MECHANISMS REGULATING CELL RESISTANCE TO ADRIAMYCIN

HL6O/ADR细胞的多药耐药(MDR)似乎与 MRP基因的过度表达和由此导致的MRP编码的增加 蛋白P190。因此，有证据表明，P19O在 非P-糖蛋白多药耐药的新模式。因此这种可能性是存在的 MRP的过表达可能有助于肿瘤细胞的多药耐药 正在接受化疗的病人。本研究的一个主要重点是 对蛋白质P190的结构和功能进行了详细的研究。至 鉴定这种蛋白质将制备合适的探针，这些探针将 包括抗P19O单抗和多克隆抗血清 针对该蛋白质推导出的序列的多肽。单克隆化 抗体将用于组织化学和电子显微镜研究 明确确定P19O在细胞内的定位。最近的证据 表明P19O是磷酸化的。我们会进行研究，以 确定磷酸化在生物功能中的作用 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。立地 特定的抗血清将被用来绘制磷酸盐沿 多肽链。一个长期目标将是确定序列 含有磷酸化氨基酸的PEP潮汐，并使用这个 定点突变中的信息。体外系统能够 将制备磷酸化P19O和能够 这种蛋白质的磷酸化将会被识别出来。一项重大努力将是 用来纯化P19O并确定该蛋白质是否含有某些 酶活性。此外，我们亦会进行广泛研究，以 检测调节MRP表达的分子机制。推动者 已对MRP区进行克隆和测序，并获得了证据 正负两种元素都能调节 转录活性。定点突变将被用来 确定该区域的序列要求和凝胶迁移率变化分析 和DNase1保护试验将用于检测核蛋白 这可能与调节启动子活性有关。核蛋白 它们调节了MRP的表达，并且以前没有 将被分离和鉴定。研究以考察 启动子活性的组织特异性也将被详细检测。

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.