MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

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Multidrug resistance (mdr) of HL6O/ADR cells appears to be related to an overexpression of the MRP gene and a resulting increase in the MRP encoded protein P19O. Evidence thus indicates that P19O plays a central role in a new mode of non-P-glycoprotein mdr. The possibility therefore exists that overexpression of MRP may contribute to an mdr in tumor cells of patients undergoing chemotherapy. A major focus of the present study will be to examine in detail the structure and function of protein P19O. To characterize this protein suitable probes will be prepared and these will include monoclonal antibodies against P19O and also polyclonal antisera against peptides of the deduced sequence of this protein. The monoclonal antibodies will be used in histochemical and electron microscopic studies to clearly define the intracellular location of P19O. Recent evidence indicates that P19O is phosphorylated. Studies will be conducted to determine the involvement of phosphorylation in the biological function of the protein. Agents capable of altering P19O phosphorylation will be examined in detail for an effect on the drug resistant phenotype. Site specific antisera will be used to map the distribution of phosphate along the polypeptide chain. A long term goal will be to identify the sequence of pep tides which contain phosphorylated amino acids and to use this information in site directed mutagenesis. In vitro systems capable of phosphorylating P19O will be prepared and kinases capable of phosphorylating this protein will be identified. A major effort will be made to purify P19O and to determine if the protein contains certain enzymatic activities. Extensive studies will also be carried out to examine molecular mechanisms regulating the expression of MRP. A promoter region of MRP has been cloned and sequenced and evidence has been obtained that both positive and negative elements are capable of modulating transcriptional activity. Site specific mutagenesis will be used to define sequence requirements of this region and gel mobility shift assays and DNase 1 protection assays will be used to examine nuclear proteins which may be involved in regulating promoter activity. Nuclear proteins which modulate MRP expression and which have not been previously identified will be isolated and characterized. Studies to examine the tissue specificity of promoter activity will also be examined in detail.