The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3′-fluoro-3′-deoxythymidine in human B-lymphoblast cells
The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3′-fluoro-3′-deoxythymidine in human B-lymphoblast cells
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DOI：
10.1016/j.nucmedbio.2011.03.009
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发表时间：
2011-10-01
影响因子：
3.1
通讯作者：
Schwartz, Jeffrey L.
中科院分区：
文献类型：
--
作者：
Plotnik, David A.;McLaughlin, Lena J.;Schwartz, Jeffrey L.
最近在人腺癌细胞系A549中的研究发现，细胞生长依赖性平衡核苷转运蛋白-1 （hENT1）是3'-氟-3'-脱氧胸腺嘧啶（FLT）摄取和保留的修饰剂。在本研究中，我们利用分离胸苷激酶1 （TK1）缺乏的人淋巴母细胞克隆的能力来研究代谢和核苷转运如何影响FLT的摄取和保留。方法：在人淋巴母细胞样细胞系TK6和从TK6分离的8个克隆中测定FLT的转运和代谢。4个克隆是tk1精通型，4个是tk1缺陷型。在可以区分集中运输和平衡运输的条件下，测量了FLT的内流和外流。结果：钠依赖的高浓度FLT转运主导了平衡转运机制，虽然抑制hENT1降低了FLT摄取，但hENT1水平的克隆变异与FLT摄取之间没有相关性。TK6细胞中FLT的浓度有TK1的绝对要求。在与FLT孵育60分钟后，FLT摄取达到峰值，之后细胞内FLT水平和FLT代谢物下降。外排迅速，并与FLT及其每一种代谢物的减少有关。在外排缓冲液中发现了FLT和FLT-单磷酸。结论：FLT的初始摄取速率是集中转运蛋白和平衡转运蛋白的函数。TK1活性是FLT积累的绝对必要条件。保留依赖于核苷/核苷酸外排和FLT核苷酸的逆行代谢。Elsevier Inc.出版。
Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention.Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished.Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer.Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides. Published by Elsevier Inc.