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.     

Evidence that cyclin D1 mediates both growth and proliferation downstream of TOR in hepatocytes

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regeneration in vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.     

Cyclin D3 is dispensable for human diffuse large B-cell lymphoma survival and growth: evidence for redundancy with cyclin E

Genomic changes disrupting the expression of cyclin D3 are associated with aberrant growth of several human B-lymphoid malignancies. We demonstrate that the human diffuse large B-cell lymphoma (DLBCL), OCI-LY18 (LY18) expresses cyclin D3 but not cyclins D1 and D2. RNA interference was used to deplete cyclin D3 from LY18 cells. Surprisingly, knockdown of cyclin D3 did not inhibit pRb phosphorylation on cdk4/6- and cdk2-specific residues or measurably affect viability and proliferation. These results suggest that cyclin D3 is dispensable in LY18 cell proliferation and survival. Similar results were obtained following depletion of cyclin E. By contrast, combined knockdown of cyclins D3 and E had substantial consequences leading to G1-phase arrest and inhibition of proliferation. Whereas cell cycle distribution was not affected following individual depletion of cdk4, cdk6, or cdk2, the combined knockdown of cdk4 and cdk6 led to accumulation of LY18 cells in G1-phase of the cell cycle and inhibition of proliferation. Likewise treatment of LY18 cells with 2-Bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, a selective inhibitor of cdk4/6, led to inhibition of proliferation. Taken together, these results uncover a built-in redundancy with cyclins D3 and E for G1-S progression. Moreover these findings highlight the rationale for simultaneous disruption of cdk4/6 as a potential therapeutic cancer strategy.     

Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2

Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin''s practical value as an anti-tumor drug.     

Short term cyclin D1 overexpression induces centrosome amplification, mitotic spindle abnormalities, and aneuploidy

In normal cells, cyclin D1 is induced by growth factors and promotes progression through the G(1) phase of the cell cycle. Cyclin D1 is also an oncogene that is thought to act primarily by bypassing the requirement for mitogens during the G(1) phase. Studies of clinical tumors have found that cyclin D1 overexpression is associated with chromosome abnormalities, although a causal effect has not been established in experimental systems. In this study, we found that transient expression of cyclin D1 in normal hepatocytes in vivo triggered dysplastic mitoses, accumulation of supernumerary centrosomes, abnormalities of the mitotic spindle, and marked chromosome changes within several days. This was associated with up-regulation of checkpoint genes p53 and p21 as well as hepatocyte apoptosis in the liver. Transient transfection of cyclin D1 also induced centrosome and mitotic spindle abnormalities in breast epithelial cells, suggesting that this may be a generalized effect. These results indicate that cyclin D1 can induce deregulation of the mitotic apparatus and aneuploidy, effects that could contribute to the role of this oncogene in malignancy.     

Cyclin A2/cyclin-dependent kinase 1-dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish

Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1~(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A~(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1~(-/-)and ccna2~(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

cAMP-dependent positive control of cyclin A2 expression during G1/S transition in primary hepatocytes.

cAMP positively and negatively regulates hepatocyte proliferation but its molecular targets are still unknown. Cyclin A2 is a major regulator of the cell cycle progression and its synthesis is required for progression to S phase. We have investigated whether cyclin A2 and cyclin A2-associated kinase might be one of the targets for the cAMP transduction pathway during progression of hepatocytes through G1 and G1/S. We show that stimulation of primary cultured hepatocytes by glucagon differentially modulated the expression of G1/S cyclins. Glucagon indeed upregulated cyclin A2 and cyclin A2-associated kinase while cyclin E-associated kinase was unmodified. In conclusion, our study identifies cyclin A2 as an important effector of the cAMP transduction network during hepatocyte proliferation.     

Regulation of MyoD function in the dividing myoblast

Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing. Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation. Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation. Co-expression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter. Similarly, transfection of myoblasts with the cyclin-dependent kinase (cdk) inhibitors p16 and p21 supports some muscle-specific gene expression even in growth medium. Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks. We review evidence coupling growth status, the cell cycle and myogenesis. We describe a novel mitogen-sensitive mechanism that involves the cyclin D1-dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen-sensitive manner.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.