MECHANISMS OF ARSENITE RESISTANCE AND TOLERANCE

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Arsenite is a human carcinogen whose mechanism of action is unknown. Previously, we demonstrated that arsenite is an inhibitor of DNA repair, especially of the ligation step. Recently, a unique aspect of arsenite's activity became apparent in our finding that arsenite treatment induces amplification of the endogenous dhfr gene, but not of SV4O sequences. This finding, along with the known ability of arsenite to activate a number of regulons (e.g. hsp, MDR) led to our current interest in arsenite-inducible genes which confer arsenite tolerance. A number of stable arsenite- resistant and sensitive sublines of V79 cells have been isolated. Both wild-type and variant lines can be induced by arsenite to a transient arsenite-tolerant state, a process requiring de novo mRNA and protein synthesis. Preliminary data shows that arsenite resistant line As/R28A has a unique phenotype. It is cross-resistant to antimonite but not to other metals, and contains normal levels of GSH. Using cell fusion techniques, the dominance of arsenite resistance over sensitivity (using sensitive line As/S14A) was ascertained. Arsenite-induced As/R28A cells will be used in creating a cDNA subtractive library (cDNA from arsenite-induced As/R28A minus cDNA from uninduced As/S14A). Vector pMSG, which contains the selectable gpt marker and in which inserted sequences are under control of the glucocorticoid promoter, will be used. Recombinant pMSG will be transfected into As/SI4A. Transfectants will be selected with HAT + dexamethasone + arsenite. The cDNA insert will be rescued from the transfectants by PCR amplification and sequenced. The phenotypes of other arsenite-resistant and sensitive sublines will be compared to those of As/R28A and As/S14A. Analysis of the gene products responsible for arsenite resistance and tolerance will increase our understanding of arsenite toxicity and may give important insights into arsenic-induced carcinogenesis as well.