Lai Yurong, Tse Chung-Ming, Unadkat Jashvant D
Department of Pharmaceutics, University of Washington, Seattle, Washington 98195, USA.
J Biol Chem. 2004 Feb 6;279(6):4490-7. doi: 10.1074/jbc.M307938200. Epub 2003 Nov 7.
Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [14C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nm nitrobenzylthioinosine. Moreover, FIAU (50 microm) produced significant mitochondrial toxicity ( approximately 70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.
许多抗病毒药物（如菲阿尿苷；FIAU）会产生具有临床意义的线粒体毒性，这限制了它们的剂量或使其无法在临床上使用。由于大多数核苷类药物亲水性过强，无法穿过高度不可渗透的线粒体膜，我们推测它们必须被转运到线粒体中才能产生毒性。为了验证这一假设，我们试图确定核苷转运体，即人平衡核苷转运体1（hENT1）或人浓缩核苷转运体1（hCNT1），当作为黄色荧光融合蛋白（YFP）在Madin-Darby犬肾细胞中稳定表达时，是否定位于线粒体。通过使用细胞器选择性染料和共聚焦显微镜，我们发现hENT1-YFP定位于线粒体以及质膜，而hCNT1-YFP主要位于质膜上。hENT1-YFP并不定位于核膜、内体、溶酶体或高尔基体复合体。蛋白质印迹法分别证实了从表达hENT1-YFP的细胞和人肝脏中分离的线粒体中存在hENT1-YFP或内源性hENT1。与这些定位数据一致，[14C]FIAU能够有效地转运到表达hENT1-YFP的细胞的线粒体中，但不能转运到表达hCNT1-YFP的细胞的线粒体中。即使质膜上的hENT1活性被10纳米的硝基苄硫肌苷选择性阻断，hENT1-YFP仍会增强FIAU对Madin-Darby犬肾细胞的线粒体毒性。此外，当FIAU（50微摩尔）与从表达hENT1的细胞中分离的线粒体直接孵育时，会产生显著的线粒体毒性（线粒体DNA合成下降约70%）。总之，我们首次确定hENT1在线粒体膜上表达，并且这种表达增强了核苷类药物如FIAU的线粒体毒性。hENTs在线粒体中的表达可能解释了齐多夫定、司他夫定和去羟肌苷等抗HIV核苷类药物所导致的具有临床意义的线粒体毒性。
