MULTIDRUG RESISTANCE IN ANIMAL CELLS

对化疗剂的耐药性的发展是在化疗中观察到的。 临床，并已在组织培养细胞中进行了广泛研究。 一 不寻常的表型是多药耐药，其中细胞是 用任何一种细胞毒性药物攻击产生耐药性 不仅对选择性试剂而且对其它试剂也具有交叉抗性， 看似无关的化合物 这种多药耐药（MDR）表型 在许多情况下，这与一个小家庭的过度生产有关。 被称为P-糖蛋白（Pgp）的膜糖蛋白被认为 作为ATP激活的外排泵。 在仓鼠的三个家庭成员中， 它是人mdr 1蛋白的同源物（已知在人mdr 1中过表达）， 人肿瘤），pgp 1，其最常参与肿瘤的发生。 组织培养中多药耐药性建立与维持 细胞系，并且是本申请的主题。 知之甚少 关于归因于PGP 1的外排机制或 建立交叉抗性模式。 此外，尚不清楚 pgp 1是单独发挥作用还是与其他因素结合发挥作用， 持续流出。 除了这些关于功能的问题， pgp 1基因表达的变化似乎是复杂的， 转录和翻译水平。 结合使用 重组DNA技术，基因转染试验，核导入 实验和原位诱变技术。 我们建议：（1）执行 pgp 1跨膜区6的分子遗传学研究 转运蛋白，已知参与介导 交叉抗性; 2）建立分子基础和功能 已经发现的pgp 1 mRNA的选择性剪接形式的意义 存在于MDR细胞中并可能编码新的Pgp 1; 3）阐明 是否需要额外的因子来维持PGP 1介导的外排， 解决仓鼠成纤维细胞中pgp 1过表达是否 降低这些细胞的致癌潜力，以及4）证明， 扩增的pgp 1基因可以在转录水平上调节， 与基因拷贝数无关。

The development of resistance to chemotherapeutic agents is observed in the clinic, and has been studied extensively in tissue culture cells. One unusual phenotype is that of multidrug resistance, in which cells that are challenged with any one of a variety of cytotoxic drugs develop resistance not only to the selective agent but also cross-resistance to other, seemingly, unrelated, compounds. This multidrug-resistant (mdr) phenotype is associated in many cases with the overproduction of a small family of membrane glycoproteins called p-glycoproteins (pgp's), which are thought to act as ATP-activated efflux pumps. Of the three family members in hamster, it is the homolog of the human mdr1 protein (known to be overexpressed in human tumors), pgp1, that is most frequently involved with the establishment and maintenance of multidrug-resistance in tissue culture cell lines, and is the subject of this application. Little is known concerning the efflux mechanism attributed to pgp1 or the manner in which cross-resistance patterns are established. Moreover it remains unclear whether pgp1 functions alone or in combination with other factors to sustain efflux. In addition to these questions about function, regulation of pgp1 gene expression appears to be complex and to occur at both the transcriptional and translational levels. Using a combination of recombinant DNA technology, gene transfection assays, nuclear run-on experiments and in situ mutagenesis techniques. We propose to 1) carry out a molecular genetic study of transmembrane domain 6 of the pgp1 transporter, a region of the protein known to be involved with mediation of cross-resistance; 2) establish the molecular basis and functional significance of alternate splicing forms of pgp1 mRNAs that have been found to exist in mdr cells and to potentially encode novel pgp1's; 3) clarify whether additional factors are required to sustain pgp1 mediated efflux and address the question of whether pgp1 overexpression in hamster fibroblasts decreases the oncogenic potential of these cells, and 4) demonstrate that amplified pgp1 genes may be regulated at the level of transcription, independent of gene copy number.