MICRODETECTION ASSAY FOR DRUG-RESISTANT TUMORS

了解多药耐药（MDR）具有明显的临床意义 重要性 MDR的典型特征是一种“标记物”的过量。 蛋白质，称为“P-糖蛋白”或gp 180”，及其外观 可以追溯到基因的扩增和/或过表达 一些病人的肿瘤样本已经被证明 通过印迹分析来获得该蛋白并过表达其mRNA。 然而，gp 180并不总是MDR的标志物。 最近的工作 从这个实验室已经确定了“非典型”MDR（at-MDR）， 人类白血病细胞对以下物质具有抗性和交叉抗性： 蒽环类和表鬼臼毒素，但对长春花敏感 生物碱 这种at-MDR具有潜在的临床重要性，因为 许多治疗方案涉及这些类别的组合 毒品 At-MDR细胞不过度表达MDR标志物 蛋白质或其mRNA;因此，可用的抗体和cDNA “经典”MDR的探针将无法检测到这种形式的MDR， 临床标本 该提案的一个具体目标是， 单克隆抗体和cDNA探针，将区分在- MDR细胞从它们的药物敏感对应物中分离出来， 临床微量检测测定中的合适试剂。 提高 面对肿瘤细胞异质性的MDR检测， 抗原调制，我们打算开发单细胞微- MDR-和at-MDR-相关蛋白的检测试验， mRNA，使用免疫组织化学和原位杂交 方法论。 这种测定将应用于各种肿瘤 标本和正常组织，结果与 临床疾病特征和治疗结果。 最后 MDR标记蛋白gp 180可能与MDR的耐药机制不同， 与药物结合、细胞生长和 细胞毒 该假设将通过开发直接测试 一组抗体来识别这些位点。 此类抗体 可用于微量检测分析，并可能 具有治疗益处。 总而言之， 建议的工作是发展一个可靠的临床 用于MDR和at-MDR存在的微量检测测定， 最终将允许开发更具体的治疗方法。 要做到这一点，就必须开发抗体和核酸 由于现有试剂将 仅检测“经典”MDR。 这些试剂将用于 开发一组敏感和特异的临床有用的单- 细胞和印迹分析，确定是前瞻性的， 回顾性研究。

Understanding multiple drug resistance (MDR) has obvious clinical importance. MDR is typified by the overabundance of a "marker" protein, known as "P-glycoprotein" or gp180", and its appearance can be traced to amplification and/or overexpression of the gene coding for it. Some tumor samples from patients have been shown by blotting assays to have this protein and overexpress its mRNA. However, gp180 is not invariably a marker of MDR. Recent work from this laboratory has identified "atypical" MDR (at-MDR) in human leukemic cells that are resistant and cross-resistant to anthracyclines and epipodophyllotoxins, but are sensitive to vinca alkaloids. This at-MDR is of potential clinical importance, since many treatment protocols involve combinations of these classes of drugs. At-MDR cells do not overexpress the MDR marker protein, or its mRNA; hence, the available antibody and cDNA probes for "classic" MDR will fail to detect this form of MDR in clinical specimens. A specific aim of this proposal is to develop monoclonal antibody and cDNA probes that will distinguish at- MDR cells from their drug-sensitive counterparts and will be suitable reagents in a clinical microdetection assay. To improve detection of MDR in the face of tumor cell heterogeneity and antigen modulation, we intend to develop single cell micro- detection assays for MDR- and at-MDR-associated proteins and mRNAs, using immunohistochemical and in situ hybridization methodologies. Such assays will be applied to a variety of tumor specimens and normal tissues, and the results correlated with clinical disease features and treatment outcome. Finally, the MDR marker protein, gp180, may have different functional domains associated with drug binding, cell growth and cytotoxicity. The hypothesis will be tested directly by developing panels of antibodies that will identify these sites. Such antibodies may be useful in a microdetection assay and potentially could have therapeutic benefit. In summary, the long term-goal of the proposed work is the development of a reliable clinical microdetection assay for the presence of MDR and at-MDR that will ultimately permit the development of more specific therapy. To do so, it will be necessary to develop antibody and nucleic acid reagents that will identify at-MDR, since existing reagents will only detect "classic" MDR. These reagents will be used to develop a panel of sensitive and specific clinically useful single- cell and blotting assays, as determined is prospective and retrospective studies.