ROLE OF POLY (ADP-RIBOSE) POLYMERASE AND COPPER HOMEOSTASIS FACTOR, ANTIOXIDANT PROTEIN IN THE MAINTENANCE OF GENOMIC INTEGRITY. LAKSHMIDEVI BALAKRISHNAN B.SC. (HONS.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements “For your thoughtfulness and generosity, from you I have learned much of life’s philosophy. Thank you sincerely.” - Author Unknown I would like to express my most sincere gratitude to my supervisor, Associate Professor M. Prakash Hande, whose attitude to continual learning and many other qualities worthy of emulating greatly motivated my decision to pursue a doctorate. Thank you for your patience, mentorship and support over the past years and hope to have your continual guidance in the years to come. My heartfelt thanks also go to my fellow lab mates, past and present who made the difficult times tolerable and the joyous times more memorable. Special thanks to Dr. Swaminathan Sethu for his critical review of the thesis, Dr. Grace Low for her efforts with PCR, Mr. Shriram Venkatesan and Ms. Kalpana Gopalakrishnan for their help in experiments. Thanks also due to friends from the ROS and Tumour laboratory, Cancer and Metastasis laboratory, Cytokine Biology laboratory, Molecular and Cellular Immunology laboratory, Dr. Taneja’s laboratory and Dr. Martin Lee’s laboratory for the many occasions they have enabled my research with equipment, reagents, scientific suggestions and words of support. I would also like to thank Mr J. Manikandan for all his invaluable help with microarray analysis. Heartfelt thanks to Mr. Ganesan Arasapam for his efforts in PCRarray and Ms. Cynthia and Mr. Ghee Chong from the National Cancer Centre who accommodated my multiple requests for radiation time slots. Sincere thank you to Ms. Lee Shu Ying, Mr. Zhang Jie, Mr. Toh Kok Tee and Ms. Saw Marlar from the NUMI confocal microscopy and flow cytometry units for their many useful suggestions that greatly assisted my experiments. Thank you Prof Zhao-Qi Wang and Prof Jonathan Gitlin for kindly providing the cell lines required for my study. Special thanks are in also in order to Ms.Yasaswini Sampath Kumar, Mr. Dulesh Peris, Dr. Peter Pushparaj, Dr. Jude Aarthi, Dr. Pratiba Kurupati and Mr. Gireedhar Venkatachalam for their invaluable help and support in my project. For their ready support and encouragement for my graduate studies, my warmest thank you to Dr. Martin Lee and Dr. Deng Yuru. My sincere appreciation also goes out to Dr. Srividya Swaminathan and Dr. Deng Lih Wen for taking time out to review my progress as part of the TAC committee. For clearing the many administrative hurdles, thank you to Ms. Asha Das, Ms. Jeanie Ong, Ms. Kamsitah, Ms. Vasantha Nathan, Ms. Kumari and Ms. Eileen Kuan. I cannot thank enough my friend, Dr. Anuradha Poonepalli, who was there for me in so many ways throughout my doctorate. I am also deeply thankful for the unconditional love, support and understanding from my parents, sisters, in laws, friends and my better half, Dr. Vinoth Kumar without whom my PhD would not have been possible. I thank the examiners for taking time to evaluate my thesis. Last but not least, I thank the National University of Singapore, Yong Loo Lin School of Medicine and the Department of Physiology for the opportunity to pursue my doctorate. i Table of Contents Acknowledgements .i Table of Contents .ii Summary vi List of Tables .viii List of Figures .ix List of publications xv List of conference presentations .xvii Chapter 1: Introduction 1.1 1.2 1.3 1.4 Review of Literature 1.1.1 Genomic Instability 1.1.1.1 Telomere mediated genomic instability .2 1.1.1.1.1 Telomeres .2 1.1.1.1.2 Telomere dysfunction and tumourigenesis 1.1.1.1.3 DNA repair proteins in telomere maintenance .8 1.1.2 Inducers of genomic instability 10 1.1.2.1 Oxidative stress 10 1.1.2.2 Arsenic-induced oxidative stress .14 1.1.2.3 Radiation 18 1.1.3 Mechanisms for preventing genomic instability 20 1.1.3.1 Poly (ADP-ribose) polymerase (PARP-1) 21 1.1.3.1.1 Role of PARP-1 at the telomeres .24 1.1.4 Copper metabolism and disease .27 1.1.4.1 PARP-1 and Copper metabolism .29 1.1.5 Copper chaperone, Antioxidant protein (ATOX1) .31 Rationale and thesis objectives 33 Significance of the study 36 Aims .37 ii Chapter 2: Methods and Materials 38 2.1 Cell lines used in this study .38 2.1.1 Mouse embryonic fibroblasts .38 2.1.2 Human cell lines 38 2.2 Chemicals utilised 39 2.2.1 Sodium arsenite 39 2.2.2 Hydrogen peroxide .39 2.2.3 Gamma radiation 40 2.2.4 Copper pre-treatment .40 2.2.5 Bathocuproine sulphonate pre-treatment .40 2.3 Cytotoxicity Assays .40 2.3.1 Crystal violet assay 40 2.3.2 3-(4, 5-Dimethylthiazol-2-yl)-2, Diphenyltetrazolium assay (MTT assay) .41 2.4 DNA damage analysis 42 2.4.1 Alkaline Single Cell Gel Electrophoresis Assay (Comet assay) .42 2.4.2 γH2AX foci quantitation 43 2.5 Genotoxicity Assays 44 2.5.1 Chromosomal aberration analysis 44 2.5.1.1 Metaphase preparation .44 2.5.1.2 Peptide Nucleic Acid Fluorescence In Situ Hybridisation (PNA-FISH) .44 2.5.2 Cytokinesis Blocked Micronucleus Assay (CBMN assay) 45 2.6 Gene expression analysis .46 2.6.1 Microarray analysis 46 2.6.2 Real Time Reverse Transcriptase Polymerase Chain Reaction Array 46 2.6.2.1 RNA extraction 47 2.6.2.2 cDNA synthesis .47 2.6.2.3 Real time PCR 47 2.6.2.4 Analysis of PCR data .48 2.7 Superoxide measurement .49 2.8 Cell cycle analysis 49 2.9 Telomere length analysis by Flow-FISH .50 iii 2.10 Bioinformatics analysis 51 2.10.1 Functional gene annotation analysis 51 2.10.2 Identification of ATOX1 consensus sequences and binding motifs in promoter sequences of potential target genes .52 2.11 Real time Reverse Transcriptase Polymerase Chain Reaction for ATOX1 and PARP-1 .53 Statistical analysis 54 2.13 Chapter 3: Results 55 3.1 Role of PARP-1 in regulating telomere-mediated genomic stability following arsenite-induced oxidative stress .55 3.1.1 Cells lacking PARP-1 displayed elevated DNA damage 55 3.1.2 Absence of PARP-1 enhances chromosomal instability 58 3.1.3 Arsenite-induced telomere attrition was greater in PARP-1-/- mouse embryonic fibroblasts .63 3.1.4 PARP-1-/- MEFs are more sensitive to arsenite-induced cell death .65 3.1.5 Differential gene expression patterns in PARP-1+/+ and PARP-1-/cells after arsenite treatment 68 3.1.6 DNA damage and oxidative stress pathway specific analysis of gene expression