Search

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21 WAF1/CIP1 and pRB/p16 INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3 EcoR ) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence., (© 2021. The Author(s).)

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.

Lack of detectable expression of p27kip1 cyclin dependent kinase inhibitor has previously been correlated with high degree of malignancy in human breast, colorectal, gastric and small cell lung carcinomas. Here we demonstrate that an inverse correlation between p27kip1 expression and tumour malignancy also exists in most types of human B cell lymphomas examined. A clear exception was Burkitt's lymphoma (BL), a highly malignant tumour which often expresses high levels of p27kip1. Analysis of p27kip1 derived from Burkitt's lymphoma cell lines expressing high levels of p27kip1, BL40 and BL41, in a cyclin E/cdk2 kinase inhibition assay demonstrated that p27kip1 is not permanently inactivated since heat treatment can restore the inhibitory activity of p27kip1. However, p27kip1 expressed in these two cell lines is largely sequestered in inactive complexes and we have no evidence that c-myc or Epstein-Barr virus are responsible for the sequestration of p27kip1 in these two cell lines although c-myc and EBV are two oncogenic agents often associated with Burkitt's lymphomas. Interestingly, we observed that high level p27kip1 expression often correlated with cyclin D3 overexpression both in vivo and in BL cell lines. The majority of p27kip1 in BL40 cells was complexed with cyclin D3 indicating that overexpressed cyclin D3 may at least be part of the sequestering activity for the inhibitory function of p27kip1. Furthermore, cyclinD3/cdk4 complex could sequester p27kip1 in a cyclin E/cdk2 kinase assay in vitro. Finally, we show that cyclin D3 transfected into an inducible p27kip1 cell line could overcome the G1 arrest mediated by p27kip1. These results argue that in addition to down-regulation of p27kip1 expression, some tumour cells can sequester and tolerate the antiproliferative function of p27kip1. They also suggest a novel role for the overexpression of D-type cyclins as one pathway allowing tumour cells to overcome the antiproliferative function of p27kip1.

The Kaposi's sarcoma-associated human herpesvirus 8 (KSHV/HHV8) encodes a protein similar to cellular cyclins. This cyclin is most closely related to cellular D-type cyclins, but biochemically it behaves atypically in various respects. Complexes formed between the viral cyclin and the cyclin-dependent kinase subunit, cdk6, can phosphorylate a wider range of substrates and are resistant to cdk inhibitory proteins. We show here that the KSHV-cyclin-cdk6 complex phosphorylates p27(Kip) on a C-terminal threonine that is implicated in destabilization of this cdk inhibitor. Expression of the viral cyclin in tissue culture cells overcomes a cell cycle block by p27(Kip). However, full cell-cycle transit of these cells appears to depend on C-terminal phosphorylation of p27(Kip) and seems to involve transactivation of other cellular cyclin-dependent kinases. A p27(Kip)-phosphorylating cdk6 complex exists in cell lines derived from primary effusion lymphoma and in Kaposi's sarcoma, this indicating that virally induced p27(Kip) degradation may occur in KSHV-associated tumours.

The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53.

Progression of cells into S phase is controlled by the retinoblastoma protein (pRB) and relies on the functional inactivation of this tumour suppressor in late G1 via protein phosphorylation. We provide evidence here that, besides controlling entry of cells into S phase, pRB can operate to inhibit S phase completion. Differential arrays of phosphorylation appear to regulate these different events, suggesting that cycle progression at these two stages of the cell cycle may be achieved via activation of distinct downstream pRB effector pathways. In agreement with this hypothesis, pRB's ability to prevent S phase entry, but not its ability to inhibit S phase completion, correlates with repression of E2F-regulated promoters. Furthermore, ectopic expression of E2F or the E2F-regulated cyclin E gene promote S phase entry in cells expressing phosphorylation-defective pRB but neither is sufficient to trigger completion of S phase. Our findings raise the possibility that pRB, in addition to its well-established role in controlling a checkpoint in late G1, could be involved in the control of a further checkpoint operating during S phase and that implementation of this checkpoint relies on an as yet unidentified pRB effector distinct from E2F.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.

The retinoblastoma protein (pRB) and the pRB-related pocket proteins p130 and p107, when bound to DNA via the E2F family of transcription factors, suppress transcription and through this may mediate growth arrest. We show here that in HaCat cells arrested by treatment with TGFbeta the only pocket protein associating with DNA-bound E2F is pRB. This contrasts the situation in HaCat cells arrested via growth factor withdrawal, where we find that both pRB and p130 can bind. The above implies that p130 participates in regulating E2F-dependent genes upon growth factor deprivation but not upon TGFbeta arrest. More importantly perhaps, in TGFbeta-arrested cells pRB alone in association with its partner E2Fs may be in charge and sufficient to control E2F-dependent gene transcription. Although p130 is not associated with a DNA-binding E2F complex in TGFbeta-treated cells, it is present in such cells in its underphosphorylated form. We provide evidence for a serum-induced process that may regulate p130 by a mechanism independent of p130 hyperphosphorylation.