The major goal of this proposal is to elucidate the molecular basis by which compounds such as arsenic alter differentiation processes in human keratinocytes, a major target cell type. The conceptual framework for this work includes the hypotheses that (1) protein kinase C activity is important for keratinocyte differentiation, (2) this kinase ordinarily activates one or more DNA binding proteins responsible for differentiation- specific transcription, (3) chronic TPA treatment of sensitive target cells depletes them of the kinase and (4) arsenate (but not arsenite) prevents phosphorylation reaction(s) necessary for transcription factor activation. First, the molecular basis for arsenate suppression of a differentiation marker (involucrin) in cultured human keratinocytes will be explored. To determine whether transcription of the gene for this marker protein is suppressed, effects on mRNA stability and transcription factor action will be investigated. Second, effects of arsenate on protein kinase C will be examined. Measurements will include total activity, isozyme distribution and intracellular localization, possible alteration of differentiation specific substrates, and comparative action on keratinocytes at various stages of neoplasia. Third, to determine the importance of arsenic oxidation state in target epithelia, the rate of arsenate/arsenite interconversion will be compared in cells which do or do not show a sensitivity to arsenate suppression of differentiated function. Finally, extension of the above target cell analysis to combustion products will be explored. The actions of metal aerosols, as well as specific metal oxides and carbon-containing compounds, on epithelial cell differentiation and protein kinase C will be measured. In addition, the degree to which these combustion products, particularly those derived from chlorinated organics such as trichloroethylene, prevent keratinocyte growth will be measured. Whether this occurs by undergoing biotransformation to reactive metabolites or by stimulating the metabolism of other agents to which the keratinocytes are thereby sensitized will be determined. The results are anticipated to provide a basis for rational assessment of the carcinogenic risk posed to humans from exposure to arsenic and combustion products.
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