profiles in PARP-1 deficient MEFs under conditions of arsenite-induced oxidative stress .71 3.1.7 Copper containing genes were differentially expressed in PARP-1 deficient MEFs .80 3.2 Role of copper in DNA damage response 84 3.2.1 Copper supplementation reduced levels of double strand breaks following genotoxic damage in normal MEFs .84 3.2.2 Copper metabolism diseases display increased susceptibility to DNA double strand breaks .88 3.2.3 Copper supplementation and chelation affected susceptibility of cells with copper metabolism defects to DNA damage .91 3.2.4 Menkes disease lymphoblastoid cells displayed increased genomic instability 104 3.3 Role of copper chaperone, antioxidant protein (ATOX1) in DNA damage response 106 3.3.1 ATOX1 levels are reduced in PARP-1 deficient MEFs 106 3.3.2 ATOX1 deficient MEFs display increased sensitivity to arseniteinduced DNA damage 108 iv 3.3.3 ATOX1 deficient MEFs displayed increased MN formation upon As3+ and radiation exposure .111 3.3.4 ATOX1 deficient MEFs display increased levels of chromosomal aberrations following As3+ treatment and radiation exposure .116 3.3.5 ATOX1 deficient MEFs sustained increased levels of double strand breaks as evidenced by increased γH2AX foci formation in response to DNA damaging agents 121 3.3.6 ATOX1 deficiency is associated with increased superoxide formation 122 3.3.7 ATOX1 deficiency causes differences in survival upon DNA damage in MEFs 128 3.3.8 Absence of ATOX1 causes changes in gene expression for genes in the DNA damage and oxidative stress pathways .131 3.3.8.1 Genes in the antioxidant defense pathway were differentially expressed between ATOX1 proficient and deficient cells and following radiation exposure 131 3.3.8.2 Genes involved in DSB repair were significantly upregulated upon DNA damage in ATOX1 deficient cells 132 3.3.9 ATOX1 consensus sequences present in some genes involved in DNA damage response and antioxidant defense .133 Chapter 4: Discussion 137 4.1 PARP-1 is an important factor in the maintenance of chromosome-genome stability in response to arsenite-induced damage .137 4.2 Copper homeostasis may affect the response to DNA damaging agents .141 4.3 ATOX1 is important for the maintenance of chromosomal stability in the presence of DNA damaging agents 145 Chapter 5: Conclusions and future directions .153 Chapter 6: Bibliography .156 v Summary Telomeres are the terminal nucleoprotein structures of chromosomes, protecting chromosomal ends from nuclease attack and recombination. Dysfunctional telomeres trigger genomic instability that underlies tumourigenesis. Poly (ADP-Ribose) Polymerase (PARP-1), an important player in the base excision repair pathway, is a regulator of telomere length and telomeric end-capping function. In this study, we wanted to investigate the role of PARP-1 at the telomeres under conditions of DNA damage. Sodium arsenite, the DNA damaging agent used in this study, is a potent environmental toxicant and a known inducer of oxidative damage. We identified that PARP-1 is a critical factor required for mouse cells to withstand arsenite-induced chromosomal aberrations and cell death. PARP-1 was also observed to have an essential function in defence against telomere attrition and resultant genomic instability. Interestingly, our microarray analysis revealed differential expression of copper metabolism and copper binding proteins following arsenite-induced DNA damage. Additionally, a link between copper metabolism and PARP-1 has been recently demonstrated where, copper was able to inhibit PARP-1 activity. Copper is a key component of enzymatic anti-oxidative defence systems yet under conditions of copper excess, it can be a key inducer of ROS. Defects in copper homeostasis are implicated in pathophysiologies such as cancer. Gene set enrichment analysis indicated that genes involved in copper metabolism were significantly differentially expressed in the absence of PARP-1 and following arsenite treatment. We thus investigated if copper metabolism may directly have a role in DNA damage response in mammalian cells. Copper supplementation reduced the levels of double strand breaks induced by genotoxicants in normal MEFs. Yet, in copper metabolism disease conditions such as Menkes and Wilson’s vi diseases, patient lymphoblastoid cells displayed increased levels of DSBs and genomic instability. These findings reiterate the importance of tight regulation of copper levels in the cellular milieu for proper biological function. We then further explored if specific factors in the copper metabolism pathway may affect the susceptibility to DNA damage. Antioxidant protein (ATOX1), a copper chaperone, was down regulated in PARP-1 deficient MEFs. Furthermore, ATOX1 was recently established to be a copper-dependent transcription factor. While the antioxidant effects of ATOX1 have been demonstrated, its role in DNA damage response or the maintenance of genomic stability has not been clearly elucidated. We identified that Atox1 mRNA levels rose in response to hydrogen peroxide and arsenite exposure. Hence, we investigated the effect of ATOX1 deficiency in MEFs under conditions of genotoxicant-induced DNA damage. Increased DNA damage was observed in Atox1 deficient MEFs when challenged with sodium arsenite and radiation. The absence of ATOX1 was also responsible for increased levels of ROS as well as DSB sustained by the cells. In addition, genes in the DNA damage signalling, oxidative stress and anti-oxidant defence pathways were differentially expressed in the absence of ATOX1. Given that oxidative processes are major sources of DNA damage, we propose that the antioxidant properties of ATOX1 may protect genomic integrity. Although the nature of PARP-1 and ATOX1 interaction has not yet been elucidated, this study proposes a new paradigm for how copper metabolism impacts cellular oxidation state and genome stability. vii List of Tables • Table 1: Effect of PARP-1 deficiency on telomere maintenance and chromosomegenomic instability. • Table 2: Chromosomal aberrations observed in PARP-1+/+ and PARP-1-/- MEFs following arsenite treatment. • Table 3: Differentially expressed genes in the oxidative stress and antioxidant defense pathway from PARP-1+/+ and PARP-1-/- MEFs after arsenite treatment by microarray. • Table 4: Differentially expressed genes in the DNA