Ifergan Ilan, Shafran Assaf, Jansen Gerrit, Hooijberg Jan Hendrik, Scheffer George L, Assaraf Yehuda G
Department of Biology, The Technion, Haifa 32000, Israel.
J Biol Chem. 2004 Jun 11;279(24):25527-34. doi: 10.1074/jbc.M401725200. Epub 2004 Mar 26.
Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX). Here we explored the relationship between cellular folate status and BCRP expression. Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF. These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression. Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells. In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid. Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively. Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively. Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of [(3)H]folic acid. Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions. This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.
乳腺癌耐药蛋白（BCRP/ABCG2）是目前唯一一种能转运叶酸和甲氨蝶呤（MTX）的单谷氨酸和多谷氨酸盐的ABC转运蛋白。在此，我们探究了细胞叶酸状态与BCRP表达之间的关系。为此，我们选取了BCRP水平低且多药耐药蛋白1（MRP1/ABCC1）水平中等的MCF-7乳腺癌细胞，以及BCRP过表达且MRP1水平低的米托蒽醌（MR）耐药MCF-7/MR亚系细胞，将它们从2.3微摩尔逐渐剥夺叶酸至3纳米，从而得到MCF-7/LF和MCF-7/MR-LF亚系细胞。这些细胞系仅表达残留的BCRP mRNA和蛋白水平，并且MRP2（ABCC2）至MRP5（ABCC5）的表达均较低。此外，相对于MCF-7/MR细胞，MCF-7/MR-LF细胞的MRP1水平也降低了5倍。相比之下，在含有2.3微摩尔叶酸的无MR培养基中培养的MCF-7/MR细胞中，BCRP过表达基本得以保留。MCF-7/LF和MCF-7/MR-LF细胞中BCRP表达的缺失导致了以下结果：（a）BCRP底物Hoechst 33342的外排显著减少；（b）流式细胞术显示MR积累增加了约2倍；这些细胞系对MR的敏感性分别增加了2.5倍和约84倍。同样，特异性BCRP抑制剂Ko143使MCF-7和MCF-7/MR细胞对MR的敏感性分别增加了2.1倍和约16.4倍。BCRP表达的缺失还导致了以下结果：（c）这些细胞系对MTX的敏感性相同，从而丧失了MCF-7/MR细胞约28倍的MTX耐药性；（d）[³H]叶酸在4小时和24小时的积累增加了约2倍。此外，MCF-7/MR-LF细胞的叶酰聚γ-谷氨酸合成酶活性显著增加。因此，与BCRP作为单谷氨酸和多谷氨酸叶酸转运蛋白的功能一致，BCRP的下调和叶酰聚γ-谷氨酸合成酶活性的增加似乎是细胞适应叶酸缺乏条件的关键组成部分。这是关于BCRP在维持细胞叶酸稳态中可能作用的首个证据。
