MULTIDRUG RESISTANCE IN MAMMALIAN CELLS

观察到对化疗药物的耐药性的发展 临床，并在组织培养细胞中得到了广泛的研究。一 不寻常的表型是多药耐药，在这种情况下， 用任何一种细胞毒药物挑战都会产生抗药性 不仅对选择剂，而且对其他的交叉抗性， 看似不相关的复合词。这种多药耐药(MDR)表型 在许多情况下与一个小家庭的生产过剩有关 被称为P-糖蛋白(PGP)的膜糖蛋白，被认为是 充当三磷酸腺苷激活的外排泵。在仓鼠的三个家庭成员中， 它是人类mdr1蛋白的同源物(已知在 人类肿瘤)，pgp1，最常与 组织培养中多药耐药性的建立与维持 细胞系，这是本申请的主题。鲜为人知 关于归因于pgp1的外流机制或 建立了交叉阻力模式。此外，目前还不清楚 Pgp1是单独发挥作用，还是与其他因素一起发挥作用 维持外排。除了这些关于功能的问题外，监管 Pgp1基因的表达似乎是复杂的，并且发生在 转录和翻译水平。使用以下组合 重组DNA技术、基因转染检测、核运行 实验和原位诱变技术。我们建议1)进行 Pgp1跨膜区6的分子遗传学研究 转运蛋白，已知的参与调节的蛋白质区域。 交叉抗性；2)建立分子基础和功能 已发现的pgp1mRNAs的不同剪接形式的意义 存在于多药耐药细胞中并可能编码新的pgp1‘S；3)阐明 是否需要额外的因素来维持pgp1介导的外流，以及 解决pgp1在仓鼠成纤维细胞中是否过表达的问题 降低这些细胞的致癌潜力，以及4)证明 扩增的pgp1基因可能在转录水平上受到调节， 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.