摘要:

We have examined the hypothesis that the polymorphic, glutathione S-transferaseGSTP1gene is a susceptibility candidate for squamous cell cancer of the oral/pharynx and larynx. We describeGSTP1genotype frequencies in 3 80 cases and 180 controls. We found a lower frequency ofGSTP1AA in the oral/pharyngeal cases compared with controls (p=0.003, odds ratio=0.47) after correction for age and gender. We used an immunohistochemical approach to show widespread expression of theGSTP1subunit throughout the pharynx and larynx. In uninfiltrated tissue, strong positivity was found throughout the squamous cell epithelium with the exception of the basal cell layer. The cilia of the respiratory epithelium of the larynx also showed positivity forGSTP1. In tumour tissue, expression ofGSTP1was similar in pharyngeal and laryngeal samples. These data are the first to show that polymorphism atGSTP1mediates susceptibility to squamous cell cancer of the upper aerodigestive tract. No significant interactions were identified between GSTP1 andGSTMl, GSTM3, GSTTland the cytochrome P450CYP1Al,CYP2D6andCYP1A1genotypes.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Many studies have been performed in an attempt to establish a link between the polymorphism of the cytochrome P45O CYP2D6 gene and the incidence of lung cancer. Nevertheless, whether or not this genetic polymorphism has a role in the development of the disease remains unclear. Recently, new advances in our knowledge of the CYP2D6 gene and its locus (CYP2D) have been achieved. In particular, CYP2D6 was found to be highly polymorphic and multiple novel mutations and allelic variants of the gene have been identified. In addition, a number of CYP2D rearrangements, including those with amplification of the gene, have been demonstrated. Taking this new information into account, we have reconsidered the potential influence of CYP2D6 polymorphism in lung cancer susceptibility by performing a comparative analysis of the overall mutational spectrum of CYP2D6 and of the rearrangements of CYP2D in 249 patients with lung cancer and in 265 control individuals matched on age, sex, hospital and residence area. For this purpose, a strategy based on SSCP analysis of the entire coding sequence of CYP2D6 and on RFLP analysis of the gene locus was carried out in DNA samples from each individual. Forty mutations occuring in various combinations on 42 alleles of the gene and 82 different genotypes were identified. No significant difference in the distribution of the mutations, alleles or genotypes was observed between the two groups, except a particular genotype (CYP2D6*lA/*2), which was more common in the sub-group of moderate smokers (< 30 pack-years) suffering from small cell carcinoma (Odds Ratio (OR) 3.6, 95% CI 1.1-11.9). When the phenotype was predicted according to genotype, only a trend toward a higher frequency of ultrarapid metabolizers in patients was obtained. In spite of a complete analysis of the CYP2D6 gene and its locus, this case-control study provides elements against an influence of the CYP2D6 polymorphism on lung cancer Susceptibility.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The polymorphic cytochrome P450 CYP2D6 catalyses the biotransformation of at least 40 drugs. The CYP2D6 genetic polymorphism is responsible for pronounced interindividual differences in plasma concentrations and, hence, in drug action and side-effects after administration of the same dose. Provided there is a close relationship between CYP2D6 genotypes and catalytic function, genotyping could be used in the clinical setting for individualization of drug dose. In the present study, we evaluated the relationship between the in-vivo enzyme activity and 3 5 different genotypes in order to determine whether genotyping can be used to predict a person's metabolic capacity for CYP2D6-catalysed drug oxidation using sparteine as a probe drug. One hundred and ninety-five Caucasian individuals were genotyped for seven nonfunctional (CYP2D6*3, *4, *5, *6, *7, *8, *26) and eight functional alleles (CYP2D6*1, *2, *2x2, *2B, *2Bx2, *9, *10, *17). The metabolic ratio distribution for sparteine showed trimodality, with 15 poor metabolizers, 21 intermediate metabolizers, and 159 extensive and ultrarapid metabolizers. AH poor metabolizers were unambiguously identified as carriers of two nonfunctional alleles. In contrast, the most frequent functional genotypes extensively overlapped and, with few exceptions, genotype was not a useful predictor of function. Gene dose effects among homozygotes and heterozygotes of the major functional alleles were not significant and could not explain the wide variations. Only a minor fraction of phenotypical ultrarapid metabolizers, arbitrarily defined as individuals with a metabolic ratio<0.2, could be identified as carriers of three functional gene copies, including duplicated CYP2D6*2x2 alleles. Similarly, only a minor fraction of the intermediate metabolizers had predictive genotypes involving alleles coding for enzyme with impaired function. Thus, genotyping correctly identifies poor metabolizers, but quantitative prediction of drug metabolism capacity among extensive metabolizers is not possible,

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Deficiencies of the glutathione transferase isoenzymes GSTM1-1 and GSTT1-1 have been shown to be risk modifiers in a number of different cancers but there have been no similar studies with GSTP1-1, the only member of the Pi class of glutathione S-transferases expressed in humans. Over-expression of GSTP1-1 in tumours suggests that it may be a significant factor in acquired resistance to certain anticancer drugs. We previously identified a cDNA clone with two amino acid substitutions (I105V, A,114V). This clone suggests that the GSTP1 gene is polymorphic and it is possible that the different genotypes may be associated with altered cancer risk or drug resistance. In the present study, we report methods for genotyping individuals at codons 105 and 114 of GSTP1 and demonstrate that these two loci are polymorphic in several different racial groups. We also detected significant linkage disequilibrium between these two loci. To determine if either of the alleles at these two loci were associated with altered cancer susceptibility, we genotyped individuals with colorectal cancer or lung cancer. A total of 131 colorectal and 184 lung cancer patients were compared with 199 control individuals. Overall, there were no significant associations between the GSTP1 polymorphisms and either form of cancer

