核苷酸切除修复酶基因多态性与晚期胃癌化疗敏感性的关系研究

目的探讨核苷酸切除修复酶基因118密码子(ERCC 1118)和751密码子(XPD 751)多态性与晚期胃癌患者对5-氟尿嘧啶(5-Fu)/铂类药物化疗敏感性的关系。方法 91例患者给予5-Fu/铂类药物方案治疗,化疗前检测ERCC1 118和XPD 751基因型。观察化疗疗效与ERCC1 118和XPD 751多态性的关系。结果 本组患者化疗有效率为37.4%。ERCC1 118的3种基因型化疗有效率比较差异无统计学意义(P>0.05);XPD 751的杂合子Lys/Gln基因型的化疗有效率为11.1%,低于纯合子Lys/Lys和Gln/Gln基因型的38.0%和100.0%,差异有统计意义(P<0.01)。结论核苷酸切除修复酶基因XPD 751基因多态性与晚期胃癌对5-Fu/铂类药物化疗疗效有关。     

The DNA repair gene XPD/ERCC2 polymorphisms Arg156Arg (exon 6) and Lys751Gln (exon 23) are closely associated

In the context of a molecular epidemiology study dealing with the effects of individual genetic susceptibility on childhood respiratory morbidity, DNA repair genotypes for the XPD/ERCC2 gene in exon 6 (Arg156Arg) and exon 23 (Lys751Gln) have been analyzed by PCR/RFLP assays in DNA samples isolated from the fetal parts of placentas. The study was performed using a cohort of 729 children born in 1994-1998 in two districts of the Czech Republic. On the basis of these data, we tested the association between the two genotypes. The principal finding of this study is that the exon 6 and exon 23 polymorphisms in the XPD/ERCC2 gene are tightly associated, with persons who are homozygous CC in exon 23 being mostly (81%) homozygous CC in exon 6, and persons homozygous AA in exon 6 mostly (88%) homozygous AA in exon 23. This strong association may have serious consequences for the interpretation of cancer susceptibility and other molecular epidemiology studies dealing with the XPD6 and XPD23 genotypes, since the observed effects of the silent XPD6 polymorphism might be, in fact, the result of XPD23 polymorphism, which is connected with an amino acid substitution in the resulting XPD protein.     

Polymorphisms and haplotypes of DNA repair and xenobiotic metabolism genes and risk of DNA damage in Chinese vinyl chloride monomer (VCM)-exposed workers

In this case-control study, we investigated the association between DNA damage and genetic susceptibility among vinyl chloride monomer (VCM)-exposed workers. The cumulative exposure dose of VCM was calculated based on the workers' duration of exposure and the geometric mean concentration of VCM in the workplace. DNA damage to peripheral blood lymphocytes was measured by single cell gel electrophoresis (SCGE) assay, and single nucleotide-polymorphisms (SNPs) in xenobiotic metabolism and DNA repair genes were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Univariate analysis showed that the CYP2E1 c1c2/c2c2 and XPD751 Lys/Gln and Gln/Gln genotypes were significantly associated with the levels of DNA damage (P<0.01 and 0.05, respectively). Further logistic regression analysis showed a significant association between CYP2E1 c1c2/c2c2 and DNA damage, and risk of having increased levels of DNA damage was more pronounced in those individuals having XRCC1 194 mutant genotypes and/or XPD751 Lys/Gln and Gln/Gln genotypes. Although most of the XPD and XRCC1 haplotypes did not show any significant difference, the XRCC1 haplotype Trp194-Arg280 was significantly over-represented in the case group (P<0.05; OR 2.09; 95% CI: 1.07-4.06) than in controls. Overall, our data suggest that the genotypes of CYP2E1, XRCC1 194, and XPD 751 were associated with the level of DNA damage and may contribute to individual sensitivity to DNA damage induced by VCM in the workplace.     

