correlation of Rad18 staining with age, sex, tumor thickness, ulceration and histological subtypes, strong Rad18 staining was significantly reduced at sun-exposed area (8%) compared to sun-protected area (30%) (P=0.001) (Table 5.1). It suggests that Rad18 might play a different role in the etiology of melanoma at sun- exposed sites.  92 Figure 5.1 Rad18 expression is reduced in melanoma (A,B) Immunohistochemical staining of Rad18 in dysplastic nevi with weak Rad18 staining (A), and metastatic melanoma with strong Rad18 staining (B). Bar, 20 µm. Rad18 expression was significantly increased in primary melanoma (PM) and metastatic melanoma (MM) compared to dysplastic nevi (DN). (C) Significant increase in Rad18 expression was observed in PM compared to DN (P=0.030, χ2); and in MM compared to DN (P=0.005, χ2), but not in PM compared to MM (P=0.228, χ2).   93 Table 5.1 Correlations of Rad18 staining with clinicopathological parameters of 146 cases of primary melanoma patients  Variables  Rad18 staining         Neg-to-Mod            Strong         Total               P Age ≤61  61 (85%) 11 (15%) 72 (49%) 0.324 >61  58 (78%) 16 (22%) 74 (51%) Sex Male  83 (86%) 14 (14%) 97 (37%) 0.075 Female  36 (74%) 13 (26%) 49 (63%) Tumor thickness ≤2mm  69 (81%) 16 (19%) 85 (58%) 0.903 >2mm  50 (82%) 11 (18%) 61 (42%) Ulceration Positive  22 (76%) 7 (24%) 29 (20%) 0.382 Negative  97 (83%) 20 (17%) 117 (80%) Location* Sun-exposed  70 (92%) 6 (8%) 76 (52%) 0.001 Sun-protected  49 (70%) 21 (30%) 70 (48%) Subtype Superficial spreading  25 (71%) 10 (29%) 35 (40%) 0.558 Lentigo maligna  12 (75%) 4 (25%) 16 (18%) Nodular  12 (86%) 2 (14%) 14 (16%) Unspecified  16 (73%) 6 (27%) 22 (25%) *Sun-protected sites: trunk, arm, leg and feet; Sun-exposed sites: head and neck.  94 5.2.2 Rad18 expression is correlated with melanoma patient survival We asked if Rad18 staining correlates with patient survival in primary melanoma. The Kaplan-Meier curve showed that patients in strong Rad18 staining group have poorer 5-year survival (63%) compared to the group with negative-to-moderate Rad18 staining (74%). However, the difference is not significant (P=0.243 for overall survival, P=0.071 for disease- specific survival, log rank test) (Figure 5.2A and B). Since we observed that Rad18 staining is significantly higher in sun-protected area, we investigated if Rad18 staining correlated with patient survival in melanoma cases found in the sun-protected sites. Strikingly, we found that patients with strong Rad18 expression significantly correlated with worse overall and disease-specific survival (P=0.001 for both, log rank test). The mean overall survival time is 59 months in the strong Rad18 group compared to 83 months for the negative-to- moderate Rad18 group (P=0.003, t-test) (Figure 5.2 C and D). Interestingly, the correlation between Rad18 expression and patient survival at sun-exposed sites was reversed. The Kaplan-Meier curve showed that patients in strong Rad18 staining group have better 5-year survival, in which all patients survived, compared to the group with negative-to-moderate Rad18 staining (64% for overall survival and 75% for disease specific survival) (Figures 5.2 E and F). However, the result was not significant possibly due to the limited number of cases with strong Rad18 staining in this group (n=6). Nevertheless, it suggests that Rad18 might play a differential role in melanoma development at the sun-exposed and sun-protected area. We sought to verify the significant correlation between Rad18 expression and patient survival in sun-protected sites by the univariate Cox regression analysis. We found that strong Rad18 expression posed significantly higher risk for the patient survival (RR=1.7 for overall survival, P=0.003; RR=5.1 for disease-specific survival, P=0.004), while two best  95 known prognostic factors for melanoma, tumor thickness and presence of ulceration, also significantly correlated with higher risk for overall and disease-specific survival (Table 5.2).  96 Figure 5.2 Correlation of Rad18 expression with melanoma patient survival Kaplan-Meier curves were generated to compare the correlation of strong and negative to moderate Rad18 staining with overall (A) and disease-specific survival (B) in 146 cases of primary melanoma; for overall (C) and disease-specific survival (D) in 70 cases of sun- protected melanoma; and for overall (E) and disease-specific survival (F) in 76 cases of sun- exposed melanoma.   97 Table 5.2 Univariate Cox regression analysis on overall and disease-specific 5-year survival of 70 cases of primary melanoma at sun-protected sites  Variables  Overall survival  Disease-specific survival  Relative risk 95% CI P  Relative risk 95% CI P Rad18  1.707 1.707-12.995 0.003  5.115 1.709-15.314 0.004 Age  2.979 1.034-8.582 0.043  2.439 0.816-7.286 0.110 Sex  1.308 0.487-3.513 0.594  1.262 0.438-3.636 0.667 Thickness  4.457 1.617-12.284 0.004  4.826 1.614-14.428 0.005 Ulceration  3.887 1.444-10.465 0.007  4.999 1.748-14.295 0.003 Location  3.065 1.284-7.314 0.012  3.112 1.223-7.914 0.017   98 5.2.3 Rad18 is an independent prognostic factor for melanoma at the sun-protected sites Since Rad18 expression correlates with melanoma patient survival at the sun-protected sites, we further performed multivariate Cox regression analysis to see if Rad18 expression is an independent prognostic marker for the sun-protected melanoma. Rad18, tumor thickness, ulceration, age and sex were included in the analysis. Strikingly, similar to tumor thickness, Rad18 is an independent prognostic factor for melanoma patient survival at the sun-protected sites (P=0.020 and 0.022 for overall and disease-specific survival, respectively) (Table 5.3).  99 Table 5.3 Multivariate Cox regression analysis on overall and disease-specific 5-year survival of 70 cases of primary melanoma at sun-protected sites.  Variables  Overall survival  Disease-specific survival  Relative risk 95% CI P  Relative risk 95% CI P Rad18  1.310 1.229-11.182 0.020  4.039 1.220-13.376 0.022 Thickness  3.729 1.483-9.374 0.005  3.879 1.211-12.420 0.022 Ulceration  2.100 0.726-6.071 0.171  2.748 0.878-8.606 0.083 Sex  1.110 0.392-3.140 0.039  1.080 0.352-3.313 0.892 Age  1.539 0.507-4.669 0.447  1.161 0.367-3.670 0.799   100 5.2.4 Rad18 regulates melanoma cell proliferation We further investigated the role of Rad18 in melanoma cell proliferation in vitro. We examined the expression of Rad18 in a panel of melanoma cell lines compared to immortalized melanocytes by Western blot. As previously reported, we detected two bands using Rad18 antibody, 75 and 85 kDa, which correspond to the unmodified and monoubiquitinated forms of Rad18 (Miyase et al, 2005). We found that Rad18 expression was increased in 7 out of 9 melanoma cell lines compared to melanocytes, from 1.9-fold in MMAN cells to 5-fold in MMLH cells; while Rad18 expression was similar in SK-mel-3 cells and reduced in SK-mel-110 cells (Figure 5.3A). We then examined the biological effect of Rad18 depletion. We transfected small interfering RNA (siRNA) into immortalized melanocytes and melanoma cell lines, RPM-MC, SK-mel-3 and MMRU. Rad18 siRNA efficiently knocked down the expression of endogenous Rad18 by over 60% (Figure 5.3B). We used the sulphorhodamine B assay to evaluate the effect of Rad18 on cell proliferation. We found that melanoma cell proliferation was significantly reduced in melanoma cell lines but not in melanocytes upon Rad18 knockdown (Figure 5.3B). Furthermore, in a clonogenic assay, we observed that the number of colonies formed was significantly reduced in melanoma cell lines upon Rad18 knockdown compared to the control, by 67% in RPM-MC, 74% in SK-mel-3 cells and 50% in MMRU cells (Figure 5.4A). The reduced colony formation upon Rad18 knockdown was not due to the difference in cell adhesion since the amount of cells attached to the culture plates was comparable in the control and Rad18 knockdown cells (Figure 5.4B). Consistent with this observation, Rad18 overexpression significantly stimulated cell proliferation in SK-mel-3 cells compared to the vector control (Figure 5.5). These data suggest that Rad18 regulates melanoma cell proliferation.  101 Figure 5.3 Rad18 enhances melanoma cell proliferation (A) Rad18 expression in immortalized melanocytes and melanoma cell lines. (B) Rad18 knockdown inhibits melanoma cell proliferation. Immortalized melanocytes and melanoma cell lines, RPM-MC, SK-mel-3 and MMRU, were transfected with control or Rad18 siRNA. 24 h after transfection, same number of cells was seeded on 24-well plate for 6 h. The cells were fixed at various time points after plating for SRB assay. Data was obtained from three independent experiments and presented as mean ± SD.   102 Figure 5.4 Rad18 knockdown inhibits colony formation of melanoma cells (A) RPM-MC, SK-mel-3 and MMRU cells were transfected with control or Rad18 siRNA. 24 h after transfection, 1000 cells were seeded into 60 mm dish in triplicates and cultured for 20 days. Cells were fixed with 1% formaldehyde and stained with 0.05% crystal violet. The number of colonies was quantified by Image J software. (B) Rad18 knockdown does not affect melanoma cell adhesion. RPM-MC, SK-mel-3 and MMRU cells were transfected with control or Rad18 siRNA. 24 h after transfection, 1000 cells were seeded into 24 well plates. 6 h after seeding, cells were fixed for SRB assay. Data was obtained from three independent experiments and presented as mean ± SD.    103 Figure 5.5 Rad18 overexpression stimulated melanoma cell growth independent of its E3 ligase activity SK-mel-3 cells were transfected with empty vector, Rad18 or Rad18 C28F expression plasmids. Overexpression of Rad18 was confirmed by Western blotting (left panel). Cells were harvested at indicated time for SRB assay (right panel). Data was obtained from three independent experiments and presented as mean ± SD.   104 5.2.5 Rad18 regulates pAkt and cyclin D1 expression We further investigated the mechanism by which Rad18 regulates cell proliferation. Rad18 is known to be important for its function as an E3 ligase for PCNA monoubiquitination which regulates the lesion bypass pathway in resolving stalled replication (Kannouche et al, 2004; Tateishi et al, 2000; Watanabe et al, 2004). We first hypothesized that Rad18 might be important for resolving stalled replication during normal DNA replication. We looked for the signs of stalled replication upon Rad18 knockdown, including induction of γH2AX, pChk1, and RPA hyperphosphorylation and foci formation. To our surprise, we did not observe significant change in these parameters upon Rad18 knockdown indicating that there was no extensive stalled replication in these cells compared to the control (Figure 5.6A and B). Moreover, the basal level of PNCA monoubiquitnation was undetectable on the Western blot (Figure 5.6A). When we overexpressed the dominant negative mutant of Rad18, the C28F mutant, which abrogates the E3 ligase activity of Rad18 for PCNA monoubiquitination (Kannouche et al, 2004; Tateishi et al, 2000), we were still able to observe the stimulation effect on cell proliferation compared to the vector control (Figure 5.5). We concluded that Rad18 regulates cell proliferation independent of its E3 ligase function and PCNA monoubiquitination.  