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol 1-1culture by Fe2+- CO versus Fe2+difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax3.30 nmol min-1mg-1protein; Km11.1 µM) in isolated membrane fractions. This Km value was similar to the Km determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 ± 0.4 min-1versus 5.7 ± 0.2 min-1at 50 JAM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P 4 5 0 isoenzymes.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The aromatase P450 (coded by the CYP19 gene) is responsible for the rate limiting step in the metabolism of C19 steroids to estrogens and is expressed in most breast carcinomas. A polymorphic tetranucleotide repeat (TTTA)nin intron 5, about 80 nucleotides downstream of exon 4 has previously been described. The allele frequencies of the polymorphic repeat were studied in series of 182 sporadic and 185 familial breast cancer patients as well as in 252 healthy control individuals. Five different alleles containing 7, 8, 9, 11 and 12 -TTTA-repeats were detected. A relatively rare allele (Al) containing the longest repeat (TTTA)12was found significantly more frequently in breast cancer patients than in control individuals. This indicates that individuals carrying the A1 allele of CYP19 may have an increased risk of developing breast cancer, OR 2.42 (95% confidence interval [CI] 1.03-5.80). The higher frequency was observed in both sporadic and familial patients, although when each of the groups was compared to the control group only a borderline significance was seen. A higher frequency of A 2 allele carriers was also found in the group of patients with positive estrogen receptor and progesterone receptor positive tumors. These data suggest that the CYP19 gene may be involved as a low penetrance gene in breast cancer Susceptibility.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The first rate-limiting step in the biosynthesis of all mammalian steroid hormones is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme, P450scc. The human CYP11A1 gene, encoding the mitochondrial P450scc, has been previously assigned by in situ hybridization to 15q23-q24 region. To map the CYP11A1 gene with linkage analysis, a novel TAAAA repeat polymorphism, found in its promoter region, was genotyped in the eight largest CEPH reference families, which included 91 children, 16 parents and 26 grandparents. Two-point linkage analysis was performed between this tetra-allelic polymorphism and the chromosome 15 microsatellite markers of Genethon as well as the tetranucleotide polymorphism of the CYP19 gene and a Ms/il RFLP of the CYP1A1 gene. A close linkage was observed with Dl 5S204 (Z max=27.33; 0 max=0.009) and CYP1A1 gene (Z max=6.62; 9 max=0.001), but not with the CYPJ9 gene (Z max=4.06; 9 max=0.26). The CYP19 gene that encodes the P450arom was rather closely linked with D15S123 (Z max=31.04; 9 max=0.001). A framework map, including Genethon markers flanking the polymorphic CYP11A1 and CYP19 genes, was built by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the 15ql5-q25.3 region was constructed, yielding to the following order: cen-D15S214 - [D15S123; CYP19] -D15S117- D15S159-D15S153 -D15S983 -[D15S204; CYP11A1; CYP1A1] - Dl 5S211 -D15S152- D15S199-tel. Thus, the CYP11A1 gene is closely linked with the CYP1A1 gene, whereas it is located approximately 2 7. 4 CM telomeriC tO the CYP19 gene.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT 1 -selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to JV-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium®. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n=144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n=19) of this sample. Urinary and plasma ratios correlated moderately (r=0.74, p<0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent Kmfor PAS and a 90-fold decreased Fmaxfor AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1*14 and NAT1*15, in case 244, as well as one variant, NAT1*11, which has been observed in previous investigations. NAT1 *24 contained a missense mutation (G560A) that is predicted to change a single amino acid (Arg187GIn), as well as two 3' non-coding region mutations (T1088A and C1095A) that have previously been observed in the NATl*10 allelic variant. NAT1*15 had a single nonsense mutation (C559T; Arg187stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1*11, NAT1*14 and NAT1*15 were 0.021, 0.028 and 0.014 (n=288 alleles), respectively, suggesting that they are relatively rare in Our predominantly Caucasian Sample.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Human JV-acetyltransferase type 1 (NAT1) catalyses the JV- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a C190T or GS60A base substitution located in the protein encoding region of the NAT1 gene. The C190T base substitution changed a highly conserved Arg64, which others have shown to be essential for fully functional NAT1 protein. The C190T mutation has not been reported previously and we have named it NAT1*17. The G560A mutation is associated with the base substitutions previously observed in the NAT1*1O allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the C190T (NAT1*17) and G560A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for Cancer risk in individual Subjects.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The Dark Agouti rat has been proposed as a model for the human debrisoquine 4-hydroxyIase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the Dark Agouti rat is caused by the absence of the expression of CYP2D1 mRNA. Although CYP2D1 is the major enzyme catalyzing debrisoquine 4-hydroxyIation, other reports have indicated the involvement of a CYP2D, purified from rat hepatic microsomes and presumed to be CYP2D2, which also exhibits this activity. The levels of CYP2D1 and CYP2D2 mRNAs were markedly lower in Dark Agouti as compared to Sprague Dawley rats. Using a baculovirus expression system, recombinant CYP2D1 and CYP2D2 from Spodopterafrugiperda insect cells were examined and were found to both forms catalize debrisoquine 4-hydroxyIase activity. These results suggest that reduced debrisoquine 4-hydroxylase activity in the Dark Agouti rat is caused by the low level expression not only of CYP2D1, but also of CYP2D2. Interestingly, bunitrolol 4-hydroxylation was catalyzed by recombinant CYP2D2, while CYP2D1 was inactive toward this substrate. Thus, the low bunitrolol 4-hydroxylation in Dark Agouti rats was caused by the low level of CYP2D2 expression in this rat strain.

ISSN:0960-314X    

出版商:OVID    

年代:1998

数据来源: OVID