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Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression
J Lukas1, B O Petersen, K Holm
1Division of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
Abstract:
The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.
Insights
The E2F transcription factors control cell cycle progression. Different E2F subsets, associated with pRB or p107/p130 proteins, have distinct roles in regulating cell cycle entry and overcoming growth arrest.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- E2F transcription factors regulate genes critical for mammalian cell cycle progression.
- Their activity is modulated by binding to retinoblastoma tumor suppressor protein (pRB) and its homologs (p107, p130).
Purpose of the Study:
- To investigate the distinct contributions of various E2F family members to cell cycle control.
- To elucidate the mechanisms by which different E2Fs regulate cell cycle progression and respond to inhibitory signals.
Main Methods:
- Generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4.
- Microinjection of expression plasmids for all E2F family members into quiescent fibroblasts.
- Assessment of S-phase entry and G1 progression.
- Analysis of G1 arrest mediated by p16INK4 or anti-cyclin D1 antibody.
Main Results:
- pRB-associated E2Fs (E2F-1, E2F-2, E2F-3) induce S-phase entry and overcome p16INK4-mediated G1 arrest independently.
- p107/p130-associated E2Fs (E2F-4, E2F-5) require co-expression with DP-1 to promote S-phase entry and overcome p16INK4-induced G1 arrest.
- E2F-1 overrides a G1 block induced by anti-cyclin D1 antibody.
Conclusions:
- E2F activity is rate-limiting for G1 progression and sufficient for S-phase entry.
- pRB-associated E2Fs are likely downstream effectors in the p16-cyclin D-pRB pathway.
- Distinct E2F subsets are regulated by different mechanisms and possess distinct functions in cell cycle control, potentially explaining the rarity of p107/p130 aberrations in cancer.