hOGG1基因多态性与胃癌遗传易感性的关系

目的探讨人类8-羟基鸟嘌呤DNA糖苷酶1（hOGG1）基因多态性与胃癌遗传易感性的关系。方法收集河南郑州和开封地区98例胃癌患者和80例非肿瘤对照组志愿者外周血样,应用聚合酶链反应一限制性片段长度多态性（PCR—RFLP）检测法检测胃癌人群的外周血中DNA损伤修复酶基因多态性,分析其与肿瘤遗传易感性的关系。结果hOGGISer326Cys基因的各基因型频率在胃癌组和对照组间的分布差异有统计学意义（P〈0．05）。携带Cys326Cys基因型者胃癌的发病风险增加1．7倍（OR=I．706,95％CI=0．341～2．462,P=0．002）。hOGGlSer326Cys基因多态性与酒的交互作用增加胃癌的发病风险（S〉1,API=0．38）。结论Cys326Cys基因型是胃癌发病的危险基因型,携带Cys易感基因与饮酒交互作用时可能增加患胃癌的易感性。     

Determination of ERCC2 Lys751Gln and GSTP1 Ile105Val gene polymorphisms in colorectal cancer patients: relationships with treatment outcome

INTRODUCTION: Glutathione S-transferase P1 (GSTP1) and excision-repair cross-complementing repair deficiency group 2 protein (ERCC2 or XPD) may modulate the activity of platinum derivatives. The SNPs, Ile105Val for GSTP1 and Lys751Gln for ERCC2, may affect the efficiency of oxaliplatin in patients treated with an oxaliplatin-based regimen for metastatic colorectal carcinoma. PATIENTS & METHODS: A total of 107 patients treated with first-line chemotherapy, 59 with an oxaliplatin-based regimen and 48 with an irinotecan-based regimen, were included retrospectively. GSTP1 and ERCC2 genotypes were identified on DNA samples extracted from paraffin blocks containing either normal tissue (nodes) or tumor tissue. We analyzed treatment response, event-free and overall survival. RESULTS: GSTP1 genotype distribution was Ile/Ile 58%, Ile/Val 35% and Val/Val 7%. ERCC2 genotype distribution was Lys/Lys 49%, Lys/Gln 44%, Gln/Gln 7%. Event-free and overall survivals were not significantly different as a function of the GSTP1 genotype, whatever the treatment received. Event-free survival was significantly different as a function of the ERCC2 genotype only in patients receiving oxaliplatin: patients having at least one variant allele had a shorter median event-free survival (6 months) than those having no variant allele (11.6 months, p = 0.008). This difference was maintained for median overall survival (15.6 vs 25.3 months, p = 0.016). Using univariate analysis, ERCC2 genotype, hemoglobinemia and carbohydrate antigen 19.9 plasma levels were significantly related to overall and event-free survival in patients receiving oxaliplatin. CONCLUSION: The ERCC2 genotype appears as an important predictive factor of the survival of patients treated with oxaliplatin in first-line therapy for metastatic colorectal cancer.     

Relationships between genetic polymorphisms and anticancer drug cytotoxicity vis-à-vis the NCI-60 panel

INTRODUCTION: The National Cancer Institute (NCI)-60 panel consists of 60 human tumor cell lines initially established for screening thousands of molecules for antiproliferative activity. It has been powerful for deciphering the relationships between anticancer drug cytotoxicity and cell molecular characteristics. We tested its potential interest for establishing relationships between the polymorphism of genes involved in drug metabolism and transport or in DNA repair, and drug cytotoxicity extracted from NCI databases. METHODS: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques, three frequent single nucleotide polymorphisms (SNPs) were analyzed: Lys751Gln in the Xeroderma pigmentosum complementation group D (XPD, ERCC2) gene, Asp1104His in the Xeroderma pigmentosum complementation group G (XPG, ERCC5) gene and Ile105Val in the glutathione S-transferase P1 (GSTP1) gene. RESULTS: The allelic frequencies of the variants were 33% for ERCC2, 23% for ERCC5 and 39% for GSTP1. The ERCC2 polymorphism appeared to be a strong determinant of the in vitro cytotoxicity of most anticancer agents, with lower half maximal inhibitory concentration (IC50) values in variant homozygous lines than in common homozygous or heterozygous cell lines. Unexpectedly, the cytotoxicity of taxanes appeared markedly dependent upon the ERCC2 genotype, with threefold lower mean IC50 values in variant homozygous cell lines. The ERCC5 genotype appeared to be important only for taxanes, with fourfold higher IC50 values in variant homozygous cell lines. The GSTP1 polymorphism was related to the cytotoxicity of several drug classes, especially topoisomerase inhibitors, antimetabolites and N7 alkylating agents. CONCLUSION: The NCI-60 panel is capable of providing clues and tracks for the establishment of clinically useful relationships between a given genotype and the cytotoxicity of an anticancer agent.     

