CARRIER MEDIATED ANTIFOLATE TRANSPORT AND RESISTANCE

该建议是该实验室正在进行的工作的延续，以确定叶酸载体RFC1介导的叶酸和抗叶酸转运的分子基础。目的是描述作为底物结合和易位决定因素的载体元件，并表征突变体RFC1的功能特性，这些特性是由于药物运输受损导致的新一代抗叶酸药物耐药性的基础。将采取一些实验方法，这些方法有支持出版物、初步数据和已证明的专业知识：(1)该实验室已经开发了26个克隆L1210白血病细胞系，其中RFC1有明确的突变，导致MTX转运受损和耐药性。在其中的一些细胞系中，转运变化是高度特异性的。我们将评估选定的胸苷酸合成酶（TS）和GARFT抑制剂的转运特性，以进一步探索这些突变的功能后果、底物特异性和交叉抗性模式。(2) RFC1突变会导致对TS和GARFT抑制剂的原发性转运相关抗性，通过化学诱变增强单步抗叶酸选择，强调5-甲酰基四氢叶酸是唯一的叶酸来源。(3) ALL患者淋巴细胞DNA中发生的RFC1突变将通过单链构象多态性和可疑区域测序来筛选突变。(4)位点定向诱变将用于进一步表征RFC1残基的功能作用，RFC1残基的替代导致转运改变，但具有高度的底物特异性，正如许多小鼠和人类RFC1氨基酸残基所证明的那样。该方法还将用于探索RFC1区域的功能作用，并确定不同预测跨膜结构域中高度保守的相反电荷氨基酸之间潜在的离子配对相互作用。在转染到转运缺陷小鼠和人白血病细胞系后，将对突变小鼠和人rfc1介导的叶酸/抗叶酸转运特性进行全面分析。这将包括无机和有机阴离子对RFC1介导的通量和跨膜梯度的影响，以确定阴离子敏感和/或参与阴离子交换的载体元素。最后，在原生和突变体，小鼠和人类中，RFC1将在功能上重组为蛋白脂质体，以评估在囊内成分确定和缺乏复杂平行运输途径的条件下的载体运输特性。

This proposal is a continuation of ongoing work in this laboratory to define the molecular basis for the transport of folates and antifolates mediated by the reduced folate carrier, RFC1. The objectives are to delineate carrier elements that are determinants of substrate binding and translocation and to characterize the functional properties of mutant RFC1 that underlie antifolate resistance to new generation antifolates due to impaired drug transport. A number of experimental approaches will be undertaken for which there are supporting publications, preliminary data and demonstrated expertise: (1) This laboratory has developed a panel of 26 clonal L1210 leukemia cell lines with defined mutations in RFC1 that result in impaired MTX transport and drug resistance. In several of these lines transport alterations are highly substrate specific. We will assess transport properties of selected thymidylate synthase (TS) and GARFT inhibitors to further explore the functional consequences of these mutations, substrate specificity and cross-resistance patterns. (2) RFC1 mutations will be identified that result in primary transport-related resistance to TS and GARFT inhibitors using single-step antifolate selection augmented by chemical mutagenesis emphasizing 5-formyltetrahydrofolate as the sole folate source. (3) RFC1 mutations that occur in the DNA from lymphoblasts of patients with ALL will be screened for mutations by single-strand conformation polymorphism followed by sequencing of suspect regions. (4) Site-directed mutagenesis will be employed to further characterize the functional role of RFC1 residues the substitution of which results in altered transport but with a high degree of substrate specificity, as has been demonstrated for a number of murine and human RFC1 amino acid residues. This approach will also be applied to explore the functional role of RFC1 regions and to identify potential ion- pairing interactions between highly conserved oppositely charged amino acids in different predicted transmembrane domains. A comprehensive analysis of folate/antifolate transport properties mediated by mutant murine and human RFC1s from all the preceeding studies will be undertaken after transfection into transport- deficient murine and human leukemia cell lines. This will include the effects of inorganic and organic anions on RFC1- mediated fluxes and transmembrane gradients to identify carrier elements that are anion-sensitive and/or participate in anion exchange. Finally, native and mutant, mouse and human, RFC1 will be functionally reconstituted into proteoliposomes to assess carrier transport properties under conditions in which the intravesicular composition is defined and in the absence of complicating parallel transport pathways.