Role In Base Excision Repair Research Articles

Investigation on XRCC1 genetic polymorphism and its relationship with breast cancer risk factors in Chinese women

Breast cancer (BC) remains one of the most common cancers among women. The human X-ray repair cross-complementing 1 (XRCC1) gene plays key roles in base excision repair, and genetic polymorphisms of XRCC1 may be associated with the susceptibility to BC. This study aimed to evaluate the relationship between the XRCC1 genetic polymorphisms and BC susceptibility. A total of 354 BC patients and 366 cancer-free controls were enrolled in this study. Data about the risk factors of BC were collected using questionnaires. The XRCC1 genetic polymorphism was determined using created restriction site-polymerase chain reaction (CRS-PCR) and DNA sequencing methods. No significant differences in the allelic and genotypic frequencies of c.1804C>A genetic polymorphism were detected between cases and controls. The distributions of BC patients' risk factors were not significantly different between CC, CA, and AA genotypes. These findings indicate that the c.1804C>A genetic polymorphism of XRCC1 gene is not significantly associated with BC susceptibility in the Chinese women.

DNA Repair Gene XRCC1 Polymorphisms, Smoking, and Bladder Cancer Risk: A Meta-Analysis

Background and ObjectiveThe X-ray repair cross-complementing group 1 (XRCC1) protein plays a crucial role in base excision repair (BER) pathway by acting as a scaffold for other BER enzymes. Variants in the XRCC1 gene might alter protein structure or function or create alternatively spliced proteins which may influence BER efficiency and hence affect individual susceptibility to bladder cancer. Recent epidemiological studies have shown inconsistent associations between these polymorphisms and bladder cancer. To clarify the situation, a comprehensive meta-analysis of all available studies was performed in this study.MethodsPubMed, EMBASE, and Chinese Biomedical Literature database (CBM) databases have been systematically searched to identify all relevant studies for the period up to February 2013. Data were abstracted independently by two reviewers and Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Subgroup analyses were performed mainly by ethnicity and smoking status.ResultsA total of 26 case-control studies, including 24 studies for R399Q polymorphism, 15 studies for R194W polymorphism, and 7 studies for R280H polymorphism met the inclusion criteria and were selected. With respect to R399Q polymorphism, significantly decreased bladder cancer risk was found among smokers (AA vs. GG: OR=0.693, 95%CI= 0.515-0.932, P=0.015 and recessive model AA vs. GA+GG: OR=0.680, 95%CI= 0.515-0.898, P=0.007, respectively). With respect to R194W and R280H polymorphism, significantly increased bladder cancer risk were observed among Asians (TT+CT vs. CC:OR = 1.327, 95% CI 1.086-1.622, P=0.006 for R194W, and AA+GA vs. GG: OR=2.094, 95% CI 1.211–3.621, P=0.008 for R280H, respectively).ConclusionsThis meta-analysis suggests that the XRCC1 R399Q polymorphism may play a protective role against bladder cancer among smokers. However, the XRCC1 R194W and R280H polymorphisms were both associated with increased bladder cancer risk among Asians. Further studies with larger sample sizes are needed to validate our finds.

Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage

During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3′→5′ exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg2+ and Ca2+ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3′→5′ exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.

Abnormal Base Excision Repair at Trinucleotide Repeats Associated with Diseases: A Tissue-Selective Mechanism

More than fifteen genetic diseases, including Huntington’s disease, myotonic dystrophy 1, fragile X syndrome and Friedreich ataxia, are caused by the aberrant expansion of a trinucleotide repeat. The mutation is unstable and further expands in specific cells or tissues with time, which can accelerate disease progression. DNA damage and base excision repair (BER) are involved in repeat instability and might contribute to the tissue selectivity of the process. In this review, we will discuss the mechanisms of trinucleotide repeat instability, focusing more specifically on the role of BER.

