BACKGROUND: The APOBEC gene family of cytidine deaminases plays important roles in DNA repair and mRNA editing. In many cancers, APOBEC3B increases the mutation load, generating clusters of closely spaced, single-strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested a possible involvement of APOBEC3A, REV1, UNG, and FHIT in molecular processes affecting APOBEC mutagenesis. It is important to understand how mutagenic processes linked to the activity of these genes may affect sensitivity of cancer cells to treatment.
RESULTS: We used information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine associations of the prevalence of APOBEC-like motifs and mutational loads with expression of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT and with cell line chemosensitivity to 255 antitumor drugs. Among the five genes, APOBEC3B expression levels were bimodally distributed, whereas expression of APOBEC3A, REV1, UNG, and FHIT was unimodally distributed. The majority of the cell lines had low levels of APOBEC3A expression. The strongest correlations of gene expression levels with mutational loads or with measures of prevalence of APOBEC-like motif counts and kataegis clusters were observed for REV1, UNG, and APOBEC3A. Sensitivity or resistance of cell lines to JQ1, palbociclib, bicalutamide, 17-AAG, TAE684, MEK inhibitors refametinib, PD-0325901, and trametinib and a number of other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancers or across cancer types.
CONCLUSIONS: We observed correlations of expression levels of the five candidate genes in cell line models with sensitivity to cancer drug treatment. We also noted suggestive correlations between measures of abundance of APOBEC-like sequence motifs with drug sensitivity in small samples of cell lines from individual cancer categories, which require further validation in larger datasets. Molecular mechanisms underlying the links between the activities of the products of each of the five genes, the resulting mutagenic processes, and sensitivity to each category of antitumor agents require further investigation.

Cisplatin exert its anticancer effect by creating intrastrand and interstrand DNA cross-links which block DNA replication and is a major drug used to treat lung cancer. However, the main obstacle of the efficacy of treatment is drug resistance. Here, we show that expression of translesion synthesis (TLS) polymerase Q (POLQ) was significantly elevated by exposure of lung cancer cells A549/DR (a cisplatin-resistant A549 cell line) to cisplatin. POLQ expression correlated inversely with homologous recombination (HR) activity. Co-depletion of BRCA2 and POLQ by siRNA markedly increased sensitivity of A549/DR cells to cisplatin, which was accompanied with impairment of double strand breaks (DSBs) repair reflected by prominent cell cycle checkpoint response, increased chromosomal aberrations and persistent colocalization of p-ATM and 53BP1 foci induced by cisplatin. Thus, co-knockdown of POLQ and HR can efficiently synergize with cisplatin to inhibit A549/DR cell survival by inhibiting DNA DSBs repair. Similar results were observed in A549/DR cells co-depleted of BRCA2 and POLQ following BMN673 (a PARP inhibitor) treatment. Importantly, the sensitization effects to cisplatin and BMN673 in A549/DR cells by co-depleting BRCA2 and POLQ was stronger than those by co-depleting BRCA2 and other TLS factors including POLH, REV3, or REV1. Our results indicate that there is a synthetic lethal relationship between pol θ-mediated DNA repair and HR pathways. Pol θ may be considered as a novel target for lung cancer therapy.

DNA damage response failure may influence the efficacy of DNA-damaging treatments. We determined the expression of 16 genes involved in distinct DNA damage response pathways, in association with the response to standard therapy. Twenty patients with locoregionally advanced, squamous cell head and neck carcinoma were enrolled. The treatment included induction chemotherapy (iChT) with docetaxel, cisplatin and 5-fluorouracil followed by concomitant chemoradiotherapy (ChRT) or radiotherapy (RT) alone. The volumetric metabolic therapeutic response was determined by [18F]FDG-PET/CT. In the tumor and matched normal tissues collected before treatment, the gene expressions were examined via the quantitative real-time polymerase chain reaction (qRT-PCR). The down-regulation of TP53 was apparently associated with a poor response to iChT, its up-regulation with complete regression in 2 cases. 7 cases with down-regulated REV1 expression showed complete regression after ChRT/RT, while 1 case with REV1 overexpression was resistant to RT. The overexpression of WRN was an independent predictor of tumor relapse. Our results suggest that an altered expression of REV1 predicts sensitivity to RT, while WRN overexpression is an unfavorable prognostic factor.

