CHEMOTHERAPEUTIC INDUCTION OF SOMATIC MUTATION

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We have recently developed a system for detecting mutations in somatic cells which is particularly efficient in detecting and quantitating such events when the number of surviving mutagen exposed cells (or clones from those cells) is small (less than 1,000). The system has been worked out in cultured -cells and is based on the electrophoretic detection of mutants at over 40 enzyme loci. The multiple locus screen indicated a wide range in the susceptibility of loci (some producing mutants at frequencies greater than 10 to the -2 to mutagens. Results also implied that loci exquisitely sensitive to some mutagens may be comparatively unaffected by others. Subclone analysis of multiply marked variant clones not only established the mutant nature of the variants, but also revealed mutant sectors most likely caused by a delayed genetic effect of the chemical. Considering the impact of such results on such fundamental problems as the role of somatic mutation in neoplastic transformation and the genetic basis of tumor cell escape from chemotherapeutic cytotoxicity, these studies need to be extended, verified, and applied in vivo. Here we shall apply newly developed methods to clone T cells directly out of peripheral blood lymphocytes of chemotherapeutically exposed humans. These clones will then be put through our electrophoretic system to screen for enzyme locus mutants. These data will then be used to investigate: the validity of the in vitro mutation model, effects of chemotherapeutic agents in inducing cell variability, and the consequences of such variability on human health and biological origin of tumor and relapse cells. The research should also be effective in developing systems for assaying genetic toxicity in animal models and man. Both the electrophoretic screening approach for enzyme locus mutants and the T cell cloning procedures have now reached concordant development to the extent that they, in combination, are uniquely efficient and capable of approaching these questions.