Lack of Poly(ADP-Ribose) Polymerase-1 Gene Product Enhances Cellular Sensitivity to Arsenite Anuradha Poonepalli, 1,2 Lakshmidevi Balakrishnan, 1 Aik Kia Khaw, 1 Grace Kah Mun Low, 1 Manikandan Jayapal, 3 Rabindra N. Bhattacharjee, 4,5 Shizuo Akira, 4,5 Adayabalam S. Balajee, 6 and M. Prakash Hande 1,2 1 Genome Stability Laboratory, Department of Physiology; 2 Oncology Research Institute; 3 Molecular and Cellular Immunology Laboratory, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 4 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University; 5 Exploratory Research for Advanced Technology, Japan Science and Technology Corp., Osaka, Japan; and 6 Center for Radiological Research, Columbia University, New York, New York Abstract Arsenite (As 3+ ) 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 carcino- genicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymer- ase (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-1 À/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-1 À/À MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1 À/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 mainte- nance. 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 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 cytotox- icity 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-1 À/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 [As 3+ ; 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 5 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. 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, 2 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 10977 Cancer Res 2005; 65: (23). December 1, 2005 Research Article Research. on March 1, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from