Next, we asked if Rad18 regulates other key pathways involved in melanoma cell proliferation. The PI3K-Akt is a key signaling cascade involved in melanoma cell proliferation (Madhunapantula & Robertson, 2009). We investigated if Rad18 regulates the PI3K-Akt pathway. We observed that pAkt was significantly reduced upon Rad18 knockdown when compared to the control (Figure 5.7A). Moreover, we observed a concomitant reduction in cyclin D1 expression (Figure 5.7A). In agreement with this  105 observation, pAkt and cyclin D1 were upregulated when we overexpressed both the wild- type and C28F mutant Rad18 in SK-mel-3 cells (Figure 5.7B), again, suggesting an E3-ligase independent role of Rad18 in regulating cell proliferation through pAkt and cyclin D1.  106 Figure 5.6 Rad18 knockdown does not induce replication stress in melanoma cells under non-stress condition (A) RPM-MC and SK-mel-3 cells were transfected with control and Rad18 siRNA for 72 h. Cells were harvested for Western blot analysis.  (B) RPM-MC and SK-mel-3 cells were transfected with control and Rad18 siRNA for 72 h. Cells were fixed and immnuofluorescent staining was performed using RPA antibody. Thirty images were obtained from each group. Percentage cells showing positive RPA foci were quantified. Data was obtained from three independent experiments and presented as mean ± SD.   107 Figure 5.7 Rad18 knockdown inhibited pAkt and cyclin D1 in cells. (A) RPM-MC and MMRU cells were transfected with control and Rad18 siRNA for 72 h. Cells were harvested for Western blot analysis. (B) Upregulation of pAkt and cyclin D1 in Rad18 overexpressed cells. SK-mel-3 cells were transfected with empty vector, Rad18 or Rad18 C28F expression plasmids. Cells were harvested for Western blot 24 h after transfection. The experiment was repeated for three times. Representative images were shown.    108 5.3  Discussion Our TMA data suggest that Rad18 plays an oncogenic role during melanoma development. We observed an increase in Rad18 expression from the precancerous lesion, dysplastic nevi, to primary and metastatic melanoma, but no significant difference between primary and metastatic melanoma (Figure 5.1). It suggests that Rad18 may be involved in the transformation step during the early stage of melanoma development. Our in vitro data support this idea. We found that Rad18 regulates melanoma cell proliferation (Figure 5.3 and 5.4). Enhanced cell proliferation is one of the most fundamental features of malignancies (Hanahan & Weinberg, 2011) and has to be sustained early on in transformation from benign melanocytic lesions into various growth phases of melanoma (Gray-Schopfer et al, 2007). In search for the mechanism by which Rad18 regulates cell proliferation, we looked into the known functions of Rad18. Rad18 is well studied for its function as the E3 ligase for PCNA monoubiquitination which is a critical event in activating the lesion bypass mechanism (Kannouche et al, 2004; Watanabe et al, 2004). There is accumulating evidence that cancer cells have high endogenous level of replication stress (Bartkova et al, 2010; Bartkova et al, 2005; Gorgoulis et al, 2005) including melanoma (Warters et al, 2005; Wasco et al, 2008).  We first hypothesized that elevated level of Rad18 may be an adaptation for cancer cells to deal with the endogenous replication stress through PCNA monoubiquitination. Contrary to our expectation, we did not find any sign of enhanced replication stress in Rad18 knockdown cells nor could we detect PCNA monoubiquitination at non-stress condition in melanoma cells (Figure 5.6). Recently, it was shown that another E3 ligase, CRL4 Cdt2 , was responsible for PCNA monoubiquitination and resolving endogenous replication stress independent of Rad18 (Terai et al, 2010). It is possible Rad18  109 is dispensable for this function. Meanwhile, we found that Rad18 regulated pAkt (Figure 5.7). PI3K-Akt pathway is one of the key signaling cascades involved in cell proliferation and cell survival. Akt inhibition reduces melanoma growth and induces apoptosis (Karst et al, 2006; Robertson, 2005; Tran et al, 2008).  Activated Akt phosphorylates GSK3β at Ser9 and Ser21 to inhibit its activity thereby promoting cell cycle progression from G1 to S phase through stabilizing cyclin D1 (Fresno Vara et al, 2004; Ramaswamy et al, 1999; Woodgett, 2005). Consistent with these reports, we observed a reduction of pAkt with a concomitant reduction in cyclin D1 expression in Rad18 knockdown cells (Figure 5.7A) whereas pAkt and cyclin D1 expression was increased in Rad18 overexpressed cells (Figure 5.7B). We for the first time showed that Rad18 regulates pAkt and cyclin D1 expression. Apparently, this is independent of the function of Rad18 as an E3 ligase since overexpression of the E3 ligase dominant negative mutant of Rad18, C28F mutant, still stimulated cell proliferation and enhanced pAkt and cyclin D1 expression same as the wild type Rad18. However, more studies are warranted to understand the mechanism by which Rad18 regulates the PI3K-Akt pathway. UV is the major environmental risk factor for melanoma and UV lesions can cause genomic instability through replication stalling. Despite the importance of Rad18 in resolving stalled replication caused by UV lesion and in preserving genomic stability (Jansen et al, 2007; Putnam et al, 2010; Tateishi et al, 2000), our observation appears to be counterintuitive at first because we found that Rad18 expression was increased in melanoma (Figure 5.1). However, when we took a closer look at our data, we noticed that Rad18 expression was indeed significantly reduced in melanoma at the sun-exposed sites (Table 1). We found that there was not a single patient died within 5 years in the strong Rad18 staining  110 group, while 36% of patients died within 5 years in negative-to-moderate Rad18 group (Figure 5.2E and F). It suggests that Rad18 plays a tumor suppressive role in the sun-exposed sites. This is consistent with the function of Rad18 in lesion bypass. The presence of UV lesion at the replication fork stalls DNA replication which can result in collapse of the replication fork and DNA double strand breaks (DSBs) (Limoli et al, 2002) and eventually leads to genomic instability (Kunkel, 2004; van Gent et al, 2001). Reduced Rad18 expression at the sun-exposed sites may be the driver for the genetic alterations in the melanoma. Indeed, we were able to observe increased γH2AX which appears upon DNA DSBs and a reduction in PCNA monoubiquitination in Rad18 knockdown cells after UV irradiation (Figure 5.8).  111 Figure 5.8 Rad18 knockdown enhances DNA double stress breaks after UV RPM-MC cells were transfected with control and Rad18 siRNA. 72 h after transfection, cells were irradiated with 10 J/m 2 . Cells were harvested for Western blot analysis 12 h after UV.   112 Meanwhile, we found that Rad18 is indeed oncogenic in the sun-protected sites. Strong Rad18 expression significantly correlated with worse 5-year patient survival when compared to the negative-to-moderate Rad18 group as analyzed by both the Kaplan-Meier curve (Figure 5.2C and D) and univariate Cox regression in the sun-protected area (Table 5.2). More importantly, Rad18 is an independent prognostic factor for patient survival in the sun-protected area. Although it is known that sun exposure contributes to melanoma development, there are very few studies focusing on differentiating the molecular etiology of melanoma developed at different sites with various degree of sun exposure. Bastian and colleagues attempted to explain the heterogeneity in different subtypes of melanoma. They found that there are distinct sets of genetic alterations identified in melanoma at different sites with different levels of sun exposure (Curtin et al, 2005) suggesting differential mechanisms in development of different subsets of melanoma. We speculate that a homeostatic level of Rad18 is important to prevent melanoma development. A reduction of Rad18 level can lead to genomic instability in the sun-exposed site whereas overexpression of Rad18 in sun-protected sites can promote cell proliferation through pAkt. This study provides a novel insight into the roles of Rad18 in melanoma development at the sun- exposed and sun-protected sites. It improves our understanding of the distinct molecular pathogenesis of melanoma which has implications in discovering new strategies to target different subsets of melanoma.  113 Chapter 6: Conclusions 6.1 Summary of Findings In Chapter 3, we analyzed the physiological role of ING1b and discovered a novel tumor suppressive function of ING1b in preserving genomic stability in response to UV- induced replication stress. It was shown that cells deficient in tumor suppressor ING1b are hypersensitive to DNA damaging agents via unknown mechanism (Kichina et al, 2006; Kuo et al, 2007; Tallen et al, 2008). We observed that cells depleted with endogenous level of ING1b have defects in S phase recovery and enhanced genomic instability after UV irradiation. The lesion bypass pathway, which is regulated by monoubiquitination of PCNA, is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. We found that ING1b knockdown cells were more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b was required for the E3 ligase Rad18-mediated PCNA monoubiquitination and Polε functions implicating the role of ING1b in regulating the lesion bypass pathway. Interestingly, ING1b- mediated PCNA monoubiquitination was associated with the regulation of histone H4 acetylation. We observed that ING1b depleted cells displayed hypoacetylation of histone H4 at S phase and that restoration of histone acetylation rescued PCNA-Ub and Rad18 binding to chromatin in ING1b knockdown cells. For the first time, we have shown that chromatin remodeling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.  Previously, our group showed that ING1b is phosphorylated by the S phase checkpoint effector kinase Chk1 (Garate et al, 2007; Garate et al, 2008).  It suggests that ING1b probably functions downstream of the S phase checkpoint. Since the S phase  114 components were shown to play a role in PCNA monoubiquitination and genomic stability, in Chapter 4, we explored if ING1b cooperated with Chk1 in such regulation. We showed that ING1b interacted with Chk1 in an UV-inducible manner. The ING1b-Chk1 interaction required ING1b S126 residue but not the kinase activity of Chk1. ING1b and Chk1 were epistatic in the regulation of PCNA-Ub suggesting that they function in the same pathway in PCNA-Ub. Moreover, ING1b knockdown did not affect Chk1 activation by phosphoryltion upon UV indicating that Chk1 is probably upstream of ING1b. Reconstitution of ING1b expression with exogenous WT ING1b restored PCNA-Ub in ING1b KD cells but reconstitution of ING1b S126A failed to rescue PCNA-Ub. Furthermore, we found that Pol foci formation following UV was abrogated in cells reconstituted with ING1b S126A when compared to the WT cells. ING1b S126A cells showed enhanced chromosomal aberrations after treating with UV compared to the WT cells. These data suggest that ING1b cooperates with Chk1 through the S126 residue in mediating PCNA-Ub, lesion bypass and genomic stability.  The E3 ligase Rad18 is a key regulator for the lesion bypass pathway which plays an important role in genomic stability. In Chapter 5, we evaluated the expression of Rad18 in different stages of melanocytic lesions and the prognostic significance of Rad18 expression on melanoma patient survival. We further investigated the mechanism by which Rad18 regulates melanoma cell proliferation. Immunohistochemical staining was performed on melanoma tissue microarray (TMA) containing 237 melanocytic lesions using anti-Rad18 antibody. We found that Rad18 expression was significantly upregulated in primary melanoma and metastatic melanoma compared to dysplastic nevi. Rad18 expression was significantly reduced in sun-exposed sites compared to the sun-protected sites. Strong Rad18  115 expression correlated with worse 5-year patient survival and was an independent prognostic factor for melanoma found in the sun-protected sites. Interestingly, we found an opposite role of Rad18 on patient survival in the sun-exposed sites. Furthermore, we showed that melanoma cell proliferation and the expression of pAkt and cyclin D1 were reduced upon Rad18 knockdown. 