DNA Repair Proficiency Research Articles

Nanotechnology in Cancer Therapy: Targeting the Inhibition of Key DNA Repair Pathways

Cancer therapy has been changing over the decades as we move away from the administration of broad spectrum cytotoxic drugs and towards the use of therapy targeted for each tumor type. After the induction of DNA damage through chemotherapeutic agents, tumor cells can survive due to their proficient DNA repair pathways, some of which are dysregulated in cancer. Latest improvements in nanotechnology and drug discovery has led to the discovery of some very unique, highly specific and innovative drugs as inhibitors of various DNA repair pathways like base excision repair and double strand break repair. In this review we look at the efficacy and potency of these small chemical molecules to target the processing of DNA damage induced by standard therapeutic agents. Emphasis is given to those drugs currently under clinical trials. We also discuss the future directions of using this nanotechnology to increase the therapeutic ratio in cancer treatment.

Sensitization to Radiation and Alkylating Agents by Inhibitors of Poly(ADP-ribose) Polymerase Is Enhanced in Cells Deficient in DNA Double-Strand Break Repair

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Wip1 Directly Dephosphorylates γ-H2AX and Attenuates the DNA Damage Response

The integrity of DNA is constantly challenged throughout the life of a cell by both endogenous and exogenous stresses. A well-organized rapid damage response and proficient DNA repair, therefore, become critically important for maintaining genomic stability and cell survival. When DNA is damaged, the DNA damage response (DDR) can be initiated by alterations in chromosomal structure and histone modifications, such as the phosphorylation of the histone H2AX (the phosphorylated form is referred to as gamma-H2AX). gamma-H2AX plays a crucial role in recruiting DDR factors to damage sites for accurate DNA repair. On repair completion, gamma-H2AX must then be reverted to H2AX by dephosphorylation for attenuation of the DDR. Here, we report that the wild-type p53-induced phosphatase 1 (Wip1) phosphatase, which is often overexpressed in a variety of tumors, effectively dephosphorylates gamma-H2AX in vitro and in vivo. Ectopic expression of Wip1 significantly reduces the level of gamma-H2AX after ionizing as well as UV radiation. Forced premature dephosphorylation of gamma-H2AX by Wip1 disrupts recruitment of important DNA repair factors to damaged sites and delays DNA damage repair. Additionally, deletion of Wip1 enhances gamma-H2AX levels in cells undergoing constitutive oncogenic stress. Taken together, our studies show that Wip1 is an important mammalian phosphatase for gamma-H2AX and shows an additional mechanism for Wip1 in the tumor surveillance network.

Regulation of Hematopoietic Stem Cell Quiescence by Ku70.

Abstract 3613Poster Board III-549Genomic integrity is essential for organism development and longevity, and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is essential for DNA double-strand break repair in all cells and for VDJ processing in B and T cells. NHEJ is also critical for hematopoietic stem cell (HSC) maintenance and function, but the mechanisms by which the NHEJ pathway regulates HSC function are not known. Ku70 is a key component of NHEJ; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Previously we have shown that HSC from Ku70-deficient mice are defective in repopulation, self-renewal and competitive repopulation. Also, mice defective in other NHEJ components, Ku80 or ligase 4, display impaired HSC function and reduced HSC numbers during aging. Here we used Ku70-deficient mice to investigate the role of the NHEJ pathway in HSC function.Ku70-deficient mice show comparable HSC (Sca1+,c-Kit+,Lin-) frequency to Wild-type (WT) mice, and HSCs from Ku70-deficient mice display similar apoptosis rates as HSCs from WT mice. SKL cells from both Ku70-deficient and WT mice proliferate at similar rates in vitro in the presence of IL3, SCF, Flt3-L and Tpo. However, a greater percentage of HSCs from Ku70-deficient mice are actively cycling, and fewer are quiescent than those obtained from WT mice. Further, recipients of Ku70-deficient BM are more sensitive to 5-FU treatment than those receiving WT BM. In addition, WT HSCs efficiently replace endogenous Ku70-deficient HSCs when WT BM cells are transplanted into Ku70-deficient mice in the absence of any conditioning.Together, our data indicate that loss of quiescence in Ku70-deficient HSCs correlates with dramatic defects observed in repopulation, self-renewal and competitive repopulation. These results suggest that the NHEJ pathway, either through the processes of DNA repair and genome maintenance, or through a stress response signaling pathway, is critical for maintaining HSCs in a quiescent state, avoiding age-related physiological stress, and allowing functional stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Probing Nucleosome Function: A Highly Versatile Library of Synthetic Histone H3 and H4 Mutants

Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

Stem Cell–Related “Self-Renewal” Signature and High Epidermal Growth Factor Receptor Expression Associated With Resistance to Concomitant Chemoradiotherapy in Glioblastoma

Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

XRCC1 polymorphisms and severe toxicity in lung cancer patients treated with cisplatin-based chemotherapy in Chinese population

Cisplatin kills tumor cells through DNA cross linking. Alterations in the function of DNA repair genes may affect DNA repair proficiency and influence cancer patients' response to cisplatin. The predictability of DNA repair XRCC1 (X-ray repair cross-complementing group 1 protein) single nucleotide polymorphisms (SNPs) for cisplatin-based grades 3 and 4 chemotherapy-related toxicity in patients with newly diagnosed advanced lung cancer was evaluated. The genotypes of XRCC1 at the Arg194Trp, and Arg399Gln sites were determined by PCR-based restriction fragment length polymorphism (RFLP) methods. There was no statistically significant association between either the Arg194Trp or the Arg399Gln polymorphisms and hematologic grade 3 or 4 toxicity. However, carrying at least one variant XRCC1 Arg399Gln allele (399Arg/Gln or 399Gln/Gln) was associated with a significantly increased risk of overall grade 3 or 4 toxicity (odds ratio, 2.05; 95% confidence interval, 1.02-4.10; p=0.04); and grade 3 or 4 gastrointestinal toxicity (odds ratio, 2.53; 95% confidence interval, 1.06-6.03; p=0.03). Our results suggested that patients carrying at least one variant XRCC1 Arg399Gln allele have a 2.5-fold increased risk of grade 3 or 4 gastrointestinal toxicity when treated with first-line cisplatin-based chemotherapy.

Six Genes Associated with the Clinical Phenotypes of Individuals with Deficient and Proficient DNA Repair

Xeroderma pigmentosum (XP) is a genetic disorder characterised by hypo-/hyperpigmentation, increased sensitivity to ultraviolet (UV)-radiation and an up to 2000-fold increased skin cancer risk. Cells from XP-patients are defective in nucleotide excision repair (NER) responsible for repair of UV-induced DNA damage. This defect accounts for their mutator phenotype but does not predict their increased skin cancer risk. Therefore, we carried out array analysis to measure the expression of more than 1000 genes after UVB-irradiation in XP cells from three complementation groups with different clinical severity (XP-A, XP-D, XP-F) as well as from patients with normal DNA repair but increased skin cancer risk (≥2 basal or squamous cell carcinoma at age <40yrs). Of 144 genes investigated, 20 showed differential expression with p < 0.05 after irradiation of cells with 100 mJ/cm(2) of UVB. A subset of six genes showed a direct association of expression levels with clinical severity of XP in genes affecting carcinogenesis relevant pathways. Genes identified in XP cells could be confirmed in cells from patients with no known DNA repair defects but increased skin cancer risk. Thus, it is possible to identify a small gene subset associated with clinical severity of XP patients also applicable to individuals with no known DNA repair defects.

Gene-Diet Interaction: DNA Repair Gene, Folate Status and Breast Cancer Risk

Folate levels have been inversely associated with breast cancer risk. Because folate deficiency can cause DNA damage such as uracil misincorporation, single-strand breaks, and double-strand breaks, genetic polymorphisms in base excision repair and double-strand break repair genes may lead to variation in DNA repair proficiency and modify the effect of folate on breast cancer risk. We present two examples of interaction between plasma folate levels and DNA repair genetic variants in a nested case-control study within the Nurses’ Health Study (712 case-control pairs). Compared with the reference group of non-carriers in the lowest quartile of plasma folate, the reduction in risk (66%) was statistically significant among XRCC1 194Trp carriers in the highest quartile (multivariate odds ratio, 0.34; 95% confidence interval, 0.16–0.72). The inverse association between XRCC1 194Trp and breast cancer risk was attenuated by lower plasma folate status. The inverse association between plasma folate level and breast cancer risk was stronger among 194Trp carriers (P, trend = 0.01) than non-carriers (P, trend = 0.09). We also observed that the positive association between the XRCC2 188His allele and breast cancer risk was significant only in women in the lowest plasma folate quartile (carriers versus non-carriers; multivariate odds ratio, 2.04; 95% confidence interval, 1.05–3.97), and this excess risk was abolished among those with higher plasma folate levels. Moreover, the inverse association between plasma folate level and breast cancer risk was stronger among XRCC2 188His carriers (P, trend = 0.004) than non-carriers (P, trend = 0.09). Although none of the statistical tests for interaction was significant, these data give some support to the hypothesis that genetic variations in DNA repair genes modify the relation between plasma folate level and breast cancer risk.

