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.
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.
Coppock
D L
DL
Oncology Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA.
Buffolino
P
P
Kopman
C
C
Nathanson
L
L
D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins; D019938:Cyclin D1; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008545:Melanoma; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D016196:S Phase / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D013755:Tetradecanoylphorbol Acetate