The role of glutathione in the regulation of nucleotide excision repair during oxidative stress

Nucleotide excision repair (NER) mainly repairs bulky DNA adducts and helix distorting lesions, but is additionally considered to be a back-up system for base excision repair to remove oxidative stress induced DNA damage. Therefore, it can be speculated that NER is up-regulated or primed by oxidative stress. Exposure of human pulmonary epithelial cells (A549) to non-toxic doses of 100muM H(2)O(2) indeed showed a 2 to 4.5-fold increase in expression of XPA, XPC, ERCC4, and ERCC5, whereas the expression of ERCC1 was 5-fold decreased. Phenotypical assessment of NER capacity (i.e. recognition and incision of benzo[a]pyrene-DNA adducts) showed a significant decrease to less than 50% after H(2)O(2) exposure, which paralleled the effects of H(2)O(2) on ERCC1 expression. To study the possible involvement of glutathione (GSH) in the regulation of NER, cells were pre-incubated with 0.5mM BSO, resulting in total GSH depletion and increased intracellular oxidative stress. In GSH-depleted cells, the down-regulation of ERCC1 expression by H(2)O(2) was completely abolished and the up-regulation of ERCC4 expression was potentiated from 2.5-fold to >10-fold. Similarly, the H(2)O(2)-induced decrease in NER capacity was absent in GSH-depleted cells. Overall, our data suggest that NER capacity as well as the expression of NER related genes can be modulated by oxidative stress. ERCC1 expression and NER capacity correlated strongly (R(2)=0.85, P<0.01) after oxidant exposure, indicating ERCC1 as a specific target for oxidative stress induced modification of NER.     

The role of nucleotide excision repair and loss of p53 in mutagenesis and carcinogenesis

Xpa mice, which have a completely defective nucleotide excision repair (NER) pathway, have a cancer predisposition when exposed to several carcinogens. NER is one of the major DNA repair pathways in the mammalian cell, and is involved in the removal of a wide variety of DNA lesions, such as those induced by UV light, bulky adducts and DNA crosslinks. To study the role of NER in both mutagenesis and carcinogenesis, NER-defective Xpa mice were crossed with transgenic lacZ/pUR288 mutation-indicator mice. Furthermore, the relationship between the tumor suppressor gene p53, NER, induction of mutations and tumor development was studied in Xpa/p53^+/−/lacZ triple transgenic mice. Using the genotoxic carcinogens benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF), it is shown that mutations in the inactive (non-transcribed) lacZ reporter gene reliably predict cancer risk. In tissues at risk for the development of tumors, increased mutant frequencies could be found at much earlier stages. A heterozygous loss of p53 appears to act synergistically to a NER defect, both in mutation- as well as tumor-induction. Surprisingly, however, the effect of a heterozygous loss of p53 appeared to be tissue-restricted, being apparent in the bladder but absent in liver and spleen.     

Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant known to cause DNA damage, which may be repaired through nucleotide excision repair (NER). However, little is known about dynamic changes in levels of DNA damage and their correlations with levels of NER proteins in cells exposed to BaP. In a series of experiments using the human bronchial epithelia cells (16HBE) exposed to different concentrations of BaP for different times, we measured dynamic changes in levels of DNA damage and expression of NER subunit xeroderma pigmentosum (XP) groups A, C, F, G (XPA, XPC, XPF, XPG) and excision repair cross-complementing 1 (ERCC1), and analyzed their possible correlations. We found that in vitro exposure to BaP reduced cell viability in a dose-dependent manner ranging from 2 to 64microM and increased DNA damage in a dose- and time-dependent manner. Our results showed that levels of these NER proteins significantly increased and peaked at 12th or 24th, 8th or 12th and 4th or 8th hours in cells exposed to 2, 8 and 16microM BaP, respectively. ERCC1 expression increased by 2.4-, 4.2- and 19.3-fold for exposure to 2, 8 and 16microM BaP, respectively, compared with control group. Moreover, levels of ERCC1 in cells exposed 16microM BaP significantly higher than those in 2 and 8microM BaP from 2nd to 48th hours. In addition, there was a significant positive correlation between Olive tail moments and relative ratios of ERCC1 in cells exposed to BaP. Our results suggested ERCC1 may be an important limiting factor for NER, but the mechanisms underlying this observation needs further investigation.     

