MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

HL-60细胞对阿霉素的耐药性是多药耐药的 并且在药物的细胞积累方面有缺陷。 这些细胞不 然而过表达mdr 1且不含可检测水平P- 糖蛋白 最近的证据表明，HL 60/Adr细胞的耐药性 与一种新的多药耐药基因的过度表达有关， 编码190 kd（P190）膜相关ATP结合蛋白。 一个主要 本研究的重点将是分子克隆和表征一个 P190基因的cDNA。 与P190高度反应性的抗体将是 在cDNA克隆过程中用作探针。 的核苷酸序列 将确定cDNA。 如果可以获得全长cDNA， 将材料置于表达载体中，并用于表达 敏感细胞 将检查转染细胞的性质。 cDNA也将用于研究基因的扩增和表达。 P190基因在各种耐药菌株和诱导细胞中的表达 分化 将进行额外的研究以纯化P190 使用不同类型的亲和列，然后确定 该蛋白质具有ATP酶或蛋白激酶活性。 HL 60/Adr细胞含有150 kd的膜蛋白（P150），其代表了一种细胞膜蛋白。 药物敏感细胞中所含蛋白质的高度磷酸化形式。 在本研究中，P150将被部分纯化，并且该材料将 用于制备抗该蛋白的单克隆抗体。 的 抗体将用于免疫沉淀实验，以进一步 表征P150磷酸化以及该事件与 耐药性的发展。 膜相关蛋白激酶类 参与P150磷酸化的蛋白质将被分离和鉴定。 P150单克隆抗体也将用作探针以克隆P150 cDNA。 该材料将被表征，并且核苷酸序列 测定 一种能够磷酸化P-糖蛋白的蛋白激酶活性， 从分离的HL 60细胞膜中部分纯化， 对长春新碱耐药。 该酶将被进一步纯化， 其特征在于，其与P-糖蛋白作为底物的性质将被 详细检查。

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.