p53 Mutation and Functional Analyses by Using Yeast Functional Assay / 대한암학회지

PURPOSE: Mutation of the p53 tumor suppressor gene is the most common genetic defect in all human tumors. Because of the widespread mutations and polymorphism in the p53 gene, the conventional screening methods cannot distinguish between polymorphisms or functionally silent mutations and inactivating mutations. It is well known that plasmids can be generated by homologous recombination in vivo in the yeast by cotransforming the PCR product with a linearized yeast expression vector encoding part of a gene and a selectable marker gene. The aim of this study is to develop more easy and reliable method for functional assay of p53 mutation. MATERIALS AND METHODS: We constructed a gap vector which can reliably and conveniently be used to screen p53 mutations in a simple yeast growth assay. The gap vector was constructed as follows: About 100 bp DNA fragments containing parts of N- and C- terminal portion of p53 were cloned into XbaI/SmaI and HindIII/XhoI sites of yeast expressing vector, respectively. The gap vector was obtained by double cutting with SmaI and HindIII followed by gel elution. Yeast was transformed with the reporter vector containing three tandem copies of the consensus p53 binding site by lithium acetate-mediated method. RT-PCR amplification of p53 transcripts from cell lines or tumor tissues was carried out. To investigate whether p53 gene is mutated or not, yeast containing reporter gene was cotransformed with PCR product and linearized gap vector, plated on SD medium minus histidine, and incubated for 3 days. The colonies on selective media were isolated and characterized. RESULTS: The tumor tissues examined were one hepatocellular carcinoma, three breast cancers, two stomach cancers and two colon cancers. One hepatocellular carcinoma tissue had mutation in both alleles of the p53 gene, and 7 cancer tissues had heterozygous mutations in the p53 gene. The result of functional assay was well correlated with mutational analysis by sequencing. CONCLUSION: p53 functional assay system might be easy and reliable method for functional screening of p53 on tumor tissues and this might be used for screening of other mutated gene. This technique, FASAY, requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

A Study on the Loss of Heterozygosity of the p53 Gene in Primary Uterine Cervical Carcinomas / 대한암학회지

PURPOSE: Allelic deletion of p53 tumor suppressor gene have been observed frequently in a variety of human tumors. These losses are believed to contribute to the development of human cancers. But the loss of heterozygosity (LOH) data on chromosome 17p are rare and controversial with respect to cervical carcinomas. So, we tried to elucidate the frequency of p53 locus LOH in primary cervical carcinoma and compared the LOH data with clinicopathological parameters. MATERIALS AND METHODS: In order to detect LOH within one of the well-known tumor suppressor gene, p53, three intragenic polymorphisms (exon 1, exon 4, and intron 6) and one microsatellite distal to the p53 gene (D17S5) were examined. Paired DNA samples from 55 primary uterine cervical carcinomas and normal bloods were studied for the chromosomal allelic loss of p53 gene locus by polymerase chain reaction (PCR), the presence of human papilloma virus (HPV), and the presence of p53 gene point mutation by PCR-single conformation polymorphism (SSCP) analysis. And the relationships between allelic losses of this gene and conventional clinicopathological parameters were evaluated. RESULTS: We could increase the heterozygosity of the p53 gene up to 1 (100%). The observed allelic loss rate of the p53 locus in informative cases was 5.5% (3/55) and the observed allelic loss rate of the D17S5 locus in informative cases was 8.7% (4/46) . Only one of the four patients with LOH at the D17S5 locus showed a concomittant allelic loss of the p53 gene. The overall LOH incidence of the chromosomal region comprising 17p13.1 (p53) to 17p13.3 (D13S5) was 10.9% (6/55). All the samples contained at least one of the oncogenic HPV type 16 and/or 18 sequences. No shifted bands were observed in the PCR-SSCP analysis of the p53 gene. The LOH of the p53 gene was not related to other parameters including clinical stage, histological type, and degree of differentiation. CONCLUSION: Concerning with the results above, we conclude that the allelic imbalance of the p53 gene itself is not implicated as a major contributing factor in the malignant transformation or the tumor progression in HPV-positive cervical cancers. Another putative tumor suppressor gene which has more important function than p53 gene in cervical carcinogenesis might exist between these two loci [p53 (17p13.1) and D17S5 (17p13.3)].

Glutathione S-Transferase Polymorphisms and Genetic Susceptibility to Cervical Cancer / 대한암학회지

PURPOSE: The identification of genetic traits that predispose individuals to environmentally induced cancers is one of the challanges in the assessment of individual cancer risk. The genetically determined differences in metabolism, related to glutathione S-transferases (GSTs) have been reported to be associated with various cancer susceptibility. The present study was set up to establish the frequencies of the polymorphic genotypes of two GST (GST- mu and GST-theta) isozymes in Korea, to evaluate a possible increased incidence of the genotypes associated with higher cervical cancer risks among Korean cervical cancer patients. MATERIALS AND METHODS: In this study, extracted DNAs from cervical cancer patients (228 for GST-mu and 241 for GST-theta genotypes) and normal controls (360 for GST-mu and 353 for GST-theta genotypes) were analysed with the polymerase chain reaction (PCR). RESULTS: The overall genotype distribution of the GST-theta polymorphisms was not statistically different between the patients and control groups. But, in the GST-mu null genotypes, there were remarkable differences between patients and control groups when the cervical cancer patients were devided into subgroups with respect to the age. The frequency of GST-mu null polymorphisms in the cervical cancer patients under the 40 years old was significantly higher compared to the patients above the 40 years old (0.01