damage signalling pathway from PARP-1+/+ and PARP-1-/- MEFs after arsenite treatment by microarray. • Table 5: Expression of genes in the copper metabolism pathway from PARP-1+/+ and PARP-1-/- MEFs by microarray. • Table 6: Chromosomal aberrations observed in ATOX1+/+ and ATOX1-/- MEFs following sodium arsenite treatment by PNA-FISH. • Table 7: Chromosomal aberrations observed in ATOX1+/+ and ATOX-/- MEFs following radiation by PNA-FISH. • Table 8: Differentially expressed genes in the DNA damage signalling pathway from ATOX1+/+ and ATOX1-/- MEFs after arsenite and radiation treatment by PCRarray. • Table 9: Differentially expressed genes in the oxidative stress and antioxidant defense pathway from ATOX1+/+ and ATOX1-/- MEFs after arsenite and radiation treatment by PCRarray. • Table 10: Bioinformatics search of Atox1 consensus sequences and response elements in the promoter of genes involved in DNA damage response and antioxidant defense. viii List of Figures • Figure 1: Telomere structure. • Figure 2: The telomeric end replication problem. • Figure 3: Breakage-fusion-bridge cycles. • Figure 4: Model of telomere-mediated genomic instability. • Figure 5: ROS levels determine cellular outcomes. • Figure 6: Hypothesis for induction of oxidative DNA adducts and protein cross-links by arsenic. • Figure 7: Induction of DNA damage by radiation. • Figure 8: Intracellular uptake and transport of copper. • Figure 9: SYBR Green–stained comets in PARP-1+/+ and PARP-1-/- MEFs following arsenite treatment by comet assay. • Figure 10: DNA damage as measured by the comet assay in PARP-1+/+ and PARP-1-/MEFs following arsenite exposure for: • - (A) 30 minutes - (B) 24 hours Figure 11: Binucleated cells from PARP-1+/+ and PARP-1-/- MEFs following arsenite treatment by cytokinesis-blocked micronucleus assay. • Figure 12: Micronuclei induction measured by the cytokinesis-blocked micronucleus assay in PARP-1+/+ MEFs and PARP-1-/- MEFs following arsenite treatment for: - (A) 24 hours - (B) 48 hours ix Milligan, S.A., Owens, M.W., and Grisham, M.B. 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Prakash Hande Genome Stability Laboratory, Department of Physiology; 2Oncology Research Institute; 3Molecular and Cellular Immunology Laboratory, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Host Defense, Research Institute for Microbial Diseases, Osaka University; 5Exploratory Research for Advanced Technology, Japan Science and Technology Corp., Osaka, Japan; and 6Center for Radiological Research, Columbia University, New York, New York Abstract 3+ Arsenite (As ) has long been known to induce cancer and other degenerative diseases. Arsenite exerts its toxicity in part by generating reactive oxygen species. Identification of genetic factors that contribute to arsenic mutagenicity and carcinogenicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymerase (PARP) is critical for genomic DNA stability, role of PARP-1 was evaluated in arsenic-induced cytotoxic and genotoxic effects. Our study revealed that telomere attrition, probably owing to arsenite-induced oxidative stress, was much more pronounced in PARP-1À/À mouse embryonic fibroblasts (MEF; 40%) compared with PARP-1+/+ MEFs (10-20%). Correlation observed between telomere reduction and apoptotic death in PARP-1 null cells strongly indicates that the telomere attrition might be a trigger for enhanced apoptotic death after arsenite treatment. Elevated DNA damage detected by alkaline comet assay points to an impaired repair ability of arsenite-induced DNA lesions in PARP-1À/À MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1À/À MEFs. Microarray analysis has revealed that arsenite treatment altered the expression of about 311 genes majority of which have known functions in cellular responses to stress/external stimulus and cell growth and/or maintenance. Our results suggest an important role for PARP-1 gene product in the maintenance of chromosome-genome stability in response to arsenite-induced DNA damage. (Cancer Res 2005; 65(23): 10977-83) Introduction Poly(ADP-ribose) polymerase-1 (PARP-1), the best-characterized member of the PARP family is an abundant nuclear zinc finger protein found in most eukaryotes. PARP-1 primarily functions as a DNA damage sensor (1, 2) by recognizing and binding with high affinity to both ssDNA and dsDNA breaks that arise directly or indirectly as byproducts of ongoing DNA repair process (1, 2). Further PARP-1 also facilitates the access of other DNA repair factors Note: A. Poonepalli and L. Balakrishnan contributed equally to this work. Requests for reprints: M. Prakash Hande, Genome Stability Laboratory, Department of Physiology, Faculty of Medicine, National University of Singapore, Block MD9, Medical Drive, Singapore 117597, Singapore. Phone: 65-6874-3664; Fax: 65-6778-8161; E-mail: phsmph@nus.edu.sg. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2336 www.aacrjournals.org to the sites of DNA damage (3, 4). PARP-1 is also known for its ability to modulate the cellular responses either to survive or to undergo apoptotic death, depending on the extent of DNA damage (5). Arsenite is a significant environmental concern worldwide especially in some parts of the United States as well as in Argentina, Canada, India, Japan, Thailand, Taiwan, and Bangladesh. Chronic exposure to inorganic arsenite is associated with hepatic injury, peripheral neuropathy, and a wide variety of cancers (6). Many different modes of arsenite-induced genotoxicity have been identified, including oxidative stress, altered DNA repair and methylation mechanisms, altered cell proliferation, and abnormal gene amplification (6). Recently, very low concentrations of arsenite have been shown to inhibit poly(ADP) ribosylation of proteins in mammalian cells (7). Our previous study showed that the PARP-1-deficient mice had drastically shortened telomeres with high chromosomal instability (8). In addition, PARP-1 deficiency also induced telomere dysfunction and tumor development in mice with a p53 mutant background (9). PARP-1 thus seems to function in regulating telomere length as well as telomeric end capping. In view of its importance in both DNA repair and chromosome stability, the present study was undertaken to determine whether PARP-1 is an important genetic factor responsible for arsenic-induced cytotoxicity