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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Mutagenesis of the H-ras protooncogene and the p53 tumor suppressor gene
P Cerutti1, P Hussain, C Pourzand
1Department of Carcinogenesis, Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne.
Abstract:
Point mutations in ras protooncogenes and in the p53 tumor suppressor gene are common in many forms of human cancer. The identification of carcinogens which are responsible for their induction in humans is of great interest because it may suggest measures for disease prevention. Furthermore, the load of somatic mutations in cancer-related genes in premalignant tissues may become a useful parameter for risk assessment. For the measurement of such mutations, highly sensitive genotypic mutation systems are required which avoid the selection and clonal expansion of cells on the basis of a mutated phenotype. We have developed the restriction fragment length polymorphism/polymerase chain reaction method for genotypic mutation analysis and applied it to the study of the mutability of hot-spot codons in c-H-ras1 and p53 genes with human carcinogens. In particular, we studied the mutability of codons 247-250 of p53 with the mycotoxin aflatoxin B1 (AFB1) in human hepatocytes. AFB1 preferentially induced the transversion of guanosine to thymidine in the third position of codon 249, generating the same mutation which is found in a large fraction of hepatocellular carcinomas from regions of the world with AFB1-contaminated food. Our results are in support of AFB1 as an etiological factor for hepatocellular carcinoma in AFB1-contaminated areas. In an ongoing study we are comparing the load of mutations in hot-spot codon 12 of c-H-ras1 in urinary bladder carcinoma and in normal tissue, by restriction fragment length polymorphism/polymerase chain reaction. We observed moderately elevated abundances of guanosine to thymidine transversions in the middle position of codon 12 in tumor DNA. These results may reflect a mutator phenotype of the tumor tissue or they could be the consequence of the heterogeneity of the biopsies which were analyzed.
Insights
This study links aflatoxin B1 (AFB1) to liver cancer by showing it causes specific mutations in the p53 gene. It also investigates mutations in the c-H-ras1 gene in bladder cancer, suggesting potential carcinogen exposure links.
Area of Science:
- Oncology
- Molecular Biology
- Toxicology
Background:
- Point mutations in ras protooncogenes and p53 tumor suppressor genes are frequent in human cancers.
- Identifying carcinogens causing these mutations is crucial for disease prevention and risk assessment.
- Sensitive genotypic mutation systems are needed to measure somatic mutations in premalignant tissues.
Purpose of the Study:
- To develop and apply a sensitive genotypic mutation analysis method to study carcinogen-induced mutations in key cancer genes.
- To investigate the mutability of hot-spot codons in c-H-ras1 and p53 genes using human carcinogens.
- To assess the etiological role of aflatoxin B1 (AFB1) in hepatocellular carcinoma and investigate c-H-ras1 mutations in bladder cancer.
Main Methods:
- Developed and utilized the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method for genotypic mutation analysis.
- Studied p53 gene mutations in human hepatocytes exposed to the mycotoxin AFB1.
- Analyzed c-H-ras1 gene mutations in urinary bladder carcinoma and normal tissue samples.
Main Results:
- AFB1 preferentially induced a specific guanosine to thymidine transversion in codon 249 of the p53 gene in human hepatocytes, matching mutations found in AFB1-associated liver cancers.
- AFB1 is supported as an etiological factor for hepatocellular carcinoma in contaminated regions.
- Moderately elevated guanosine to thymidine transversions were observed in codon 12 of c-H-ras1 in tumor DNA compared to normal tissue, potentially indicating a mutator phenotype.
Conclusions:
- The RFLP/PCR method provides a sensitive tool for genotypic mutation analysis of cancer-related genes.
- AFB1 is strongly implicated as a causative agent for hepatocellular carcinoma in regions with contaminated food.
- Further investigation is needed to clarify the implications of observed c-H-ras1 mutations in bladder cancer, considering potential mutator phenotypes or biopsy heterogeneity.
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