CARRIER MEDIATED ANTIFOLATE TRANSPORT AND RESISTANCE

本建议是本实验室正在进行的工作的延续，以确定由还原叶酸载体RFC 1介导的叶酸和抗叶酸剂转运的分子基础。 我们的目标是描绘载体元素，是底物结合和易位的决定因素，并表征的功能特性的突变体RFC 1的基础抗叶酸剂耐新一代抗叶酸剂，由于受损的药物转运。 将采取许多实验方法，这些方法有支持性出版物、初步数据和已证明的专业知识：（1）本实验室开发了一组26个克隆L1210白血病细胞系，这些细胞系具有RFC 1中的确定突变，导致MTX转运受损和耐药性。 在这些线路中的几个运输改变是高度底物特异性的。 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，以进一步探讨这些突变，底物特异性和交叉耐药模式的功能后果。 (2)将使用通过强调5-甲酰四氢叶酸作为唯一叶酸来源的化学诱变增强的单步抗叶酸剂选择来鉴定导致对TS和GARFT抑制剂的主要转运相关抗性的RFC 1突变。 (3)ALL患者淋巴母细胞DNA中发生的RFC 1突变将通过单链构象多态性筛查突变，然后对可疑区域进行测序。 (4)将采用定点诱变来进一步表征RFC 1残基的功能作用，其取代导致转运改变，但具有高度的底物特异性，如已经证明的许多鼠和人RFC 1氨基酸残基。 这种方法也将被应用于探索RFC 1区域的功能作用，并确定不同的预测跨膜结构域中高度保守的带相反电荷的氨基酸之间的潜在离子配对相互作用。 在转染到转运缺陷型鼠和人白血病细胞系中后，将对来自所有前述研究的突变型鼠和人RFC 1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RFC 1介导的通量和跨膜梯度的影响，以识别阴离子敏感和/或参与阴离子交换的载体元素。 最后，天然和突变体，小鼠和人类，RFC 1将功能性重组到蛋白脂质体中，以评估在囊内组成被定义的条件下以及在没有复杂的平行转运途径的情况下的载体转运特性。

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.