Rothem Lilah, Aronheim Ami, Assaraf Yehuda G
Department of Biology, the Rappaport Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa 31096, Israel.
J Biol Chem. 2003 Mar 14;278(11):8935-41. doi: 10.1074/jbc.M209578200. Epub 2003 Jan 7.
The human reduced folate carrier (hRFC) is the dominant transporter mediating the uptake of reduced folate cofactors and antifolate anticancer drugs. Defective antifolate uptake due to inactivating mutations in the hRFC gene is an established mechanism of drug resistance in various tumor cells. However, while antifolate transport is frequently impaired, either no or only a single hRFC allele is inactivated, suggesting that additional mechanism(s) of resistance are operative. Here we studied the relationship between the expression and function of transcription factors and antifolate resistance in transport-defective leukemia cells that poorly express or completely lack RFC mRNA. Stable transfection with a hRFC expression construct resulted in restoration of normal RFC mRNA expression and nearly wild type drug sensitivity in these antifolate-resistant cells. The loss of RFC gene expression prompted us to explore transcription factor binding to the hRFC promoter. The hRFC promoter contains an upstream GC-box and a downstream cAMP-response element (CRE)/AP-1-like element. Electrophoretic mobility shift assays and oligonucleotide competition revealed a substantial loss of nuclear factor binding to CRE and GC-box in these drug-resistant cell lines. Consistently, antibody-mediated supershift analysis showed a marked decrease in the binding of CRE-binding protein 1 (CREB-1) and specificity protein 1 (Sp1) to CRE and GC-box, respectively. Western blot analysis revealed undetectable expression of CREB-1, decreased ATF-1 levels, parental Sp1 levels, and increased levels of the short Sp3 isoforms, recently shown to repress hRFC gene expression. Transient transfections into these antifolate-resistant cells demonstrated a marked loss of GC-box-dependent, and CRE-driven reporter gene activities and introduction of CREB-1 or Sp1 expression constructs resulted in restoration of hRFC mRNA expression. These results establish a novel mechanism of antifolate resistance that is based on altered expression and function of transcription factors resulting in transcriptional silencing of the hRFC promoter.
人还原型叶酸载体（hRFC）是介导还原型叶酸辅因子和抗叶酸抗癌药物摄取的主要转运蛋白。hRFC基因失活突变导致抗叶酸摄取缺陷是多种肿瘤细胞耐药的既定机制。然而，虽然抗叶酸转运经常受损，但要么没有hRFC等位基因失活，要么只有单个hRFC等位基因失活，这表明存在其他耐药机制。在此，我们研究了转录因子的表达与功能和抗叶酸耐药性之间的关系，这些转录因子存在于表达不良或完全缺乏RFC mRNA的转运缺陷白血病细胞中。用hRFC表达构建体进行稳定转染可使这些抗叶酸耐药细胞恢复正常的RFC mRNA表达，并使其药物敏感性接近野生型。RFC基因表达的缺失促使我们探索转录因子与hRFC启动子的结合情况。hRFC启动子包含一个上游GC盒和一个下游cAMP反应元件（CRE）/AP-1样元件。电泳迁移率变动分析和寡核苷酸竞争显示，在这些耐药细胞系中，核因子与CRE和GC盒的结合大幅减少。一致地，抗体介导的超迁移分析表明，CRE结合蛋白1（CREB-1）和特异性蛋白1（Sp1）分别与CRE和GC盒的结合显著减少。蛋白质印迹分析显示，CREB-1表达无法检测到，ATF-1水平降低，亲本Sp1水平正常，而短Sp3亚型水平增加（最近发现其可抑制hRFC基因表达）。将这些构建体瞬时转染到这些抗叶酸耐药细胞中，结果显示GC盒依赖性和CRE驱动的报告基因活性显著丧失，而引入CREB-1或Sp1表达构建体可恢复hRFC mRNA表达。这些结果建立了一种新的抗叶酸耐药机制，该机制基于转录因子表达和功能的改变，导致hRFC启动子转录沉默。