in mammalian cells. Our study indicates that arsenite-induced cell death, telomere attrition, and genomic instability are greatly enhanced in PARP-1 null cells, and that PARP-1 is required for cellular resistance to arsenite exposure. Materials and Methods Cell culture and sodium arsenite treatment. PARP-1+/+ and PARP-1À/À mouse embryonic fibroblasts (MEFs; kindly provided by Dr. Zhao-Qi Wang) were cultured following the procedure described earlier (10). Cells in exponential growth phase (at about 70% confluence) were exposed to doses of sodium arsenite [As3+; Sigma, St. Louis, MO; 1.5 Ag/mL (11.5 Amol/L) or 3.0 Ag/mL (23 Amol/L)], and the cells were treated for 24 or 48 hours. These same doses were used for all the experiments described below. Several earlier studies have shown that doses in the range of to 20 Amol/L of sodium arsenite were found to show moderate effect level for the induction of sister chromatid exchanges and micronuclei in mammalian systems (11). Doses in the range of 1.5 and 10 Ag/mL were required to induce chromosome aberrations in mouse lymphoma cells (12). Additionally, a dose of 1.5 Ag/mL is considered pertinent to our study, as the arsenic level is quite high in some Asian countries. A higher dose of 3.0 Ag/mL was used to induce sufficient oxidative damage (13, 14) for enabling us to show the effect in a DNA repair–deficient system. Two independent sets of MEFs for each genotype were used in the experiments. The data were pooled and presented. 10977 Cancer Res 2005; 65: (23). December 1, 2005 Cancer Research Figure 1. Telomere length measurements by flow FISH in PARP-1+/+ (white columns ) and PARP-1À/À (black columns) MEFs treated with sodium arsenite for (A) 24 hours and (B ) 48 hours. Cells were treated with two different doses of arsenite [11.5 Amol/L (1.5 Ag/mL) and 23 Amol/L (3.0 Ag/mL)]. Sham-treated cells served as control. Columns, means of two experiments; bars, SD. There is greater telomere loss (P < 0.05) in PARP-1À/À MEFs (f40%) compared with the normal MEFs (f10-20%) at 48 hr after treatment with arsenite. MESF, molecules equivalent of soluble fluorochromes. *, P < 0.05, statistically significant when comparing the response of PARP-1À/À cells with the PARP-1+/+ cells (two-way ANOVA test and Student’s t test). Flow fluorescence in situ hybridization for telomere measurement. After a continuous treatment with As3+ for 24 or 48 hours, cells were trypsinized and washed with 1Â PBS/0.1% bovine serum albumin (BSA). Cells were then probed with a telomere sequence–specific peptide nucleic acid (PNA) by fluorescence in situ hybridization (FISH). Telomere length was measured by flow cytometry as explained elsewhere (15). Alkaline single-cell gel electrophoresis (Comet) assay. Cells were treated with As3+ for 30 minutes and 24 hours with the doses mentioned earlier. The treated cells were harvested by trypsinization, washed in icecold 1Â PBS, and resuspended in HBSS with 10% DMSO with EDTA. The cells were then suspended in (0.75%) molten low melting point agarose (at 37jC) and immediately pipetted onto the comet slides (Trevigen, Gaithersburg, MD). Electrophoresis was done as per vendor’s suggestions. After electrophoresis, slides were briefly rinsed in neutralization buffer (500 mmol/L Tris-HCl, pH 7.5), air-dried, and stained with SYBR green dye. The tail moment of the comets was generated using the Metasystems (Altlussheim, Germany) analysis software ‘‘Comet imager version 1.2.’’ Fifty randomly chosen comets were analyzed per sample. The extent of DNA damage observed was expressed as tail moment, which corresponded to the fraction of the DNA in the tail of the comet. Fluorescence in situ hybridization analysis of chromosomes. After As3+ treatment for the specified time intervals, cells were released from the treatment and allowed to grow for 24 hours in the absence of arsenite. Cells were arrested at mitosis by treatment with colcemid (0.1 Ag/mL). The cells were then incubated with a hypotonic solution of sodium citrate at 37jC for 20 minutes followed by fixation in Carnoy’s fixative. FISH was done using telomere-specific PNA probe labeled with Cy3, and the cells were counterstained with 4V,6-diamidino-2-phenylindole (Vectashield; refs. 8, 15). Fifty metaphases were captured using the Zeiss Axioplan imaging fluorescence microscope and analyzed using the in situ imaging software (Metasystems). Cytokinesis-blocked micronucleus assay. Cells, after treatment for 24 and 48 hours with As3+, were incubated with cytochalasin B (Sigma, Ag/ mL) for an additional 22 hours. The cells were then trypsinized and subsequently fixed using a combination of both Carnoy’s fixative (acetic acid/methanol, 1:3) and three to four drops of formaldehyde (to fix the Cancer Res 2005; 65: (23). December 1, 2005 cytoplasm). Fixed cells were dropped onto clean slides and stained with Ag/mL of acridine orange, which differentially stains cytoplasm and nucleus (16, 17). One thousand binucleated cells were scored for each sample. Cell cycle analysis. Control and As3+-treated cells were washed with 1Â PBS/0.1% BSA and fixed in 70% ethanol. The fixed cells were later stained with propidium iodide/RNase A. Samples were then analyzed by flow cytometry at 488-nm excitation E and 610-nm emission E. Approximately 10,000 events per sample were collected, and the data was analyzed using WINMDI software. Microarray gene chip analysis. PARP-1+/+ and PARP-1À/À cells were treated with 1.5 Ag/mL of As3+ for 24 hours. Total RNA was extracted (RNeasy kit, Qiagen, Hilden, Germany), and double-stranded cDNA was synthesized from Ag of total RNA using Superscript system (Invitrogen, Carlsbad, CA) primed with T7-(dT)-24 primer. For biotin-labeled cRNA synthesis, in vitro transcription reaction was done in the presence of T7 RNA polymerase and biotinylated ribonucleotides (Enzo Diagnostics, Farmingdale, NY). The cRNA product was purified (RNeasy kit, Qiagen), fragmented, and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 in a Gene chip hybridization oven 640 (Affymetrix, Inc., Santa Clara, CA) as per the Gene Chip Expression Analysis manual (Affymetrix). After 16 hours of hybridization, the gene chips were washed and stained using the Affymetrix Fluidic station and scanned by Gene Array Scanner (Affymetrix). Image data were normalized and statistically analyzed