6.2 New Insights into the Mechanisms of Lesion Bypass Proteins involved in regulating PCNA monoubiquitination and lesion bypass are of great importance in maintaining genomic stability since this process is essential for recovery from stalled replication and thus plays significant role in cancer development (Friedberg, 2005; Jansen et al, 2007). It is exemplified by a higher incidence of sunlight-induced skin cancers in patients with Xeroderma Pigmentosum variant who are deficient in the translesion DNA polymerase, Polε (Moriwaki & Kraemer, 2001). ING family proteins are classified as type II tumor suppressors. ING2 and ING5 are shown to be involved in the normal DNA replication process (Doyon et al, 2006; Larrieu et al, 2009). In this study, we defined a unique and novel role of ING1b in regulating PCNA monoubiquitination and preserving genomic stability upon replication stress (Figure 6.1).  We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination, and to our knowledge, this is first study linking histone acetylation to the regulation of PCNA monoubiquitination. It concurs with the accumulating publications in supporting the notion that chromatin remodeling enzymes are involved in maintaining replication stability upon stress. The ATP-dependent chromatin remodeling enzyme Ino80 is implicated in stabilizing stalled replication forks and is required for replication restart after stalling (Falbo et al, 2009; Papamichos-Chronakis & Peterson, 2008; Shimada et al, 2008).  116 In fact, histone acetylation has also been implicated in DNA replication stress response. The Rtt109 HAT in yeast acetylates H3K56 specifically in S phase and cells altered in H3K56 or lacking Rtt109 are sensitive to genotoxic agents (Han et al, 2007; Masumoto et al, 2005). The human orthologue for Rtt109 was recently identified to be p300/CBP which is also responsible for H3K56 acetylation in S phase (Das et al, 2009).  However, it still remains elusive of why these chromatin remodeling factors participate in maintaining genomic integrity upon stress. It is well accepted that chromatin remodeling plays an important role in other processes such as transcription and DNA repair. Due to the compact structure of chromatin, the chromatin has to be relaxed before these processes could be carried out (Allard et al, 2004; Li et al, 2007; Smerdon, 1991; van Attikum & Gasser, 2005). We previously showed that ING1b regulates histone H4 acetylation and chromatin relaxation to provide accessibility for XPA in nucleotide excision repair (Kuo et al, 2007). We propose that ING1b remodels the chromatin structure through H4 acetylation to provide accessibility for factors involved in lesion bypass. This is in part supported by our observation that Rad18 binding to chromatin is reduced in ING1b knockdown cells (Figure 3.10). However, one intriguing fact is that the replication fork is devoid of histones and the double stranded strand DNA structure is opened up by MCM helicase. Why is accessibility required for the lesion bypass machinery? We speculate that histone acetylation may play a role in stabilization of stalled forks or it is required for the recruitment of additional factors that are crucial for this function. In fact, it is known that various protein complexes are recruited to the site of stalled replication during activation of the ATR-Chk1 pathway (Cimprich & Cortez, 2008). Many of these factors, such as TOPBP1, BRCA1, claspin and Chk1, play a direct role in stabilization of stalled forks (Branzei &  117 Foiani, 2010; Flynn & Zou, 2011). ING1b may be required for recruitment of these factors to stalled forks.  An alternative hypothesis is that histones may not be the only target affected by acetylation (Batta et al, 2007; Glozak et al, 2005). Polβ was found to be acetylated and acetylation in Polβ affects its function in the base excision repair pathway (Hasan et al, 2002). ING1b can also be involved in acetylation of other factor(s) that is involved in lesion bypass through cooperation with HATs or HDACs. These are testable hypotheses and require more work to dissect the role of ING1b in lesion bypass and genomic stability.  It is known that S phase checkpoint regulates genomic stability through multiple ways (Branzei & Foiani, 2010; Flynn & Zou, 2011). However, the mechanism of how the S phase checkpoint leads to the stabilization of stalled forks is not clear. Since we found that ING1b S126 was required for PCNA-Ub, TLS and genomic stability, we propose that ING1b connects the S phase checkpoint in regulation of genome integrity. It was shown that ING1b possesses a PIP box through which ING1b binds PCNA and such binding is increased after UV (Scott et al, 2001b). Moreover, they observed that ING1b colocalized with PCNA foci after UV (Scott et al, 2001b). Therefore, it is possible that ING1b may be recruited to the sites of replication blockage through the PIP box, presumably, through which ING1b is phosphorylated at S126 by Chk1.  118 Figure 6.1 Model for ING1b in maintaining genomic stability through regulating lesion bypass Upon stalled replication due to the presence of UV photolesion, checkpoint kinase Chk1 is activated. Chk1 in turn interacts and phosphorylates ING1b at S126 residue. Such interaction and/or phosphorylation is require for mediating Rad18-dependent PNCA monoubiquitination. ING1b regulates Rad18 through maintaining level of histone H4 acetylation. Monoubiquitinated PCNA recruits Pol to replicate across the photolesion to resolve the stalled replication and maintain genomic stability.    119 6.3 The Role of Rad18 in Melanoma Development We for the first time showed that Rad18 was involved in melanoma development. On one hand, we showed that Rad18 played an oncogenic role in the sun-protected melanoma through regulating melanoma cell proliferation and pAkt; on the other hand, Rad18 possibly played a tumor suppressive role in the sun-exposed area through protecting cells from genomic instability due to UV photolesions. Interestingly, we found that the stimulation effect of Rad18 on cell proliferation was independent of its E3 ligase activity while its E3 ligase function for PCNA monoubiquitination was required to prevent DSBs and thus genomic instability after UV. Therefore, we speculate that a homeostatic level of Rad18 is essential to prevent melanoma development. We propose that Rad18 plays a dual role, the E3 ligase dependent and independent, in the sun-exposed and sun-protected melanomas. It potentially has tremendous implications in the etiology of melanoma from different locations with various degree of sun exposure. It can lead to the design of different therapeutic strategies in treating melanoma from different origins. 6.4 Future Directions We have discovered a novel function of ING1b in regulation of PCNA monoubiquitination and lesion bypass. In future, we will continue to investigate the role of ING1b in genomic stability. First, we propose to identify ING1b complex in S phase during stalled replication. We observed that ING1b regulated histone H4 acetylation in S phase, however the mechanism is not known. Previously, it has been identified that ING1b is associated with both HATs and HDACs (Skowyra et al, 2001; Vieyra et al, 2002). The isolation of ING1b complex was done in non-stress condition and in asynchronous cells. We hypothesize that ING1b might cooperate with specific HAT(s) or HDAC(s) during stalled  120 replication. In order to assess this, we would need to isolate ING1b complex immunoprecipitation in cells synchronized in S phase using double thymidine block treated with or without UV. Then, we will identify the interaction partners by mass spectrometry. We will further characterize the involvement of these interaction partners in PCNA-Ub and lesion bypass.  ING1b carries a PIP box and ING1b interaction with PCNA is increased upon UV through its PIP box (Scott et al, 2001b). We hypothesize that ING1b might be recruited to the sites of replication through the PIP box upon replication stalling. Therefore, we would need to evaluate if ING1b colocalize with PCNA, RPA and BrdU after UV which label the sites of stalled replication. We will also have to study if this domain is required for PCNA monoubiquitination and genomic stability. To study the role of ING1b in photocarcinogenesis in vivo, we propose to induce tumor in wild type and p37ING1 knockout mice. UV irradiation in mice is known to induce skin cancer (Li et al, 1995). We hypothesize that UV-induced tumor will be accelerated in ING1 knockout mice. To understand if genomic instability is enhanced in ING1 knockout mice, tumors will have to be isolated from the wild type and ING1 knockout mice and signs of genomic instability will be measured by CGH. We expect that tumors from ING1 knockout mice will carry more genome rearrangement, and genome gains and losses. We hypothesize that the ING1b-Rad18-PCNA-Ub and the S phase checkpoint pathways play important role in cancer development. Therefore, the expression of these factors, ING1b, PCNA-Ub, pChk1 and claspin, in different stages of melanoma will have to be evaluated in melanoma tissue microarray to investigate if they correlate with the clinicopathological parameters and patient survival of melanoma.  121 We will have to further investigate the mechanism by which Rad18 regulates melanoma cell proliferation and pAkt. We found that Rad18 regulates melanoma cell proliferation in an E3 ligase-independent manner. Different mutants of Rad18 could be generated to study which domain is required for Rad18-stimulated cell proliferation. 6.5 Targeting Cancer Susceptibility Pathways It is known that components of the DNA damage response pathway are commonly abrogated in cancer (Bartkova et al, 2005; Fackenthal & Olopade, 2007; Frebourg & Friend, 1992; Lavin & Shiloh, 1997; Lehmann, 2003; Nevanlinna & Bartek, 2006; O'Driscoll et al, 2003; Shiloh, 2003; Taylor et al, 2004). Therefore, it can be exploited in cancer therapy. It has been shown that cancer cells that are defective in certain component of the DNA repair pathway become more dependent on other pathways for survival. Inhibition of these pathways that cancer cells rely on can selectively kill these cells. This idea is known as synthetic lethality (Al-Ejeh et al, 2010; Martin et al, 2010; Reinhardt et al, 2009).  For instance, BRCA1 and BRCA2 mutations are commonly found in breast and ovarian cancer (Bryant et al, 2005; Farmer et al, 2005). These cells are defective in homologous recombination. As a result, the BRCA1 and 2 deficient cells are more sensitive to PARP-1 inhibition, an essential component in the base excision repair (BER), because the unrepaired BER substrate can leads to DNA DSBs which cannot be repaired efficiently in the BRCA1 and 2 deficient cells. Another example is the targeting for the p53-deficient tumor. It was observed that upon treatment with genotoxic agent such as doxorubicin, the p53-deficient cells are dependent on ATM/Chk2 pathway to activate the G2/M checkpoint to prevent mitotic catastrophe while the p53 proficient cells can activate the G1 checkpoint for cell  122 survival (Jiang et al, 2009). Therefore, inhibition of ATM/Chk2 greatly increases sensitivity of p53-deficient tumors to doxorubicin (Fedier et al, 2003).  