This study explored candidate causal single nucleotide polymorphisms (SNPs) to clarify the biological mechanism of prostate cancer (PCa). Identify candidate Causal SNPs and Pathways (ICSNPathway) analysis was applied using a PCa genome-wide association study (GWAS) dataset that included 473,736 SNPs in 1151 cases of PCa and 1156 controls of European ancestry. Five candidate causal SNPs, three candidate causal genes, and two candidate causal pathways were identified using integrating linkage disequilibrium analysis, functional SNP annotation, and pathway-based analysis. The ICSNPathway analysis suggested three hypothetical mechanisms of PCa. The first was rs13112390, rs13112358, rs2048074 to nei-like DNA glycosylase 3 (NEIL3) gene to damaged DNA binding. The second was rs3087386 to REV1, DNA directed polymerase (REV1) gene to damaged DNA binding. The third was rs1063134 to potassium channel, inwardly rectifying subfamily J, member 4 (KCNJ4) gene to inward rectifier potassium channel activity.

REV7 is a multifunctional protein involved in DNA damage tolerance, cell-cycle regulation, gene expression, and carcinogenesis. Although its expression is reportedly associated with poor prognosis in human solid tissue cancers, the significance of REV7 expression in hematopoietic malignancies is unclear. This study evaluated the prognostic significance of REV7 expression in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-combined chemotherapy. Using immunohistochemistry, we analyzed 83 specimens of de novo DLBCL [38 germinal center B-cell-like (GCB) and 45 non-GCB DLBCLs] treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone for REV7 expression. Aberrant REV7 expression was detected in DLBCL cell nuclei. High REV7 expression was associated with significantly shorter overall survival (OS) and progression-free survival (PFS) using Kaplan-Meier analysis and log-rank tests (P < 0.01 and P < 0.01, respectively). Multivariate analysis revealed that REV7 expression is an independent prognostic factor for both OS and PFS. Additionally, when patients were divided into four groups using a combination of REV7 expression and international prognostic index (IPI) or Bcl-2 expression, REV7(High)/IPI(Poor) and REV7(High)/Bcl-2(High) patients showed the poorest outcome. These results indicate that REV7 may be a useful biomarker to predict the prognosis of patients with DLBCL treated with rituximab.

Cytotoxic activity of most chemotherapeutic agents is based on their ability to induce DNA damage. Interstrand crosslinks are among the most detrimental forms of DNA damage as both DNA strands are affected. As translesion polymerases participate in their repair, they may be important for response to chemotherapeutic agents that induce such lesions, including commonly used cisplatin. Altered expression of translesion polymerase genes REV1 and REV3L may modify sensitivity to cisplatin. As osteosarcoma patients are commonly treated with cisplatin-based chemotherapy, our aim was to investigate if REV1 and REV3L polymorphisms influence survival of osteosarcoma patients treated with cisplatin-based chemotherapy. We determined the genotypes of common functional tag REV1 and REV3L polymorphisms in 66 osteosarcoma patients. Cox regression was used for survival analysis. Carriers of at least one polymorphic REV1 rs3087403 allele had significantly shorter EFS and overall survival (OS) (p = 0.004; HR = 3.79; 95%CI = 1.53-9.35 and p < 0.001; HR = 4.44; 95%CI = 1.92-10.27, respectively). Combination of REV1 rs3087403 and REV3L rs462779 polymorphisms was also significantly associated with shorter OS (ptrend<0.001) and shorter EFS (ptrend = 0.003). The results of this first study on polymorphisms in translesion polymerase genes in osteosarcoma suggest they could help predict outcome of cisplatin-based chemotherapy in osteosarcoma patients.