using Gene Spring 7.2 (Silicon Genetics, Redwood City, CA). There were 311 differentially (P < 0.05, one-way ANOVA) expressed genes, and they were annotated according to Gene Ontology: Biological Process. Subsequent data analysis involved agglomerative average-linkage hierarchical clustering for finding different patterns and levels of gene expression. Statistical analysis. Statistical comparisons between and among the groups were made using two-way ANOVA, Student’s t test, and contingency tables analysis (m2 test and Fisher’s exact test) using Microsoft Excel 2003 (Microsoft Corp., Redmond, WA). The difference was considered to be statistically significant when P < 0.05. Figure 2. Histogram of micronuclei induction measured by the cytokinesis-blocked micronucleus assay after 24 hours (A ) and 48 hours (B ) of sodium arsenite treatment. The micronuclei formation, a biomarker of chromosomal instability, is shown in PARP-1+/+ MEFs (white columns) and PARP-1À/À MEFs (black columns): 11.5 Amol/L (1.5 Ag/mL) and 23 Amol/L (3.0 Ag/mL) of sodium arsenite treatment for both cell lines compared with the untreated samples. A total of 1,000 binucleated cells were scored. The micronuclei containing binucleated cells after arsenite treatment showed a 2-fold increase in PARP-1À/À cells compared with PARP-1+/+ cells. *, P < 0.05, m2 test and Fisher’s exact test. **, P < 0.05, statistically significant when compared with respective untreated samples as well as with As3+-treated PARP-1+/+ MEFs. 10978 www.aacrjournals.org PARP-1 Deficiency and Cellular Sensitivity to Arsenite Results Arsenite-induced telomere attrition was greater in PARP-1À/À mouse embryonic fibroblasts. Arsenite has been suggested to be a potent inducer of oxidative stress and DNA damage (14), and telomere shortening has been attributed to oxidative stress (18, 19). Our earlier studies have implicated PARP-1 in telomere maintenance (8, 9). We therefore investigated the effect of arsenite treatment on telomere length in the absence of the PARP-1 gene product. There was no significant difference in telomere length in both the cell types studied (Fig. 1A and B) at 24 hours after treatment, but the extent of telomere attrition was significantly (P < 0.05, two-way ANOVA) greater in PARP-1À/À cells (30-40% loss) compared with the PARP-1+/+ cells (10-20% loss; Fig. 1A and B) at 48 hours after treatment. This interesting observation led us further to examine whether the telomere loss triggered by As3+ treatment enhances the chromosome instability. Figure 4. DNA damage as measured by the comet assay in PARP-1+/+ (white columns ) and PARP-1À/À (black columns ) MEFs after 11.5 Amol/L (1.5 Ag/mL) and 23 Amol/L (3.0 Ag/mL) of sodium arsenite treatment. Representative of SYBR Green–stained comets prepared from control (A ) and arsenic-treated PARP-1 null cells (B). The extent of DNA damage measured as tail moment (product of tail length and fraction of DNA) differed between PARP-1-deficient and PARP-1proficient cells. Columns, mean; bars, SD. Data for tail moment for PARP-1+/+ and PARP-1À/À MEFs after treatment with arsenite for 30 minutes (C ) and for 24 hours (D ). *P, < 0.05, two-way ANOVA test and Student’s t test. **, P < 0.05, statistically significant when compared with respective untreated samples as well as with As3+-treated PARP-1+/+ MEFs. Figure 3. Telomere PNA-FISH analysis on metaphase chromosomal spreads from PARP-1+/+ and PARP-1À/À MEFs. A, chromosomal DNA was stained with DAPI (gray ) and telomeres were hybridized with Cy3-labeled telomere probe (white ). Arrow points to a Robertsonian fusion-like configuration without telomeres at the fusion point in PARP-1À/À MEFs following arsenic treatment. Partial metaphase spread. B, in a partial metaphase spread from PARP-1À/À MEFs, arrow points to a ring-like structure resulting from loss of telomeres on sister chromatids in a chromosome. C and D, compilation of chromosome analysis from PARP-1+/+ and PARP-1À/À MEFs with or without arsenic treatment. C, chromosome aberrations detected in PARP-1+/+ and PARP-1À/À MEFs following arsenic treatment for 24 hours. Frequency of chromosome aberrations per cell is given. D, chromosome aberrations detected in PARP-1+/+ and PARP-1À/À MEFs following arsenic treatment for 48 hours. Frequency of chromosome aberrations per cell is given. Chromosome alterations detected include fragments, breaks, and end-to-end fusions. *, P < 0.05, C test and Fisher’s exact test. www.aacrjournals.org To test this possibility, micronuclei analysis was done because it is a reliable indicator of chromosomal damage and genomic instability (20, 21). Micronuclei formation is due to the exclusion of chromosomes or chromosomal fragments from the daughter nuclei. Consistent with telomere loss, PARP-1À/À cells exhibited a 2- to 3-fold increase (P < 0.05, m2/Fisher’s exact test) in the number of micronuclei containing binucleated cells at 24 and 48 hours after sodium arsenite treatment compared with PARP-1+/+ cells (Fig. 2A and B). FISH using telomeric PNA probe was employed to analyze the chromosomal aberrations, particularly chromosome end-to-end fusions. Chromosomes with critically short telomeres are unstable and often result in end-to-end 10979 Cancer Res 2005; 65: (23). December 1, 2005 Cancer Research Figure 5. Cell cycle profile and cell viability were assessed by flow cytometry in both genotypes [PARP-1+/+ (first three columns ) and PARP-1À/À (last three columns )] after arsenite treatment with different doses (1.5 and 3.0 Ag/mL) for 24 hours (A ) and 48 hours (B). Arsenite-treated PARP-1À/À MEFs showed more cell death as indicated by the sub-G1 population of cells in a time-dependent manner compared with the normal MEFs. Percentage of cells in different cell cycle stages after arsenic treatment in PARP-1-proficient and PARP-1-deficient cells (histograms ). Percentage of cells in each phase of cell cycle was compared with the respective phase in treated samples. *, P < 0.05, compared with respective untreated sample (m2 test and Fisher’s exact test). **, P < 0.05, compared with respective untreated sample as well as with As3+-treated PARP-1+/+ MEFs. fusions and chromosomal aberrations. Higher numbers of chromosome aberrations were detected in PARP-1À/À MEFs at 1.5 Ag/mL dose at both the time points. Typical fusions, such as Robertsonian