Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.
Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.
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发表时间：
1998-10
期刊：
影响因子：
11.2
通讯作者：
J. Mackey;R. Mani;M. Selner;D. Mowles;J. Young;J. A. Belt;C. R. Crawford;C. Cass
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文献类型：
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作者：
J. Mackey;R. Mani;M. Selner;D. Mowles;J. Young;J. A. Belt;C. R. Crawford;C. Cass
吉西他滨(2‘，2’-二氟脱氧胞苷)是一种新型的嘧啶核苷类药物，对几种常见的上皮性肿瘤具有临床疗效。我们提出吉西他滨需要核苷转运体(NT)蛋白穿透质膜并显示药理活性。在人类中，已报道的七种不同的NT活性在底物特异性、钠依赖性以及对硝基苄硫肌苷(NBMPR)和双嘧达莫抑制的敏感性方面有所不同。为了确定哪些NTs是吉西他滨依赖的生长抑制所必需的，从12个质膜NT活性明确的细胞系中提取的培养物与不同浓度的吉西他滨孵育。用硫代罗丹明B比色法和细胞计数法检测细胞增殖，以确定吉西他滨抑制细胞复制50%的浓度(IC50)。NT活性是体外生长抑制的先决条件，因为：(A)核苷转运缺陷细胞对吉西他滨高度耐药；(B)仅与NBMPR或双嘧达莫保持平衡NT活性的细胞处理后，对吉西他滨的抗药性增加了39-1800倍。这些数据表明，细胞所具有的NT活性的类型可能是其对吉西他滨敏感性的重要决定因素，NT缺乏可能导致显著的吉西他滨耐药。我们分析了5种人NT活性中的每一种对[~3H]吉西他滨的摄取动力学；瞬时转染编码人集中NT蛋白的cDNA(hCNT1和hCNT2)分别用于研究cit和cif的活性。吉西他滨摄取效率在含有单一NTS的细胞系之间有显著差异：ES约=CIT>；EI>；CIB>；>；>；CIF。通过人ES-NT在蛋白脂质体中的重组，证实了[~3H]吉西他滨的转运能力，证实了吉西他滨的渗透是一个蛋白质介导的过程。
Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.