Coppock D L, Buffolino P, Kopman C, Nathanson L
Oncology Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA.
Exp Cell Res. 1995 Nov;221(1):92-102. doi: 10.1006/excr.1995.1356.
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 p34cdc2 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 p21Cip1 and p27Kip1 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 p21Cip1 and p27Kip1 kinase inhibitors and limiting the amount of cyclin A.
12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）可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。我们之前证明TPA可抑制德梅尔黑色素瘤细胞的生长，并导致细胞在G1/S和G2/M细胞周期转换阶段停滞。为了研究TPA使黑色素瘤细胞在G1/S转换阶段停滞生长的机制，我们检测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CDK2激酶活性激活的影响。在G1期添加TPA可阻止p34cdc2 mRNA水平的升高，但不影响CDK2 mRNA水平。当在G1期添加TPA时，它抑制了CDK2的迁移率变化，这反映了磷酸化状态的改变。这与CDK2组蛋白H1激酶活性升高的抑制相对应。对CDK4水平几乎没有影响。在G1期用TPA处理导致细胞周期蛋白D1 mRNA表达增加三到四倍，但在细胞周期后期阻止了细胞周期蛋白A和细胞周期蛋白B mRNA表达的增加。TPA使细胞周期蛋白D1水平略有增加，对细胞周期蛋白E影响不大，表明这些G1期细胞周期蛋白并非限制因素。在G1期添加TPA可阻止细胞周期蛋白A水平的升高，表明细胞周期蛋白A可能在介导生长抑制中起重要作用。对CDK抑制剂p21Cip1和p27Kip1水平的检测表明，这些抑制剂的水平在G1期较高，随着细胞进入S期而下降。在TPA存在的情况下，这种下降并未发生。这些结果表明，TPA通过调节CDK2激酶的磷酸化和激活机制，在G1晚期阻止德梅尔黑色素瘤细胞的G1/S转换。这些机制包括阻止p21Cip1和p27Kip1激酶抑制剂的减少以及限制细胞周期蛋白A的量。
