CARRIER MEDIATED ANTIFOLATE TRANSPORT AND RESISTANCE

这项建议是该实验室正在进行的工作的继续，目的是确定还原叶酸载体Rfc1介导的叶酸和反叶酸运输的分子基础。目的是描述底物结合和转位的决定因素的载体元件，并表征突变的Rfc1的功能特性，该突变的Rfc1是由于药物转运障碍而导致的新一代抗叶酸抵抗的基础。将采取一些实验方法，这些方法有支持的出版物、初步数据和证明的专业知识：(1)本实验室开发了一组26个克隆L1210白血病细胞株，其Rfc1存在明确的突变，导致MTX转运受损和耐药性下降。在其中的几条线路中，运输变化是高度底物特有的。我们将评估部分胸苷酸合酶(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、底物特异性和交叉耐药模式。(2)Rfc1突变将被鉴定为导致对TS和GARFT抑制剂产生初级运输相关耐药的突变，方法是采用一步抗叶酸选择，并以5-甲酰四氢叶酸为唯一叶酸来源的化学诱变来加强。(3)ALL患者淋巴母细胞DNA中的Rfc1突变将通过单链构象多态和可疑区域测序来筛选突变。(4)定点突变将被用来进一步表征Rfc1残基的功能作用，该残基的替代导致转运改变，但具有高度的底物特异性，就像对一些鼠和人的Rfc1氨基酸残基所证明的那样。这一方法也将被用于探索Rfc1区域的功能作用，并在不同预测的跨膜结构域中识别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。在将上述所有研究中的突变小鼠和人RFC1s导入运输缺陷的小鼠和人类白血病细胞系后，将对其介导的叶酸/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，以确定对阴离子敏感和/或参与阴离子交换的载体元素。最后，天然的和突变的，小鼠和人，Rfc1将被功能性地重组为蛋白脂质体，以评估在确定囊泡内组成的条件下和在没有复杂的平行运输路径的条件下的载体运输性质。

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.