MULTIDRUG RESISTANCE AND PROGRAMMED CELL DEATH IN TUMORIGENESIS

我们希望研究分子之间可能的联系 耐药的基础和程序性细胞死亡的调节。 在涉及的几种机制中，我们选择比较它们的效果 由于MDR基因的表达与那些由表达引起的 原癌基因bcl2。为了调查可能发生的 我们认为这两种耐药机制之间的相互作用， 作为第一步，要证明在哺乳动物细胞中的表达 最近在我们实验室克隆的大鼠mdr1b基因的 具有MDR表型的患者。作为第二步，我们计划表达 人bcl2基因在含有mdr1b的耐药细胞中的表达 亲本控制细胞系，尝试(1)比较亲缘关系 这些耐药机制的效力，以及(2)看看 它们可以相互协同作用，保护细胞免受 细胞毒剂。第一个目标是通过亚克隆的cDNAs 将大鼠mdr1b基因从pGEM-11Zf()载体克隆到EcoRI酶切位点。 PBK-CMV原核-真核表达载体 巨细胞病毒启动子。PBK-CMV/mdr1b重组载体和pBK-CMV/mdr1b重组载体 然后将载体pBK-CMV单独导入NIH3T3小鼠胚胎 成纤维细胞与脂联胺。Mdr1b赋予的抗性水平 在这些实验中比以前的研究中显示的要高 对转染人MDR1的NIH3T3细胞进行同样的检测 在RSV启动子控制下的基因。因此，NIH/3T3-mdr1b 细胞被认为是比较耐多药的一个很好的模型 具有新的bcl2依赖耐药机制的表型。

We wish to investigate the possible connections between the molecular basis of drug resistance and the regulation of programmed cell death. Among several mechanisms involved, we have chosen to compare the effects due to the expression of mdr genes with those caused by the expression of the protooncogene bcl-2. In order to investigate the possible interactions between these two mechanisms of drug resistance we decided, as a first step, to demonstrate that the expression in mammalian cells of the rat mdr1b gene, recently cloned in our laboratory, would provide them with the MDR phenotype. As a second step, we plan to express the human bcl-2 gene in the resistant cells containing mdr1b and in the parental control cell line, attempting (1) to compare the relative potency of these mechanisms of drug resistance, and (2) to see whether they can act synergistically with each other in protecting cells against cytotoxic agents. The first goal was achieved by subcloning the cDNA of the rat mdr1b gene from the pGEM-11Zf(+) plasmid into the EcoRI site of the pBK-CMV prokaryotic-eukaryotic expression vector, under the control of the CMV promoter. The pBK-CMV/mdr1b recombinant plasmid and the vector pBK-CMV alone were then transfected into the NIH3T3 mouse embryo fibroblasts with lipofectamine. The resistance levels conferred by mdr1b in these experiments was higher than the one shown in previous studies by using the same assay on NIH3T3 cells transfected with the human MDR1 gene under the control of the RSV promoter. Therefore, the NIH/3T3-mdr1b cells are thought to be a good model for comparing the mdr resistant phenotype with the novel bcl-2 dependent drug-resistant mechanism.