MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

耐阿霉素的HL60细胞对多药耐药 并且在细胞内药物积累方面存在缺陷。这些细胞不会 然而，过度表达mdr1并且不包含可检测到的P- 糖蛋白。最近的证据表明，HL60/ADR细胞中的耐药性 与一种新的多药耐药基因的过度表达有关 编码一个190kd(P190)的膜相关ATP结合蛋白。一位少校 本研究的重点将是从分子上克隆和鉴定一种 P190基因的c DNA序列。与P190高度反应的抗体将是 在cdna克隆过程中用作探针。该病毒的核苷酸序列 将确定其cDNA值。如果可以获得全长的cdna， 材料将被放置在表达载体中并用于转基因 敏感细胞。将对转基因细胞的特性进行检测。 该基因还将被用于研究基因的扩增和表达。 P190基因在各种耐药株和诱导细胞中的表达 差异化。将进行更多的研究来纯化P190 使用不同类型的亲和层析柱，然后确定 该蛋白质含有ATPase或蛋白激酶活性。 HL60/ADR细胞含有一个150kd的膜蛋白(P150)，它代表一个 药物敏感细胞中蛋白质的过度磷酸化形式。 在本研究中，P150将被部分提纯，该材料将 可用于制备抗该蛋白的单抗。这个 抗体将用于免疫沉淀实验，以进一步 表征P150的磷酸化以及这一事件与 耐药性的发展。膜相关蛋白激酶 参与P150磷酸化的基因将被分离和鉴定。 P150单抗也将被用作克隆P150的探针 CDNA.这种材料将被表征和核苷酸序列 下定决心。 一种能够磷酸化P-糖蛋白的蛋白激酶活性 是从分离的HL60细胞膜中部分纯化出来的 对长春新碱耐药。该酶将进一步纯化并 以P-糖蛋白为底物的表征及其性质 仔细检查了一下。

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.