Keszler Gergely, Szikla Karoly, Kazimierczuk Zygmunt, Spasokoukotskaja Tatjana, Sasvari-Szekely Maria, Staub Maria
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
Biochem Pharmacol. 2003 Feb 15;65(4):563-71. doi: 10.1016/s0006-2952(02)01557-5.
Deoxycytidine kinase (dCK) catalyses the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity [Biochem Pharmacol 56 (1998) 1175], improving the efficacy of the cytostatic therapy. Here, we measured the effect of the 5'-thiosulphate (5'-TS) derivatives of four deoxyribonucleosides (deoxyadenosine, deoxycytidine (dCyd), azidothymidine, thymidine) and two ribonucleosides (ribopurine, ribouridine (Urd)) on the activity of the two main salvage deoxynucleoside kinases, and on the salvage of dCyd and deoxythymidine (dThd). It turned out that only 2'-deoxythymidine-5'-thiosulphate (dThd-5'-TS) can potentiate the dCK activity, without influencing the thymidine kinase isoenzymes during short-time treatments of human peripheral blood and tonsillar lymphocytes. The enhancement of dCK activity by dThd-5'-TS can be reversed by dCyd, but dThd had no effect on the enzyme activation in cells. Neither dThd-5'-TS nor Urd-5'-TS had any effect on the dCK and thymidine kinase activities tested in cell-free extracts. The stimulation of dCK activity in cells was accompanied by an imbalance in the dThd and dCyd metabolism. The incorporation of 3H-dThd into DNA was suppressed by 90% in cells by dThd-5'-TS, while Urd-5'-TS only slightly influenced the same process. The 3H-dCyd incorporation into DNA was inhibited only to 50% of the control, while the 3H-dCyd labelling of the nucleotide fraction was enlarged in dThd-5'-TS-treated cells, as a consequence of the increased dCK activity. We suggest that the enhancement of dCK activity is a compensatory mechanism in cells that might be induced by different "inhibitors" of DNA synthesis leading to damage of DNA. The increased dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells; this suggestion seems to be supported by the counteracting effect of extracellular dCyd, too.
脱氧胞苷激酶（dCK）催化三种天然脱氧核糖核苷以及几种治疗性核苷类似物补救途径的限速步骤，这些核苷类似物进而可以增强其酶活性[《生物化学与药物学》56(1998)1175]，提高细胞生长抑制疗法的疗效。在此，我们测定了四种脱氧核糖核苷（脱氧腺苷、脱氧胞苷（dCyd）、叠氮胸苷、胸苷）和两种核糖核苷（核糖嘌呤、尿苷（Urd））的5'-硫代硫酸盐（5'-TS）衍生物对两种主要的补救脱氧核苷激酶活性以及dCyd和脱氧胸苷（dThd）补救途径的影响。结果表明，在对人外周血和扁桃体淋巴细胞进行短期处理时，只有2'-脱氧胸苷-5'-硫代硫酸盐（dThd-5'-TS）能够增强dCK活性，而不影响胸苷激酶同工酶。dThd-5'-TS对dCK活性的增强作用可被dCyd逆转，但dThd对细胞中的酶激活没有影响。dThd-5'-TS和Urd-5'-TS对无细胞提取物中测试的dCK和胸苷激酶活性均无任何影响。细胞中dCK活性的刺激伴随着dThd和dCyd代谢的失衡。dThd-5'-TS使细胞中3H-dThd掺入DNA的量被抑制了90%，而Urd-5'-TS仅对同一过程有轻微影响。3H-dCyd掺入DNA的量仅被抑制至对照的50%，而在dThd-5'-TS处理的细胞中，由于dCK活性增加，核苷酸部分的3H-dCyd标记有所增加。我们认为，dCK活性的增强是细胞中的一种补偿机制，可能由导致DNA损伤的不同DNA合成“抑制剂”诱导。dCK活性的增加能够为静止细胞中的DNA修复提供脱氧核苷三磷酸；细胞外dCyd的抵消作用似乎也支持了这一观点。
