The human T-cell cloning assay: identifying genotypes susceptible to drug toxicity and somatic mutation.

Humans exhibit marked genetic polymorphisms in drug metabolism that contribute to high incidence of adverse effects in susceptible individuals due to altered balance between metabolic activation and detoxification. The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyl transferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T-lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of the mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis, for investigating the functional impact of common polymorphism in metabolism and repair genes, and for identifying risk genotypes for drug-induced toxicity and mutagenicity. This chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis.

The (CCTTT)n microsatellite polymorphism in the NOS2 gene may influence lung cancer risk and long-term survival, especially in non-smokers.

We analyzed the associations of the NOS2 (CCTTT)n promoter polymorphism to lung cancer risk and tumor histology in smokers and non-smokers. We also investigated lung cancer long-term survival in relation to the polymorphism, smoking data, histology, age at diagnosis, and gender. One hundred eighty-five lung-cancer patients and 164 matched controls, where non-smokers were enriched among the lung cancer cases, were genotyped by fragment analysis and sequencing. Genotypes were combined with information on histology, patient smoking status, and cancer-specific death, using a 20-year follow-up. We divided the (CCTTT)n alleles into short (n ≤ 10), intermediate (n = 11-12), and long (n ≥ 13). Patients homozygous for short repeats had significantly increased risk of lung cancer (p = 0.030) compared to carriers of two long alleles (LL). Lack of long allele was associated with a significantly increased lung cancer risk overall (p = 0.011), especially among non-smokers (p = 0.001). A significantly higher lung cancer survival was seen in non-smokers compared to smokers (p = 0.046) and in low-dose smokers compared to high-dose smokers at the time of diagnosis (p = 0.028). Moreover, non-smoking patients with squamous cell carcinoma (p = 0.015) or adenocarcinoma (p = 0.024) showed a significantly lower survival compared to other lung carcinomas. Nitric oxide can induce proliferation as well as apoptosis depending on cellular context. Our results suggest that the (CCTTT)n NOS2 microsatellite may influence the risk of developing lung cancer, especially in non-smokers, possibly by affecting intracellular nitric oxide levels. Our results also give additional information about the yet poorly understood etiological and prognostic differences between lung cancer in non-smokers and smokers.

Polymorphisms in the DNA repair genes XRCC1, APEX1, XRCC3 and NBS1, and the risk for lung cancer in never- and ever-smokers.

This case-control study examines the association between lung cancer and genetic polymorphisms in two base excision repair (BER) genes, XRCC1 and APEX1 and two genes involved in homologous recombination repair (HR), XRCC3 and NBS1. Never-smoking lung cancer patients were recruited, and also the next diagnosed ever-smoking case of the same gender and age group. Controls were recruited from the regional population register, frequency matched to cases by hospital catchment area, gender, age group and smoking category. As a result more than 70% of the study population were women. A total of 331 individuals were analysed. Presence of the XRCC1 399Gln allele was associated with a significantly decreased risk for lung cancer among non-smoking women (odds ratio (OR) 0.4, 95% confidence interval (CI) 0.2-0.9). No significant effect was seen with the APEX1 polymorphism. Women smokers carrying the XRCC3 241Met allele showed a significantly decreased risk for lung cancer (OR 0.3, CI 0.2-0.7). The NBS1 185Gln allele was significantly associated with an increased risk for lung cancer among non-smoking women (OR 2.2, CI 1.0-4.8) and low-dose smoking women (OR 4.8, CI 1.5-15.7). The protective effect of the variant XRCC3 241Met allele was strengthened when combined with the low-risk Glu185 allele of the NBS1 gene. Smokers (OR 0.38, CI 0.16-0.90) and women (OR 0.42, CI 0.21-0.85) with at least three low-risk alleles in these two HR genes showed a significantly decreased risk for lung cancer. Thus, in spite of a relatively small study population, this study, including a comparatively large number of never-smokers and women, presents several novel aspects on genetic susceptibility to lung cancer. Our results show that the genetic variation in XRCC1, XRCC3 and NBS1 influence lung cancer susceptibility among women, and that combinations of risk alleles in the two HR genes can enhance the effects.

Influence of polymorphism in DNA repair and defence genes on p53 mutations in bladder tumours.

We studied the effects of polymorphisms in nine genes involved in DNA repair and detoxification on occurrence and type of p53 mutation in 327 bladder cancer patients. The included polymorphisms are XPC(Lys939Gln), XPD(Lys751Gln), XPG(Asp1104His), XRCC1(Arg3999Gln), XRCC3(Thr241Met), NBS1(Glu185Gln), cyclin D1(Pro241Pro), MTHFR(Ala222Val and Glu429Ala) and NQO1(Arg139Trp and Pro187Ser). We found increased risk for p53 mutation among cyclin D1 variant allele homozygotes (OR 2.4 CI 0.8-6.7). Among non-smokers, 75% (3/4) with p53 mutation but only 12.5% (3/24) without p53 mutations were XRCC3 241Met homozygotes (P=0.03). Among smokers, all p53 transversions (3/3), but only 41.7% (5/12) of p53 transitions were found among carriers of the XPC 939Gln allele. Individuals carrying the NQO1 187Ser allele showed increased risk for p53 transversions (OR 4.7, CI 0.9-26.1). All (2/2) NQO1 139Trp allele carriers but only 17.5% (7/40) of the Arg139 homozygotes had p53 transversions. Our findings suggest that altered repair and detoxification due to genetic polymorphism may influence the occurrence of p53 mutations in bladder cancer.