DNA polymerase beta overexpression correlates with poor prognosis in esophageal cancer patients

Gene of DNA polymerase beta (polβ) plays an important role in base excision repair, DNA replication and translesion synthesis. This study aims to investigate the expression and prognostic significance of DNA polβ in esophageal cancer. DNA polβ expression was analyzed using real-time quantitative PCR (RT-qPCR) and immunohistochemical staining on tissue samples from a consecutive series of 114 esophageal squamous carcinoma patients who underwent resections between 2002 and 2006. Polβ expression was investigated on its correlation to clinico-pathological factors and survival. RT-qPCR results showed higher expression of DNA polβ mRNA in tumor tissue than in its matched adjacent non-tumor tissue sample, different expression of DNA polβ mRNA was noticed with significance between tumors with and without lymph node metastasis. Immunohistochemistry staining results indicated the polβ strong-positive rate was 44.73% (51/114) in tumor tissue samples and 0.00% in matched adjacent non-tumor tissue samples, with significant difference. Kaplan-Meier survival curves revealed that high expression of pol? was associated with tumor metastasis and poor prognosis in esophageal cancer patients. Our data suggests that polβ plays an important role in tumor progression and that high polβ expression predicts an unfavorable prognosis in esophageal squamous carcinoma patients.

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

XRCC1 codon 280 polymorphism and susceptibility to lung cancer: a meta-analysis of the literatures

The X-ray repair cross-complementation group 1 (XRCC1) protein plays an important role in base excision repair, and the genetic polymorphisms in the XRCC1 gene influence its function. XRCC1 codon 280 polymorphism is an Arg-His change in the XRCC1 gene. Many studies were published to investigate the association between XRCC1 codon 280 polymorphism and risk of lung cancer, but the results were inconsistent. We performed a meta-analysis of 16 studies with a total of 18,660 subjects (8,736 cases and 9,924 controls). The pooled odds ratios (OR) and corresponding 95 % confidence intervals (95 % CI) for the gene-disease association were calculated. Overall, there was a significant association between XRCC1 codon 280 polymorphism and increased risk of lung cancer (HisHis vs. ArgArg: OR = 1.53, 95 % CI 1.08-2.16, P = 0.016; HisHis vs. ArgArg/ArgHis: OR = 1.55, 95 % CI 1.10-2.19, P = 0.012). However, subgroup analysis by race failed to confirm the obvious association in Europeans and Asians. Therefore, there is a significant association between XRCC1 codon 280 polymorphism and increased risk of lung cancer. More studies with a large sample are needed to further evaluate the possible race-specific effect in the association above.

NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks

Recent evidence suggests a role for base excision repair (BER) proteins in the response to DNA interstrand crosslinks, which block replication and transcription, and lead to cell death and genetic instability. Employing fluorescently tagged fusion proteins and laser microirradiation coupled with confocal microscopy, we observed that the endonuclease VIII-like DNA glycosylase, NEIL1, accumulates at sites of oxidative DNA damage, as well as trioxsalen (psoralen)-induced DNA interstrand crosslinks, but not to angelicin monoadducts. While recruitment to the oxidative DNA lesions was abrogated by the anti-oxidant N-acetylcysteine, this treatment did not alter the accumulation of NEIL1 at sites of interstrand crosslinks, suggesting distinct recognition mechanisms. Consistent with this conclusion, recruitment of the NEIL1 population variants, G83D, C136R, and E181K, to oxidative DNA damage and psoralen-induced interstrand crosslinks was differentially affected by the mutation. NEIL1 recruitment to psoralen crosslinks was independent of the nucleotide excision repair recognition factor, XPC. Knockdown of NEIL1 in LN428 glioblastoma cells resulted in enhanced recruitment of XPC, a more rapid removal of digoxigenin-tagged psoralen adducts, and decreased cellular sensitivity to trioxsalen plus UVA, implying that NEIL1 and BER may interfere with normal cellular processing of interstrand crosslinks. While exhibiting no enzymatic activity, purified NEIL1 protein bound stably to psoralen interstrand crosslink-containing synthetic oligonucleotide substrates in vitro. Our results indicate that NEIL1 recognizes specifically and distinctly interstrand crosslinks in DNA, and can obstruct the efficient removal of lethal crosslink adducts.