We propose that we can target ING1b deficient tumors with agents that lead to replication stress such as cisplatin, methyl methanesulfonate and hydroxyurea because ING1b deficient cells are defective in PCNA monoubiquitination and lesion bypass. Treatment of these cells will increase the burden of replication and lead to the „stress overload‟ (Luo et al, 2009) in ING1b deficient tumor cells while the normal cells with competent lesion bypass mechanism will tolerate these stress better. Moreover, we can continue to investigate other survival pathway(s) that ING1b deficient tumors rely on upon replication. Inhibition of these pathways should potentiate the toxicity of replication stress agents on ING1b deficient tumors (Figure 6.2).  We have discovered a novel tumor suppressor function of ING1b in genomic stability and the role of Rad18 in melanoma development. It sheds light on the mechanism of lesion bypass pathway and the role of this pathway in melanoma tumorigenesis. This knowledge might eventually lead us to designing new strategies in cancer treatment.  123 Figure 6.2 Targeting ING1 deficient tumor ING1b deficient cancer cells display enhanced genomic instability due to defective lesion bypass. Upon treatment with agents that induce replication stress such as hydroxyurea and aphidicolin, it causes replication fork collapse and cell death. Meanwhile, normal cells can better tolerate the replication stress and survive.    124 Bibliography  Aasland R, Gibson TJ, Stewart AF (1995) The PHD finger: implications for chromatin- mediated transcriptional regulation. Trends Biochem Sci 20: 56-59  Abad M, Moreno A, Palacios A, Narita M, Blanco F, Moreno-Bueno G, Palmero I (2011) The tumor suppressor ING1 contributes to epigenetic control of cellular senescence. Aging Cell 10: 158-171  Acharya N, Yoon JH, Gali H, Unk I, Haracska L, Johnson RE, Hurwitz J, Prakash L, Prakash S (2008) Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA synthesis. Proc Natl Acad Sci U S A 105: 17724-17729  Agar NS, Halliday GM, Barnetson RS, Ananthaswamy HN, Wheeler M, Jones AM (2004) The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc Natl Acad Sci U S A 101: 4954-4959  Aguilera A, Gomez-Gonzalez B (2008) Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet 9: 204-217  Aguissa-Toure AH, Wong RP, Li G (2010) The ING family tumor suppressors: from structure to function. Cell Mol Life Sci  Al-Ejeh F, Kumar R, Wiegmans A, Lakhani SR, Brown MP, Khanna KK (2010) Harnessing the complexity of DNA-damage response pathways to improve cancer treatment outcomes. Oncogene 29: 6085-6098  Allard S, Masson JY, Cote J (2004) Chromatin remodeling and the maintenance of genome integrity. Biochim Biophys Acta 1677: 158-164  Andreassen PR, Ho GP, D'Andrea AD (2006) DNA damage responses and their many interactions with the replication fork. Carcinogenesis 27: 883-892  Arlt MF, Durkin SG, Ragland RL, Glover TW (2006) Common fragile sites as targets for chromosome rearrangements. DNA Repair (Amst) 5: 1126-1135  Armstrong JD, Kunz BA (1990) Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast. Proc Natl Acad Sci U S A 87: 9005-9009  Bailly V, Lamb J, Sung P, Prakash S, Prakash L (1994) Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin- conjugating activity to DNA damage sites. Genes Dev 8: 811-820   125 Bao S, Tibbetts RS, Brumbaugh KM, Fang Y, Richardson DA, Ali A, Chen SM, Abraham RT, Wang XF (2001) ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature 411: 969-974  Bartek J, Lukas C, Lukas J (2004) Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5: 792-804  Barth A, Wanek LA, Morton DL (1995) Prognostic factors in 1,521 melanoma patients with distant metastases. J Am Coll Surg 181: 193-201  Bartkova J, Hamerlik P, Stockhausen MT, Ehrmann J, Hlobilkova A, Laursen H, Kalita O, Kolar Z, Poulsen HS, Broholm H, Lukas J, Bartek J (2010) Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas. Oncogene 29: 5095-5102  Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J (2005) DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434: 864-870  Batista LF, Kaina B, Meneghini R, Menck CF (2009) How DNA lesions are turned into powerful killing structures: insights from UV-induced apoptosis. Mutat Res 681: 197-208  Batta K, Das C, Gadad S, Shandilya J, Kundu TK (2007) Reversible acetylation of non histone proteins: role in cellular function and disease. Subcell Biochem 41: 193-212  Bauer J, Bastian BC (2006) Distinguishing melanocytic nevi from melanoma by DNA copy number changes: comparative genomic hybridization as a research and diagnostic tool. Dermatol Ther 19: 40-49  Bell DW, Varley JM, Szydlo TE, Kang DH, Wahrer DC, Shannon KE, Lubratovich M, Verselis SJ, Isselbacher KJ, Fraumeni JF, Birch JM, Li FP, Garber JE, Haber DA (1999) Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome. Science 286: 2528- 2531  Berardi P, Russell M, El-Osta A, Riabowol K (2004) Functional links between transcription, DNA repair and apoptosis. Cell Mol Life Sci 61: 2173-2180  Berdasco M, Esteller M (2010) Aberrant epigenetic landscape in cancer: how cellular identity goes awry. Dev Cell 19: 698-711  Bi X, Barkley LR, Slater DM, Tateishi S, Yamaizumi M, Ohmori H, Vaziri C (2006) Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint- mediated arrest. Mol Cell Biol 26: 3527-3540   126 Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR, Hofmann K, Dikic I (2005) Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310: 1821-1824  Bienko M, Green CM, Sabbioneda S, Crosetto N, Matic I, Hibbert RG, Begovic T, Niimi A, Mann M, Lehmann AR, Dikic I (2010) Regulation of translesion synthesis DNA polymerase eta by monoubiquitination. Mol Cell 37: 396-407  Bienz M (2006) The PHD finger, a nuclear protein-interaction domain. Trends Biochem Sci 31: 35-40  Binda O, Nassif C, Branton PE (2008) SIRT1 negatively regulates HDAC1-dependent transcriptional repression by the RBP1 family of proteins. Oncogene 27: 3384-3392  Boddy MN, Shanahan P, McDonald WH, Lopez-Girona A, Noguchi E, Yates IJ, Russell P (2003) Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60. Mol Cell Biol 23: 5939-5946  Boissy RE, Nordlund JJ (1997) Molecular basis of congenital hypopigmentary disorders in humans: a review. Pigment Cell Res 10: 12-24  Bourre F, Renault G, Sarasin A (1987) Sequence effect on alkali-sensitive sites in UV- irradiated SV40 DNA. Nucleic Acids Res 15: 8861-8875  Bowman GD, O'Donnell M, Kuriyan J (2004) Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 429: 724-730  Branzei D, Foiani M (2005) The DNA damage response during DNA replication. Curr Opin Cell Biol 17: 568-575  Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11: 208-219  Brash DE (1997) Sunlight and the onset of skin cancer. Trends Genet 13: 410-414  Brash DE, Haseltine WA (1982) UV-induced mutation hotspots occur at DNA damage hotspots. Nature 298: 189-192  Bromidge T, Lynas C (2002) Relative levels of alternative transcripts of the ING1 gene and lack of mutations of p33/ING1 in haematological malignancies. Leuk Res 26: 631-635  Brown EJ, Baltimore D (2000) ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 14: 397-402   127 Bruls WA, Slaper H, van der Leun JC, Berrens L (1984) Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40: 485-494  Bruno M, Flaus A, Stockdale C, Rencurel C, Ferreira H, Owen-Hughes T (2003) Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities. Mol Cell 12: 1599-1606  Brush GS, Morrow DM, Hieter P, Kelly TJ (1996) The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast. Proc Natl Acad Sci U S A 93: 15075- 15080  Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T (2005) Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434: 913-917  Burgers PM, Koonin EV, Bruford E, Blanco L, Burtis KC, Christman MF, Copeland WC, Friedberg EC, Hanaoka F, Hinkle DC, Lawrence CW, Nakanishi M, Ohmori H, Prakash L, Prakash S, Reynaud CA, Sugino A, Todo T, Wang Z, Weill JC, Woodgate R (2001) Eukaryotic DNA polymerases: proposal for a revised nomenclature. J Biol Chem 276: 43487-43490  Busino L, Donzelli M, Chiesa M, Guardavaccaro D, Ganoth D, Dorrello NV, Hershko A, Pagano M, Draetta GF (2003) Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature 426: 87-91  Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B (1998) Mutations of mitotic checkpoint genes in human cancers. Nature 392: 300-303  Campos EI, Cheung KJ, Jr., Murray A, Li S, Li G (2002) The novel tumour suppressor gene ING1 is overexpressed in human melanoma cell lines. Br J Dermatol 146: 574-580  Campos EI, Chin MY, Kuo WH, Li G (2004a) Biological functions of the ING family tumor suppressors. Cell Mol Life Sci 61: 2597-2613  Campos EI, Martinka M, Mitchell DL, Dai DL, Li G (2004b) Mutations of the ING1 tumor suppressor gene detected in human melanoma abrogate nucleotide excision repair. Int J Oncol 25: 73-80  Canella KA, Seidman MM (2000) Mutation spectra in supF: approaches to elucidating sequence context effects. Mutat Res 450: 61-73  Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates fragile site stability. Cell 111: 779-789   128 Cha RS, Kleckner N (2002) ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones. Science 297: 602-606  Chang DJ, Cimprich KA (2009) DNA damage tolerance: when it's OK to make mistakes. Nat Chem Biol 5: 82-90  Chang DJ, Lupardus PJ, Cimprich KA (2006) Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities. J Biol Chem 281: 32081-32088  Chen B, Campos EI, Crawford R, Martinka M, Li G (2003a) Analyses of the tumour suppressor ING1 expression and gene mutation in human basal cell carcinoma. Int J Oncol 22: 927-931  Chen L, Matsubara N, Yoshino T, Nagasaka T, Hoshizima N, Shirakawa Y, Naomoto Y, Isozaki H, Riabowol K, Tanaka N (2001) Genetic alterations of candidate tumor suppressor ING1 in human esophageal squamous cell cancer. Cancer Res 61: 4345-4349  Chen S, Paul P, Price BD (2003b) ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response. Oncogene 22: 6332-6339  Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. J Biol Chem 267: 166-172  Cheung KJ, Jr., Bush JA, Jia W, Li G (2000) Expression of the novel tumour suppressor p33(ING1)is independent of p53. Br J Cancer 83: 1468-1472  Cheung KJ, Jr., Li G (2002) p33(ING1) enhances UVB-induced apoptosis in melanoma cells. Exp Cell Res 279: 291-298  Cheung KJ, Jr., Mitchell D, Lin P, Li G (2001) The tumor suppressor candidate p33(ING1) mediates repair of UV-damaged DNA. Cancer Res 61: 4974-4977  Chin MY, Ng KC, Li G (2005) The novel tumor suppressor p33ING2 enhances UVB- induced apoptosis in human melanoma cells. Exp Cell Res 304: 531-543  Chiu RK, Brun J, Ramaekers C, Theys J, Weng L, Lambin P, Gray DA, Wouters BG (2006) Lysine 63-polyubiquitination guards against translesion synthesis-induced mutations. PLoS Genet 2: e116  Choy JS, Kron SJ (2002) NuA4 subunit Yng2 function in intra-S-phase DNA damage response. Mol Cell Biol 22: 8215-8225  Chudnovsky Y, Khavari PA, Adams AE (2005) Melanoma genetics and the development of rational therapeutics. J Clin Invest 115: 813-824  129  Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 9: 616-627  Clark WH, Jr., Elder DE, Guerry Dt, Epstein MN, Greene MH, Van Horn M (1984) A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma. Hum Pathol 15: 1147-1165  Cleaver JE (1972) Xeroderma pigmentosum: variants with normal DNA repair and normal sensitivity to ultraviolet light. J Invest Dermatol 58: 124-128  Cobb JA, Bjergbaek L, Shimada K, Frei C, Gasser SM (2003) DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. Embo J 22: 4325-4336  Cohen C, Zavala-Pompa A, Sequeira JH, Shoji M, Sexton DG, Cotsonis G, Cerimele F, Govindarajan B, Macaron N, Arbiser JL (2002) Mitogen-actived protein kinase activation is an early event in melanoma progression. Clin Cancer Res 8: 3728-3733  Coles AH, Liang H, Zhu Z, Marfella CG, Kang J, Imbalzano AN, Jones SN (2007) Deletion of p37Ing1 in mice reveals a p53-independent role for Ing1 in the suppression of cell proliferation, apoptosis, and tumorigenesis. Cancer Res 67: 2054-2061  Coles AH, Marfella CG, Imbalzano AN, Steinman HA, Garlick DS, Gerstein RM, Jones SN (2008) p37Ing1b regulates B-cell proliferation and cooperates with p53 to suppress diffuse large B-cell lymphomagenesis. Cancer Res 68: 8705-8714  Cortez D, Glick G, Elledge SJ (2004) Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci U S A 101: 10078- 10083  Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Brocker EB, LeBoit PE, Pinkel D, Bastian BC (2005) Distinct sets of genetic alterations in melanoma. N Engl J Med 353: 2135-2147  Dahle J, Kvam E (2003) Induction of delayed mutations and chromosomal instability in fibroblasts after UVA-, UVB-, and X-radiation. Cancer Res 63: 1464-1469  Dai DL, Martinka M, Li G (2005) Prognostic significance of activated Akt expression in melanoma: a clinicopathologic study of 292 cases. J Clin Oncol 23: 1473-1482  Das C, Lucia MS, Hansen KC, Tyler JK (2009) CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459: 113-117  Dauda MM, Shehu SM (2005) Malignant melanoma: a review. Niger Postgrad Med J 12: 125-130   130 Davies AA, Huttner D, Daigaku Y, Chen S, Ulrich HD (2008) Activation of ubiquitin- dependent DNA damage bypass is mediated by replication protein a. Mol Cell 29: 625-636  Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA (2002) Mutations of the BRAF gene in human cancer. Nature 417: 949-954  Delacroix S, Wagner JM, Kobayashi M, Yamamoto K, Karnitz LM (2007) The Rad9-Hus1- Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev 21: 1472-1477  Despras E, Daboussi F, Hyrien O, Marheineke K, Kannouche PL (2010) ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells. Hum Mol Genet 19: 1690-1701  Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK, Zhou MM (1999) Structure and ligand of a histone acetyltransferase bromodomain. Nature 399: 491-496  Dimitrova DS, Gilbert DM (2000) Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat Cell Biol 2: 686-694  Doyon Y, Cayrou C, Ullah M, Landry AJ, Cote V, Selleck W, Lane WS, Tan S, Yang XJ, Cote J (2006) ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol Cell 21: 51-64  Doyon Y, Selleck W, Lane WS, Tan S, Cote J (2004) Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans. Mol Cell Biol 24: 1884-1896  Draviam VM, Xie S, Sorger PK (2004) Chromosome segregation and genomic stability. Curr Opin Genet Dev 14: 120-125  Errico A, Costanzo V, Hunt T (2007) Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts. Proc Natl Acad Sci U S A 104: 14929-14934  Esteller M (2007) Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 8: 286-298  Fackenthal JD, Olopade OI (2007) Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations. Nat Rev Cancer 7: 937-948   131 Falbo KB, Alabert C, Katou Y, Wu S, Han J, Wehr T, Xiao J, He X, Zhang Z, Shi Y, Shirahige K, Pasero P, Shen X (2009) Involvement of a chromatin remodeling complex in damage tolerance during DNA replication. Nat Struct Mol Biol 16: 1167-1172  Falbo KB, Shen X (2006) Chromatin remodeling in DNA replication. J Cell Biochem 97: 684-689  Fang JM, Arlt MF, Burgess AC, Dagenais SL, Beer DG, Glover TW (2001) Translocation breakpoints in FHIT and FRA3B in both homologs of chromosome 3 in an esophageal adenocarcinoma. Genes Chromosomes Cancer 30: 292-298  Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A (2005) Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434: 917-921  Fedier A, Schlamminger M, Schwarz VA, Haller U, Howell SB, Fink D (2003) Loss of atm sensitises p53-deficient cells to topoisomerase poisons and antimetabolites. Ann Oncol 14: 938-945  Feng X, Bonni S, Riabowol K (2006) HSP70 induction by ING proteins sensitizes cells to tumor necrosis factor alpha receptor-mediated apoptosis. Mol Cell Biol 26: 9244-9255  Feng X, Hara Y, Riabowol K (2002) Different HATS of the ING1 gene family. Trends Cell Biol 12: 532-538  Fitzpatrick TB, Szabo G (1959) The melanocyte: cytology and cytochemistry. J Invest Dermatol 32: 197-209  Flynn RL, Zou L (2011) ATR: a master conductor of cellular responses to DNA replication stress. Trends Biochem Sci 36: 133-140  Frampton J, Irmisch A, Green CM, Neiss A, Trickey M, Ulrich HD, Furuya K, Watts FZ, Carr AM, Lehmann AR (2006) Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol Biol Cell 17: 2976-2985  Frebourg T, Friend SH (1992) Cancer risks from germline p53 mutations. J Clin Invest 90: 1637-1641  Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M (2004) PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 30: 193-204  Friedberg EC (2005) Suffering in silence: the tolerance of DNA damage. Nat Rev Mol Cell Biol 6: 943-953   132 Friedberg EC, Feaver WJ, Gerlach VL (2000) The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance. Proc Natl Acad Sci U S A 97: 5681- 5683  Friedberg EC, Lehmann AR, Fuchs RP (2005) Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 18: 499-505  Friedberg EC, Wagner R, Radman M (2002) Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science 296: 1627-1630  Friedel AM, Pike BL, Gasser SM (2009) ATR/Mec1: coordinating fork stability and repair. Curr Opin Cell Biol 21: 237-244  Frohling S, Dohner H (2008) Chromosomal abnormalities in cancer. N Engl J Med 359: 722- 734  Garate M, Campos EI, Bush JA, Xiao H, Li G (2007) Phosphorylation of the tumor suppressor p33(ING1b) at Ser-126 influences its protein stability and proliferation of melanoma cells. Faseb J 21: 3705-3716  Garate M, Wong RP, Campos EI, Wang Y, Li G (2008) NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33(ING1b). EMBO Rep 9: 576-581  Garg P, Burgers PM (2005) Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1. Proc Natl Acad Sci U S A 102: 18361-18366  Garkavtsev I, Grigorian IA, Ossovskaya VS, Chernov MV, Chumakov PM, Gudkov AV (1998) The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control. Nature 391: 295-298  Garkavtsev I, Kazarov A, Gudkov A, Riabowol K (1996) Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation. Nat Genet 14: 415-420  Garkavtsev I, Riabowol K (1997) Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor. Mol Cell Biol 17: 2014-2019  Giacca M, Zentilin L, Norio P, Diviacco S, Dimitrova D, Contreas G, Biamonti G, Perini G, Weighardt F, Riva S, et al. (1994) Fine mapping of a replication origin of human DNA. Proc Natl Acad Sci U S A 91: 7119-7123  Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and deacetylation of non- histone proteins. Gene 363: 15-23   133 Gohler T, Sabbioneda S, Green CM, Lehmann AR (2011) ATR-mediated phosphorylation of DNA polymerase eta is needed for efficient recovery from UV damage. J Cell Biol 192: 219- 227  Gong W, Suzuki K, Russell M, Riabowol K (2005) Function of the ING family of PHD proteins in cancer. Int J Biochem Cell Biol 37: 1054-1065  Gonzalez L, Freije JM, Cal S, Lopez-Otin C, Serrano M, Palmero I (2006) A functional link between the tumour suppressors ARF and p33ING1. Oncogene 25: 5173-5179  Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere M, Ditullio RA, Jr., Kastrinakis NG, Levy B, Kletsas D, Yoneta A, Herlyn M, Kittas C, Halazonetis TD (2005) Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434: 907-913  Graham CH, Forsdike J, Fitzgerald CJ, Macdonald-Goodfellow S (1999) Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression. Int J Cancer 80: 617-623  Gray-Schopfer V, Wellbrock C, Marais R (2007) Melanoma biology and new targeted therapy. Nature 445: 851-857  Groth A, Corpet A, Cook AJ, Roche D, Bartek J, Lukas J, Almouzni G (2007a) Regulation of replication fork progression through histone supply and demand. Science 318: 1928-1931  Groth A, Rocha W, Verreault A, Almouzni G (2007b) Chromatin challenges during DNA replication and repair. Cell 128: 721-733  Guo C, Fischhaber PL, Luk-Paszyc MJ, Masuda Y, Zhou J, Kamiya K, Kisker C, Friedberg EC (2003) Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis. EMBO J 22: 6621-6630  Guo C, Kosarek-Stancel JN, Tang TS, Friedberg EC (2009) Y-family DNA polymerases in mammalian cells. Cell Mol Life Sci 66: 2363-2381  Guo C, Sonoda E, Tang TS, Parker JL, Bielen AB, Takeda S, Ulrich HD, Friedberg EC (2006) REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo. Mol Cell 23: 265-271  Guo Z, Kumagai A, Wang SX, Dunphy WG (2000) Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev 14: 2745-2756  Haass NK, Smalley KS, Herlyn M (2004) The role of altered cell-cell communication in melanoma progression. J Mol Histol 35: 309-318   134 Halazonetis TD, Gorgoulis VG, Bartek J (2008) An oncogene-induced DNA damage model for cancer development. Science 319: 1352-1355  Han J, Zhou H, Horazdovsky B, Zhang K, Xu RM, Zhang Z (2007) Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication. Science 315: 653-655  Han X, Feng X, Rattner JB, Smith H, Bose P, Suzuki K, Soliman MA, Scott MS, Burke BE, Riabowol K (2008) Tethering by lamin A stabilizes and targets the ING1 tumour suppressor. Nat Cell Biol 10: 1333-1340  Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57-70  Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646- 674  Hanawalt PC (2007) Paradigms for the three rs: DNA replication, recombination, and repair. Mol Cell 28: 702-707  Haracska L, Kondratick CM, Unk I, Prakash S, Prakash L (2001) Interaction with PCNA is essential for yeast DNA polymerase eta function. Mol Cell 8: 407-415  Hasan S, El-Andaloussi N, Hardeland U, Hassa PO, Burki C, Imhof R, Schar P, Hottiger MO (2002) Acetylation regulates the DNA end-trimming activity of DNA polymerase beta. Mol Cell 10: 1213-1222  Haushalter KA, Kadonaga JT (2003) Chromatin assembly by DNA-translocating motors. Nat Rev Mol Cell Biol 4: 613-620  He GH, Helbing CC, Wagner MJ, Sensen CW, Riabowol K (2005) Phylogenetic analysis of the ING family of PHD finger proteins. Mol Biol Evol 22: 104-116  Hibi K, Takahashi T, Yamakawa K, Ueda R, Sekido Y, Ariyoshi Y, Suyama M, Takagi H, Nakamura Y (1992) Three distinct regions involved in 3p deletion in human lung cancer. Oncogene 7: 445-449  Higgins NP, Kato K, Strauss B (1976) A model for replication repair in mammalian cells. J Mol Biol 101: 417-425  Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S (2002) RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135- 141  Hsu MY, Meier F, Herlyn M (2002) Melanoma development and progression: a conspiracy between tumor and host. Differentiation 70: 522-536   135 Huang TT, Nijman SM, Mirchandani KD, Galardy PJ, Cohn MA, Haas W, Gygi SP, Ploegh HL, Bernards R, D'Andrea AD (2006) Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat Cell Biol 8: 339-347  Hyrien O (2000) Mechanisms and consequences of replication fork arrest. Biochimie 82: 5- 17  Ito K, Kinjo K, Nakazato T, Ikeda Y, Kizaki M (2002) Expression and sequence analyses of p33(ING1) gene in myeloid leukemia. Am J Hematol 69: 141-143  Jager D, Stockert E, Scanlan MJ, Gure AO, Jager E, Knuth A, Old LJ, Chen