Influence of GSTM1, GSTT1, GSTP1 and NAT2 genotypes on the p53 mutational spectrum in bladder tumours.

Genetic polymorphisms affecting expression or activity of the corresponding enzymes can influence the risk of acquiring gene mutations and various cancers. We have studied 327 bladder cancer patients with regard to the functionally related polymorphisms of GSTM1, GSTT1, GSTP1 and NAT2 and analysed the p53 mutational status of their tumours. Fifty p53 mutations, 26% transversions and 74% transitions, were detected in 44 patients. P53 mutation frequency was significantly higher in higher-grade tumours than in low-grade tumours (OR = 2.09, 95% CI 1.44-3.02, adjusted for age and sex). Also, a significant association was found between tumour stage (Tis and T2+ vs. Ta and T1) and presence of the GSTP1 val allele (adjusted OR = 2.00, CI 1.14-3.52). Overall, there was no significant difference in frequency of p53 mutation among patients with different genotypes. Among patients with p53 mutation, transversions were significantly more frequent in GSTM1-negative as compared to GSTM1-positive individuals (OR = 5.18, CI 1.07-25.02, adjusted for age, sex and tumour stage). With one exception, all tumours with the most common type of transversion, G:C-C:G, occurred in GSTM1-negative patients. Among smokers, all transversions (3 of 3), but only 2 of 13 transitions, were found among carriers of the GSTP1 variant allele, and samples carrying at least 1 variant GSTP1 allele had more transitions at CpG sites than wild-type samples (adjusted OR = 4.61, CI 0.82-26.04). No significant associations were found for the NAT2 gene. Our results suggest that impaired glutathione conjugation may affect the mutation spectrum in critical target genes.

Methods for detecting somatic mutations in vitro: the human T-cell cloning assay selecting for HPRT mutants.

The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of their mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis and for investigating the functional impact of common polymorphisms in metabolism and repair genes. The present chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis.

Dietary fruit and vegetables protect against somatic mutation in vivo, but low or high intake of carotenoids does not.

Epidemiological studies have demonstrated protective effects of vegetables and fruit on risk of cancer, but underlying mechanisms remain unclear. Intervention studies have in some cases contradicted previous epidemiological evidence, e.g. for beta-carotene supplementation and lung cancer, emphasizing the need for mechanistic data. We assessed in vivo mutagenic effects of several dietary items using the HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene assay with T-lymphocytes from 312 individuals (158 lung cancer cases, 154 population controls), who provided information on diet and smoking habits. HPRT mutant frequency (MF) was significantly decreased in relation to intake of vegetables, citrus fruits and berries, respectively, as well as calculated vitamin C intake from diet. There was a significant U-shaped association with dietary carotenoid intake, with lowest MF near population average carotenoid intakes and higher mutation frequencies both at low and high intakes, and a similar borderline significant association was observed for beta-carotene. Our study is consistent with known diet-cancer associations and provides novel human in vivo mechanistic support for a cancer-protective effect of vegetables and fruit by modulation of somatic mutagenesis. Our results also provide support for the increase in lung cancer risk observed particularly in smokers in studies of beta-carotene supplementation.

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes.

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

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.

The XPD variant alleles are associated with increased aromatic DNA adduct level and lung cancer risk.

The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is involved in the nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. In order to study the functional impact of the common polymorphisms in XPD exon 10 (G > A, Asp312Asn) and exon 23 (A > C, Lys751Gln), we have genotyped 185 Swedish lung cancer cases (97 smokers and 88 never-smokers) and 162 matched population controls (83 smokers and 79 never-smokers). Presence of one or two variant alleles was associated with increased risk for lung cancer among never-smokers only, in particular younger (<70 years) never-smokers [odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.1-6.5 for exon 10; OR = 3.2, 95% CI = 1.3-8.0 for exon 23, adjusted for age, gender and environmental tobacco smoke]. Aromatic DNA adduct level (AL) in peripheral lymphocytes was found to be similar between cases and controls, but significantly increased by current or recent smoking. Overall, there was a significant trend for increasing AL with increasing number of variant alleles in exon 10 (P = 0.02) or in exon 23 (P = 0.001). In addition, subjects with the combined exon 10 AA and exon 23 CC genotype showed a significantly higher AL compared with all those with any of the other genotypes (P = 0.02). We conclude that the XPD variant alleles may be associated with reduced repair of aromatic DNA adducts in general and increased lung cancer risk among never-smokers.