Hentosh P, Tibudan M
Department of Pharmacology and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064, USA.
Mol Pharmacol. 1997 Apr;51(4):613-9. doi: 10.1124/mol.51.4.613.
2-Chloro-2'-deoxyadenosine [CldAdo (cladribine)], a novel effective antileukemic agent, was examined for its effects on cellular mitochondrial function and DNA content after long term (< or = 7-day) incubation of cultured CCRF-CEM human leukemia cells. Dideoxycytidine (ddC), which is known to have a delayed effect on mitochondrial DNA content, was used as a positive control to monitor mitochondrial dysfunction. CldAdo at 6-16 nM was toxic to cells within 24 hr, which is in contrast to 300 nM ddC, which had no effect on cell growth for the first 4 days of treatment. Cellular lactic acid production was used to monitor concomitant perturbations in oxidative phosphorylation during drug treatment. Unlike the delayed increase in lactate observed with ddC exposure, CldAdo-treated cells exhibited a 2-2.4-fold increase in lactate levels after 2 days of exposure to 16 nM CldAdo. By days 4 and 7, however, lactate production returned to control levels. Shorter incubations with CldAdo revealed that lactate levels began to increase within 12 hr of drug exposure, paralleling cytotoxicity. We also examined mitochondrial DNA content during drug treatment by competitive polymerase chain reaction. ddC (300 nM) reduced mitochondrial DNA levels from approximately 1000 copies/untreated cell to approximately 130 copies/cell after 7 days of exposure. In contrast, cytotoxic doses of CldAdo had little or no effect on mitochondrial DNA content during the 1-week incubation. Thus, the early CldAdo-induced perturbation of mitochondrial function was not associated with a loss of mitochondrial DNA per cell. In addition, no evidence of DNA laddering, indicative of cellular apoptosis, was detected at these dosage levels and treatment times.
2-氯-2'-脱氧腺苷[CldAdo（克拉屈滨）]是一种新型有效的抗白血病药物，在体外培养的CCRF-CEM人白血病细胞长期（≤7天）孵育后，研究了其对细胞线粒体功能和DNA含量的影响。已知对线粒体DNA含量有延迟影响的双脱氧胞苷（ddC）用作监测线粒体功能障碍的阳性对照。6-16 nM的CldAdo在24小时内对细胞有毒性，这与300 nM的ddC形成对比，后者在治疗的前4天对细胞生长没有影响。细胞乳酸产生用于监测药物治疗期间氧化磷酸化的伴随扰动。与ddC暴露后观察到的乳酸延迟增加不同，暴露于16 nM CldAdo 2天后，CldAdo处理的细胞乳酸水平增加了2-2.4倍。然而，在第4天和第7天，乳酸产生恢复到对照水平。用CldAdo进行较短时间的孵育表明，药物暴露后12小时内乳酸水平开始增加，与细胞毒性平行。我们还通过竞争性聚合酶链反应检查了药物治疗期间的线粒体DNA含量。ddC（300 nM）在暴露7天后将线粒体DNA水平从约1000拷贝/未处理细胞降低至约130拷贝/细胞。相比之下，在1周的孵育期间，细胞毒性剂量的CldAdo对线粒体DNA含量几乎没有影响。因此，早期CldAdo诱导的线粒体功能扰动与每个细胞线粒体DNA的丢失无关。此外，在这些剂量水平和治疗时间未检测到指示细胞凋亡的DNA梯状条带的证据。