Short-term starvation or fasting can augment cancer treatment efficacy and can be effective in delaying cancer progression in the absence of chemotherapy, but the underlying molecular mechanisms of action remain elusive. Here, we describe the role of REV1, a specialized DNA polymerase involved in DNA repair, as an important signaling node linking nutrient sensing and metabolic control to cell fate. We show that REV1 is a novel binding partner of the tumor suppressor p53 and regulates its activity. Under starvation, REV1 is modified by SUMO2/3, resulting in the relief of REV1's inhibition of p53 and enhancing p53's effects on proapoptotic gene expression and apoptosis in breast cancer and melanoma cells. Thus, fasting in part through its effect on REV1 is a promising nontoxic strategy to increase p53-dependent cell death and to enhance the efficacy of cancer therapies.

AIM: We evaluated the influence of genetic variability in translesion polymerases REV1 and REV3L on the outcome of cisplatin treatment in malignant mesothelioma patients.
MATERIALS & METHODS: In total, 139 malignant mesothelioma patients were genotyped for seven tag SNPs in REV1 and REV3L. Logistic regression and Cox regression were used to assess the influence of SNPs on treatment outcome.
RESULTS: Polymorphic REV1 rs3087403 allele and REV1 TGT haplotype were associated with increased risk for leukopenia (p = 0.013 and p = 0.047, respectively) and neutropenia (p = 0.048 and p = 0.024, respectively). REV3L rs465646, rs462779 and REV3L CCGG haplotype were significantly associated with longer overall survival (p = 0.007, p = 0.022 and p = 0.013, respectively).
CONCLUSION: Our results suggest for the first time that REV1 and REV3L SNPs might serve as potential predictive markers of outcome of cisplatin-based chemotherapy. Original submitted 7 October 2013; Revision submitted 15 January 2014.

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies.

PURPOSE: Translesion DNA synthesis (TLS) plays an important role in promoting replication through DNA lesions. Genetic polymorphisms in TLS genes may have potential roles in lung cancer development in humans.
METHODS: We evaluated the association between genetic variants in six TLS genes and the risk and survival of lung cancer in a case-control study in China. Included in the study are 224 lung cancer patients and 448 healthy controls.
RESULTS: Carriers of the G allele of POLκ rs5744724 had significantly reduced risk of lung cancer (odds ratio (OR)=0.62, 95% confidence interval (CI): 0.44-0.89), comparing with those carrying the C allele, and the AA genotype of PCNA rs25406 was also associated with significantly decreased cancer risk compared with the major homozygote alleles (OR=0.47, 95% CI: 0.25-0.86). Haplotype analysis showed that subjects with the POLκ C-G (rs5744533-rs5744724) haplotype had decreased risk of lung cancer (OR=0.69, 95% CI: 0.49-0.98), comparing with those carrying the C-C haplotype. Besides, the heterozygote of REV1 rs3087386 and rs3792136 were independent prognostic factors for lung cancer survival with hazard radio (HR) 1.54 (95% CI: 1.12-2.12) and 1.44 (95% CI: 1.06-1.97) respectively.
CONCLUSIONS: Our findings suggested that genetic variants in POLκ and PCNA genes may play roles in the susceptibility of lung cancer, and REV1 gene may have roles in lung cancer survival in Chinese men.

INTRODUCTION: Black/white disparities in lung cancer incidence and mortality mandate an evaluation of underlying biological differences. We have previously shown higher risks of lung cancer associated with prior emphysema in African American compared with white patients with lung cancer.
METHODS: We therefore evaluated a panel of 1440 inflammatory gene variants in a two-phase analysis (discovery and replication), added top genome-wide association studies (GWAS) lung cancer hits from white populations, and 28 single-nucleotide polymorphisms (SNPs) from a published gene panel. The discovery set (477 self-designated African Americans cases, 366 controls matched on age, ethnicity, and gender) were from Houston, Texas. The external replication set (330 cases and 342 controls) was from the EXHALE study at Wayne State University.
RESULTS: In discovery, 154 inflammation SNPs were significant (p < 0.05) on univariate analysis, as was one of the gene panel SNPs (rs308738 in REV1, p = 0.0013), and three GWAS hits, rs16969968 p = 0.0014 and rs10519203 p = 0.0003 in the 15q locus and rs2736100, in the HTERT locus, p = 0.0002. One inflammation SNP, rs950286, was successfully replicated with a concordant odds ratio of 1.46 (1.14-1.87) in discovery, 1.37 (1.05-1.77) in replication, and a combined odds ratio of 1.40 (1.17-1.68). This SNP is intergenic between IRF4 and EXOC2 genes. We also constructed and validated epidemiologic and extended risk prediction models. The area under the curve (AUC) for the epidemiologic discovery model was 0.77 and 0.80 for the extended model. For the combined datasets, the AUC values were 0.75 and 0.76, respectively.
CONCLUSIONS: As has been reported for other cancer sites and populations, incorporating top genetic hits into risk prediction models, provides little improvement in model performance and no clinical relevance.