fusion-like structures and ring like structures (Fig. 3A and B), were detected in the As3+ -treated PARP-1À/À MEFs, which are best indicators of telomere loss and dysfunction. However, arsenite treatment did not increase the frequency of chromosome endto-end fusions in PARP-1 null cells as expected, but the frequency of gross chromosome aberrations (which includes end-to-end fusions, chromosome breaks, and fragments) observed was higher in PARP-1À/À cells than PARP-1+/+ cells (Fig. 3C and D). Statistically significant increase (P < 0.05, m2 test) in the total number of chromosome aberrations was observed at a dose of 1.5 Ag/mL in PARP-1À/À MEFs at both 24 and 48 hours following treatment. Cells lacking PARP-1 displayed elevated DNA damage. The extent of arsenic induced DNA damage and repair was estimated by alkaline single-cell gel electrophoresis popularly known as comet assay (Fig. 4A and B). The comet assay was done under alkaline condition (pH >13), to estimate all types of DNA damage, including double-strand breaks, single-strand breaks, and alkali labile sites. PARP-1À/À cells reflected enhanced DNA damage induction compared with PARP-1+/+ cells after arsenite treatment (Fig. 4C and D). DNA damage induced by 30 minutes of treatment of As3+ was 5- to 6-fold (P < 0.001, two-way ANOVA test and Student’s t test) more in PARP-1À/Àcells than PARP-1+/+ cells. Similarly, arsenic exposure for 24 hours resulted in an increased tail Cancer Res 2005; 65: (23). December 1, 2005 moment, indicating the impaired repair efficiency of arseniteinduced DNA damage in PARP-1 null cells (Fig. 4D). PARP-1À/À mouse embryonic fibroblasts are more sensitive to arsenite induced cell death. The dependence of elevated persistent DNA damage and telomere attrition on cell viability was next evaluated by flow cytometry. Although no marked differences were observed between the two cell types at 24 hours after arsenic treatment, PARP-1 null cells showed a 2-fold increase over PARP-1-proficient cells in apoptotic death at 48 hours after arsenite treatment (P < 0.05, m2 Testand Fisher’s exact test; Fig. 5A and B) illustrative of a delayed cell killing effect. This increased cell death was further verified by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay and crystal violet assay (data not shown). Cell cycle analysis is useful for detecting early events of DNA damage/repair response, and cells deficient in DNA repair invariably exhibit abnormal cell cycle checkpoint regulation. To determine whether the increased cell death of PARP-1À/À cells is due to loss or abnormal checkpoint regulation, cell cycle analysis was done after arsenic treatment in both PARP-1+/+ and PARP-1À/À cells. Both cell lines showed a dose-dependent increase (P < 0.05) in apoptosis visualized as sub-G1 population of cells and the fraction of sub-G1 population being higher in PARP-1À/À cells than PARP-1+/+ cells. There was a corresponding decrease (P < 0.05) in G1 cell population in both PARP-1-proficient and PARP-1-deficient MEFs at 24 and 48 hours of treatment (Fig. 5A and B). At 48 hours after treatment with As3+, the percentage of G2-M phase cells had greatly reduced (P < 0.05) in PARP-1À/À MEFs compared with PARP-1+/+ MEFs. It is likely that mitotic Figure 6. Gene expression data were obtained using Affymetrix Mouse Genome 430 2.0 GeneChip. Cluster diagram, 311 differentially expressed probes with P < 0.05 and fold change of >2.0 in one of the experimental conditions. Column, a single experiment condition; row, a single gene. Expression levels (red) for up-regulation and (blue ) for down-regulation according to the color scale. 10980 www.aacrjournals.org PARP-1 Deficiency and Cellular Sensitivity to Arsenite Table 1. Functional categorization of differentially expressed genes in PARP-1+/+ and PARP-1À/À MEFs after arsenite treatment Gene name Genbank ID Response to stress and external stimuli Prdx1 NM_011034 Hspcb BI154147 Daf1 NM_010016 Gadd45a NM_007836 Sod1 BC002066 Hspa1b M12573 Hspa1a AW763765 Ifi30 NM_023065 Ii BC003476 Cell growth and maintenance Afp NM_007423 Ube1c NM_011666 Calm1 AU079514 Ets1 BC010588 Ube2b AK010432 Arhgap5 BM248774 Btg1 L16846 Mt2 AA796766 Prkar1b BB274009 Ikbkg BB821318 Slc9a3r1 BB805362 Rab3d BB349707 Myrip BB429683 Cell death Mcl1 BC003839 Hspa1b M12573 Elmo3 AI481208 Sod1 BC002066 DNA metabolism Foxc1 BB759833 Uox M27695 Rfx3 BC017598 Lbh AK007400 Hoxd8 BF785056 Ncor1 BM239260 Ash1l BG694892 Zfp99 AK009842 Prps2 BM934034 PARP-1+/+ treated* PARP-1À/À c treated PARP-1À/À b untreated PARP-1À/À treatedx 8.78 5.86 À41.28 5.29 5.96 2.47 17.88 4.51 3.42 À1.53 1.3 5.84 1.52 À1.52 9.08 230.82 À2.39 15.5 À3.7 À4.25 À3.45 À1.67 À1.81 À2.35 À14.21 1.83 À4.51 À49.71 À19.09 69.9 À5.79 À16.4 1.56 À1.1 À5.89 1.01 10.16 4.71 4.84 À2.17 4.63 4.8 4.59 11.85 7.27 5.17 7.34 À1.67 11.03 48.71 À2.58 1.98 4.69 À6.53 1.23 À2.1 1.84 3.91 À1.08 À1.25 60.42 À1.12 À100 À1.73 À4.59 À1.15 À1.32 3.49 À1.35 1.82 82.96 À1.71 5.72 À47.56 19.61 À30.34 À21.08 À11.22 8.86 À39.99 À1.12 À13.01 À3.53 44.58 À9.56 À1.6 2.12 1.59 4.52 27.13 4.17 5.96 1.02 24.35 À1.21 À1.52 1.22 1.43 4.21 À1.81 À3.65 1.28 À1.19 À16.4 0.85 0.95 0.04 4.27 2.21 7.93 2.12 4.38 4.16 9.41 10.66 2.06 À1.08 À24.02 1.63 À1.94 À1.35 À2.06 À11.5 À6.9 À1.49 3.32 7.5 7.42 6.76 4.27 6.68 À1.03 1.63 32.13 À1.39 À7.08 1.52 1.64 À1.39 À1.28 NOTE: Expression profile of selected genes, which were differentially expressed to a fold change of z2.0 in one of the treatments is given. Data indicate the fold increase (positive values) or decrease (negative values) in treated samples compared with corresponding controls. *Compared with untreated PARP-1+/+ MEFs. cCompared with untreated PARP-1À/À MEFs. bCompared with untreated PARP-1+/+ MEFs. x Compared with treated PARP-1+/+ MEFs. catastrophe occurs increasingly in PARP-1À/À cells, leading to increased apoptotic death detected by an increase in sub-G1 cell population. Differential gene expression patterns in PARP-1+/+ and PARP-1À/À cells after arsenite treatment. To study the differential gene expression patterns in PARP-1+/+ and PARP-1À/À MEFs with or without arsenite treatment, microarray technology was employed. Using this technology, the expression pattern of over 34,000 genes was analyzed and compared with untreated controls. PARP-1+/+ and PARP-1À/À MEFs were treated with 1.5 Ag/mL of As+3 for 24 