MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

HL 60/ADR细胞的多药耐药（mdr）可能与其耐药机制有关。 MRP基因的过表达和由此产生的MRP编码的 P19 O蛋白。 因此，有证据表明，P19 O在细胞凋亡中起着核心作用。 一种新的非P-糖蛋白mdr模式。 因此， MRP的过度表达可能导致肿瘤细胞的MDR， 正在接受化疗的患者。 本研究的一个主要重点是 详细研究P19 O蛋白的结构和功能。 到 将制备合适的探针来表征该蛋白质，并且这些探针将 包括抗P19 O单克隆抗体以及多克隆抗血清 针对该蛋白质的推导序列的肽。 单克隆 抗体将用于组织化学和电子显微镜研究 以明确P19 O的细胞内定位。 最近的证据 表明P19 O被磷酸化。 将进行研究， 确定磷酸化在生物学功能中的参与， 蛋白质。 能够改变P19 O磷酸化的试剂将被 详细检查了对耐药表型的影响。 网站 将使用特异性抗血清绘制磷酸盐沿着分布图 多肽链。 一个长期的目标将是确定序列 含有磷酸化氨基酸的肽， 定点诱变中的信息。 体外系统能够 将制备磷酸化P19 O的激酶， 将鉴定磷酸化该蛋白质。 一项重大努力将是 纯化P19 O并确定蛋白质是否含有某些 酶活性 还将进行广泛的研究， 研究调节MRP表达的分子机制。 启动子 已克隆并测序了MRP区域，并获得了证据 正负两种元素都能调节 转录活性 位点特异性诱变将用于 定义该区域的序列要求和凝胶迁移率变动测定 和DNA酶1保护试验将用于检查核蛋白 其可能参与调节启动子活性。 核蛋白 其调节MRP表达，并且其以前没有被 将被分离并表征。 研究以审查 启动子活性的组织特异性也将被详细检查。

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.