Loss of Ku70 Results in a HSC Repopulating Defect.

Genomic integrity is essential for organism development and longevity and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is a specific pathway essential for DNA double-strand break repair in all cells and VDJ processing in B and T cells; and it is crucial for maintenance of mammalian genome. We hypothesize that NHEJ is also critical for hematopoietic stem cells (HSC) maintenance and function. Ku70 is a key component of the NHEJ pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Ku70-deficient mice were used to assess the importance of NHEJ pathway in HSC function. Ku70-deficient mice have decreased bone marrow cells (BMC), and significant reductions in the frequency of colony-forming units in culture (CFU-C) in the presence of cytokines including IL-3, IL-6, SCF and Epo. SKL (Sca1+, c-Kit+, Lin−) cells from Ku70-deficeint mice showed a similar proliferation rate compared with SKL cells from WT mice when cultured in vitro in the presence of cytokines; single cell culture experiment showed that SKL cells from Ku70-deficient mice and WT mice have similar clonogenic ability. Thus, Ku70-deficient SKL cells retain proliferation capacity although there is a deficiency in overall hematopoietic homeostasis. To address Ku70-deficient HSC function in vivo, Ku70-deficient BMCs were serially transplanted into lethally irradiated congenic wild-type mice. These Ku70-deficient marrow cells were able to reconstitute the myeloid lineages in both primary and secondary recipients, while over 95% of the T cells in the recipients were derived from the host. However, the tertiary whole marrow recipients died within 2 weeks of transplantation, while the WT BMCs recipients survived tertiary transplantation. Competitive repopulation assays were also performed between WT and Ku70-deficient BMCs. The results showed that when Ku70-deficient BMCs were mixed with WT competitor BMCs at a 1:1 ratio, Ku70-deficient BMCs were completely outcompeted by the WT BMCs. These data demonstrate that Ku70, a key component of NHEJ, is required for HSC function. Ku70 deficiency in the hematopoietic compartment results in a long-term repopulating defect and loss of competitive repopulation ability.

Bronchial malondialdehyde DNA adducts, tobacco smoking, and lung cancer

Tobacco smoking is a major risk factor for lung cancer causing, among other effects, oxidative stress and lipid peroxidation. Malondialdehyde (MDA)–DNA adducts can be induced by direct DNA oxidation and by lipid peroxidation. We measured the relationship between bronchial MDA–DNA adducts and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing a bronchoscopic examination for diagnostic purposes. MDA–DNA adducts were higher in current smokers than in never smokers (frequency ratio (FR) = 1.51, 95% confidence interval (CI) 1.01–2.26). MDA–DNA adducts were also increased in lung cancer cases with respect to controls, but only in smokers (FR = 1.70, 95% CI 1.16–2.51). Subjects with GA and AA cyclin D1 (CCND1) genotypes showed higher levels of MDA–DNA adducts than those with the wild-type genotype (FR = 1.51 (1.04–2.20) and 1.45 (1.02–2.07)). Lung cancer cases with levels of MDA–DNA adducts over the median showed a worse, but not statistically significant, survival, after adjusting for age, gender, and packyears (hazard ratio = 2.48, 95% CI 0.65–9.44). Our findings reinforce the role of smoking in lung carcinogenesis through oxidative stress. Subjects who carry at least one variant allele of the CCND1 gene could accumulate DNA damage for altered cell-cycle control and reduced DNA repair proficiency.

DNA-Repair Gene Polymorphisms Predict Favorable Clinical Outcome Among Patients With Advanced Squamous Cell Carcinoma of the Head and Neck Treated With Cisplatin-Based Induction Chemotherapy

