Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway

TY - JOUR

T1 - Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway

AU - Santoni-Rugiu, E

AU - Falck, J

AU - Mailand, Niels

AU - Bartek, J

AU - Lukas, J

PY - 2000/5

Y1 - 2000/5

N2 - 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.

AB - 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.

KW - CDC2-CDC28 Kinases

KW - Carrier Proteins

KW - Cell Cycle Proteins

KW - Clone Cells

KW - Cyclin E

KW - Cyclin-Dependent Kinase 2

KW - Cyclin-Dependent Kinases

KW - DNA Replication

KW - DNA-Binding Proteins

KW - E2F Transcription Factors

KW - E2F1 Transcription Factor

KW - G1 Phase

KW - Gene Expression Regulation

KW - Interphase

KW - Models, Biological

KW - Protein-Serine-Threonine Kinases

KW - Proto-Oncogene Proteins c-myc

KW - Retinoblastoma Protein

KW - Retinoblastoma-Binding Protein 1

KW - S Phase

KW - Transcription Factor DP1

KW - Transcription Factors

KW - Transgenes

KW - Tumor Cells, Cultured

KW - cdc25 Phosphatases

U2 - 10.1128/MCB.20.10.3497-3509.2000

DO - 10.1128/MCB.20.10.3497-3509.2000

M3 - Journal article

C2 - 10779339

SN - 0270-7306

VL - 20

SP - 3497

EP - 3509

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

IS - 10

ER -