YT (1999) Cancer-testis antigens and ING1 tumor suppressor gene product are breast cancer antigens: characterization of tissue-specific ING1 transcripts and a homologue gene. Cancer Res 59: 6197-6204  Jansen JG, Fousteri MI, de Wind N (2007) Send in the clamps: control of DNA translesion synthesis in eukaryotes. Mol Cell 28: 522-529  Jhappan C, Noonan FP, Merlino G (2003) Ultraviolet radiation and cutaneous malignant melanoma. Oncogene 22: 3099-3112  Jiang H, Reinhardt HC, Bartkova J, Tommiska J, Blomqvist C, Nevanlinna H, Bartek J, Yaffe MB, Hemann MT (2009) The combined status of ATM and p53 link tumor development with therapeutic response. Genes Dev 23: 1895-1909  Jin J, Shirogane T, Xu L, Nalepa G, Qin J, Elledge SJ, Harper JW (2003) SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. Genes Dev 17: 3062-3074  Johnson RE, Washington MT, Haracska L, Prakash S, Prakash L (2000) Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature 406: 1015-1019  Jones DR, Bultsma Y, Keune WJ, Halstead JR, Elouarrat D, Mohammed S, Heck AJ, D'Santos CS, Divecha N (2006) Nuclear PtdIns5P as a transducer of stress signaling: an in vivo role for PIP4Kbeta. Mol Cell 23: 685-695  Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128: 683-692  Kai M, Boddy MN, Russell P, Wang TS (2005) Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. Genes Dev 19: 919- 932  Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, 3rd, Johnson BE, Skolnick MH (1994) A cell cycle regulator potentially involved in genesis of many tumor types. Science 264: 436-440   136 Kameyama K, Huang CL, Liu D, Masuya D, Nakashima T, Sumitomo S, Takami Y, Kinoshita M, Yokomise H (2003) Reduced ING1b gene expression plays an important role in carcinogenesis of non-small cell lung cancer patients. Clin Cancer Res 9: 4926-4934  Kannouche P, Lehmann A (2006) Localization of Y-family polymerases and the DNA polymerase switch in mammalian cells. Methods Enzymol 408: 407-415  Kannouche PL, Wing J, Lehmann AR (2004) Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol Cell 14: 491-500  Kanzaki H, Ouchida M, Hanafusa H, Sakai A, Yamamoto H, Suzuki H, Yano M, Aoe M, Imai K, Date H, Nakachi K, Shimizu K (2007) Single nucleotide polymorphism in the RAD18 gene and risk of colorectal cancer in the Japanese population. Oncol Rep 18: 1171- 1175  Kanzaki H, Ouchida M, Hanafusa H, Yamamoto H, Suzuki H, Yano M, Aoe M, Imai K, Date H, Nakachi K, Shimizu K (2008) The association between RAD18 Arg302Gln polymorphism and the risk of human non-small-cell lung cancer. J Cancer Res Clin Oncol 134: 211-217  Karst AM, Dai DL, Cheng JQ, Li G (2006) Role of p53 up-regulated modulator of apoptosis and phosphorylated Akt in melanoma cell growth, apoptosis, and patient survival. Cancer Res 66: 9221-9226  Kerzendorfer C, O'Driscoll M (2009) Human DNA damage response and repair deficiency syndromes: Linking genomic instability and cell cycle checkpoint proficiency. DNA Repair (Amst) 8: 1139-1152  Kichina JV, Zeremski M, Aris L, Gurova KV, Walker E, Franks R, Nikitin AY, Kiyokawa H, Gudkov AV (2006) Targeted disruption of the mouse ing1 locus results in reduced body size, hypersensitivity to radiation and elevated incidence of lymphomas. Oncogene 25: 857- 866  Kim JK, Choi BS (1995) The solution structure of DNA duplex-decamer containing the (6-4) photoproduct of thymidylyl(3'-->5')thymidine by NMR and relaxation matrix refinement. Eur J Biochem 228: 849-854  Kim JK, Patel D, Choi BS (1995) Contrasting structural impacts induced by cis-syn cyclobutane dimer and (6-4) adduct in DNA duplex decamers: implication in mutagenesis and repair activity. Photochem Photobiol 62: 44-50  Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11: 725-732   137 Korkola J, Gray JW (2010) Breast cancer genomes--form and function. Curr Opin Genet Dev 20: 4-14  Kraemer KH, Lee MM, Scotto J (1987) Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol 123: 241-250  Krishnamurthy J, Kannan K, Feng J, Mohanprasad BK, Tsuchida N, Shanmugam G (2001) Mutational analysis of the candidate tumor suppressor gene ING1 in Indian oral squamous cell carcinoma. Oral Oncol 37: 222-224  Kumagai A, Dunphy WG (2000) Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol Cell 6: 839-849  Kunkel TA (2004) DNA replication fidelity. J Biol Chem 279: 16895-16898  Kuo WH, Wang Y, Wong RP, Campos EI, Li G (2007) The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA. Exp Cell Res 313: 1628-1638  Kusewitt DF, Applegate LA, Ley RD (1991) Ultraviolet radiation-induced skin tumors in a South American opossum (Monodelphis domestica). Vet Pathol 28: 55-65  Kuzmichev A, Zhang Y, Erdjument-Bromage H, Tempst P, Reinberg D (2002) Role of the Sin3-histone deacetylase complex in growth regulation by the candidate tumor suppressor p33(ING1). Mol Cell Biol 22: 835-848  Lambert S, Carr AM (2005) Checkpoint responses to replication fork barriers. Biochimie 87: 591-602  Lambert WC, Kuo HR, Lambert MW (1995) Xeroderma pigmentosum. Dermatol Clin 13: 169-209  Larrieu D, Pedeux R (2009) SharING out the roles in replicatING DNA. Cell Cycle 8: 3623- 3624  Larrieu D, Ythier D, Binet R, Brambilla C, Brambilla E, Sengupta S, Pedeux R (2009) ING2 controls the progression of DNA replication forks to maintain genome stability. EMBO Rep 10: 1168-1174  Lavin MF (1999) ATM: the product of the gene mutated in ataxia-telangiectasia. Int J Biochem Cell Biol 31: 735-740  Lavin MF, Shiloh Y (1997) The genetic defect in ataxia-telangiectasia. Annu Rev Immunol 15: 177-202   138 Lee J, Kumagai A, Dunphy WG (2007) The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. J Biol Chem 282: 28036-28044  Lehmann AR (2003) DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85: 1101-1111  Lehmann AR (2006) Translesion synthesis in mammalian cells. Exp Cell Res 312: 2673- 2676  Lengauer C, Kinzler KW, Vogelstein B (1997) Genetic instability in colorectal cancers. Nature 386: 623-627  Leung KM, Po LS, Tsang FC, Siu WY, Lau A, Ho HT, Poon RY (2002) The candidate tumor suppressor ING1b can stabilize p53 by disrupting the regulation of p53 by MDM2. Cancer Res 62: 4890-4893  Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. Cell 128: 707-719  Li G, Ho VC, Berean K, Tron VA (1995) Ultraviolet radiation induction of squamous cell carcinomas in p53 transgenic mice. Cancer Res 55: 2070-2074  Li G, Tang L, Zhou X, Tron V, Ho V (1998) Chemotherapy-induced apoptosis in melanoma cells is p53 dependent. Melanoma Res 8: 17-23  Li J, Li G (2010) Cell cycle regulator ING4 is a suppressor of melanoma angiogenesis that is regulated by the metastasis suppressor BRMS1. Cancer Res 70: 10445-10453  Li J, Martinka M, Li G (2008) Role of ING4 in human melanoma cell migration, invasion and patient survival. Carcinogenesis 29: 1373-1379  Li J, Wang Y, Wong RP, Li G (2009) The role of ING tumor suppressors in UV stress response and melanoma progression. Curr Drug Targets 10: 455-464  Limoli CL, Giedzinski E, Bonner WM, Cleaver JE (2002) UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization. Proc Natl Acad Sci U S A 99: 233-238  Lin JR, Zeman MK, Chen JY, Yee MC, Cimprich KA (2011) SHPRH and HLTF Act in a Damage-Specific Manner to Coordinate Different Forms of Postreplication Repair and Prevent Mutagenesis. Mol Cell 42: 237-249  Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362: 709- 715   139 Liu JS, Kuo SR, Melendy T (2006a) Phosphorylation of replication protein A by S-phase checkpoint kinases. DNA Repair (Amst) 5: 369-380  Liu P, Barkley LR, Day T, Bi X, Slater DM, Alexandrow MG, Nasheuer HP, Vaziri C (2006b) The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism. J Biol Chem 281: 30631-30644  Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14: 1448- 1459  Loewith R, Smith JS, Meijer M, Williams TJ, Bachman N, Boeke JD, Young D (2001) Pho23 is associated with the Rpd3 histone deacetylase and is required for its normal function in regulation of gene expression and silencing in Saccharomyces cerevisiae. J Biol Chem 276: 24068-24074  Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, Muzi-Falconi M, Newlon CS, Foiani M (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412: 557-561  Lopez-Girona A, Tanaka K, Chen XB, Baber BA, McGowan CH, Russell P (2001) Serine- 345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast. Proc Natl Acad Sci U S A 98: 11289-11294  Lucca C, Vanoli F, Cotta-Ramusino C, Pellicioli A, Liberi G, Haber J, Foiani M (2004) Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing. Oncogene 23: 1206-1213  Ludwig S, Klitzsch A, Baniahmad A (2011) The ING tumor suppressors in cellular senescence and chromatin. Cell Biosci 1: 25  Luo J, Solimini NL, Elledge SJ (2009) Principles of cancer therapy: oncogene and non- oncogene addiction. Cell 136: 823-837  Lupardus PJ, Byun T, Yee MC, Hekmat-Nejad M, Cimprich KA (2002) A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev 16: 2327-2332  Madhunapantula SV, Robertson GP (2009) The PTEN-AKT3 signaling cascade as a therapeutic target in melanoma. Pigment Cell Melanoma Res 22: 400-419  Majka J, Burgers PM (2004) The PCNA-RFC families of DNA clamps and clamp loaders. Prog Nucleic Acid Res Mol Biol 78: 227-260   140 Marrett LD, King WD, Walter SD, From L (1992) Use of host factors to identify people at high risk for cutaneous malignant melanoma. CMAJ 147: 445-453  Martin DG, Baetz K, Shi X, Walter KL, MacDonald VE, Wlodarski MJ, Gozani O, Hieter P, Howe L (2006) The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3. Mol Cell Biol 26: 7871-7879  Martin SA, Hewish M, Lord CJ, Ashworth A (2010) Genomic instability and the selection of treatments for cancer. J Pathol 220: 281-289  Masumoto H, Hawke D, Kobayashi R, Verreault A (2005) A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response. Nature 436: 294-298  Masutani C, Kusumoto R, Yamada A, Dohmae N, Yokoi M, Yuasa M, Araki M, Iwai S, Takio K, Hanaoka F (1999) The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399: 700-704  Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316: 1160-1166  Matsuura S, Tauchi H, Nakamura A, Kondo N, Sakamoto S, Endo S, Smeets D, Solder B, Belohradsky BH, Der Kaloustian VM, Oshimura M, Isomura M, Nakamura Y, Komatsu K (1998) Positional cloning of the gene for Nijmegen breakage syndrome. Nat Genet 19: 179- 181  Mattera L, Escaffit F, Pillaire MJ, Selves J, Tyteca S, Hoffmann JS, Gourraud PA, Chevillard-Briet M, Cazaux C, Trouche D (2009) The p400/Tip60 ratio is critical for colorectal cancer cell proliferation through DNA damage response pathways. Oncogene 28: 1506-1517  Melixetian M, Klein DK, Sorensen CS, Helin K (2009) NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint. Nat Cell Biol 11: 1247-1253  Melo JA, Cohen J, Toczyski DP (2001) Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev 15: 2809-2821  Menendez C, Abad M, Gomez-Cabello D, Moreno A, Palmero I (2009) ING proteins in cellular senescence. Curr Drug Targets 10: 406-417  Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der Horst CM, Majoor DM, Shay JW, Mooi WJ, Peeper DS (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436: 720-724   141 Middleton MR, Grob JJ, Aaronson N, Fierlbeck G, Tilgen W, Seiter S, Gore M, Aamdal S, Cebon J, Coates A, Dreno B, Henz M, Schadendorf D, Kapp A, Weiss J, Fraass U, Statkevich P, Muller M, Thatcher N (2000) Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J Clin Oncol 18: 158-166  Miller AJ, Mihm MC, Jr. (2006) Melanoma. N Engl J Med 355: 51-65  Mirkin SM (2007) Expandable DNA repeats and human disease. Nature 447: 932-940  Mitchell DL, Haipek CA, Clarkson