MULTIDRUG RESISTANCE AND PROGRAMMED CELL DEATH IN TUMORIGENESIS

我们希望研究分子之间的可能联系 耐药性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 由于MDR基因的表达与那些由表达 原癌基因bcl-2 为了调查可能的 这两种耐药机制之间的相互作用， 作为第一步，证明在哺乳动物细胞中的表达 我们实验室最近克隆的大鼠mdr 1b基因， 他们有MDR表型。 作为第二步，我们计划表达 人bcl-2基因在含mdr 1b的耐药细胞和 亲本对照细胞系，试图（1）比较相对 这些耐药机制的效力，和（2）看看是否 它们可以相互协同作用， 细胞毒性剂。 第一个目标是通过亚克隆 将大鼠mdr 1b基因从pGEM-11 Zf（+）质粒中插入pGEM-11 Zf（+）质粒的EcoRI位点， pBK-CMV原核-真核表达载体， CMV启动子。 pBK-CMV/mdr 1b重组质粒和 然后将载体pBK-CMV单独转染到NIH 3 T3小鼠胚胎中 用脂质体转染成纤维细胞。 mdr 1b产生的耐药水平 在这些实验中， 通过对转染人MDR 1的NIH 3 T3细胞使用相同的测定， RSV启动子控制下的基因。 因此，NIH/3 T3-mdr 1b 细胞被认为是比较MDR抗性的良好模型 具有新的bcl-2依赖性耐药机制的表型。

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.