A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance
A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance
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DOI：
10.1074/jbc.273.46.30189
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发表时间：
1998-11-13
影响因子：
4.8
通讯作者：
Assaraf, YG
中科院分区：
文献类型：
--
作者：
Jansen, G;Mauritz, R;Assaraf, YG
CEM/MTX是人CCRF-CEM白血病细胞的亚系，其由于通过还原叶酸载体（RFC）的缺陷性转运而显示出对甲氨蝶呤（MTX）>200倍的抗性。CEM/MTX-低叶酸（LF）细胞，通过在CEM/MTX细胞的细胞培养基中从2.3 μ M至2 nM（LF）逐渐剥夺叶酸而衍生，导致结构改变的RFC的>20倍过表达，其特征在于：1）MTX转运的野生型K-m值，但叶酸和亚叶酸的K-m值分别低31倍和9倍，相对于野生型RFC; 2）RFC 1基因扩增沿着增加10倍，主要3.1-脱氢酶RFC 1 mRNA的表达增加>20倍; 3）阴离子对MTX转运的显著刺激（即氯化物）;和4）在CEM/MTX-LF和CEM/MTX中RFC cDNA的核苷酸227处的G -> A突变，导致在预测位于人RFC第一跨膜结构域内的氨基酸残基45处赖氨酸取代谷氨酸。当将CEM/MTX-LF细胞转移到叶酸充分培养基（2.3 μ M叶酸）时，CEM/MTX-LF细胞中更有效的叶酸摄取导致与CEM和CEM/MTX细胞相比，总叶酸库分别升高7倍和24倍（分别为500 pmol/mg蛋白质对69和21 pmol/mg蛋白质）。这种显著升高的细胞内叶酸池通过消除其多聚谷氨酰化和规避靶酶抑制，赋予了对多聚谷氨酸（例如ZD 1694、DDATHF和AG 2034）和亲脂性抗叶酸剂（例如曲美曲塞和乙胺嘧啶）的新耐药机制。
CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 mu M to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type K-m value for MTX transport but a 31-fold and 9-fold lower K-m values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G --> A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate replete medium (2.3 mu M folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.