JM (1985) (6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers. Mutat Res 143: 109-112  Miyase S, Tateishi S, Watanabe K, Tomita K, Suzuki K, Inoue H, Yamaizumi M (2005) Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination. J Biol Chem 280: 515-524  Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129: 665-679  Moraes TF, Edwards RA, McKenna S, Pastushok L, Xiao W, Glover JN, Ellison MJ (2001) Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nat Struct Biol 8: 669-673  Moriwaki S, Kraemer KH (2001) Xeroderma pigmentosum--bridging a gap between clinic and laboratory. Photodermatol Photoimmunol Photomed 17: 47-54  Moschos SJ, Edington HD, Land SR, Rao UN, Jukic D, Shipe-Spotloe J, Kirkwood JM (2006) Neoadjuvant treatment of regional stage IIIB melanoma with high-dose interferon alfa-2b induces objective tumor regression in association with modulation of tumor infiltrating host cellular immune responses. J Clin Oncol 24: 3164-3171  Motegi A, Liaw HJ, Lee KY, Roest HP, Maas A, Wu X, Moinova H, Markowitz SD, Ding H, Hoeijmakers JH, Myung K (2008) Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks. Proc Natl Acad Sci U S A 105: 12411-12416  Motegi A, Sood R, Moinova H, Markowitz SD, Liu PP, Myung K (2006) Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination. J Cell Biol 175: 703-708  Mu JJ, Wang Y, Luo H, Leng M, Zhang J, Yang T, Besusso D, Jung SY, Qin J (2007) A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints. J Biol Chem 282: 17330-17334  142  Myung K, Datta A, Kolodner RD (2001) Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell 104: 397- 408  Nagashima M, Shiseki M, Pedeux RM, Okamura S, Kitahama-Shiseki M, Miura K, Yokota J, Harris CC (2003) A novel PHD-finger motif protein, p47ING3, modulates p53-mediated transcription, cell cycle control, and apoptosis. Oncogene 22: 343-350  Nakamura T, Ishikawa S, Koga Y, Nagai Y, Imamura Y, Ikeda K, Mori T, Nomori H, Baba H (2009) Mutation analysis of Rad18 in human cancer cell lines and non small cell lung cancer tissues. J Exp Clin Cancer Res 28: 106  Negrini M, Monaco C, Vorechovsky I, Ohta M, Druck T, Baffa R, Huebner K, Croce CM (1996) The FHIT gene at 3p14.2 is abnormal in breast carcinomas. Cancer Res 56: 3173- 3179  Negrini S, Gorgoulis VG, Halazonetis TD (2010) Genomic instability--an evolving hallmark of cancer. Nat Rev Mol Cell Biol 11: 220-228  Nevanlinna H, Bartek J (2006) The CHEK2 gene and inherited breast cancer susceptibility. Oncogene 25: 5912-5919  Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM, Kunkel TA (2008) Division of labor at the eukaryotic replication fork. Mol Cell 30: 137-144  Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368: 753-756  Nouman GS, Anderson JJ, Mathers ME, Leonard N, Crosier S, Lunec J, Angus B (2002a) Nuclear to cytoplasmic compartment shift of the p33ING1b tumour suppressor protein is associated with malignancy in melanocytic lesions. Histopathology 40: 360-366  Nouman GS, Anderson JJ, Wood KM, Lunec J, Hall AG, Reid MM, Angus B (2002b) Loss of nuclear expression of the p33(ING1b) inhibitor of growth protein in childhood acute lymphoblastic leukaemia. J Clin Pathol 55: 596-601  Nouman GS, Angus B, Lunec J, Crosier S, Lodge A, Anderson JJ (2002c) Comparative assessment expression of the inhibitor of growth 1 gene (ING1) in normal and neoplastic tissues. Hybrid Hybridomics 21: 1-10  O'Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA (2003) A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet 33: 497-501   143 Oakley GG, Loberg LI, Yao J, Risinger MA, Yunker RL, Zernik-Kobak M, Khanna KK, Lavin MF, Carty MP, Dixon K (2001) UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein. Mol Biol Cell 12: 1199-1213  Ohgi T, Masaki T, Nakai S, Morishita A, Yukimasa S, Nagai M, Miyauchi Y, Funaki T, Kurokohchi K, Watanabe S, Kuriyama S (2002) Expression of p33(ING1) in hepatocellular carcinoma: relationships to tumour differentiation and cyclin E kinase activity. Scand J Gastroenterol 37: 1440-1448  Ohmori H, Friedberg EC, Fuchs RP, Goodman MF, Hanaoka F, Hinkle D, Kunkel TA, Lawrence CW, Livneh Z, Nohmi T, Prakash L, Prakash S, Todo T, Walker GC, Wang Z, Woodgate R (2001) The Y-family of DNA polymerases. Mol Cell 8: 7-8  Ohmori M, Nagai M, Tasaka T, Koeffler HP, Toyama T, Riabowol K, Takahara J (1999) Decreased expression of p33ING1 mRNA in lymphoid malignancies. Am J Hematol 62: 118- 119  Oki E, Maehara Y, Tokunaga E, Kakeji Y, Sugimachi K (1999) Reduced expression of p33(ING1) and the relationship with p53 expression in human gastric cancer. Cancer Lett 147: 157-162  Omholt K, Krockel D, Ringborg U, Hansson J (2006) Mutations of PIK3CA are rare in cutaneous melanoma. Melanoma Res 16: 197-200  Pages V, Santa Maria SR, Prakash L, Prakash S (2009) Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast. Genes Dev 23: 1438-1449  Palacios A, Garcia P, Padro D, Lopez-Hernandez E, Martin I, Blanco FJ (2006) Solution structure and NMR characterization of the binding to methylated histone tails of the plant homeodomain finger of the tumour suppressor ING4. FEBS Lett 580: 6903-6908  Papamichos-Chronakis M, Peterson CL (2008) The Ino80 chromatin-remodeling enzyme regulates replisome function and stability. Nat Struct Mol Biol 15: 338-345  Parker JL, Ulrich HD (2009) Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5. EMBO J 28: 3657-3666  Pascual J, Martinez-Yamout M, Dyson HJ, Wright PE (2000) Structure of the PHD zinc finger from human Williams-Beuren syndrome transcription factor. J Mol Biol 304: 723-729  Pedeux R, Sengupta S, Shen JC, Demidov ON, Saito S, Onogi H, Kumamoto K, Wincovitch S, Garfield SH, McMenamin M, Nagashima M, Grossman SR, Appella E, Harris CC (2005) ING2 regulates the onset of replicative senescence by induction of p300-dependent p53 acetylation. Mol Cell Biol 25: 6639-6648  144  Pena PV, Davrazou F, Shi X, Walter KL, Verkhusha VV, Gozani O, Zhao R, Kutateladze TG (2006) Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2. Nature 442: 100-103  Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H (1997) Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277: 1501-1505  Peris K, Chimenti S, Fargnoli MC, Valeri P, Kerl H, Wolf P (1999) UV fingerprint CDKN2a but no p14ARF mutations in sporadic melanomas. J Invest Dermatol 112: 825-826  Peterson CL, Cote J (2004) Cellular machineries for chromosomal DNA repair. Genes Dev 18: 602-616  Pichiorri F, Ishii H, Okumura H, Trapasso F, Wang Y, Huebner K (2008) Molecular parameters of genome instability: roles of fragile genes at common fragile sites. J Cell Biochem 104: 1525-1533  Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka J, Salem G, Pohida T, Heenan P, Duray P, Kallioniemi O, Hayward NK, Trent JM, Meltzer PS (2003) High frequency of BRAF mutations in nevi. Nat Genet 33: 19-20  Prakash S, Johnson RE, Prakash L (2005) Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu Rev Biochem 74: 317-353  Putnam CD, Hayes TK, Kolodner RD (2010) Post-replication repair suppresses duplication- mediated genome instability. PLoS Genet 6: e1000933  Ramaswamy S, Nakamura N, Vazquez F, Batt DB, Perera S, Roberts TM, Sellers WR (1999) Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 96: 2110-2115  Reinhardt HC, Jiang H, Hemann MT, Yaffe MB (2009) Exploiting synthetic lethal interactions for targeted cancer therapy. Cell Cycle 8: 3112-3119  Remmele W, Stegner HE (1987) [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 8: 138-140  Ries LA, Wingo PA, Miller DS, Howe HL, Weir HK, Rosenberg HM, Vernon SW, Cronin K, Edwards BK (2000) The annual report to the nation on the status of cancer, 1973-1997, with a special section on colorectal cancer. Cancer 88: 2398-2424   145 Robertson GP (2005) Functional and therapeutic significance of Akt deregulation in malignant melanoma. Cancer Metastasis Rev 24: 273-285  Runger TM, Kappes UP (2008) Mechanisms of mutation formation with long-wave ultraviolet light (UVA). Photodermatol Photoimmunol Photomed 24: 2-10  Russell MW, Soliman MA, Schriemer D, Riabowol K (2008) ING1 protein targeting to the nucleus by karyopherins is necessary for activation of p21. Biochem Biophys Res Commun 374: 490-495  Sabbioneda S, Bortolomai I, Giannattasio M, Plevani P, Muzi-Falconi M (2007) Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1. DNA Repair (Amst) 6: 121-127  Sabbioneda S, Gourdin AM, Green CM, Zotter A, Giglia-Mari G, Houtsmuller A, Vermeulen W, Lehmann AR (2008) Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases. Mol Biol Cell 19: 5193-5202  Sabbioneda S, Green CM, Bienko M, Kannouche P, Dikic I, Lehmann AR (2009) Ubiquitin- binding motif of human DNA polymerase eta is required for correct localization. Proc Natl Acad Sci U S A 106: E20; author reply E21  Sabbioneda S, Minesinger BK, Giannattasio M, Plevani P, Muzi-Falconi M, Jinks-Robertson S (2005) The 9-1-1 checkpoint clamp physically interacts with polzeta and is partially required for spontaneous polzeta-dependent mutagenesis in Saccharomyces cerevisiae. J Biol Chem 280: 38657-38665  Saito A, Furukawa T, Fukushige S, Koyama S, Hoshi M, Hayashi Y, Horii A (2000) p24/ING1-ALT1 and p47/ING1-ALT2, distinct alternative transcripts of p33/ING1. J Hum Genet 45: 177-181  Salk JJ, Fox EJ, Loeb LA (2010) Mutational heterogeneity in human cancers: origin and consequences. Annu Rev Pathol 5: 51-75  Sanchez-Carbayo M, Socci ND, Lozano JJ, Li W, Charytonowicz E, Belbin TJ, Prystowsky MB, Ortiz AR, Childs G, Cordon-Cardo C (2003) Gene discovery in bladder cancer progression using cDNA microarrays. Am J Pathol 163: 505-516  Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ (1997) Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277: 1497-1501  Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle P, Elder DE, Herlyn M (2003) Constitutive mitogen-activated protein kinase activation in melanoma is  146 mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res 63: 756-759  Sauter ER, Yeo UC, von Stemm A, Zhu W, Litwin S, Tichansky DS, Pistritto G, Nesbit M, Pinkel D, Herlyn M, Bastian BC (2002) Cyclin D1 is a candidate oncogene in cutaneous melanoma. Cancer Res 62: 3200-3206  Sayan BS, Sayan AE, Yang AL, Aqeilan RI, Candi E, Cohen GM, Knight RA, Croce CM, Melino G (2007) Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis. Proc Natl Acad Sci U S A 104: 10871-10876  Scott M, Boisvert FM, Vieyra D, Johnston RN, Bazett-Jones DP, Riabowol K (2001a) UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences. Nucleic Acids Res 29: 2052-2058  Scott M, Bonnefin P, Vieyra D, Boisvert FM, Young D, Bazett-Jones DP, Riabowol K (2001b) UV-induced binding of ING1 to PCNA regulates the induction of apoptosis. J Cell Sci 114: 3455-3462  Sedgwick B, Bates PA, Paik J, Jacobs SC, Lindahl T (2007) Repair of alkylated DNA: recent advances. DNA Repair (Amst) 6: 429-442  Serra-Pages C, Kedersha NL, Fazikas L, Medley Q, Debant A, Streuli M (1995) The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co- localize at focal adhesions. EMBO J 14: 2827-2838  Setlow RB, Woodhead AD, Grist E (1989) Animal model for ultraviolet radiation-induced melanoma: platyfish-swordtail hybrid. Proc Natl Acad Sci U S A 86: 8922-8926  Sharpless E, Chin L (2003) The INK4a/ARF locus and melanoma. Oncogene 22: 3092-3098  Shi X, Hong T, Walter KL, Ewalt M, Michishita E, Hung T, Carney D, Pena P, Lan F, Kaadige MR, Lacoste N, Cayrou C, Davrazou F, Saha A, Cairns BR, Ayer DE, Kutateladze TG, Shi Y, Cote J, Chua KF, Gozani O (2006) ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature 442: 96-99  Shiloh Y (2003) ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3: 155-168  Shimada H, Liu TL, Ochiai T, Shimizu T, Haupt Y, Hamada H, Abe T, Oka M, Takiguchi M, Hiwasa T (2002) Facilitation of adenoviral wild-type p53-induced apoptotic cell death by overexpression of p33(ING1) in T.Tn human esophageal carcinoma cells. Oncogene 21: 1208-1216   147 Shimada K, Oma Y, Schleker T, Kugou K, Ohta K, Harata M, Gasser SM (2008) Ino80 chromatin remodeling complex promotes recovery of stalled replication forks. Curr Biol 18: 566-575  Shimada Y, Saito A, Suzuki M, Takahashi E, Horie M (1998) Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor. Cytogenet Cell Genet 83: 232- 235  Shinoura N, Muramatsu Y, Nishimura M, Yoshida Y, Saito A, Yokoyama T, Furukawa T, Horii A, Hashimoto M, Asai A, Kirino T, Hamada H (1999) Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas. Cancer Res 59: 5521-5528  Shiseki M, Nagashima M, Pedeux RM, Kitahama-Shiseki M, Miura K, Okamura S, Onogi H, Higashimoto Y, Appella E, Yokota J, Harris CC (2003) p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity. Cancer Res 63: 2373-2378  Shridhar R, Shridhar V, Rivard S, Siegfried JM, Pietraszkiewicz H, Ensley J, Pauley R, Grignon D, Sakr W, Miller OJ, Smith DI (1996) Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck. Cancer Res 56: 5576-5578  Shridhar V, Wang L, Rosati R, Paradee W, Shridhar R, Mullins C, Sakr W, Grignon D, Miller OJ, Sun QC, Petros J, Smith DI (1997) Frequent breakpoints in the region surrounding FRA3B in sporadic renal cell carcinomas. Oncogene 14: 1269-1277  Skowyra D, Zeremski M, Neznanov N, Li M, Choi Y, Uesugi M, Hauser CA, Gu W, Gudkov AV, Qin J (2001) Differential association of products of alternative transcripts of the candidate tumor suppressor ING1 with the mSin3/HDAC1 transcriptional corepressor complex. J Biol Chem 276: 8734-8739  Slominski A, Tobin DJ, Shibahara S, Wortsman J (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84: 1155-1228  Smalley KS, Herlyn M (2005) Targeting intracellular signaling pathways as a novel strategy in melanoma therapeutics. Ann N Y Acad Sci 1059: 16-25  Smerdon MJ (1991) DNA repair and the role of chromatin structure. Curr Opin Cell Biol 3: 422-428  Sogo JM, Lopes M, Foiani M (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297: 599-602  Soliman MA, Riabowol K (2007) After a decade of study-ING, a PHD for a versatile family of proteins. Trends Biochem Sci 32: 509-519   148 Sotillo R, Garcia JF, Ortega S, Martin J, Dubus P, Barbacid M, Malumbres M (2001) Invasive melanoma in Cdk4-targeted mice. Proc Natl Acad Sci U S A 98: 13312-13317  Stahl JM, Cheung M, Sharma A, Trivedi NR, Shanmugam S, Robertson GP (2003) Loss of PTEN promotes tumor development in malignant melanoma. Cancer Res 63: 2881-2890  Stahl JM, Sharma A, Cheung M, Zimmerman M, Cheng JQ, Bosenberg MW, Kester M, Sandirasegarane L, Robertson GP (2004) Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res 64: 7002-7010  Stary A, Kannouche P, Lehmann AR, Sarasin A (2003) Role of DNA polymerase eta in the UV mutation spectrum in human cells. J Biol Chem 278: 18767-18775  Stelter P, Ulrich HD (2003) Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425: 188-191  Stokes MP, Rush J, Macneill J, Ren JM, Sprott K, Nardone J, Yang V, Beausoleil SA, Gygi SP, Livingstone M, Zhang H, Polakiewicz RD, Comb MJ (2007) Profiling of UV-induced ATM/ATR signaling pathways. Proc Natl Acad Sci U S A 104: 19855-19860  Stratton MR (2011) Exploring the genomes of cancer cells: progress and promise. Science 331: 1553-1558  Sutherland GR (1977) Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 197: 265-266  Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25: 3553-3562  Takahashi M, Seki N, Ozaki T, Kato M, Kuno T, Nakagawa T, Watanabe K, Miyazaki K, Ohira M, Hayashi S, Hosoda M, Tokita H, Mizuguchi H, Hayakawa T, Todo S, Nakagawara A (2002) Identification of the p33(ING1)-regulated genes that include cyclin B1 and proto- oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NMuMG. Cancer Res 62: 2203-2209  Tallen UG, Truss M, Kunitz F, Wellmann S, Unryn B, Sinn B, Lass U, Krabbe S, Holtkamp N, Hagemeier C, Wurm R, Henze G, Riabowol KT, von Deimling A (2008) Down-regulation of the inhibitor of growth 1 (ING1) tumor suppressor sensitizes p53-deficient glioblastoma cells to cisplatin-induced cell death. J Neurooncol 86: 23-30  Tateishi S, Niwa H, Miyazaki J, Fujimoto S, Inoue H, Yamaizumi M (2003) Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells. Mol Cell Biol 23: 474-481   149 Tateishi S, Sakuraba Y, Masuyama S, Inoue H, Yamaizumi M (2000) Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc Natl Acad Sci U S A 97: 7927-7932  Taylor AM, Groom A, Byrd PJ (2004) Ataxia-telangiectasia-like disorder (ATLD)-its clinical presentation and molecular basis. DNA Repair (Amst) 3: 1219-1225  Terai K, Abbas T, Jazaeri AA, Dutta A (2010) CRL4(Cdt2) E3 ubiquitin ligase monoubiquitinates PCNA to promote translesion DNA synthesis. Mol Cell 37: 143-149  Tercero JA, Diffley JF (2001) Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412: 553-557  Thomas NE, Berwick M, Cordeiro-Stone M (2006) Could BRAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet radiation? J Invest Dermatol 126: 1693-1696  Thompson JF, Scolyer RA, Kefford RF (2005) Cutaneous melanoma. Lancet 365: 687-701  Tissier A, Frank EG, McDonald JP, Iwai S, Hanaoka F, Woodgate R (2000) Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota. EMBO J 19: 5259-5266  Tokunaga E, Maehara Y, Oki E, Kitamura K, Kakeji Y, Ohno S, Sugimachi K (2000) Diminished expression of ING1 mRNA and the correlation with p53 expression in breast cancers. Cancer Lett 152: 15-22  Toyama T, Iwase H, Watson P, Muzik H, Saettler E, Magliocco A, DiFrancesco L, Forsyth P, Garkavtsev I, Kobayashi S, Riabowol K (1999) Suppression of ING1 expression in sporadic breast cancer. Oncogene 18: 5187-5193  Tran MA, Gowda R, Sharma A, Park EJ, Adair J, Kester M, Smith NB, Robertson GP (2008) Targeting V600EB-Raf and Akt3 using nanoliposomal-small interfering RNA inhibits cutaneous melanocytic lesion development. Cancer Res 68: 7638-7649  Trenz K, Errico A, Costanzo V (2008) Plx1 is required for chromosomal DNA replication under stressful conditions. EMBO J 27: 876-885  Trenz K, Smith E, Smith S, Costanzo V (2006) ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks. EMBO J 25: 1764-1774  Tsang FC, Po LS, Leung KM, Lau A, Siu WY, Poon RY (2003) ING1b decreases cell proliferation through p53-dependent and -independent mechanisms. FEBS Lett 553: 277-285  Unk I, Hajdu I, Fatyol K, Szakal B, Blastyak A, Bermudez V, Hurwitz J, Prakash L, Prakash S, Haracska L (2006) Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent  150 polyubiquitylation of proliferating cell nuclear antigen. Proc Natl Acad Sci U S A 103: 18107-18112  Unoki M, Shen JC, Zheng ZM, Harris CC (2006) Novel splice variants of ING4 and their possible roles in the regulation of cell growth and motility. J Biol Chem 281: 34677-34686  Unsal-Kacmaz K, Chastain PD, Qu PP, Minoo P, Cordeiro-Stone M, Sancar A, Kaufmann WK (2007) The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement. Mol Cell Biol 27: 3131-3142  van Attikum H, Gasser SM (2005) The histone code at DNA breaks: a guide to repair? Nat Rev Mol Cell Biol 6: 757-765  van Bueren KL, Bennetts JS, Fowles LF, Berkman JL, Simpson F, Wicking C (2007) Murine embryonic expression of the gene for the UV-responsive protein p15(PAF). Gene Expr Patterns 7: 47-50  van Gent DC, Hoeijmakers JH, Kanaar R (2001) Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2: 196-206  Vieyra D, Loewith R, Scott M, Bonnefin P, Boisvert FM, Cheema P, Pastyryeva S, Meijer M, Johnston RN, Bazett-Jones DP, McMahon S, Cole MD, Young D, Riabowol K (2002) Human ING1 proteins differentially regulate histone acetylation. J Biol Chem 277: 29832- 29839  Vieyra D, Senger DL, Toyama T, Muzik H, Brasher PM, Johnston RN, Riabowol K, Forsyth PA (2003) Altered subcellular localization and low frequency of mutations of ING1 in human brain tumors. Clin Cancer Res 9: 5952-5961  Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 132: 363-374  Walworth NC, Bernards R (1996) rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science 271: 353-356  Wang H, Guan J, Perrault AR, Wang Y, Iliakis G (2001) Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia- mutated and DNA-dependent protein kinase. Cancer Res 61: 8554-8563  Wang J, Chin MY, Li G (2006a) The novel tumor suppressor p33ING2 enhances nucleotide excision repair via inducement of histone H4 acetylation and chromatin relaxation. Cancer Res 66: 1906-1911   151 Wang Y, Tan XH, DiGiovanna JJ, Lee CC, Stern JB, Raffeld M, Jaffe ES, Kraemer KH (2010) Genetic diversity in melanoma metastases from a patient with xeroderma pigmentosum. J Invest Dermatol 130: 1188-1191  Wang Y, Wang J, Li G (2006b) Leucine zipper-like domain is required for tumor suppressor ING2-mediated nucleotide excision repair and apoptosis. FEBS Lett 580: 3787-3793  Wang Y, Woodgate R, McManus TP, Mead S, McCormick JJ, Maher VM (2007) Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. Cancer Res 67: 3018-3026  Warters RL, Adamson PJ, Pond CD, Leachman SA (2005) Melanoma cells express elevated levels of phosphorylated histone H2AX foci. J Invest Dermatol 124: 807-817  Wasco MJ, Pu RT, Yu L, Su L, Ma L (2008) Expression of gamma-H2AX in melanocytic lesions. Hum Pathol 39: 1614-1620  Washington MT, Minko IG, Johnson RE, Haracska L, Harris TM, Lloyd RS, Prakash S, Prakash L (2004a) Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta. Mol Cell Biol 24: 6900-6906  Washington MT, Minko IG, Johnson RE, Wolfle WT, Harris TM, Lloyd RS, Prakash S, Prakash L (2004b) Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa. Mol Cell Biol 24: 5687- 5693  Watanabe K, Tateishi S, Kawasuji M, Tsurimoto T, Inoue H, Yamaizumi M (2004) Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination. Embo J 23: 3886-3896  Wolchok JD, Saenger YM (2007) Current topics in melanoma. Curr Opin Oncol 19: 116-120  Wong RP, Lin H, Khosravi S, Piche B, Jafarnejad SM, Chen DW, Li G (2011) Tumour suppressor ING1b maintains genomic stability upon replication stress. Nucleic Acids Res  Woodgett JR (2005) Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol 17: 150-157  Wu H, Goel V, Haluska FG (2003) PTEN signaling pathways in melanoma. Oncogene 22: 3113-3122  Yang XH, Shiotani B, Classon M, Zou L (2008) Chk1 and Claspin potentiate PCNA ubiquitination. Genes Dev 22: 1147-1152   152 Yoo HY, Shevchenko A, Dunphy WG (2004) Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses. J Biol Chem 279: 53353- 53364  Yu SL, Johnson RE, Prakash S, Prakash L (2001) Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts. Mol Cell Biol 21: 185-188  Yunis JJ, Soreng AL (1984) Constitutive fragile sites and cancer. Science 226: 1199-1204  Zachos G, Rainey MD, Gillespie DA (2005) Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. Mol Cell Biol 25: 563-574  Zhang JT, Wang DW, Li QX, Zhu ZL, Wang MW, Cui DS, Yang YH, Gu YX, Sun XF (2008) Nuclear to cytoplasmic shift of p33(ING1b) protein from normal oral mucosa to oral squamous cell carcinoma in relation to clinicopathological variables. J Cancer Res Clin Oncol 134: 421-426  Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542-1548  Zuo L, Weger J, Yang Q, Goldstein AM, Tucker MA, Walker GJ, Hayward N, Dracopoli NC (1996) Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nat Genet 12: 97-99   

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0072283/manifest

Comment

Related Items