Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway.
The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.
Santoni-Rugiu
E
E
Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen O., Denmark.
Falck
J
J
Mailand
N
N
Bartek
J
J
Lukas
J
J
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002999:Clone Cells; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016193:G1 Phase / Q000502:physiology; D005786:Gene Expression Regulation; D007399:Interphase / Q000502:physiology*; D008954:Models, Biological; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase / Q000502:physiology; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D019076:Transgenes; D014407:Tumor Cells, Cultured; D020687:cdc25 Phosphatases / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C517007:Cdc25a protein, rat; D020687:cdc25 Phosphatases