hours and then subjected to gene www.aacrjournals.org expression studies. Microarray analysis was focused on genes that were altered with a >2-fold change with P < 0.05. A one-way ANOVA test was done to find distinct groups of genes that were significantly changed. Analysis has indicated that there are about 311 genes, which are differentially expressed among the different groups (control versus treated, PARP-1À/À versus PARP-1+/+). These genes belong to different biological processes, such as apoptosis/ cell death, physiologic processes, and response to stress/external agents. The data are presented as a hierarchical clustering in Fig. for the genes with differential expression in arsenic-treated 10981 Cancer Res 2005; 65: (23). December 1, 2005 Cancer Research samples. It is clear from the data that there are several genes, which are up-regulated in both PARP-1+/+ and PARP-1À/À following arsenic treatment. Differentially expressed genes were classified according to Gene Ontology: Biological Process. Expression profile of selected genes, which were differentially expressed to a fold change of z2.0 in one of the treatments, is given in Table 1. We have also identified candidate genes, which are in apoptosis and cell cycle pathways following treatment with arsenic. Differences observed in the expression patterns of these genes (data not shown) between the PARP-1-proficient and PARP-1-deficient cells warrant further investigation. Discussion In this study, PARP-1-deficient cells showed a greater telomere attrition than in PARP-1-proficient cell lines at doses of 11.5 and 23 Amol/L. Use of low concentrations (1.0 Amol/L) used in this study have shown a rapid and dramatic loss of telomeric DNA, leading to apoptotic cell death. Although inhibition of telomerase alone by arsenite may cause telomere shortening over numerous population doublings, rapid telomere loss was observed in PARP-1À/À cells after only 24 to 48 hours of sodium arsenite treatment. There are several possible explanations for this observation. Rapid telomere loss may be due to the susceptibility of the hexametric repeat structure of telomeric DNA (triple-G-containing structures) to oxidative damage (23, 24), as available evidences indicate that oxidative stress has the potency to break polyguanosine sequences in telomeric repeats, leading to telomere loss (19, 25). Arsenite, being one of the efficient inducers of oxidative stress (13, 14, 26), can also break polyguanosine sequences in telomeric repeats, resulting in rapid telomere loss (27). Additionally, DNA repair in the telomeric DNA was shown to be less efficient than the rest of the genome (25), further exacerbating the telomere attrition in the absence of the PARP-1 gene product. In view of the interactions of PARP-1 with the participants of the base excision repair pathway (BER; refs. 28, 29), it is likely that PARP-1 deficiency may be a contributing factor to the rapid telomere shortening due to perturbations in the cellular processes of DNA damage recognition and repair. Consistent with this, deficiencies in both shortlong- and long-patch BER pathways have been reported in PARP-1-deficient cells (28). This study (28) shows that both uracil and 8-oxoguanine are poorly repaired in PARP-1deficient cells compared with wild-type cells. Arsenite has been shown to induce a wide variety of oxidized base lesions, including 8-hydroxyguanine (30). Furthermore, acute arsenic treatment inhibits the activity of human 8-hydroxyguanine glycosylase (OGG1) enzyme (30). Hence, a fundamental BER deficiency in the absence of PARP-1 is presumably responsible for the enhanced sensitivity of PARP-1 null cells to arsenite treatment. Recently, arsenic mutagenicity has been shown to involve mitochondrial DNA damage (31), and it is presently unclear whether PARP-1 is also involved in BER process in mitochondrial DNA. Earlier studies have implicated telomere shortening as an important checkpoint to limit the potential of human cells to proliferate (32). Shortening of telomeres triggers an apoptotic response in a p53-dependent manner (33). Telomere dysfunction coupled with chromosome instability due to inefficient DNA repair might be responsible for increased cell death in PARP-1-deficient Cancer Res 2005; 65: (23). December 1, 2005 cells. The cellular hypersensitivity of PARP-1 null cells therefore unequivocally projects the role of PARP-1 as a survival factor against arsenic exposure. One of the earliest responses to ionizing radiation– and alkylating agent–induced DNA damage is the poly(ADP) ribosylation of many nuclear proteins, which is mediated chiefly by PARP-1 and to a lesser extent by the PARP family of proteins (2, 34). Arsenite was shown to inhibit poly(ADP) ribosylation of proteins (7) in mammalian cells, and it is presently unclear whether the lack of poly(ADP) ribosylation of proteins affects the DNA repair process. As PARP-1 is implicated in diverse repair pathways, particularly BER, it is reasonable to assume that the increased cell death, telomere attrition, and genomic instability observed in PARP-1 null cells is due to inefficient repair of oxidized base lesions. Telomere length maintenance has also been implicated in cancer development due to reactivation of telomerase in tumor cells. Telomerase inhibition is therefore emerging as a promising approach in cancer chemotherapy (35, 36). Treatment of cancer cell lines with telomerase inhibitors induces telomere shortening and halts cellular proliferation (35). The importance of PARP-1 in telomere integrity suggests that PARP-1 inhibitors may present a myriad of potential therapeutic applications, especially in cancer treatment (37, 38). Seimiya et al. (37) showed that the combination of inhibitors to the PARP family protein found at the telomeres, tankyrase, might serve as an effective anticancer therapy approach (36, 37). Because PARP-1 deficiency increases the cytotoxicity of arsenite treatment, PARP-1 inhibitors could also be used in combination with other DNA-damaging agents to increase the cytotoxicity of cancer cells (39). Gene expression studies have revealed differential expression of >300 genes following arsenic treatment in PARP-1+/+ and PARP-1À/À MEFs. Genes that are up-regulated or down-regulated following treatment with arsenic in both cell types include genes that participate