Proteomic studies to date have had limited use as an investigative tool in the skin's response to UV radiation. These studies used cell lines and reconstructed skin and have shown evidence of cell injury with oxidative damage and stress induced heat shock proteins. Others changes included altered cytokeratin and cytoskeletal proteins with enhanced expression of TRIM29 as the keratinocytes regenerate. The associated DNA repair requires polη, Rad18/Rad16 and Rev1. In the whole animal these events would be associated with inflammation, remodelling of the epidermis and modulation of the immune response. Longer term changes include ageing and skin cancers such as melanoma, squamous cell carcinoma and basal cell carcinoma. In the future proteomics will be used to explore these important aspects of photobiology. Better characterisation of the proteins involved should lead to a greater understanding of the skin's response to UV radiation.

Cellular DNA is continuously assaulted by chemical and physical agents that arise from both endogenous metabolic processes as well as exogenous insults. Commonly encountered environmental agents include polyaromatic hydrocarbons, polycyclic aromatic amines, the ultraviolet component of sunlight, and ionizing radiation, among many others. Although the kinds of damages and the mechanisms involved in their interaction with DNA vary widely, genotoxic agents alter the structure of DNA in ways that may result in permanent alterations in the DNA sequence or in cell death. To avoid these consequences, cells have evolved countermeasures to reduce the biological consequences of DNA damage. These mechanisms are highly conserved and are present in all eukaryotic cells. In general, cellular responses include the detection of damage, signal transduction to halt cell cycle progression, and the recruitment of repair mechanisms that are tailored to the specific kind of damage. If replication-blocking damage remains when cells enter S-phase, then tolerance mechanisms in the form of complex recombination mechanisms or translesion DNA synthesis using accessory DNA polymerases exist. These mechanisms complete the replication of damaged genomes and suppress cytotoxicity, but at the potential cost of mutagenesis and genomic instability. This review focuses on error-prone mechanisms, including a discussion of the Y-family of DNA polymerases, current concepts of DNA polymerase switching mechanisms, and their relevance to cancer and cancer prevention.

Chromosomal instability is a known hallmark of many cancers. DNA polymerases represent a group of enzymes that are involved in the mechanism of chromosomal instability as they have a central function in DNA metabolism. We hypothesized that genetic variation in the polymerase genes may affect gene expression or protein configuration and by that cancer risk and clinical outcome. We selected four genes encoding for the catalytic subunits of the polymerases β, δ, θ and ζ (POLB, POLD1, POLQ and REV3L, respectively) and two associated proteins (MAD2L2 and REV1) because of their previously reported association with chromosomal instability and/or tumorigenesis. We selected potentially functional and most informative tagging single nucleotide polymorphisms (SNPs) for genotyping in a population-based series of 783 Swedish breast cancer (BC) cases and 1562 controls. SNPs that showed a significant association in the Swedish population were additionally genotyped in a Polish population consisting of 506 familial/early onset BC cases and 568 controls. SNPs in all three polymerase ζ subunit genes associated either with BC risk or prognosis. Two SNPs in REV3L and one SNP in MAD2L2 associated with BC risk: rs462779 (multiplicative model: OR 0.79, 95% CI 0.68-0.92), rs3204953 (dominant model: OR 1.28, 95% CI 1.05-1.56) and rs2233004 (recessive model: OR 0.49, 95% CI 0.28-0.86). Homozygous carriers of the minor allele C of the third SNP in REV3L, rs11153292, had significantly worse survival compared to the TT genotype carriers (HR 2.93, 95% CI 1.34-6.44). Minor allele carriers of two REV1 SNPs (rs6761391 and rs3792142) had significantly more often large tumours and tumours with high histological grade and stage. No association was observed for SNPs in POLB, POLQ and POLD1. Altogether, our data suggest a significant role of genetic variation in the polymerase ζ subunit genes regarding the development and progression of BC.

