MOLECULAR MECHANISM MULTIDRUG RESISTANCE--MAMMALIAN CELL

恶性肿瘤细胞对细胞毒药物耐药性的发展 细胞是化疗失败的主要原因。 癌症的管理。 这种抵抗力的发展 与结构蛋白或DNA相互作用的细胞毒性药物 不仅发生在选择性试剂上， 其他看似无关的化合物 建立 组织培养细胞中的多药耐药（MDR）代表了 与基因扩增相关的复杂过程 以及一种质膜糖蛋白的过度表达， P-糖蛋白 该研究旨在分析 P-糖蛋白基因的表达在 多药耐药细胞耐药性的建立和维持 亲本中国仓鼠肺（CHL）细胞的耐药亚系 DC-3F 描述这些的实验策略 基因转录本将首先在pUC 9中制备cDNA文库 来自含有过表达基因的MDR亚系的载体。 这些图书馆将在一定条件下建造， 产生全长cDNA并用切口翻译的P- 从药物制备的糖蛋白cDNA探针 抗性品系 对应于未扩增的 来自亲本系DC-3F的转录本将在 λ gt 10载体，这些文库将用 类似的探针。 在这样的筛选后分离的全长cDNA 将使用精细限制性作图相互比较，并且 如有必要，进行测序研究，以确定 多药耐药相关基因异质性 表达式存在于各个子行之间。 转染 来自抗性亚系的功能性全长MST-cDNA， 将进行药物敏感的母体线，以评估实际的 的作用，其他实验设计 使用反义RNA来抑制 多基因家族 这种反义抑制的作用 将通过评估耐药性的可能变化进行测定， 通过克隆形成测定期间的ED 50值确定。 最后为 确定差异基因表达是否被控制在 5'调控序列，将进行实验， 分离和表征DC-3F启动子序列， 其抗性亚系DC-3F/ADXC。 的人员和 斯隆-凯廷研究所的实验室设施设备齐全 承担这个项目，这样的基础研究可能会导致 最终制定战略，以克服多药耐药问题， 在临床上的抵抗力。

Development of drug resistance to cytotoxic agents by malignant cells is a major reason for failur to present chemotherapy in the management of cancer. Development of such resistance to cytotoxic drugs that interact with structural proteins or DNA occurs not only to the selective agents but also to a wide variety of other, seemingly unrelated, compounds. The establishment of multidrug-resistance (MDR) in tissue culture cells represents a complicated process that is associated with gene amplificiation and over-expression of a plasma-membrane glycoprotein, termed P-glycoprotein. The proposed study is designed to analyze the role expression of P-glycoprotein gene(s) plays in the establishment and maintenance of drug resistance in multidrug- resistant sublines of the parent Chinese hamster lung (CHL) cell line, DC-3F. The experimental strategy to characterize these geen transcripts will be to first prepare cDNA libraries in pUC9 vectors from MDR sublines containing overexpressed genes. These libraries wil be constructed under conditions that will generate full-length cDNAs and screended with nick-translated P- glycoprotein cDNA probes that have been prepared from drug resistant lines. cDNAs corresponding to the nonamplified transcripts from the parent line, DC-3F, will be constructed in lambda gt10 vectors and these libraries will be screened with similar probes. Full-length cDNAs isolated after such screening will be compared to each other using fine restriction mapping, and if necessary, sequencing studies to determine the extent to which heterogeneity of multidrug-resistant-associated (MDRA) gene expression exists between the various sublines. Transfection of a functional full-length MDRA cDNA from a resistant subline to the drug-sensitive parent line will be carried out to assess the actual role the MDRA gene expression, other experiments are designed to use anti-sense RNAs to inhibit expression of members of the MDRA multigene family. Effects of such anti-sense inhibition will be assayed by assessing possible changes in drug-resistance as determined by ED50 values during clonogenic assays. Finally, to determine if differential gene expression is controlled at the level of 5' regulatory sequences, experiments will be carried out to isolate and characterize promoter sequences of DC-3F and one of its resistant sublines, DC-3F/ADXC. The personnel and laboratory facilities of Sloan-Ketting Institute are well equipped to undertake this project and such basic research may lead to the eventual development of strategies to overcome multidrug- resistance in the clinical setting.