Lawrence T S, Canman C E, Maybaum J, Davis M A
Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor 48109-0010.
Cancer Res. 1989 Sep 1;49(17):4775-9.
We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.
我们发现，强心苷哇巴因对Na⁺,K⁺-泵的阻断作用可保护人A549细胞和仓鼠V79细胞免受拓扑异构酶II毒药依托泊苷的细胞毒性作用。与A549细胞相比，保护V79细胞需要浓度高一千倍的哇巴因。由于这种差异与先前测得的泵敏感性差异相似，这表明保护作用是通过直接阻断泵介导的。哇巴因既不影响依托泊苷的细胞内流入也不影响其流出。然而，泵阻断确实减少了依托泊苷诱导的DNA-拓扑异构酶II可切割复合物的形成，使用碱性和中性洗脱法将其评估为单链和双链DNA断裂。为了确定这种减少是否是由泵阻断产生的离子环境变化的直接影响，我们进行了分离细胞核和部分纯化拓扑异构酶II的实验。在模拟正常细胞内离子条件或哇巴因产生的条件的缓冲液中评估依托泊苷诱导的可切割复合物形成和拓扑异构酶介导的解连环作用。与模拟正常细胞内离子条件的缓冲液相比，模拟泵阻断产生的条件的缓冲液在分离的细胞核中产生的依托泊苷介导的可切割复合物更少，部分纯化的拓扑异构酶II的解连环活性更低。这些发现表明，抑制Na⁺,K⁺-泵会导致细胞内离子环境发生改变，从而降低拓扑异构酶II的活性，进而减少依托泊苷诱导的可切割复合物形成和细胞毒性。由于在细胞周期进程中以及在经历转化的细胞中正常细胞内会发生离子变化，这些数据表明细胞内离子环境在决定正常细胞和恶性细胞对这类化疗药物的敏感性方面发挥着作用。