There are many various chromosomal and gene mutations in human cancer cells. The total mutation rate in normal human cells is 2·10(-7) mutations/gene/division. From 6 to 12 carcinogenic mutations can arise by the end of the life, and these can affect the structure of ~150 protooncogenes and genes encoding suppressors of tumor growth. However, this does not explain the tens and hundreds of thousands of mutations detectable in cancer cells. Mutation is any change of nucleotide sequence in cellular DNA. Gene mutations are mainly consequences of errors of DNA polymerases, especially of their specialized fraction (inaccurate DNA polymerases β, ζ, η, θ, ι, κ, λ, µ, σ, ν, Rev1, and terminal deoxynucleotidyl transferase, and only polymerases θ and σ manifest a slight 3'-exonuclease activity) and also consequences of a decrease in the rate of repair of these errors. Inaccurate specialized human polymerases are able to synthesize DNA opposite lesions in the DNA template, but their accuracy is especially low during synthesis on undamaged DNA. In the present review fundamental features of such polymerases are considered. DNA synthesis stops in the area of its lesion, but this block is overcome due to activities of inaccurate specialized DNA polymerases. After the lesion is bypassed, DNA synthesis is switched to accurate polymerases α, δ, ε, or γ. Mechanisms of direct and reverse switches of DNA polymerases as well as their modifications during carcinogenesis are discussed.

BACKGROUND: Papillary thyroid carcinoma (PTCs), the most frequent thyroid cancer, is usually not life threatening, but may recur or progress to aggressive forms resistant to conventional therapies. A more detailed understanding of the signaling pathways activated in PTCs may help to identify novel therapeutic approaches against these tumors. The aim of this study is to identify signaling pathways activated in PTCs.
METHODOLOGY/PRINCIPAL FINDINGS: We examined coordinated gene expression patterns of ligand/receptor (L/R) pairs using the L/R database DRLP-rev1 and five publicly available thyroid cancer datasets of gene expression on a total of 41 paired PTC/normal thyroid tissues. We identified 26 (up) and 13 (down) L/R pairs coordinately and differentially expressed. The relevance of these L/R pairs was confirmed by performing the same analysis on REarranged during Transfection (RET)/PTC1-infected thyrocytes with respect to normal thyrocytes. TGFA/EGFR emerged as one of the most tightly regulated L/R pair. Furthermore, PTC clinical samples analyzed by real-time RT-PCR expressed EGFR transcript levels similar to those of 5 normal thyroid tissues from patients with pathologies other than thyroid cancer, whereas significantly elevated levels of TGFA transcripts were only present in PTCs. Biochemical analysis of PTC cell lines demonstrated the presence of EGFR on the cell membrane and TGFA in conditioned media. Moreover, conditioned medium of the PTC cell line NIM-1 activated EGFR expressed on HeLa cells, culminating in both ERK and AKT phosphorylation. In NIM-1 cells harboring BRAF mutation, TGFA stimulated proliferation, contributing to PI3K/AKT activation independent of MEK/ERK signaling.
CONCLUSIONS/SIGNIFICANCE: We compiled a reliable list of L/R pairs associated with PTC and validated the biological role of one of the emerged L/R pair, the TGFA/EGFR, in this cancer, in vitro. These data provide a better understanding of the factors involved in the biology of PTCs and would be useful in developing combination therapeutic approaches against these cancers.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine in cooked foods, and is both mutagenic and carcinogenic. It has been suspected that the carcinogenicity of PhIP is derived from its ability to form DNA adducts, principally dG-C8-PhIP. To shed further light on the molecular mechanisms underlying the induction of mutations by PhIP, in vitro DNA synthesis analyses were carried out using a dG-C8-PhIP-modified oligonucleotide template. In this template, the dG-C8-PhIP adduct was introduced into the second G of the TCC GGG AAC sequence located in the 5' region. This represents one of the mutation hot spots in the rat Apc gene that is targeted by PhIP. Guanine deletions at this site in the Apc gene have been found to be preferentially induced by PhIP in rat colon tumors. DNA synthesis with A- or B-family DNA polymerases, such as Escherichia coli polymerase (pol) I and human pol delta, was completely blocked at the adducted guanine base. Translesional synthesis polymerases of the Y-family, pol eta, pol iota, pol kappa, and REV1, were also used for in vitro DNA synthesis analyses with the same templates. REV1, pol eta, and pol kappa were able to insert dCTP opposite dG-C8-PhIP, although the efficiencies for pol eta and pol kappa were low. pol kappa was also able to catalyze the extension reaction from the dC opposite dG-C8-PhIP, during which it often skipped over one dG of the triple dG sequence on the template. This slippage probably leads to the single dG base deletion in colon tumors.

