MOLECULAR REGULATION OF FOLATE AND ANTIFOLATE TRANSPORT

这个项目的目标是描述结构和规则的特征 “经典”甲氨蝶呤/四氢叶酸辅助因子膜 人类肿瘤细胞中的转运蛋白。这一系统对于 甲氨蝶呤及其相关抗叶酸酯的抗肿瘤作用。为 甲氨蝶呤，一种广泛用于人类癌症化疗的药物， 运输系统的调节或生物合成方面的缺陷导致 在肿瘤细胞内抗叶酸蓄积减少，似乎是 影响耐药发展的关键因素。 在早期的实验中，K562人红白血病细胞数量增加 对甲氨蝶呤和还原叶酸的摄取能力进行了选择和 鉴定了一种高度糖化的转运蛋白(GP-MTX)，并 纯净的。最近，制备了抗GP-MTX的抗血清，并制备了几种 人淋巴细胞白血病c DNA文库中可能的GP-MTX c DNA 都是孤立的。 基于这些初步结果，我们建议(I)验证和表征 体外构建GP-MTX的全长cDNA 转录/翻译、双脱氧测序和真核表达。 此外，我们建议(Ii)鉴定和表征生物化学 以及耐药时甲氨蝶呤转运受损的分子基础 K562红白血病和CCRF-CEM淋巴细胞白血病细胞变异体 GP-MTX的结构、生物合成和/或调控水平。 这些研究为研究监管提供了一个框架 甲氨蝶呤和四氢叶酸辅助因子在两者体内的转运系统 对药物敏感和耐药的人类肿瘤细胞。他们应该澄清 甲氨蝶呤转运性耐药的机制基础。在……里面 这种方式，鉴定膜连接的生化或 甲氨蝶呤体外敏感性或耐药性的分子“标记物” 应促进该药和相关药物的更有效的临床应用 药物，包括耐药肿瘤的检测和治疗。

The goal of this project is to characterize the structure and regulation of the "classical" methotrexate/tetrahydrofolate cofactor membrane transporter in human tumor cells. This system is critical to the antitumor effectiveness of methotrexate and related antifolates. For methotrexate, an agent widely used in the chemotherapy of human cancers, defects in the regulation or biosynthesis of the transport system result in decreased antifolate accumulation within tumor cells and appear to be key factors in the development of drug resistance. In earlier experiments, K562 human erythroleukemia cells with increased uptake capacities for methotrexate and reduced folates were selected and a highly glycosylated transporter protein (GP-MTX) was identified and purified. Most recently, antiserum to GP-MTX was prepared, and several putative GP-MTX cDNAs from a human lymphoblastic leukemia cDNA library were isolated. Based on these initial results, we propose to (i) verify and characterize the putative full length cDNAs for GP-MTX by in vitro transcription/translation, dideoxy sequencing, and eukaryotic expression. In addition, we propose to (ii) identify and characterize the biochemical and molecular bases for impaired methotrexate transport in drug resistant K562 erythroleukemia and CCRF-CEM lymphoblastic leukemia cell variants at the levels of GP-MTX structure, biosynthesis, and/or regulation. These studies provide a framework for the study of regulation of the transport system for methotrexate and tetrahydrofolate cofactors in both drug sensitive and resistant human tumor cells. They should clarify the mechanistic bases for transport-mediated resistance to methotrexate. In this fashion, the identification of membrane-linked biochemical or molecular "markers" for methotrexate sensitivity or resistance in vitro should promote the more effective clinical use of this and related agents, including the detection and treatment of drug resistant tumors.