DHFR MEDIATED ANTIFOLATE RESISTANCE IN MAMMALIAN CELLS

哺乳动物细胞中 DHFR 介导的抗叶酸耐药性

• 批准号: 3193707
• 负责人: Peter William Melera
• 金额: $ 18.52万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193707
• 关键词: affinity chromatography; alleles; complementary DNA; dihydrofolate reductase; drug resistance; electrofocusing; enzyme mechanism; gel electrophoresis; gene expression; hamsters; immunocytochemistry; methotrexate; monoclonal antibody; southern blotting; tissue /cell culture

The wide use of the antifolate drug methotrexate (NM) as a chemotherapeutic agent requires that knowledge of its target enzyme, dihydrofolate reductase (DHFR), be extensive and that the enzymes' biochemical and genetic characteristics be understood. Moreover, since the effectiveness of MTX is often comprimised by intrinsic or acquired resistance, a better understanding of the mechanism(s) of such resistance might provide insights into more effective treatment protocols. It has been found that the Chinese hamster lung cell line DC-3F is heterozygous at the DHFR locus and contains 2 polymorphic DHFR alleles. Among 58 independently derived, gene amplification mediated DHFR overproducing MTX-resistant sublines, 44 amplify and overexpress one of the 2 allelic forms of DHFR and 14 the other. Since gene amplification is thought to be a random phenomenon, one of the goals of this proposal is to determine, by use of pulse field gel electrophoresis, Southern blotting and cDNA transfection analysis, the genetic basis for the observed differential allelic amplification. In addition, recent determination that commonly observed minor isoelectric forms of DHFR (which can account for 10-15% of the cellular enzyme) are derived by N-terminal acetylation of the major DHFR proteins, suggests that these forms may serve a specific biological function. With a combination of NM-affinity column and isoelectrofocusing purification techniques, enzyme kinetic analysis and monoclonal antibody preparation for immunocytochemical detection we propose to clarify the biological significance of those long recognized but poorly understood DHFRS.

抗叶酸药物甲氨蝶呤(NM)作为化疗药物的广泛应用 试剂需要了解其目标酶二氢叶酸还原酶 (DHFR)，是广泛的，酶的生化和遗传 要了解他们的特点。此外，由于甲氨喋呤的有效性是 通常伴随着固有的或后天的阻力，更好的 了解这种抗性的机制(S)可能会提供一些见解 变成更有效的治疗方案。据发现，中国人 仓鼠肺细胞系DC-3F是DHFR基因的杂合子，含有 2多态DHFR等位基因。在58个独立衍生的基因中， 扩增介导的DHFR高表达MTX抗性亚系，44 扩增并过度表达DHFR和14的2种等位基因之一 其他的。由于基因扩增被认为是一种随机现象，因此 这项提议的目标之一是通过使用脉冲场凝胶来确定 电泳法、Southern blotting法和cDNA转染法分析 观察到的差异等位基因扩增的遗传基础。在……里面 此外，最近的测定表明，通常观察到的次等电点 脱氢表雄酮受体的形式(可占细胞酶的10%-15%)有 由主要的DHFR蛋白的N-末端乙酰化而来的，表明 这些形式可能具有特定的生物学功能。通过组合使用 NM亲和层析和等电聚焦纯化技术、酶 免疫细胞化学动力学分析及单抗制备 我们提出的检测是为了阐明这些长的 公认但对DHFRS知之甚少。