Cisplatin kills tumor cells through DNA cross linking. Alterations in the function of DNA repair genes may affect DNA repair proficiency and influence cancer patients' response to cisplatin. We studied whether single nucleotide polymorphisms (SNPs) of DNA repair genes predict the response to cisplatin or prognosis in patients with squamous cell carcinoma of the head and neck (SCCHN). A polymerase chain reaction-restriction fragment length polymorphism (RFLP) approach was used to determine the frequency of the SNPs: XPD-Asp312Asn, XPD-Lys751Gln, ERCC1-C8092A, and XRCC1-Arg399Gln in DNA from peripheral lymphocytes of 103 stage IV SCCHN patients. The frequencies of the distinct genotypes were, respectively, for the homozygous common allele, heterozygous and homozygous polymorphic variant: 53%, 40%, and 7% for ERCC1; 50%, 42%, and 8% for XPD-312; 35%, 57%, and 8% for XPD751; and 35%, 51%, and 13% for XRCC1. Patients with only common alleles at all the SNPs tested had a median overall survival of 5.1 months (range, 4.3 to 6.0 months) as compared with not reached for patients with at least one polymorphic variant (P < .001). Estimates from Cox's multivariate analysis suggest that the accumulation of each polymorphic variant decreases the probability of dying by a factor of 2.1 (P < .001; the presence of seven polymorphic variants confers a 175-fold protection). The accumulation of polymorphic variants increases by 2.94-fold the probability of achieving a complete response to treatment (P = .041). Using a multivariate model, the presence of polymorphic variants in DNA-repair genes are powerful prognosis factors and response to cisplatin predictors among SCCHN patients.

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 μl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate – that starts at zero and accelerates to the maximal rate ( μ m) – and by the lag time ( λ). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2Δ and rpd3Δ cells were found to be more resistant than the WT, while gcn5Δ and spt10Δ cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.

Mutations in Mre11 Phosphoesterase Motif I That Impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 Complex Stability in Addition to Nuclease Activity

The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited <2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.

Interaction between Genetic Variations in DNA Repair Genes and Plasma Folate on Breast Cancer Risk

Abstract Folate status has been inversely associated with breast cancer risk. Because folate deficiency can cause DNA damage, such as uracil misincorporation, single strand breaks, and double strand breaks, genetic polymorphisms in base excision repair and double strand break repair genes may lead to variation in DNA repair proficiency and modify the effect of folate on breast cancer risk. We prospectively investigated the a priori hypothesized interaction between plasma folate levels and five nonsynonymous polymorphisms in the XRCC1, XRCC2, and XRCC3 genes on breast cancer risk in a nested case-control study within the Nurses' Health Study (712 case-control pairs). Suggestive evidence of interaction was seen for two of these polymorphisms. Compared with the reference group of non-carriers in the lowest quartile of plasma folate, the reduction in risk (66%) was statistically significant among XRCC1 194Trp carriers in the highest quartile (multivariate odds ratio, 0.34; 95% confidence interval, 0.16–0.72). The inverse association between XRCC1 194Trp and breast cancer risk was attenuated by lower plasma folate status. The inverse association between plasma folate level and breast cancer risk was stronger among 194Trp carriers (P, trend = 0.01) than non-carriers (P, trend = 0.09). We also observed that the positive association between the XRCC2 188His allele and breast cancer risk was only significant in women in the lowest plasma folate quartile (carriers versus non-carriers; multivariate odds ratio, 2.04; 95% confidence interval, 1.05–3.97), and this excess risk was abolished among those with higher plasma folate levels. Moreover, the inverse association between plasma folate level and breast cancer risk was stronger among XRCC2 188His carriers (P, trend = 0.004) than non-carriers (P, trend = 0.09). Although none of the statistical tests for interaction was significant, these data give some support for the hypothesis that genetic variations in DNA repair genes may modify the relation between plasma folate level and breast cancer risk.

Lack of association between DNA base excision repair gene XRCC1 Gln399Arg polymorphism and risk of malignant lymphoma in Japan

Growing evidence suggests that the polymorphism of DNA base excision repair gene XRCC1 Arg399Gln is associated with altered DNA repair proficiency and subsequent cancer susceptibility; however, no evidence is available for malignant lymphoma. We therefore conducted a case-control study (372 cases, 500 controls) to evaluate links with malignant lymphoma risk in Japan. The risk was evaluated in terms of odds ratio (OR) and 95% confidence interval (CI) adjusted for age and sex in an unconditional logistic regression model. There was no statistical risk change with the Arg/Gln (adjusted OR 0.89; 0.65–1.23, P = 0.492) or the Gln/Gln (0.57; 0.27–1.17, P = 0.127) compared with the Arg/Arg of the XRCC1 Arg399Gln polymorphism. The results were unchanged in analyses according to histological subtype (diffuse large lymphoma, follicular lymphoma, low-grade lymphoma of mucosa-associated lymphoid tissue, and others). These data suggest that XRCC1 Gln399Arg polymorphism plays a limited role in lymphomagenesis. Further study on the interaction between the polymorphism and environmental exposure is required.

