Capranico G, Tinelli S, Zunino F
Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Chem Biol Interact. 1989;72(1-2):113-23. doi: 10.1016/0009-2797(89)90022-7.
The formation and disappearance of DNA single-strand breaks (SSB) produced by 4-demethoxydaunorubicin (4-dmDR) in P388 murine leukemia cells and in a resistant subline were examined by alkaline elution methods in relation to cellular pharmacokinetics. DNA strand breaks produced by this intercalating agent were essentially DNA lesions mediated by topoisomerase II, even at very high drug concentrations, since they were detected as protein-associated breaks by filter elution. Similarly, the appearance of delayed DNA breaks in cells exposed to high concentrations, following drug removal, showed predominance of protein-associated breaks, thus supporting a similar mechanism of breakage induction. This finding indirectly suggests that, in this experimental model, free radical production makes little (if any) contribution to DNA damage, and also that DNA effects are not the consequence of early cell death. In contrast to a rapid disappearance of protein-associated strand breaks produced by intercalating agents and topoisomerase II inhibitors of different classes, DNA breaks induced by low concentrations of the anthracycline derivative are only partially reversible following drug removal, but they persisted and even increased with high concentrations. Thus, not only the extent of DNA breaks but also their persistence may contribute to the cytotoxic potency of anthracyclines. The importance of DNA lesions to cytotoxic action of the anthracycline is also emphasized by drug effect on the resistant line. A negligible effect on DNA of resistant cells was detected at drug concentrations lethal to sensitive cells. However, exposure to equitoxic drug concentrations resulted in a comparable amount of DNA breaks in sensitive and resistant cells. Although faster DNA rejoining in resistant cells may be in part attributable to increased efflux of drug, no correlation exists between cell drug accumulation and extent of DNA lesions. With equitoxic drug concentrations cellular drug content was higher in resistant cells, suggesting an intrinsic insensitivity of this variant to DNA cleavage effects of the anthracycline.
采用碱性洗脱法，结合细胞药代动力学，研究了4-去甲氧基柔红霉素（4-dmDR）在P388小鼠白血病细胞及其耐药亚系中产生的DNA单链断裂（SSB）的形成与消失情况。这种嵌入剂产生的DNA链断裂本质上是由拓扑异构酶II介导的DNA损伤，即使在非常高的药物浓度下也是如此，因为通过滤膜洗脱检测到它们是与蛋白质相关的断裂。同样，在去除药物后，暴露于高浓度药物的细胞中延迟DNA断裂的出现显示出与蛋白质相关的断裂占主导地位，从而支持了类似的断裂诱导机制。这一发现间接表明，在该实验模型中，自由基产生对DNA损伤的贡献很小（如果有的话），并且DNA效应也不是早期细胞死亡的结果。与不同类别的嵌入剂和拓扑异构酶II抑制剂产生的与蛋白质相关的链断裂迅速消失相反，低浓度蒽环类衍生物诱导的DNA断裂在去除药物后仅部分可逆，但在高浓度下它们持续存在甚至增加。因此，不仅DNA断裂的程度，而且它们的持续性都可能有助于蒽环类药物的细胞毒性效力。蒽环类药物对耐药细胞系的作用也强调了DNA损伤对其细胞毒性作用的重要性。在对敏感细胞致死的药物浓度下，检测到对耐药细胞DNA的影响可忽略不计。然而，暴露于等毒性药物浓度下，敏感细胞和耐药细胞中产生的DNA断裂量相当。尽管耐药细胞中更快的DNA修复可能部分归因于药物外排增加，但细胞药物积累与DNA损伤程度之间不存在相关性。在等毒性药物浓度下，耐药细胞中的细胞药物含量更高，这表明该变体对蒽环类药物的DNA切割效应具有内在的不敏感性。
