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[Cancer Research 65, 10977-10983, December 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Lack of Poly(ADP-Ribose) Polymerase-1 Gene Product Enhances Cellular Sensitivity to Arsenite

Anuradha Poonepalli1,2, Lakshmidevi Balakrishnan1, Aik Kia Khaw1, Grace Kah Mun Low1, Manikandan Jayapal3, Rabindra N. Bhattacharjee4,5, Shizuo Akira4,5, Adayabalam S. Balajee6 and M. Prakash Hande1,2

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

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{at}nus.edu.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arsenite (As3+) 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-1–/– 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–/– MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1–/– 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and sodium arsenite treatment. PARP-1+/+ and PARP-1–/– 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 µg/mL (11.5 µmol/L) or 3.0 µg/mL (23 µmol/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 µmol/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 µg/mL were required to induce chromosome aberrations in mouse lymphoma cells (12). Additionally, a dose of 1.5 µg/mL is considered pertinent to our study, as the arsenic level is quite high in some Asian countries. A higher dose of 3.0 µg/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.

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 1x 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 ice-cold 1x PBS, and resuspended in HBSS with 10% DMSO with EDTA. The cells were then suspended in (0.75%) molten low melting point agarose (at 37°C) 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 µg/mL). The cells were then incubated with a hypotonic solution of sodium citrate at 37°C 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 4',6-diamidino-2-phenylindole (Vectashield; refs. 8, 15). Fifty metaphases were captured using the Zeiss Axioplan 2 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, 5 µg/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 cytoplasm). Fixed cells were dropped onto clean slides and stained with 3 µg/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 1x 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 {lambda} and 610-nm emission {lambda}. Approximately 10,000 events per sample were collected, and the data was analyzed using WINMDI software.

Microarray gene chip analysis. PARP-1+/+ and PARP-1–/– cells were treated with 1.5 µg/mL of As3+ for 24 hours. Total RNA was extracted (RNeasy kit, Qiagen, Hilden, Germany), and double-stranded cDNA was synthesized from 5 µg 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 ({chi}2 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Arsenite-induced telomere attrition was greater in PARP-1–/– 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-1–/– 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. 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-1–/– cells exhibited a 2- to 3-fold increase (P < 0.05, {chi}2/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 fusions and chromosomal aberrations. Higher numbers of chromosome aberrations were detected in PARP-1–/– MEFs at 1.5 µg/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-1–/– MEFs, which are best indicators of telomere loss and dysfunction. However, arsenite treatment did not increase the frequency of chromosome end-to-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-1–/– cells than PARP-1+/+ cells (Fig. 3C and D). Statistically significant increase (P < 0.05, {chi}2 test) in the total number of chromosome aberrations was observed at a dose of 1.5 µg/mL in PARP-1–/– MEFs at both 24 and 48 hours following treatment.



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  		Figure 1. Telomere length measurements by flow FISH in PARP-1+/+ (white columns) and PARP-1–/– (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 µmol/L (1.5 µg/mL) and 23 µmol/L (3.0 µg/mL)]. Sham-treated cells served as control. Columns, means of two experiments; bars, SD. There is greater telomere loss (P < 0.05) in PARP-1–/– MEFs (~40%) compared with the normal MEFs (~10-20%) at 48 hr after treatment with arsenite. MESF, molecules equivalent of soluble fluorochromes. *, P < 0.05, statistically significant when comparing the response of PARP-1–/– cells with the PARP-1+/+ cells (two-way ANOVA test and Student's t test).

 


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  		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-1–/– MEFs (black columns): 11.5 µmol/L (1.5 µg/mL) and 23 µmol/L (3.0 µg/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-1–/– cells compared with PARP-1+/+ cells. *, P < 0.05, {chi}2 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.

 


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  		Figure 3. Telomere PNA-FISH analysis on metaphase chromosomal spreads from PARP-1+/+ and PARP-1–/– 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-1–/– MEFs following arsenic treatment. Partial metaphase spread. B, in a partial metaphase spread from PARP-1–/– 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-1–/– MEFs with or without arsenic treatment. C, chromosome aberrations detected in PARP-1+/+ and PARP-1–/– MEFs following arsenic treatment for 24 hours. Frequency of chromosome aberrations per cell is given. D, chromosome aberrations detected in PARP-1+/+ and PARP-1–/– 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, {chi}2 test and Fisher's exact test.

 
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-1–/– 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-1–/–cells than PARP-1+/+ cells. Similarly, arsenic exposure for 24 hours resulted in an increased tail moment, indicating the impaired repair efficiency of arsenite-induced DNA damage in PARP-1 null cells (Fig. 4D).



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  		Figure 4. DNA damage as measured by the comet assay in PARP-1+/+ (white columns) and PARP-1–/– (black columns) MEFs after 11.5 µmol/L (1.5 µg/mL) and 23 µmol/L (3.0 µg/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-1–/– 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.

 
PARP-1–/– 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, {chi}2 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)-2,5-diphenyltetrazolium 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-1–/– cells is due to loss or abnormal checkpoint regulation, cell cycle analysis was done after arsenic treatment in both PARP-1+/+ and PARP-1–/– 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-1–/– 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-1–/– MEFs compared with PARP-1+/+ MEFs. It is likely that mitotic catastrophe occurs increasingly in PARP-1–/– cells, leading to increased apoptotic death detected by an increase in sub-G1 cell population.



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  		Figure 5. Cell cycle profile and cell viability were assessed by flow cytometry in both genotypes [PARP-1+/+ (first three columns) and PARP-1–/– (last three columns)] after arsenite treatment with different doses (1.5 and 3.0 µg/mL) for 24 hours (A) and 48 hours (B). Arsenite-treated PARP-1–/– 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 ({chi}2 test and Fisher's exact test). **, P < 0.05, compared with respective untreated sample as well as with As3+-treated PARP-1+/+ MEFs.

 
Differential gene expression patterns in PARP-1+/+ and PARP-1–/– cells after arsenite treatment. To study the differential gene expression patterns in PARP-1+/+ and PARP-1–/– 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-1–/– MEFs were treated with 1.5 µg/mL of As+3 for 24 hours and then subjected to gene 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-1–/– 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. 6 for the genes with differential expression in arsenic-treated samples. It is clear from the data that there are several genes, which are up-regulated in both PARP-1+/+ and PARP-1–/– 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 ≥2.0 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.



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  		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.

 

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  		Table 1. Functional categorization of differentially expressed genes in PARP-1+/+ and PARP-1–/– MEFs after arsenite treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 µmol/L. Use of low concentrations (<1.0 µmol/L) of arsenite have been shown to promote telomerase activity with or without an increment in telomere length (22). High concentrations of arsenite (>1.0 µmol/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-1–/– 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-1-deficient 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 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-1–/– 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
 
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. Hei (Columbia University, New York, NY) for his advise on the use of sodium arsenite for studying the oxidative damage, and Adam Ng Tsan Sheng for his assistance with statistical analysis.


   Footnotes
 
Note: A. Poonepalli and L. Balakrishnan contributed equally to this work.

Received 7/ 5/05. Revised 8/22/05. Accepted 9/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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