Irradiance dependence of the He–Ne laser-induced protection against UVC radiation in E. coli strains

He–Ne laser pre-irradiation-induced protection against UVC damage was investigated in wild-type E. coli K12 strain AB1157 and its isogenic DNA repair mutant strains. At a dose of 7 kJ/m 2, pre-irradiation was observed to induce protection in recA proficient strains (AB1157 and uvrA − AB1886) at both the irradiances investigated (2 and 100 W/m 2). However, at the same dose (7 kJ/m 2), while no protection was observed at 100 W/m 2 in the recA − strain, some protection appeared to be there at 2 W/m 2. Mechanistic studies carried out on these strains at the two irradiances suggest that, whereas the protection observed at 100 W/m 2 is mediated by singlet oxygen, that observed at 2 W/m 2 is not. Further, the fact that protection at 100 W/m 2 was observed only in recA proficient strains suggests that it may arise due to the induction of DNA repair processes controlled by the recA gene. The latter may arise due to the oxidative stress produced by singlet oxygen generated by He–Ne laser irradiation. In contrast, the protection observed at 2 W/m 2 appears to be independent of the DNA repair proficiency of the strain.

Influence of common XPD and XRCC1 variant alleles on p53 mutations in lung tumors.

The DNA repair proteins XPD and XRCC1 are involved in the nucleotide and base excision repair of DNA lesions induced by many tobacco and environmental carcinogens. Common variant alleles at the XPD (312Asn, 751Gln) and XRCC1 (399Gln) loci have been identified and associated with increased risk for lung cancer. We therefore investigated a possible effect of these variant alleles on the frequency and spectrum of p53 mutations in the tumors of 97 Swedish lung cancer patients (56 never-smokers and 41 age-, gender-, and hospital-matched ever-smokers). The p53 gene was mutated in 4 never-smokers (7%) and 11 ever-smokers (27%). Smoking-related transversion-type mutations predominated over transitions among smokers (8:3), but not among never-smokers (1:3). None of the variant alleles altered the overall frequency of p53 mutation. Transversions, however, were marginally increased among patients with at least one XPD variant allele compared with patients who were wild-type homozygotes (73% vs. 25% for the Asp312Asn polymorphism, P = 0.095; 78% vs. 33% for Lys751Gln, P = 0.085). Five of six women or six of seven smokers who carried at least one XPD 751Gln allele had p53 transversion. The XRCC1 variant allele did not show any effect on the p53 mutation. We conclude that the XPD variant alleles may be associated with an increased frequency of smoking-related p53 mutations in lung tumors, presumably due to reduced DNA repair proficiency.

Influence of XPD variant alleles on p53 mutations in lung tumors of nonsmokers and smokers

The DNA repair protein XPD is involved in the transcription-coupled nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. Common polymorphisms in XPD exon 10 (G>A, Asp312Asn) and exon 23 (A>C, Lys751Gln) have been identified, and lung cancer cases homozygous for the variant allele in either exon have been reported to have a reduced repair capacity against benzo[ a]pyrene-induced DNA damage. We therefore investigated a possible effect of these variant alleles on the p53 mutation frequency and spectrum among 97 Swedish lung cancer patients. Transversions were found to occur more frequently among patients with at least one variant allele than among wild type homozygotes. The XPD variant alleles may thus be associated with reduced DNA repair proficiency. This finding is not in accord with the previous report that associated the exon 23 wild type allele with increased frequency of X-ray-induced chromatid aberrations.

Evolution of colorectal cancer: change of pace and change of direction.

This review compiles evidence for an alternative to the classical adenoma-carcinoma sequence in the evolution of colorectal cancer. It is suggested that between 30 and 50 of colorectal cancers are not initiated by mutation of the tumor suppressor gene APC, but through the epigenetic silencing of genes implicated in the control of differentiation, cell cycle control and DNA repair proficiency. The precursor polyps are often characterized by a serrated architecture, and include hyperplastic polyps, admixed polyps and serrated adenomas. The alternative pathway is heterogeneous and may culminate in cancers showing low or high level DNA microsatellite instability (MSI-L and MSI-H, respectively), and in cancers that are microsatellite stable (MSS). Cancers showing DNA MSI may be characterized by an accelerated evolution. Cancers in hereditary non-polyposis colorectal cancer show features of both classical (adenoma and APC mutation) and alternative pathways (rapid evolution, MSI-H and lack of chromosomal instability).