Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.     

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.     

Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression

Cyclin D1 is required at high levels for passage through G₁ phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G₁ phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.     

Patched1 interacts with cyclin B1 to regulate cell cycle progression

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, we identified a novel interaction between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacted specifically with constitutively phosphorylated cyclin B1 derivatives and was able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells was inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to ALA: In addition, we demonstrate that endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, we propose that ptc1 participates in a G(2)/M checkpoint by regulating the localization of MPF.     

Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein

Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1- and E- dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.     

Coordinating cell cycle progression via cyclin specificity

Comment on: Pagliuca FW, et al. Mol Cell 2011; 43:406-17.     

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.     

Regulation of postembryonic G(1) cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex.

In many organisms, initiation and progression through the G(1) phase of the cell cycle requires the activity of G(1)-specific cyclins (cyclin D and cyclin E) and their associated cyclin-dependent kinases (CDK2, CDK4, CDK6). We show here that the Caenorhabditis elegans genes cyd-1 and cdk-4, encoding proteins similar to cyclin D and its cognate cyclin-dependent kinases, respectively, are necessary for proper division of postembryonic blast cells. Animals deficient for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G(1) cell cycle arrest. Moreover, ectopic expression of cyd-1 and cdk-4 in transgenic animals is sufficient to activate a S-phase reporter gene. We observe no embryonic defects associated with depletion of either of these two gene products, suggesting that their essential functions are restricted to postembryonic development. We propose that the cyd-1 and cdk-4 gene products are an integral part of the developmental control of larval cell proliferation through the regulation of G(1) progression.     

Diethylstilbestrol induces arrest of rat vascular smooth muscle cell cycle progression through downregulation of cyclin D1 and cyclin E

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1

Schlafen-1 (Slfn-1), the prototypic member of the Schlafen family of proteins, was described as an inducer of growth arrest in T-lymphocytes and causes a cell cycle arrest in NIH3T3 fibroblasts prior to the G1/S transition. How Slfn-1 exerts its effects on the cell cycle is not currently known. We report that synchronized murine fibroblasts expressing Slfn-1 do not exit G1 when stimulated with fetal calf serum, platelet-derived growth factor BB (PDGF-BB) or epidermal growth factor (EGF). The induction of cyclin D1 by these stimuli was blocked in the presence of Slfn-1 as were all downstream cell cycle processes. Overexpression of cyclin D1 in growth-arrested, Slfn-1-expressing cells induced an increase in cell growth consistent with this protein being the biological target of Slfn-1. Activation of the mitogen-activated protein kinase pathway by EGF or phorbol 12-myristate 13-acetate was unaffected by Slfn-1 expression. PDGF signaling was, however, almost completely blocked. This was due to a lack of PDGF receptor expression in Slfn-1-expressing cells consistent with Slfn-1 blocking the cell cycle in G1 where PDGF receptor expression is normally down-regulated. Finally, overexpression of Slfn-1 inhibited the activation of the cyclin D1 promoter. Slfn-1 therefore causes a cell cycle arrest during G1 by inhibiting induction of cyclin D1 by mitogens.     

Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.

Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was upregulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.