VanderWaal R P, Griffith C L, Wright W D, Borrelli M J, Roti J L
Washington University School of Medicine, Mallinckrodt Institute of Radiology, Radiation Oncology Center, Section of Cancer Biology, St. Louis, Missouri, USA.
J Cell Physiol. 2001 May;187(2):236-43. doi: 10.1002/jcp.1073.
The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.
细胞保护自身免受热休克和其他应激诱导剂致死效应的机制是众多研究的主题。我们研究了热诱导对DNA复制机制的损伤与热休克致死效应之间的关系，研究对象是处于S期的细胞，该期细胞比G1期或G2期细胞对热休克更敏感。我们发现，将细胞维持在阿非迪霉素中，可阻止细胞通过S期，这能使S期的HeLa细胞从热休克的致死效应中获救。当S期的HeLa细胞在3 microM阿非迪霉素中培养5 - 6小时时，测得的克隆形成存活率与指数生长细胞的存活率相似。已知热休克会诱导蛋白质变性或展开，使其溶解度降低，更有可能与核基质共分离。在此，我们表明，在四种不同实验条件下，参与DNA复制的蛋白质（增殖细胞核抗原、复制蛋白A和细胞周期蛋白A）与核基质的结合增强，与热休克后S期细胞的致死率相关。具体而言，当细胞恢复通过S期进程时，与核基质相关的复制蛋白A、增殖细胞核抗原和细胞周期蛋白A的量与细胞杀伤相关。热诱导的与DNA代谢其他方面相关的核蛋白（Mrell、蛋白二硫键异构酶）的结合增强，并未显示出这种相关性。这些结果支持了这样一种假说，即热诱导的与DNA复制工厂相关的蛋白质结合变化是潜在的致死性损伤，这些损伤会因S期进程而固定为致死性损伤，但如果S期进程延迟则可修复。
