III. Investigation of the ABC Half-Transporter ABCG2

我们实验室长期以来一直对非Pgp介导的耐药机制感兴趣，已经建立了几个以ABC半转运体ABCG2为重点的耐药细胞系模型。我们成功地从对米托蒽醌耐药的结肠癌细胞系S1-M1-80中克隆了ABCG2，该细胞株表现出依赖于ATP的药物积累减少。该基因由6个跨膜区和一个ATP结合区组成，被认为是活性所必需的二聚化，并且该基因编码一个半转运体分子。ABCG2的过表达使细胞对米托蒽醌、喜树碱、拓扑替康和SN-38(伊立替康的活性代谢物)具有耐药性。ABCG2的底物和抑制剂都已被加速发现，而且抑制剂的变化与P-糖蛋白的描述相竞争。越来越多的证据支持ABCG2在药物口服吸收中的作用。我们在ABCG2(R482T；R482G)中发现了一个自然发生的突变，它改变了底物和抑制剂的特异性；然后对代表全球遗传多样性的90个DNA样本进行了序列分析，并确定了ABCG2中的单核苷酸多态。然后，我们在HEK293细胞中建立了转染体。利用野生型背景(R482)，建立了表达V12M、Q141K和D620N的克隆。选择了表面表达水平相当的克隆，在携带Q141K的克隆中观察到运输受损。由于拓扑替康在胃肠道的吸收与肠上皮细胞中ABCG2的表达有关，这项工作的一个明显的含义是Q141K SNP可能与口服拓扑替康摄取的增加有关。在ABCG2参与药物排泄的程度上，这种SNP可能会增加对伊立替康和拓扑替康等底物的暴露。为了确定ABCG2的二聚化机制，我们实验室研究了跨膜螺旋1中的GXXXG二聚化基序，发现虽然它对正常的转运活性至关重要，但在表达甘氨酸到亮氨酸突变的细胞中，406和410氨基酸仍然保持着正常的交联性。进一步的突变分析正在进行中，分别基于与Michael Dean和Di Xia的合作研究，通过同源分析或分子建模确定残基中的突变。我们的目标是确定参与二聚化的残基。在与Balasz Sarkadi的合作中，我们已经生成了Sf9昆虫载体，以便在高表达昆虫系统中进行转基因。该系统对错误折叠的蛋白质具有更强的耐受性，并已被证明可以产生功能强大的ABCG2分子。这个系统将允许免疫共沉淀研究，这是确认我们的突变对二聚化的影响所需的。最近，在Di Xia生成的模型中高度保守或预测重要的其他残基正在评估中。高度保守的氨基酸残基553的突变导致哺乳动物细胞表面蛋白表达的丧失，以及昆虫细胞表面非功能蛋白的表达。用于鉴定突变体和SNPs的米托蒽醌外排定量分析部分用于临床样本。我们能够令人信服地表明，米托蒽醌的外排与mRNA和蛋白质的表达之间存在线性关系。细胞在37℃下孵育30分钟，加入或不加入ABCG2特异性抑制剂Fumitremorgin C(FTC)。然后洗涤细胞，并在37℃下，在不含米托蒽醌的培养液中孵育1小时，继续添加或不添加FTC。然后洗涤细胞，并用流式细胞仪进行荧光定量。在有或没有FTC的情况下，外排后峰值的差异，“通道移位值”与选择耐药的细胞中的mRNA和表面蛋白的表达是线性相关的。

Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, S1-M1-80, that exhibited an ATP-dependent reduction in drug accumulation. Comprised of 6 transmembrane domains and a single ATP binding domain, it is thought that dimerization is required for activity, and that the gene encodes a half-transporter molecule. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan). Both substrates and inhibitors of ABCG2 have been discovered at an accelerating pace, and the variation in inhibitors rivals that described for P-glycoprotein. There is increasing evidence supporting a role for ABCG2 in oral absorption of pharmacologic agents. We identified a naturally occurring mutation in ABCG2 (R482T; R482G) that alters substrate and inhibitor specificity; and then carried out a sequence analysis of 90 DNA samples representing a global genetic diversity and identified single nucleotide polymorphisms in ABCG2. We then established transfectants in HEK293 cells. Using the wildtype background (R482), clones expressing V12M, Q141K and D620 N were established. Clones with comparable levels of surface expression were selected, and impaired transport was observed in clones bearing the Q141K. Since gastrointestinal absorption of topotecan has been related to ABCG2 expression in the intestinal epithelium, a clear implication of this work is that the Q141K SNP could be associated with increased oral topotecan uptake. To the extent that ABCG2 is involved in drug excretion, this SNP could increase exposure to substrates such as irinotecan and topotecan. In an attempt to identify the mechanism of dimerization of ABCG2, our laboratory has studied a GXXXG dimerization motif in the transmembrane helix 1, finding that while it is critical for normal transport activity, normal cross-linking is retained in cells expressing glycine to leucine mutations at amino acids 406 and 410. Further mutational analysis is ongoing, generating mutations in residues identified by analysis of homologues or by molecular modeling, based on collaborative studies with Michael Dean and Di Xia, respectively. Our goal is to identify residues involved in the dimerization. In collaboration with Balasz Sarkadi, we have generated Sf9 insect vectors to allow transfection in the high expression insect system. This system is more tolerant of misfolded protein and has already been shown to generate functional ABCG2 molecules. This system will allow co-immunoprecipitation studies that are needed to provide confirmation of the impact of our mutations on dimerization. More recently, additional residues highly conserved or predicted to be important in the model generated by Di Xia are being evaluated. Mutation of the highly conserved amino acid residue 553 results in loss of protein expression on the mammalian cell surface, and expression of a nonfunctional protein on the insect cell surface. Quantitative mitoxantrone efflux assays used in characterizing the mutants and the SNPs were developed in part for use in clinical samples. We were able to convincingly show a linear relationship between mitoxantrone efflux and both mRNA and protein expression. Cells are incubated for 30 min at 37??C in mitoxantrone with or without the ABCG2-specific inhibitor, fumitremorgin C (FTC). The cells are then washed and incubated again at 37??C for 1 hr in mitoxantrone-free medium continuing with or without FTC. The cells are then washed and fluorescence quantitated by flow cytometry. The difference in the post-efflux peaks with or without FTC, the "channel -shift value" is linearly related to both mRNA and surface protein expression in the cells selected for drug resistance.