MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

调节细胞对阿霉素耐药的机制

• 批准号: 3175365
• 负责人: MELVIN S CENTER
• 金额: $ 10.56万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3175365
• 关键词: DNA binding protein; adenosine triphosphate; adenosinetriphosphatase; cell membrane; complementary DNA; doxorubicin; drug metabolism; drug resistance; gel electrophoresis; gene expression; glycoproteins; human tissue; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; multidrug resistance; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; nucleic acid sequence; pharmacokinetics; phosphorylation; protein kinase; vincristine

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.

HL60细胞分离以抗adrimycin具有多药耐药性 并且在药物的细胞积累中有缺陷。 这些细胞不 但是过表达MDR1，并且不包含可检测水平的P- 糖蛋白。 最近的证据表明HL60/ADR细胞的耐药性 与新的多药电阻基因的过表达有关，该基因 编码190 KD（P190）膜相关的ATP结合蛋白。 专业 本研究的重点将是分子克隆并表征 P190基因的cDNA。 p190高反应性的抗体将是 用作cDNA克隆程序中的探针。 核苷酸序列 将确定cDNA。 如果可以获得全长cDNA 材料将放在表达载体中，并用于转染 敏感细胞。 将检查转染细胞的特性。 cDNA还将用于研究扩增和表达 各种抗性分离株和细胞中的P190基因 分化。 将进行其他研究以净化P190 使用不同类型的亲和力列，然后确定是否 该蛋白质含有ATPase或蛋白激酶活性。 HL60/ADR细胞含有150 kD膜蛋白（P150），代表A 药物敏感细胞中包含的蛋白质的高磷酸化形式。 在本研究中，P150将部分纯化，该材料将 用于制备针对该蛋白的单克隆抗体。 这 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 参与P150的磷酸化将分离和表征。 P150单克隆抗体也将用作克隆P150的探针 cDNA。 该材料将被表征和核苷酸序列 决定。 能够磷酸化P-糖蛋白的蛋白激酶活性 从分离的HL60细胞的膜中部分纯化 抗vincristine的能力。 该酶将被进一步纯化，并且 特征及其具有P-糖蛋白作为底物的特性将是 详细检查。