Zhu C, Johansson M, Permert J, Karlsson A
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, S-14186 Stockholm, Sweden.
J Biol Chem. 1998 Jun 12;273(24):14707-11. doi: 10.1074/jbc.273.24.14707.
The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG), and 2',2'-difluorodeoxyguanosine (dFdG) are phosphorylated by human mitochondrial deoxyguanosine kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was approximately 4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.
细胞毒性抗癌嘌呤核苷类似物2-氯-2'-脱氧腺苷（CdA）、9-β-D-阿拉伯呋喃糖基鸟嘌呤（araG）和2',2'-二氟脱氧鸟苷（dFdG）在体外可被人线粒体脱氧鸟苷激酶（dGK）磷酸化。我们将dGK作为绿色荧光蛋白的融合蛋白在人胰腺癌细胞系PanC-1和MIA PaCa-2中过表达，以确定dGK介导的核苷类似物磷酸化的重要性。转染后的细胞呈现出线粒体荧光模式，通过用多克隆抗dGK抗体对细胞提取物进行蛋白质印迹分析，验证了内源性和过表达的dGK的线粒体定位。过表达细胞中dGK活性增加约4倍。与未转染的亲本细胞系相比，这些细胞系对CdA、araG和dFdG表现出更高的敏感性。据我们所知，这是首次证明线粒体脱氧核糖核苷激酶的活性与核苷类似物的细胞毒性之间存在相关性。我们的数据表明，在所研究的细胞系中，dGK活性是核苷类似物疗效的限速因素。