XRCC1 Arg399Gln, Arg194Trp, and Arg280His Polymorphisms in Esophageal Cancer Risk: A Meta-Analysis

The X-ray repair cross-complementation group 1 (XRCC1) protein plays an important role in base excision repair. To elucidate the role of XRCC1 Arg399Gln, Arg194Trp and Arg280His genotypes in esophageal cancer risk, all available studies were considered in the present meta-analysis. Eligible studies were identified by searching several electronic databases for relevant reports published before June 2012. According to the inclusion criteria and exclusion criteria, a total of 21 eligible studies were included in the pooled analyses. Among the 21 studies, 18 focused on Arg399Gln polymorphism, 11 described the Arg194Trp, and 4 articles investigated on Arg280His. Our analysis suggested that there was no evidence of significant association between XRCC1 Arg399Gln polymorphism and esophageal cancer risk in any genetic model. In the stratified analysis by ethnicity for Arg399Gln polymorphism and esophageal cancer, the results showed that Arg399Gln polymorphism was not associated with esophageal cancer risk. Only 4 studies analyzed the relationship between XRCC1 Arg280His polymorphism and the risk of esophageal cancer. The Arg/His and His/His genotypes were not significantly associated with increased risk of EC. A similar negative association was maintained in dominant and recessive models. However, for XRCC1 Arg194Trp polymorphism, our study showed individuals carrying the variant genotype Trp/Trp had a significant increased risk of esophageal cancer (OR=1.295, 95% CI 1.053-1.591, P=0.014). In addition, increased associations were found in recessive model (OR=1.332, 95% CI 1.093-1.624, P=0.005). Our meta-analysis suggested that Arg194Trp Trp allele might act as a risk allele in its association with esophageal cancer.

Cytotoxic mechanism of Piper gaudichaudianum Kunth essential oil and its major compound nerolidol

Piper gaudichaudianum Kunth is used in popular medicine as anti-inflamatory and against liver disorders. One of the most studied components of the plant is the essential oil for which chemical analysis revealed (E)-nerolidol as major compound. Recently, we have shown that P. gaudichaudianum essential oil possesses strong cytotoxic effects in mammalian V79 cells. The aim of this study was to analyze the cytotoxicity and mutagenicity of P. gaudichaudianum essential oil and nerolidol using Saccharomyces cerevisiae as model study. Treatment of the XV185-14c and N123 strains with essential oil and nerolidol led to cytotoxicity but did not induce mutagenicity. Our results revealed an important role of base excision repair (BER) as the ntg1, ntg2, apn1 and apn2 mutants showed pronounced sensitivity to essential oil and nerolidol. In the absence of superoxide dismutase (in sod1Δ mutant strain) sensitivity to the essential oil and nerolidol increased indicating that this oil and nerolidol are generating reactive oxygen species (ROS). The ROS production was confirmed by DCF-DA probing assay in Sod-deficient strains. From this, we conclude that the observed cytotoxicity to P. gaudichaudianum essential oil and nerolidol is mainly related to ROS and DNA single strand breaks generated by the presence of oxidative lesions.

Trinucleotide repeat deletion via a unique hairpin bypass by DNA polymerase β and alternate flap cleavage by flap endonuclease 1

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegenerative diseases and prostate cancer. Recent studies have pointed to a linkage between oxidative DNA damage, base excision repair (BER) and TNR expansion, which is demonstrated by the observation that DNA polymerase β (pol β) gap-filling synthesis acts in concert with alternate flap cleavage by flap endonuclease 1 (FEN1) to mediate CAG repeat expansions. In this study, we provide the first evidence that the repair of a DNA base lesion can also contribute to CAG repeat deletions that were initiated by the formation of hairpins on both the template and the damaged strand of a continuous run of (CAG)20 or (CAG)25 repeats. Most important, we found that pol β not only bypassed one part of the large template hairpin but also managed to pass through almost the entire length of small hairpin. The unique hairpin bypass of pol β resulted in large and small deletions in coordination with FEN1 alternate flap cleavage. Our results provide new insight into the role of BER in modulating genome stability that is associated with human diseases.