Breast cancer is the most frequent cancer in females worldwide and it has long been known that multiple genetic rearrangements correlate with complex biology and clinical behavior. In addition, copy number variations (CNVs) of DNA sequences account for a significant proportion of normal phenotypic variation and may have an important role in human pathological variation. In this study, we carried out a high-density oligonucleotide array comparative genomic hybridization (CGH) analyses in a series of breast cancer cell lines to identify novel homozygous deletion loci. The results were confirmed by quantitative PCR (Q-PCR) and 4 genes, the REV1L, ZNF14, NPAS1 and APOBEC3B genes, were selected. Analyses of 30 microdissected human breast tumors and paired normal mammary tissue samples indicated that these homozygous deletions are small-scale deletion polymorphisms. The variation in copy number at the loci of the 4 genes in blood-derived DNA demonstrated the frequency of deletions including homozygous deletions and single copy variants to be higher in breast cancer patients than healthy females. Notably, the homozygous deletion of APOBEC3B involved part of exon 5 and seemed to be cancer-specific in some patients, indicating that this is a functionally important structural variant. These copy number changes may play an important role in breast cancer and array-CGH analyses can thus be expected to provide new insight into the genetic background of breast cancer.

REV1 interacts with Y-type DNA polymerases (Pol) and Pol zeta to bypass many types of adducts that block the replicative DNA polymerases. This pathway accounts for many of the mutations induced by cisplatin (cis-diamminedichloroplatinium II, DDP). This study sought to determine how increasing human REV1 (hREV1) affects the cytotoxicity and mutagenicity of DDP. Human ovarian carcinoma 2008 cells were transfected with an hREV1 expression vector and 4 sublines developed in which the hREV1 mRNA level was increased by 6.3- to 23.4-fold and hREV1 protein by 2.7- to 6.2-fold. The sublines were 1.3- to 1.7-fold resistant to the cytotoxic effect of DDP and 2.3- to 5.1-fold hypersensitive to the mutagenic effect of DDP. The hREV1-transfected sublines were 1.5- to 1.8-fold better than the parental 2008 cells at managing DDP adducts as assessed by their ability to express Renilla reniformis luciferase from a vector that had been extensively loaded with DDP adducts before transfection. Increased hREV1 expression was associated with a 1.5-fold increase in the rate at which the whole population acquired resistance to DDP during sequential cycles of drug exposure. Increasing the abundance of hREV1 thus resulted in both resistance to DDP and a significant elevation in DDP-induced mutagenicity. This was accompanied by an enhanced capacity to synthesize a functional protein from a DDP-damaged gene and, most importantly, by more rapid development of resistance during sequential cycles of DDP exposure that mimic clinical schedules of DDP administration. We conclude that hREV1-dependent processes are important determinants of DDP-induced genomic instability and the development of resistance.

