Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression - A role for BCRP in cellular folate homeostasis
Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression - A role for BCRP in cellular folate homeostasis
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DOI：
10.1074/jbc.m401725200
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发表时间：
2004-06-11
影响因子：
4.8
通讯作者：
Assaraf, YG
中科院分区：
文献类型：
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作者：
Ifergan, I;Shafran, A;Assaraf, YG
乳腺癌耐药蛋白（BCRP/ABCG 2）是目前唯一的ABC转运蛋白，输出叶酸和甲氨蝶呤（MTX）的单谷氨酸和多谷氨酸。在这里，我们探讨了细胞叶酸状态和BCRP表达之间的关系。为此，将具有低BCRP和中等多药耐药蛋白1（MRP 1/ABCC 1）水平的MCF-7乳腺癌细胞及其具有BCRP过表达和低MRP 1水平的米托蒽醌（MR）抗性MCF-7/MR亚系从2.3 μ M至3 nM逐渐剥夺叶酸，产生亚系MCF-7/LF和MCF-7/MR-LF。这些细胞系仅表达残留的BCRP mRNA和蛋白水平，并通过MRP 5（ABCC 5）表达保留了较差的MRP 2（ABCC 2）。此外，MCF-7/MR-LF细胞也显示相对于MCF-7/MR细胞5倍降低的MRP 1水平。相反，在含有2.3 μ M叶酸的无MR培养基中生长的MCF-7/MR细胞中，BCRP的过度表达在很大程度上得以保留。MCF-7/LF和MCF-7/MR-LF细胞中BCRP表达缺失导致以下结果：（a）Hoechst 33342（一种BCRP底物）的外排显著减少;（B）流式细胞术显示MR蓄积增加至2倍;这分别伴随着这些细胞系中MR敏感性增加2.5倍和84倍。BCRP特异性抑制剂Ko 143使MCF- 7和MCF-7/MR细胞对MR的敏感性分别提高2.1倍和16.4倍。BCRP表达缺失还导致以下结果：（c）这些细胞系中MTX敏感性相同，从而丧失了与MCF-7/MR细胞相似的28倍MTX耐药性;（d）[H-3]叶酸4小时和24小时蓄积增加了相似的2倍。此外，MCF-7/MR-LF细胞表现出叶酸聚-γ-谷氨酸合成酶活性的显著增加。因此，与BCRP的单谷氨酸盐和多谷氨酸盐叶酸输出蛋白功能一致，BCRP下调和叶酸聚γ-谷氨酸合成酶活性增加似乎是细胞适应叶酸缺乏条件的关键组成部分。这是BCRP在维持细胞叶酸稳态中可能作用的第一个证据。
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 muM 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, MCF7/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 muM 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 similar to2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and similar to84-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 similar to16.4-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 similar to28-fold MTX resistance of the MCF-7/MR cells; (d) an similar to2-fold increase in the 4- and 24-h accumulation of [H-3]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.