MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

点击翻译按钮获取中文摘要

HL60 cells isolated for resistance to adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not however overexpress mdr1 and do not contain detectable levels of P- glycoprotein. Recent evidence indicates that resistance in HL60/Adr cells is related to overexpression of a new multidrug resistance gene which encodes a 190 kd (P190) membrane associated ATP binding protein. A major focus of the present study will be to molecularly clone and characterize a cDNA of the P190 gene. Antibody which is highly reactive with P190 will be used as a probe in the cDNA cloning procedure. The nucleotide sequence of the cDNA will be determined. If a full length cDNA can be obtained this material will be placed in an expression vector and used to transfect sensitive cells. The properties of the transfected cells will be examined. The cDNA will also be used to study amplification and expression of the P190 gene in various resistant isolates and in cells induced to undergo differentiation. Additional studies will be carried out to purify P190 using different types of affinity columns and to thereafter determine if the protein contains ATPase or protein kinase activities. HL60/Adr cells contain a 150 kd membrane protein (P150) which represents a hyperphosphorylated form of a protein contained in drug sensitive cells. In the present study P150 will be partially purified and this material will be used to prepare a monoclonal antibody against this protein. The antibody will be used in immunoprecipitation experiments to further characterize P150 phosphorylation and the relation of this event to the development of drug resistance. Membrane associated protein kinases involved in the phosphorylation of P150 will isolated and characterized. The P150 monoclonal antibody will also be used as a probe to clone a P150 cDNA. This material will be characterized and the nucleotide sequence determined. A protein kinase activity capable of phosphorylating P-glycoprotein has been partially purified from membranes of HL60 cells isolated for resistance to vincristine. This enzyme will be further purified and characterized and its properties with P-glycoprotein as substrate will be examined in detail.