MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

调节细胞对阿霉素耐药的机制

• 批准号: 2376778
• 负责人: MELVIN S CENTER
• 金额: $ 15.72万
• 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 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2376778
• 关键词: DNA footprinting; P glycoprotein; adenosinetriphosphatase; binding proteins; doxorubicin; electron microscopy; enzyme activity; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; intracellular transport; laboratory mouse; laboratory rabbit; monoclonal antibody; multidrug resistance; nucleoproteins; phosphorylation; protein purification; protein sequence; protein structure function; protein transport; site directed mutagenesis; tissue /cell culture; transcription factor

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.

HL6O/ADR细胞的多药电阻（MDR）似乎与 MRP基因的过表达和MRP编码的增加 蛋白质P19O。 因此，证据表明p19o在 非P-糖蛋白MDR的一种新模式。 因此存在可能性 MRP的过表达可能导致MDR的MDR 接受化疗的患者。 本研究的重点将 要详细检查蛋白质p19o的结构和功能。 到 表征该蛋白质适当探针的特征，这些探针将 包括针对P19O和多克隆抗血清的单克隆抗体 反对该蛋白的推导序列的肽。 单克隆 抗体将用于组织化学和电子显微镜研究 清楚定义p19o的细胞内位置。 最近的证据 表明P19O被磷酸化。 研究将进行 确定磷酸化参与的生物学功能 蛋白质。 能够改变P19O磷酸化的药物将是 详细检查了对耐药表型的影响。 地点 特定的抗血清将用于绘制磷酸盐的分布 多肽链。 长期目标是确定顺序 含有磷酸化氨基酸的PEP潮汐，并使用 网站中的信息定向诱变。 能够 将制备磷酸化的P19O，激酶能够 将鉴定磷酸化该蛋白。 将是一项重大努力 纯化p19o并确定蛋白质是否包含某些 酶活性。 广泛的研究也将进行 检查调节MRP表达的分子机制。 发起人 MRP区域已被克隆并进行了测序，并获得了证据 正面和负元素都可以调节 转录活动。 特定地点的诱变将用于 定义该区域和凝胶迁移率转移测定的序列要求 和DNase 1保护测定法将用于检查核蛋白 可能参与调节启动子活动。 核蛋白质 哪个调节MRP表达和以前尚未 确定的将被隔离和表征。 研究 启动子活性的组织特异性也将进行详细检查。