Schlegel R, Croy R G, Pardee A B
J Cell Physiol. 1987 Apr;131(1):85-91. doi: 10.1002/jcp.1041310113.
Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.
咖啡因已被证明能在DNA复制过程中被阻断的叙利亚仓鼠成纤维细胞（BHK细胞）中诱导有丝分裂事件（施莱格尔和帕迪，《科学》232:1264 - 1266，1986）。蛋白质合成的抑制会阻断这些咖啡因诱导的事件，而RNA合成的抑制则几乎没有影响。我们现在报告，在这些细胞中诱导有丝分裂所需的蛋白质在添加咖啡因后不久就被合成，在没有咖啡因的情况下其活性非常不稳定，并且该活性通过一种依赖ATP的机制丧失。咖啡因显著提高了这些假定蛋白质的稳定性，同时对整体蛋白质降解没有影响。用RNA合成抑制剂进行的实验表明，与有丝分裂相关的RNA在DNA复制受抑制期间积累，并且当允许复制恢复时这种RNA是不稳定的。这些结果表明，有丝分裂所需RNA的稳定性受细胞的DNA复制状态调节，并且咖啡因选择性地稳定了这种RNA的蛋白质产物。因此可以选择一些条件，使有丝分裂因子在细胞周期的不适当时间在细胞中积累。二维凝胶电泳已证明咖啡因处理导致了几种蛋白质变化；它们与有丝分裂诱导活性的相关性仍有待确定。
