ISOLATION OF A SELECTED DRUG RESISTANT GENE

选定的耐药基因的分离

• 批准号: 2683675
• 负责人: Lori A Hazlehurst
• 金额: $ 3.05万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-18 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2683675
• 关键词: MCF7 cell; adenosine triphosphate; analog; antineoplastics; artificial chromosomes; chromosome walking; complementary DNA; drug metabolism; drug resistance; fluorescent in situ hybridization; gene expression; genetic library; genetic mapping; high performance liquid chromatography; mass spectrometry; membrane transport proteins; mitoxantrone; neoplasm /cancer pharmacology; nuclear magnetic resonance spectroscopy; polymerase chain reaction; protein purification; protein sequence

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.

进一步化疗难治性肿瘤的出现是一个主要因素 癌症成功治疗的障碍。 过度表达 MDR1 与临床多药耐药性相关。 然而，临床试验表明，目前尚有其他 临床多重耐药性中发生的机制尚未明确。这 实验室已选择了米托蒽醌耐药的人类细胞系 与能量依赖性药物流出相关，但不相关 已知药物转运蛋白 MDR1 和 MRP 的过度表达。 这些细胞系提供了新型多重耐药性的模型 表型。 我们最近通过染色体转移确定 与米托蒽醌选择相关的耐药性是由于 显性遗传事件。 这为克隆提供了理论依据 负责米托蒽醌选择抗性的基因是 本提案的目标。该提案的一项主要承诺是 开发探针来筛选 cDNA 文库。我们已经调用了 还利用米托蒽醌光亲和类似物的策略 作为 ATP 光亲和类似物来识别所涉及的推定蛋白质 在抵抗中。纯化带有抗性标记的蛋白质 光亲和力类似物的细胞系最终将使我们能够 开发用于免疫筛选 cDNA 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 设计简并寡核苷酸以筛选 cDNA 文库。最后，我们 建议如果我们无法识别本节中概述的探针 建议我们将继续绘制该基因的物理图谱。这将是 通过利用从染色体生成的 YAC 文库来完成 转移杂交体以克隆米托蒽醌选择的抗性基因。

项目成果

Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line.

多种机制赋予人 8226 骨髓瘤细胞系对米托蒽醌的耐药性。

• DOI: 10.1038/bjc.1996.243
• 发表时间: 1999-03-01
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: L. Hazlehurst;Nils E. Foley;M. Gleason;M. Hacker;A. Cress;L. Greenberger;M. C. D. Jong;W. Dalton
• 通讯作者: W. Dalton