An American Brain Tumor Consortium (ABTC) Phase 1 Trial of Veliparib (ABT-888), Temozolomide (TMZ), and Concurrent Radiation Therapy (RT) for Newly Diagnosed Glioblastoma Multiforme (GBM): Need for Modification to Approach for Combining These Therapies

PARP (Poly [ADP-ribose] polymerase) enzyme has a critical role in base excision repair for DNA damage by RT and TMZ. Veliparib is an oral PARP inhibitor that potentiates RT and TMZ in preclinical glioma models. ABTC conducted a phase I safety and dose finding trial of Veliparib given concurrent with standard TMZ and RT.

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3′-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3′-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3′-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3′-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe

We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3′-phosphatase activity is necessary for repair of DNA damage, whereas the 5′-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3′-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3′-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair.

XRCC1 Arg194Trp polymorphism, risk of nonmelanoma skin cancer and extramammary Paget’s disease in a Japanese population

The X-ray repair cross-complementing groups 1 gene plays an important role in base excision repair. At least three common single nucleotide polymorphisms frequently occur in this gene (Arg399Gln, Arg194Trp and Arg280His). Recent studies reported that these polymorphisms were associated with not only risk of visceral malignancy but also that of skin cancer such as basal cell carcinoma and squamous cell carcinoma, whereas the results of previous study vary among races. In this case–control study, we investigated whether these single nucleotide polymorphisms were associated with the risk of skin cancer in a Japanese population. The study population was composed of 197 patients with skin cancer (27 actinic keratoses, 47 basal cell carcinomas, 27 squamous cell carcinomas, 29 Bowen’s diseases, 46 malignant melanomas and 21 extramammary Paget’s diseases) and 93 control subjects. We genotyped two single nucleotide polymorphisms (Arg194Trp and Arg399Gln) using polymerase chain reaction-restriction fragments length polymorphism analysis. We found a significantly increased risk for basal cell carcinoma, squamous cell carcinoma and extramammary Paget’s disease associated with Arg194Trp [adjusted odds ratio (AOR) = 2.347, 3.587, 3.741, 95 % confidence interval (CI) 1.02–5.39, 1.19–10.8, 1.15–12.2, respectively]. We also found a significantly decreased risk for basal cell carcinoma associated with Gln399Gln (AOR = 0.259, 95 % CI 0.07–0.96). Our data suggest that the Arg194Trp polymorphism could be associated with nonmelanoma skin cancer and extramammary Paget’s disease risk in a Japanese population.

Abstract 2546: A novel DNA damage detection assay reveals a critical role of XPF in the repair of endogenous damage

