Gemcitabine transport in Xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters
Gemcitabine transport in Xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters
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DOI：
10.1093/jnci/91.21.1876
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发表时间：
1999-11-03
影响因子：
10.3
通讯作者：
Young, JD
中科院分区：
文献类型：
--
作者：
Mackey, JR;Yao, SYM;Young, JD
背景资料：吉西他滨是一种脱氧胞苷的嘧啶类似物，是一种抗癌核苷药物，需要功能性质膜核苷转运蛋白才能到达其细胞内靶点并引起细胞毒性，由于研究人类细胞中核苷转运的固有技术困难，我们通过生产每一种可用的人（h）和大鼠（r）重组核苷转运蛋白（NT），方法：用体外转录的RNA显微注射非洲爪蟾卵母细胞，显微注射后3天测定[H-3]吉西他滨和[C-14]尿苷的摄取，以确定动力学常数。我们还使用双电极电压钳技术研究hCNT 1介导的吉西他滨转运的电生理学。结果如下：吉西他滨被大多数测试蛋白转运（嘌呤选择性rCNT 2和hCNT 2除外），最大的摄取发生在产生重组rCNT 1和hCNT 1的卵母细胞中。hCNT 1、hENT 1和hENT 2介导的吉西他滨流入是饱和的，符合Michaelis-Menten动力学，表观Km值分别为24、160和740 μ M。吉西他滨通过简单扩散穿过卵母细胞膜脂双层的能力有限。体外应用吉西他滨的卵母细胞产生重组hCNT 1诱导内向电流，这表明，hCNT 1的功能作为一个Na+/核苷共转运蛋白，并确认转运的能力，运输吉西他滨，结论：哺乳动物核苷转运蛋白的亲和力和能力有很大的差异，运输吉西他滨。质膜核苷转运蛋白的肿瘤和组织分布的变化可能导致吉西他滨的实体瘤活性和方案依赖性毒性作用。
Background: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity, Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes, Methods: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2, Uptake of [H-3]gemcitabine and [C-14] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. Results: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1, Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K-m values of 24, 160, and 740 mu M, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na+/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine, Conclusions: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.