INHIBITION OF THE MELANOMA CELL-CYCLE AND REGULATION AT THE G1/S TRANSITION BY 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) BY MODULATION OF CDK2 ACTIVITY
INHIBITION OF THE MELANOMA CELL-CYCLE AND REGULATION AT THE G1/S TRANSITION BY 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) BY MODULATION OF CDK2 ACTIVITY
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DOI：
10.1006/excr.1995.1356
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发表时间：
1995-11-01
影响因子：
3.7
通讯作者：
NATHANSON, L
中科院分区：
文献类型：
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作者：
COPPOCK, DL;BUFFOLINO, P;NATHANSON, L
12-O-十四酰基佛波醇-13-乙酸酯（TPA）抑制恶性黑色素瘤细胞的生长，而刺激正常黑素细胞的生长。我们以前证明，TPA抑制Demel黑色素瘤细胞的生长，并导致在G1/S和G2/M细胞周期转换的停滞。为了研究TPA在G1/S转换时阻止黑色素瘤细胞生长的机制，我们研究了其对细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）水平以及CDK 2激酶活性活化的影响。在G1中加入TPA可阻断p34（cdc 2）mRNA水平的增加，但不能阻断CDK 2 mRNA水平的增加。当在G1中加入TPA时，它抑制了反映磷酸化状态变化的CDK 2的迁移率移动。这对应于抑制CDK 2组蛋白H1激酶活性的增加。对CDK 4水平影响不大。在G1期用TPA处理导致细胞周期蛋白D1 mRNA表达增加3 - 4倍，但在细胞周期后期阻断细胞周期蛋白A和细胞周期蛋白B mRNA表达的增加。TPA引起细胞周期蛋白D1水平的小幅增加，对细胞周期蛋白E的影响很小，表明这些G1期细胞周期蛋白不是限制性的。在G1细胞中加入TPA可抑制cyclin A水平的升高，提示cyclin A可能在介导生长抑制中起重要作用。对CDK抑制剂p21（Cip 1）和p27（Kip 1）水平的检测表明，这些抑制剂的水平在G1期较高，并随着细胞进入S期而下降。在TPA的存在下，这种减少没有发生。这些结果表明，TPA通过调节CDK 2激酶的磷酸化和活化的机制，在G1晚期阻断Demel黑素瘤细胞中的G1/S转变。这些机制包括防止p21（Cip 1）和p27（Kip 1）激酶抑制剂的减少和限制细胞周期蛋白A的量。(C)出版社：Academic Press
The growth of malignant melanoma cells is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) while the growth of normal melanocytes is stimulated. We previously demonstrated that TPA inhibits the growth of Demel melanoma cells and leads to arrest at both at the G1/S and G2/M cell cycle transitions. To investigate the mechanism by which TPA arrests melanoma cell growth at the G1/S transition we have examined its effects on the levels of cyclins and cyclin dependent kinases (CDKs) and activation of CDK2 kinase activity. Addition of TPA in G1 blocked the increase in the level of p34(cdc2) mRNA, but not of CDK2 mRNA When TPA was added in G1, it inhibited the mobility shift of CDK2 reflecting a change in phosphorylation state. This corresponded to inhibition of the increase in CDK2 histone H1 kinase activity. There was little effect on the level of CDK4. Treatment with TPA during G1 caused a three to four fold increase in cyclin D1 mRNA expression, but blocked the increase in the expression of cyclin A and cyclin B mRNAs later in the cell cycle. TPA caused a small increase in levels of cyclin D1 and had little effect on cyclin E, suggesting these G1 cyclins were not limiting. Addition of TPA in G1 prevented an increase in cyclin A levels, suggesting cyclin A might play an important role in mediating the growth inhibition. Examination of the levels of the CDK inhibitors p21(Cip1) and p27(Kip1) showed that the level of these inhibitors was higher in G1 and dropped as cells entered S phase. In the presence of TPA this decrease did not occur. These results demonstrate that TPA blocks the G1/S transition in Demel melanoma cells in late G1 by mechanisms which regulate phosphorylation and activation of the CDK2 kinase. These mechanisms include preventing the decrease in p21(Cip1) and p27(Kip1) kinase inhibitors and limiting the amount of cyclin A. (C) 1995 Academic Press, Inc.