You J, Bird R C
Department of Pathobiology, Auburn University, Alabama 36849, USA.
J Cell Physiol. 1995 Aug;164(2):424-33. doi: 10.1002/jcp.1041640223.
Genes encoding cdk1 (p34cdc2), cyclin A, cyclin B, and the tumor suppressor gene Rb are fundamental regulators of cell cycle progression which associate as a complex with the transcription factor E2F. Expression of many of these proteins has previously been shown to be repressed by okadaic acid, a specific inhibitor of protein phosphatases 1/2A (PP1/PP2A), resulting in growth arrest in nontransformed but immortalized cells. We have investigated levels of mRNA encoding cdk1 (p34cdc2), cyclin A, cyclin B, Rb, GAPDH, c-myc, and histone H4 genes for sensitivity to okadaic acid in HeLa cells to determine if transformation altered their regulation. Serum starvation slowed growth and diminished mRNA levels for all genes tested except c-myc and GAPDH. When starved cells were subsequently exposed to 19 nM okadaic acid or refed 10% serum, mRNA levels of cyclin A, cyclin B, cdk1, and Rb dramatically increased while mRNA levels for c-myc and GAPDH were largely unaffected. Histone H4 mRNA levels and the rate of DNA synthesis were greatly enhanced by serum addition but not affected appreciably by okadaic acid. Okadaic acid was also effective in blocking proliferation of exponentially growing HeLa cells at G2/M and S phase. Despite the cell cycle phase-specific block, elevated mRNA levels for cdk1, cyclin A, cyclin B, Rb, and suppression of H4 mRNA levels were detected and persisted for at least 12 hr following okadaic acid removal. The results demonstrate that cell cycle progression is blocked and several cell cycle regulatory genes, encoding transcription factor E2F-associated proteins, experience elevation of mRNA levels through mechanisms sensitive to okadaic acid likely through a PP1/PP2A-sensitive mechanism. Data from transformed cells contrast with data from immortalized but nontransformed cells in which okadaic acid also blocks cell cycle progression during G2/M phase but suppresses expression of these genes. Such contrasts may be correlated with reduced growth factor dependence and transformation.
编码细胞周期蛋白依赖性激酶1（p34cdc2）、细胞周期蛋白A、细胞周期蛋白B以及肿瘤抑制基因Rb的基因是细胞周期进程的基本调节因子，它们与转录因子E2F形成复合物。此前已有研究表明，这些蛋白中的许多表达会被冈田酸（一种蛋白磷酸酶1/2A（PP1/PP2A）的特异性抑制剂）抑制，从而导致未转化但已永生化的细胞生长停滞。我们研究了编码细胞周期蛋白依赖性激酶1（p34cdc2）、细胞周期蛋白A、细胞周期蛋白B、Rb、甘油醛-3-磷酸脱氢酶（GAPDH）、c-myc和组蛋白H4基因的mRNA水平对HeLa细胞中冈田酸的敏感性，以确定转化是否改变了它们的调控。血清饥饿减缓了生长速度，并降低了除c-myc和GAPDH外所有测试基因的mRNA水平。当饥饿细胞随后暴露于19 nM冈田酸或重新添加10%血清时，细胞周期蛋白A、细胞周期蛋白B、细胞周期蛋白依赖性激酶1和Rb的mRNA水平显著增加，而c-myc和GAPDH的mRNA水平基本不受影响。血清添加极大地提高了组蛋白H4的mRNA水平和DNA合成速率，但冈田酸对此没有明显影响。冈田酸在G2/M期和S期也能有效阻断指数生长的HeLa细胞的增殖。尽管存在细胞周期阶段特异性阻断，但在去除冈田酸后至少12小时内，仍检测到细胞周期蛋白依赖性激酶1、细胞周期蛋白A、细胞周期蛋白B、Rb的mRNA水平升高，以及组蛋白H4的mRNA水平受到抑制。结果表明，细胞周期进程被阻断，几个编码与转录因子E2F相关蛋白的细胞周期调控基因，其mRNA水平通过可能对PP1/PP2A敏感的机制对冈田酸敏感的机制升高。来自转化细胞的数据与来自已永生化但未转化细胞的数据形成对比，在未转化细胞中，冈田酸也会在G2/M期阻断细胞周期进程，但会抑制这些基因的表达。这种对比可能与生长因子依赖性降低和转化有关。