Excision repair of BPDE-adducts in human lymphocytes: diminished capacity associated with ERCC1 C8092A (rs3212986) polymorphism

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a metabolite of Benzo[a]pyrene (B[a]P), is a high-risk factor for development of a number of cancers. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair system of which ERCC1 exerts an important role. We investigated whether two single nucleotide polymorphisms in ERCC1 (C19007T; rs11615 and C8092A; rs3213986) affected the repair efficacy of BPDE-DNA adducts. We collected peripheral blood of 780 healthy individuals from the northeast of China and detected the genotypes of rs11615 and rs3213986. The amount of induced BPDE-DNA adducts in lymphocytes from 117 randomly selected participants was assessed by HPLC. Presence of BPDE-DNA adducts in nucleus of lymphocytes was visualized using the modified comet assay. ERCC1 and CAST (3′ adjacent gene of ERCC1) mRNA expression levels were quantified after in vitro exposure to BPDE. We found that the minor A allele in rs3212986 was related to higher levels of BPDE-DNA adducts and holistic marking DNA damage (P < 0.01). Haplotype CA (rs11615 and rs3213986) was also associated with an elevated risk of high BPDE-DNA adduct levels (OR = 1.801, 95 % CI of OR 1.191–2.724). Interestingly, in participants with AA genotype for rs3213986, CAST mRNA level was decreased compared to individuals with the homozygous CC genotype. Our findings suggests that ERCC1 C8092A (rs3213986) is associated with a diminished capacity of repairing BPDE-DNA adducts and may be used as a valid biomarker to predict an individual’s risk to develop cancer upon exposure to environmental carcinogens.     

Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair

Tobacco smoking, alcohol drinking, and occupational exposures to polycyclic aromatic hydrocarbons are the major proven risk factors for human head and neck squamous-cell cancer (HNSCC). Major research focus on gene-environment interactions concerning HNSCC has been on genes encoding enzymes of metabolism for tobacco smoke constituents and repair enzymes. To investigate the role of genetically determined individual predispositions in enzymes of xenobiotic metabolism and in repair enzymes under the exogenous risk factor tobacco smoke in the carcinogenesis of HNSCC, we conducted a case-control study on 312 cases and 300 noncancer controls. We focused on the impact of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1, ERCC2/XPD, GSTM1, GSTP1, GSTT1, NAT2, NQO1, and XRCC1. To assess relevant main and interactive effects of polymorphic genes on the susceptibility to HNSCC we used statistical models such as logic regression and a Bayesian version of logic regression. In subgroup analysis of nonsmokers, main effects in ERCC2 (Lys751Gln) C/C genotype and combined ERCC2 (Arg156Arg) C/A and A/A genotypes were predominant. When stratifying for smokers, the data revealed main effects on combined CYP1B1 (Leu432Val) C/G and G/G genotypes, followed by CYP1B1 (Leu432Val) G/G genotype and CYP2E1 (-70G>T) G/T genotype. When fitting logistic regression models including relevant main effects and interactions in smokers, we found relevant associations of CYP1B1 (Leu432Val) C/G genotype and CYP2E1 (-70G>T) G/T genotype (OR, 10.84; 95% CI, 1.64-71.53) as well as CYP1B1 (Leu432Val) G/G genotype and GSTM1 null/null genotype (OR, 11.79; 95% CI, 2.18-63.77) with HNSCC. The findings underline the relevance of genotypes of polymorphic CYP1B1 combined with exposures to tobacco smoke.     

Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells

The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called “bulky” DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.     

Pharmacogenetics of platinum-based chemotherapy in non-small cell lung cancer: predictive validity of polymorphisms of ERCC1

Introduction: The efficacy of platinum-based chemotherapy for patients with non-small cell lung cancer (NSCLC) is limited by chemoresistance. Platinum drugs damage DNA by introducing intrastrand and interstrand crosslinks which result in cell death. Excision repair cross-complementing 1 (ERCC1) is a member of the nucleotide excision repair (NER) pathway which erases such defects. Single nucleotide polymorphisms (SNPs) in ERCC1 impair this activity and have been suggested to predict the response to chemotherapy.