Replicative bypass of many DNA adducts is dependent on the interaction of hREV1 with DNA polymerase zeta and potentially with members of the Y family of DNA polymerases. To examine the role of hREV1 in the development of cisplatin (DDP) resistance, a subline (2008-shREV1-3.3) of the ovarian carcinoma cell line 2008 was isolated in which stable expression of a short hairpin RNA suppressed hREV1 expression to 20% and reduced hREV1 protein level to 43% of that found in the parental cells. The 2008-shREV1-3.3 cells were 1.5-fold more sensitive to the cytotoxic effect of DDP but less sensitive to the mutagenic effect of DDP as evidenced by a 2.6- or 2.7-fold reduction in the ability to induce clones highly resistant to 6-thioguanine or DDP itself, respectively, in the surviving population. Reduction of hREV1 did not alter the initial rate of DDP adduct removal from DNA but did impair both spontaneous and DDP-induced extra-chromosomal homologous recombination, as measured by the recombination-sensitive reporter vector pBHRF. DDP induced an increase in hREV1 protein level. DDP resistance at the population level evolved 2.8-fold more slowly in the 2008-shREV1-3.3 cells than in the parental cells during repeated cycles of drug exposure. The results indicate that hREV1 functions to enhance both cell survival and the generation of drug-resistant variants in the surviving population. DDP up-regulates hREV1, suggesting that it may enhance its own mutagenicity. Most importantly, hREV1 controls the rate of emergence of resistance to DDP at the population level. Thus, hREV1 is an important contributor to DDP-induced genomic instability and the subsequent emergence of resistance.

Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (associated with amino acid changes) SNPs identified were assessed for associations with lung cancer risk by a case-control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest association with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double-strand break repair protein, also showed associations (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, respectively). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was associated with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages < 61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, respectively). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.

In order to identify genes whose expression is associated with resistance to the chemotherapeutic agent oxaliplatin, transcripts differentially expressed between an oxaliplatin sensitive and a stably resistant subline were compared in six independent replicates using Stanford cDNA microarrays for five cell lines. "Significance analysis of microarrays" (SAM) was used to identify genes whose expression was statistically significantly different in the sensitive versus resistant members of each cell line pair. The biochemical pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were searched to identify those pathways in which the number of SAM-identified genes exceeded the number expected. This identified four pathways in which upregulated genes were significantly associated with resistance in two of the cell line pairs, and two pathways in which the association was found in three cell line pairs. The search also identified 12 pathways in which downregulated genes were associated with resistance in two cell line pairs and one pathway in which the association reached statistical significance in three cell line pairs. Pathways identified included the ribosome pathway, the Huntington's disease pathway that includes caspase 8, and the ATP synthesis pathways. Determination of the chromosomal location of each SAM-identified gene revealed several locales within which genes lay in close proximity, including three genes (APACD, IF-2, and REV1L) located on chromosome 2 that lie immediately adjacent to each other and were significantly upregulated in three of five cell line pairs. Biochemical pathway and chromosomal mapping of genes identified by SAM as differentially expressed in related cell line pairs points to mechanisms and chromosomal sites not previously suspected of association with the oxaliplatin-resistant phenotype.

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.

Increased spontaneous mutation is associated with increased cancer risk. Here, by using a model system, we show that spontaneous mutation can be increased several hundred-fold by a simple imbalance between the first two enzymes involved in DNA base excision repair. The Saccharomyces cerevisiae MAG1 3-methyladenine (3MeA) DNA glycosylase, when expressed at high levels relative to the apurinic/apyrimidinic endonuclease, increases spontaneous mutation by up to approximately 600-fold in S. cerevisiae and approximately 200-fold in Escherichia coli. Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1/REV3/REV7 lesion bypass pathway. Comparison of the mutator activity produced by Mag1, which has a broad substrate range, with that produced by the E. coli Tag 3MeA DNA glycosylase, which has a narrow substrate range, indicates that the removal of endogenously produced 3MeA is unlikely to be responsible for the mutator effect of Mag1. Finally, the human AAG 3-MeA DNA glycosylase also can produce a small (approximately 2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.