The long term goal of this research program is to gain knowledge concerning the mechanisms involved in the response of mammalian cells to radiation, with particular reference to its carcinogenic and mutagenic effects. The approach is a multi- faceted one, and the endpoints under investigation include mutagenesis, malignant transformation, cell survival and the induction of chromosomal abnormalities. The program has a dual purpose: first, to better define the risks of low-level radiation exposure by examining the role of factors such as total dose, dose-rate, LET and the intracellular distribution of dose on mutagenesis and transformation; and second, to learn more about the cellular and molecular mechanisms for these effects.
The specific aims are: 1) To identify oncogenes activated during the process of malignant transformation by radiation. Transfection of DNA into NIH 3T3 cells will be carried out at regular intervals after irradiation of 10T1/2 cell cultures; DNA from primary and secondary transfectants will be examined by Southern blotting and reactivity to specific monoclonal antibodies. 2) To examine the direct mutagenic effects of various types of ionizing radiation on the c-Ha-ras proto-oncogene by transfection of the irradiated gene in a plasmid vector. 3) To examine factors involved in the morphological transformation and immortalization of human diploid fibroblasts. A particular focus will be on the study of a DNA fragment derived from the second intron of c-myc which induces specific morphologic alterations in a strain of partially transformed cells. We propose to identify this presumptive regulatory element by deletion analysis and sequencing, and furthur study its role in transformation. 4) To analyze the molecular structure of spontaneous and induced mutations at the autosomal tk locus in the TK6 human cell line. Appropriate probes will be obtained and restriction fragment or sequence polymorphic markers sought. Molecular biological techniques will be employed in order to determine whether the large-scale changes we have observed involve gene conversion, mitotic recombination or multi-locus deletion events and to estimate their extent. 5) To carry out cytogenetic analysis of mutant clones in order to determine whether large scale DNA structural changes are associated with visible chromosomal abnormalities. Results will be correlated with those of dosage blots. 6) To analyse the spectrum of molecular structural changes induced by different types of radiation including fast neutrons, heavy ions and Auger emitting radionuclides. 7) To continue studies of the relative biological effectiveness for transformation and mutagenesis of low dose, low dose-rate neutron exposure. 8) To investigate the role of microdistribution of dose of high LET radiation within the cell on its biologic effects by use of Auger electron emitting radionuclides. Emphasis will be on mutagenesis in human TK6 cells.
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