ISOLATION OF A SELECTED DRUG RESISTANT GENE

对进一步化疗耐药的肿瘤的出现是一个主要原因 这是成功治疗癌症的障碍。过度表达 多药耐药与临床多药耐药有关。 然而，临床试验表明，到目前为止，还有其他一些 临床上出现多药耐药的机制不明。这 实验室已经选择了对米托蒽醌耐药的人类细胞株 与能量依赖的药物外排有关，但不相关 与已知的药物转运蛋白mdr1和mrp的过度表达有关。 这些细胞系提供了一种新的多药耐药模型 表型。我们最近通过染色体转移确定了 与米托蒽醌选择相关的抗性是由于 显性遗传事件。这为克隆提供了理论基础 导致米托蒽醌产生抗性的基因 这项提议的目标是。这项提议的一项主要承诺是 用于筛选cDNA文库的探针的开发。我们已经调用了 也利用米托蒽醌的光亲和性类似物的策略 作为ATP光亲和类似物来鉴定可能涉及的蛋白质 在抵抗中。抗性标记蛋白的纯化 通过光亲和力类比的细胞系最终将允许我们 研制用于免疫筛选表达文库的抗体。 纯化的蛋白质也将被测序，这将使我们能够 设计简并寡核苷酸用于筛选cDNA文库。最后，我们 建议如果我们不能识别出本文件中概述的探测器 建议我们将继续对基因进行物理定位。这将是 利用染色体产生的YAC文库实现 转移杂交种克隆米托蒽醌抗性基因。

The emergence of tumors refractory to further chemotherapy is a major obstacle for the successful treatment of cancer. Overexpression of MDR1 has been associated with clinical multidrug resistance. However, clinical trials have indicated that other as yet unidentified mechanisms occur in clinical multidrug resistance. This laboratory has selected mitoxantrone resistant human cell lines which is associated with an energy dependent drug efflux but is not related to the overexpression of known drug transporters, MDR1 and MRP. These cell lines provide a model of a novel multidrug resistant phenotype. We have recently determined by chromosome transfer that the resistance associated with mitoxantrone selection is due to a dominant genetic event. This provides the rationale for the cloning of the gene responsible for mitoxantrone selected resistance and is the goal of this proposal. A major undertaking of this proposal is the development of probes to screen a cDNA library. We have invoked strategies that utilize photoaffinity analogs of mitoxantrone as well as ATP photoaffinity analogs to identify putative proteins involved in resistance. Purification of proteins which are tagged in resistant cell lines by the photo affinity analogs will ultimately allow us to develop antibodies for immunoscreening a cDNA expression library. Purified proteins will also be sequenced which will allow us to design degenerate oligos for screening of a cDNA library. Finally, we propose that if we are unable to identify probes as outlined in this proposal we will proceed to physically map the gene. This will be accomplished by utilizing YAC libraries generated from chromosome transfer hybrids to clone the mitoxantrone selected resistant gene.