Confluence-induced cell cycle exit involves pre-mitotic CDK inhibition by p27Kip1 and cyclin D1 downregulation

INSERM U634, France.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 08/2008; 7(13):2038-46.
Source: PubMed


Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G(1), requires pRb family members, and is driven by p27(Kip1)-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G(1), prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G(2) phase and requires the Ras-dependent induction of cyclin D1, which promotes following G(1)/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Overexpression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.

Download full-text


Available from: Vjekoslav Dulic
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cell cycle inhibitor p21(CDKN1A) induces cell cycle arrest under different conditions, including senescence and terminal differentiation. Still debated is its involvement in the reversible transition from proliferation to a non-dividing quiescent state (G(0)), in which a significant role has been attributed to cell cycle inhibitor p27(CDKN1B). Here we provide evidence showing that high p21 protein levels are necessary to enter and maintain the quiescence state following contact inhibition and growth factor withdrawal. In fact, entry into quiescence was impaired, both in human fibroblasts in which p21 gene has been deleted, or protein expression knocked-down by RNA interference. Importantly, in the absence of p21, human fibroblasts activate a DNA damage-like signalling pathway, as shown by phosphorylation of histone H2AX and Chk1 proteins. In addition, we show that in the absence of p21, checkpoint is activated by an unscheduled entry into S phase, with a reduced efficiency in DNA maturation, in the presence of high c-myc protein levels. These results highlight the role of p21 in counteracting inappropriate proliferation stimuli for genome stability maintenance.
    Full-text · Article · Feb 2009 · Cell cycle (Georgetown, Tex.)
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although contact inhibition is a fundamental process for multicellular organisms, how proliferation is inhibited at high cellular densities remains poorly characterized. Here we show that 4E-BP1, one major repressor of cap-dependent translation, plays a critical role in density-mediated cell cycle arrest. 4E-BP1 promoter is activated and 4E-BP1 protein amount increases as cells reach confluence. Conversely, a much less marked density-dependent inhibition of cell proliferation is observed upon 4E-BP1 silencing. We further show that at high density, progression through the G₁ phase of the cell cycle is faster and Cyclin D1 protein is induced in different cell types where 4E-BP1 has been either downregulated (stable shRNA expression or transient siRNA transfection) or removed (knock-out). Thus 4E-BP1 appears as an important mediator of contact inhibition.
    Full-text · Article · Apr 2010 · Cell cycle (Georgetown, Tex.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Numerous studies have shown that mammalian target of rapamycin (mTOR) inhibitor activates Akt signaling pathway via a negative feedback loop while inhibiting mTORC1 signaling. In this report, we focused on studying the role of mTORC1 and mTORC2 in rapamycin-mediated Akt and ERK phosphorylation, and the antitumor effect of rapamycin in cancer cells in combination with Akt and ERK inhibitors. Moreover, we analyzed the effect of mTORC1 and mTORC2 on regulating cell cycle progression. We found that low concentrations rapamycin increased Akt and ERK phosphorylation through a mTORC1-dependent mechanism because knockdowned raptor induced the activation of Akt and ERK, but higher doses of rapamycin inhibited Akt and ERK phosphorylation mainly via the mTORC2 signaling pathway because that the silencing of rictor led to the inhibition of Akt and ERK phosphorylation. We further showed that mTORC2 was tightly associated with the development of cell cycle through an Akt-dependent mechanism. Therefore, we combined PI3K and ERK inhibitors prevent rapamycin-induced Akt activation and enhanced antitumor effects of rapamycin. Collectively, we conclude that mTORC2 plays a much more important role than mTORC1 in rapamycin-mediated phosphorylation of Akt and ERK, and cotargeting AKT and ERK signaling may be a new strategy for enhancing the efficacy of rapamycin-based therapeutic approaches in cancer cells.
    No preview · Article · Jun 2010 · Molecular Carcinogenesis
Show more