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: 3193706
• 负责人: Peter William Melera
• 金额: $ 17.19万
• 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/3193706
• 关键词: DNA; affinity chromatography; alleles; complementary DNA; dihydrofolate reductase; drug resistance; electrofocusing; enzyme mechanism; gel electrophoresis; gene expression; hamsters; immunocytochemistry; methotrexate; monoclonal antibody; nucleic acid hybridization; 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使用 药剂需要知道其靶酶二氢叶酸还原酶 （DHFR），是广泛的，酶的生化和遗传 特点要理解。此外，由于MTX的有效性是 通常被内在或后天的抵抗力所困扰， 了解这种耐药性的机制可能有助于了解 转化为更有效的治疗方案。据了解，中国人 仓鼠肺细胞系DC-3F在DHFR基因座处是杂合的，并且含有 2个多态性DHFR等位基因。在58个独立衍生的基因中， 扩增介导的DHFR过度产生MTX耐药亚系，44 扩增并过表达DHFR的2种等位基因形式之一， 其他.由于基因扩增被认为是一种随机现象， 该建议的目标之一是通过使用脉冲场凝胶 电泳、Southern印迹和cDNA转染分析， 观察到的差异等位基因扩增的遗传基础。在 此外，最近的测定表明， DHFR的形式（可占细胞酶的10-15%）是 由主要DHFR蛋白的N-末端乙酰化衍生，表明， 这些形式可能具有特定的生物学功能。的组合 纳米亲和柱和等电聚焦纯化技术，酶 免疫细胞化学动力学分析及单克隆抗体制备 检测，我们建议澄清这些长期的生物学意义 公认但了解甚少的DHFRS。