in diverse biological processes, such as cell death, signal transduction, response to stress/external stimulus, and cell growth and/or maintenance. As many as 36 genes involved in response to external stimulus were differentially expressed in the treated samples. More importantly, about 39 genes, which are important for cell growth and/or maintenance, were altered in their expression profiles. This clearly indicates that PARP-1 has a role in the above cellular processes, which warrants further validation and investigation. Differential expression in these genes may provide biomarkers for gaining insights into mode of arsenic toxicity. Efforts are under way to identify and characterize specific DNA repair and cell cycle pathway(s) in the pathobiology of arsenic exposure. Taken together, our study identifies PARP-1 as one of the important genetic factors responsible for mediating the toxic effects of arsenite. Acknowledgments Received 7/5/2005; revised 8/22/2005; accepted 9/22/2005. Grant support: Academic Research Fund; National University of Singapore; Office of Life Sciences; National University Medical Institutes, Singapore; and National Medical Research Council, Ministry of Health, Singapore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. Zhao-Qi Wang and Wei-Min Tong (IARC, Lyon, France) for the PARP-1-proficient and PARP-1-deficient MEFs, Prof. Tom K. 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Anticancer chemosensitization and radiosensitization by the novel poly(ADP-ribose) polymerase-1 inhibitor AG14361. J Natl Cancer Inst 2004;96:56–67. Cancer Res 2005; 65: (23). December 1, 2005 [...]... 2: The telomeric end replication problem The replication forks move in opposite directions Since DNA polymerases only elongate in the 5′ to 3′ direction, each fork contains a leading and a lagging strand Lagging strand synthesis cannot be completed because the removal of primers thus causing net loss of DNA base pairs from the lagging strand (Hug and Lingner, 2006) 5 1. 1 .1. 1.2 Telomere dysfunction and. .. (Blackburn, 19 91) Telomeres are composed of guanine-rich hexameric DNA repeats and specific telomere binding proteins (Figure 1A) (Blackburn, 19 91) Telomeres prevent the natural ends of chromosomes from being recognised as double strand breaks and protect the chromosomes from degradation and chromosomal fusions (Blackburn, 19 91; Greider, 19 91) They also function in meiotic and mitotic pairing, as well... expression of Parp -1 mRNA in PARP -1+ /+ and PARP -1- /MEFs • Figure 22B: Differentially expressed copper containing genes in PARP -1+ /+ and PARP -1/ - MEFs after arsenite treatment • Figure 23: γH2AX foci staining in MEFs • Figure 24: γH2AX foci staining in normal MEFs treated with: - (B) Sodium arsenite with and without 10 µM of copper pre-treatment - (C) Hydrogen peroxide with and without 10 µM of copper pre-treatment... contributing to arsenic-induced genotoxicity (Li and Rossman, 19 89a) Part of the DNA repair inhibition properties of many arsenical compounds including arsenite, MMA(III), DMA(III), TMA(III), and dimethylarsine can be attributed to the affinity for proteins with thiol and free sulfhydryl groups, which are common in DNA-binding proteins, transcription factors, and DNA-repair proteins (Li and Rossman, 19 89b)(Delnomdedieu... 19 90; Hastie et al., 19 90; Levy et al., 19 92; Olovnikov, 19 71, 19 73) This phenomenon, known as the end replication problem (Figure 2), is due the unidirectional DNA polymerases not being able to fill the gap left by the primer on the terminal end of the lagging strand (Harley et al., 19 90; Hastie et al., 19 90; Levy et al., 19 92) The terminal gap is further enlarged by the action of putative 5' to 3'... and direct molecular modifications in dominantly transforming cellular proto-oncogenes [reviewed in (Aguilera and Gómez-González, 2008)] Molecular mechanisms maintaining genomic stability are deficient in cancer, further aggravating the accumulation of genetic mutations and deficiencies of diverse mechanisms beyond repair [reviewed in (Meeker and Argani, 2004)] 1 1 .1. 1 .1 Telomere-mediated genomic instability... to genomic instability and tumourigenesis (Hande, 2004) (Figure 4) 8 Figure 4: Model of telomere-mediated genomic instability The flow chart highlights how DNA damage-signalling molecules and DNA repair factors may play a significant role in the maintenance of telomere equilibrium and in the event of this equilibrium not being maintained, it leads to telomere-mediated chromosome -genomic instability and. .. during meiosis and mitosis, (Pandita et al., 2007) with key roles in nuclear organization and transcriptional silencing (Blackburn, 19 91; Greider, 19 90, 19 91) The physical structure of the telomeres is believed to be in the form of a telomere loop (T loop) and displacement loop (D-loop) structure where the C terminal portion of telomeres folds back on itself to form the large T-loop and the 3' G-strand... deficiencies in DNA repair factors (Finkel et al., 2007) 3 A C B Figure 1: Telomere structure The fluorescence image shows the location of a telomere within a chromosome Mammalian telomeres consist of TTAGGG repeats with a single-stranded 3’ overhang of the G-rich strand (A) Specific protein complexes bind to the double- and single-stranded telomeric DNA (B) The single-stranded overhang can invade the double-stranded... 19 96) and re-expressed in majority of cancer cells (Kim et al., 19 94) A small proportion of cancer cells utilise a telomerase independent mechanism termed alternative lengthening of telomere (ALT) employing homologous recombination for the maintenance of telomeres (Bryan et al., 19 95) In the absence of telomerase, 50 -15 0 bp of DNA is lost from the telomeres with each cell division (Harley et al., 19 90; . 1. 1.3 .1 Poly (ADP- ribose) polymerase 1 (PARP -1) 21 1. 1.3 .1. 1 Role of PARP -1 at the telomeres 24 1. 1.4 Copper metabolism and disease 27 1. 1.4 .1 PARP -1 and Copper metabolism 29 1. 1.5 Copper. 1 1. 1 .1. 1 Telomere mediated genomic instability 2 1. 1 .1. 1 .1 Telomeres 2 1. 1 .1. 1.2 Telomere dysfunction and tumourigenesis 6 1. 1 .1. 1.3 DNA repair proteins in telomere maintenance 8 1. 1.2. ROLE OF POLY (ADP- RIBOSE) POLYMERASE 1 AND COPPER HOMEOSTASIS FACTOR, ANTIOXIDANT PROTEIN 1 IN THE MAINTENANCE OF GENOMIC INTEGRITY. LAKSHMIDEVI