Area covered: Among the polymorphisms of proteins involved in uptake, metabolism, cytotoxicity and efflux of platinum drugs, codon 118 C/T and C8092A in ERCC1 are the best characterized SNPs studied for their predictive power. Here, the divergent results for studies of these markers in NSCLC are summarized and the reasons for this contradictory data discussed.

Expert opinion: Cytotoxicity of platinum compounds comprise complex cellular processes for which DNA repair may not constitute the rate limiting step. These drugs are administered as doublets to histologically diverse patients and, furthermore, the NER pathway in ERCC1 wildtype cohorts may be still impaired by the chemotherapeutics applied. At present, assessment of a limited number of polymorphism in DNA repair proteins is not reliably associated with response to treatment in NSCLC patients.     

Genetic polymorphisms in detoxification and DNA repair genes and susceptibility to glycidamide-induced DNA damage

Acrylamide (AA) is a probable human carcinogen formed in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), the AA metabolite formed by epoxidation, is considered the ultimate genotoxic agent. In this study, the in vitro genotoxic potential of AA and GA in human whole blood leukocytes was compared using the alkaline comet assay. Although AA did not induce significant DNA damage in the concentrations tested (up to 1000 μM), GA markedly increased the percentage of tail DNA at concentrations ≥250 μM. Further, this study addressed the role of genetic polymorphisms in key genes involved in metabolism and DNA repair pathways (BER, NER, HRR, and NHEJ) on GA-induced genotoxicity assessed by the alkaline comet assay. The results obtained suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val) genes, either alone or in combination.     

Compound K suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair in human keratinocytes

Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage of mammalian cells. The purpose of this study is to investigate the functional role of ginsenoside compound K on HaCaT cells (a keratinocyte-derived permanent cell line) irradiated by UV. Hoechst 33258 staining were performed in analyzing UV-induced apoptosis on keratinocytes which were treated with compound K. ImmunoDotBlot assay was used in detecting cyclobutane pyrimidine dimers, the main DNA damage. Western blot analysis was applied for analyzing XPC and ERCC1, two of the NER proteins. Compound K inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In agreement with this, compound K induced the expression of particular components of the NER complex, such as XPC and ERCC1. Our results demonstrate that compound K can protect cells from apoptosis induced by UV radiation by inducing DNA repair.     

Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and FOLFOX response in colorectal cancer patients

AIMS

To test prospectively the predictive value of germinal gene polymorphisms related to fluorouracil (FU) and oxaliplatin (Oxa) pharmacodynamics on toxicity and responsiveness of colorectal cancer (CRC) patients receiving FOLFOX therapy.

METHODS

Advanced CRC patients (n= 117) receiving FOLFOX 7 therapy were enrolled. Gene polymorphisms relevant for FU [thymidylate synthase (TYMS, 28 bp repeats including the G→C mutation + 6 bp deletion in 3''UTR), methylenetetrahydrofolate reductase (MTHFR, 677C→T, 1298A→C), dihydropyrimidine deshydrogenase (IVS14+1G→A) and Oxa: glutathione S-transferase (GST) π (105Ile→Val, 114Ala→Val), excision repair cross-complementing group 1 (ERCC1) (118AAT→AAC), ERCC2 (XPD, 751Lys→Gln) and XRCC1 (399Arg→Gln)] were determined (blood mononuclear cells).

RESULTS

None of the genotypes was predictive of toxicity. Response rate (54.7% complete response + partial response) was related to FU pharmacogenetics, with both 677C→T (P= 0.042) and 1298A→C (P= 0.004) MTHFR genotypes linked to clinical response. Importantly, the score of favourable MTHFR alleles (677T and 1298C) was positively linked to response, with response rates of 37.1, 53.3, 62.5 and 80.0% in patients bearing no, one, two or three favourable alleles, respectively (P= 0.040). Polymorphisms of genes related to Oxa pharmacodynamics showed an influence on progression-free survival, with a better outcome in patients bearing GSTπ 105 Val/Val genotype or XPD 751Lys-containing genotype (P= 0.054).

CONCLUSIONS

These results show that response to FOLFOX therapy in CRC patients may be driven by MTHFR germinal polymorphisms.