Lorico A, Boiocchi M, Rappa G, Sen S, Erba E, D'Incalci M
Division of Experimental Oncology I, Centro di Riferimento Oncologico, Aviano, Italy.
Int J Cancer. 1990 Jan 15;45(1):156-62. doi: 10.1002/ijc.2910450128.
Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.
用低浓度、无毒浓度（0.04微摩尔）的甲氨蝶呤（MTX）预处理16小时，可增强依托泊苷（VP16）对人组织细胞淋巴瘤U937细胞系的生长抑制作用和细胞毒性。当在MTX预处理后立即给予VP16 2小时，或在从MTX预处理恢复后的2至4小时或4至6小时给予VP16时，其细胞毒性显著增强。从MTX恢复后24小时，增效作用不明显。通过蛋白酶K消化后碱性洗脱法评估，VP16细胞毒性增加与药物诱导的DNA断裂增加有关。在MTX预处理后0、2和4小时给予药物时，DNA单链断裂（DNA SSB）的数量增加。MTX预处理后6至24小时药物诱导的DNA SSB与未预处理细胞中的相似。当从MTX恢复后4小时给予VP16时，其引起的DNA双链断裂（DNA DSB）数量显著增加。MTX预处理后6小时，VP16诱导的DNA DSB仍然较高，但到24小时时，它们与未用MTX处理的细胞中观察到的相似。流式细胞术分析表明，MTX预处理导致U937细胞在细胞周期的G1-S边界积累。当去除MTX时，随之出现一波同步化。使用蛋白质印迹电泳和抗拓扑异构酶II多克隆抗体，我们发现MTX预处理提高了细胞拓扑异构酶II的含量。我们的研究结果表明，低浓度、无毒的MTX预处理增强VP16对U937细胞的细胞毒性是由于较大比例的S期细胞含有较高浓度的拓扑异构酶II，而拓扑异构酶II是VP16作用的假定靶点。
