The objective is to assess and validate the value of mutation spectra in the p53 tumour suppression gene and other critical genes in the identification of environmental agents responsible for cancer induction and development in humans.
The research objective is to assess and validate the value of mutation spectra in the p53 tumour suppression gene and other critical genes in the identification of environmental agents responsible for cancer induction and development in humans.
Mutations in the p53 tumour suppressor gene are commonly found in the major human cancers and the mutational spectrum in some cancers is consistent with the genotoxic effects of the associated environmental risk factors. Thus far there is little information on p53 mutations in cancers of factory workers with a history of carcinogen exposure in the workplace. Tumours from 5 vinyl chloride exposed patients, 4 with liver angiosarcomas (ASL) and one with hepatocellular carcinoma (HCC) were examined for evidence of MDM2 protooncogene amplification of p53 mutation in exons 5 to 8. Amplification of MDM2 was not found, but in 2 of the angiosarcomas an A:T to T:A missense mutation was detected. p53 sequence analysis of vinyl chloride associated cancers may provide valuable information on the relationship between carcinogen exposure and DNA damage in cancer related genes.
Experiments are under investigation to increase the reactivity of the mutagenic metabolite. In bacteria, the first activation of quinoline (IQ) produces the hydroxylamine derivative which can react directly with deoxyribonucleic acid (DNA), but a second activation step mediated by a N-acetyltransferase produced the highly reactive N-acetoxy derivative. In contrast to what is frequently observed with unicellular systems and in mammalian cells in culture, large deletions represent the predominant type of sequence change found after mutagen exposure of postmeiotic germ cell stages. This is the uniform picture found for a whole series of monofunctional (exceptions are ENU and EMS) and crosslinking agents in both Drosophila and the mouse. The pilot results obtained so far for aflatoxin B1 support the conclusion that this carcinogen may be a further efficient deletion maker in germ cells in vivo.
Attempts are presently underway made to establish a suitable technique to measure mutations in a gene that does not allow a phenotypic selection, such as the p53 oncogene. This technique is based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) techniques.
The selection of the environmental chemicals to be examined in this proposal is based principally on the following criteria:
i) environmental relevance;
ii) identification of populations that are exposed specifically to a carcinogen/mutagen; and
iii) knowledge of the mechanisms of induction of DNA damage by the chemicals considered.
The complementary studies in this project include:
i) Determination of the mutation profile of p53 tumour suppressor gene from histological archival cancer specimens (bladder and liver) in populations exposed to specific carcinogens (aromatic amines and vinyl chloride); the advantages of studying occupationally exposed individuals to establish a link between tumour-specific cancer gene damage and exposure to a mutagenic carcinogen are the well-documented exposure levels, the clear indications of the candidate chemical agent linked to elevated risk, and the documentation of the degree of risk attributable to these exposures.
ii) Determination of mutation frequencies and molecular mutation spectra in two experimental systems (tetracycline forward mutation assay in E.coli and Drosophila in vivo) of classes of important environmental mutagens (heterocyclic amines, aromatic amines, mycotoxins) for which human exposure occurs. This data base will be essential for the critical analysis of mutation spectra in the p53 tumour suppressor gene recovered from specific collections of human tumours. In addition, the Drosophila system permits detection in somatic cells of recombination events that could be specifically induced at high frequencies by some mutagens. Evidence exists that loss of heterozygosity by recombination or gene conversion is an important event in the inactivation of p53 or other tumour suppressor genes in the carcinogenesis process.
iii) Examination in epithelial rat intestinal cultured cells of the specificity of mutations in the p53 tumour suppressor gene induced by different chemical carcinogens. The detection of mutations will be done by a non-selective method involving restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR). This permits measurement, using specific oligonucleotide probes of mutations at low frequency in any gene of known sequence. It is expected that the data resulting from this in vitro epithelial cell culture system would eventually allow the application of this approach in human tumours as well as the determination of the temporal occurrence of such mutations in the natural history of these tumours.
Funding SchemeCSC - Cost-sharing contracts
2300 RA Leiden