Abstract Introduction: DNA damage, notably that resulting from defective nucleotide excision repair (NER), can lead to a number of diseases including those associated with neurodegeneration and cancer. XPF, a gene essential for effective NER, is typically associated with the repair of chemically and UV induced bulky DNA lesions as well as crosslinks. Recently, we developed a novel DNA damage detection assay (PADDA) capable of detecting at the nucleotide level, on a high-throughput scale, endogenous and induced DNA damage. We have previously validated this assay using multiple in vitro and in vivo approaches. By applying this assay to the study of wild-type (WT) and base excision repair (BER) defective cells, we have also reported the first direct evidence for preferential strand repair of endogenous DNA damage and documented the major role of BER in this process. In this study, we used PADDA to determine the role of XPF in the repair of endogenous DNA damage. Methods: Endogenous DNA damage was detected in the CAN1 gene by PADDA in wild-type and XPF defective S. cerevisiae cells in stationary phases of growth. Levels and locations of lesions were compared between strains. Locations of DNA damage were then compared to sites of previously reported mutations in the CAN1 gene using statistical approaches based on Chi-square goodness of fit and exact nonparametric tests. Results: PADDA revealed higher levels of endogenous DNA damage in XPF defective yeast cells compared to WT. Furthermore, a significantly higher rate of polymerase by-passable DNA damage was observed in XPF defective yeast cells compared to WT. There was a high association between the sites of by-passable endogenous DNA damage found in XPF defective cells to sites of previously reported mutations in the CAN1 gene. Conclusions: Our results establish yet another novel role for XPF protein: a major function in the repair of endogenous DNA damage. Furthermore, our results suggest a key XPF role in the repair of highly mutagenic by-passable DNA damage and therefore in the prevention of mutations caused by endogenous DNA damage. Given this important role, XPF may become a biomarker of cancer risk or even a suitable target for strategies aimed at preventing cancer and other diseases associated with DNA damage. Additionally, the ability of PADDA to map and quantify many different types of DNA damage suggests it may become a very important clinical tool to assess cancer risk and response to chemotherapy. Funding: This work was supported by the National Institute of Health [R03 CA117316-01] (AR) and the OUHSC Vice President for Research Fund (LQ). L.Q. holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2546. doi:1538-7445.AM2012-2546

Abstract 4430: Acute inflammation-induced abrogation of base excision repair as a potential mechanism of cancer initiation in inflammation-mediated hepatocarcinogenesis

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic inflammation seems to be the most important risk factor. Since inflammation induces oxidative stress and DNA damage, base excision repair (BER) of oxidative damage in prevention of mutations and subsequent tumorigenesis in an inflammatory environment seems to be important, but a direct link between deficient DNA repair and carcinogenesis in the highly damaging environment of the inflamed liver has not been shown. Furthermore, the longstanding question about how and in which cells cancer initiation occurs and the roles that DNA damage and repair play in the process are larger problems that remain unanswered. In our study we utilize the Long Evans Cinnamon (LEC) rat, which develops spontaneous HCC after consecutive phases of acute and chronic hepatitis induced by the accumulation of copper due to a mutation in the copper transporter gene ATP7B, mimicking Wilson's disease in humans. We have previously shown that the LEC rat exhibits deficient BER during acute hepatitis, particularly in the activity of 8-oxoguanine DNA glycosylase (Ogg1), and activity returns during chronic hepatitis as preneoplastic foci begin to form. In order to study the role of BER in cancer initiation, we have generated three new cell lines from the animal. We report here that we have isolated and grown apparently spontaneously immortalized LEC hepatocytes from the acute hepatitis liver (LEC-AH) and the chronic hepatitis liver (LEC-CH) in long term culture. We have also developed a carcinoma cell line from a LEC liver tumor (LEC-T). The newly generated cell lines express albumin, and preliminary karyotyping analysis shows that the LEC-AH and LEC-CH cells are cytogenetically normal (average 42 chromosomes). As observed in the acute and chronic hepatitis tissues, LEC-AH cells exhibit decreased activity (-2.5 fold) of Ogg1 compared to LEC-CH cells. Furthermore, at 12 days post-confluency, we have observed spontaneous foci formation in the LEC-AH cells (96 foci per 106 cells) in culture while LEC-CH cells do not form foci (<1 foci per 106 cells). These preliminary results indicate a correlation between foci formation in culture and Ogg1 activity, demonstrating a potential direct link between decreased BER and preneoplastic foci formation. Additionally, the foci may represent an enriched population of initiated cells, providing a unique opportunity to study the mechanism of spontaneous cancer initiation in vitro and identify initiation markers. Experiments elucidating the specific roles of Ogg1, other BER enzymes, and oxidative damage in foci formation of LEC-AH cells are in progress, as is further molecular analysis of the foci-forming cell populations as they may reflect the biochemical and molecular characteristics of preneoplastic foci in vivo. (Supported by a grant from NIH/NCI RO1 CA113447) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4430. doi:1538-7445.AM2012-4430