Deregulated E2F transcriptional activity in autonomously growing melanoma cells.
Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors.
Halaban
R
R
Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06520-8059, USA. ruth.halaban@yale.edu
Cheng
E
E
Smicun
Y
Y
Germino
J
J
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D029721:Drosophila Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050703:E2F2 Transcription Factor; D050699:E2F3 Transcription Factor; D050704:E2F4 Transcription Factor; D050700:E2F5 Transcription Factor; D050701:E2F6 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008544:Melanocytes / Q000378:metabolism / Q000502:physiology; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding; D056465:Retinoblastoma-Binding Protein 1; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017077:Culture Media, Conditioned; D016213:Cyclins; D004268:DNA-Binding Proteins; C088053:Dp transcription factor, Drosophila; D029721:Drosophila Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050703:E2F2 Transcription Factor; C493761:E2F2 protein, human; D050699:E2F3 Transcription Factor; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; D050700:E2F5 Transcription Factor; D050701:E2F6 Transcription Factor; D004791:Enzyme Inhibitors; D005419:Flavonoids; D010880:Piperidines; D056465:Retinoblastoma-Binding Protein 1; C493735:TFDP1 protein, human; C094103:TFDP2 protein, human; D015534:Trans-Activators; D050680:Transcription Factor DP1; D014157:Transcription Factors; C077990:alvocidib; D018844:Cyclin-Dependent Kinases