Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.
Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.
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发表时间：
1991-07
期刊：
影响因子：
11.2
通讯作者：
K. Keyomarsi;L. Sandoval;V. Band;A. Pardee
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文献类型：
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作者：
K. Keyomarsi;L. Sandoval;V. Band;A. Pardee
哺乳动物细胞的同步化对于涉及细胞增殖的研究是必不可少的。一个简单的方法来获得同步在所有类型的细胞，通过几个周期，并与最小的整体代谢扰动，尚未获得。我们描述了一个程序同步正常以及肿瘤细胞可逆的细胞周期的G1期使用洛伐他汀，3-羟基-3-甲基戊二酰辅酶A还原酶的抑制剂。这种同步化方法对所有测试的细胞系都是成功的，包括小鼠、仓鼠和人来源的正常和肿瘤细胞。例如，当MCF-7人乳腺癌细胞与洛伐他汀同步化并通过加入甲羟戊酸（由3-羟基-3-甲基戊二酰辅酶A还原酶催化的反应产物）释放时，在90小时的细胞复制期间发生了3个相的加速胸苷掺入DNA中，对应于细胞周期的3S期。在第一个S期前18 h的初始延迟期间，胸苷掺入量降低至小于或等于4%，并且仅在6 h后达到最大掺入量。检测与增殖相关的核抗原的抗体Ki-67存在于用洛伐他汀阻滞的细胞中。这一事实，加上在初始滞后时间内缺乏胸苷掺入，表明细胞被阻滞在细胞周期的G1期而不是G 0期。此外，在同步肿瘤来源的人乳腺上皮细胞中，组蛋白H4 RNA在洛伐他汀释放后较低，并随着DNA合成的开始而增加。在2个循环中可以观察到DNA的合成和组蛋白H4 RNA的表达。由于RNA、蛋白质和初始DNA合成速率不受洛伐他汀影响，因此对一般代谢功能的干扰最小，[3 H]尿苷、[3 H]亮氨酸和初始[3 H]胸苷掺入证明了这一点。最后，虽然洛伐他汀诱导的同步被甲羟戊酸克服，但添加角鲨烯或胆固醇乙醇没有这种效果。因此，洛伐他汀似乎可以防止胆固醇途径中早期中间体的形成，这是细胞通过早期G1期进展所必需的。
Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase.