Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Coupling the cell cycle to development and regeneration of the inner ear

Cell cycle exit and acquirement of a postmitotic state is essential for the proper development of organs. In the present review, we examine the role of the cell cycle control in the sensory epithelia of the mammalian inner ear. We describe the roles of the core cell cycle regulators in the proliferation of prosensory cells and in the initiation and maintenance of terminal mitosis of the sensory epithelia. We also discuss how other intracellular signalling may influence the cell cycle. Finally, we address the question of whether manipulations of the cell cycle may have the potential to create replacement cells for the damaged inner sensory epithelia.     

The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids

By inhibiting the activity of Cdc28/Clb cyclin-dependent protein kinase (CDK) complexes, Sic1 prevents the premature initiation of S phase in the yeast Saccharomyces cerevisiae. By testing a series of Sic1 truncation mutants, we have mapped the minimal domain necessary for Cdc28/Clb inhibition in vivo to the C-terminal 70 amino acids of Sic1. Site-directed mutagenesis was used to show that a sequence that matches the zRxL motif found in mammalian CDK inhibitors is essential for Sic1 function. This motif is not found in the Schizosaccharomyces CDK inhibitor p25^rum1, which appears to be a structural and functional homolog of Sic1. Based on the mutational data and sequence comparisons, we argue that Sic1 and p25^rum1 are structurally distinct from the known mammalian CDK inhibitors, but may bind CDK complexes in a manner more closely resembling CDK substrates like the retinoblastoma and E2F proteins. Received: 3 February 1999 / Accepted: 23 April 1999     

Cyclin D1与细胞周期调控

细胞周期是细胞生命活动中一个最重要的过程,其关键是G1 期的启动.细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制因子(CKIs)是参与钿胞周期调控的主要因子.Cyclin D1是调控细胞周期G1期的关键蛋白,是一个比其他Cyclins更加敏感的指标,对细胞周期调控至关重要.综述Cyclin D1的结构和功能及其在肿瘤组织中的表达特征,初步分析Cyclin D在昆虫细胞周期调控的研究.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1

We have previously shown that the mitogenic effect of endothelin-1 (ET-1) in primary astrocytes is dependent on activation of both extracellular signal-regulated kinase (ERK)- and cytoskeleton (CSK)-dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET-1-induced expression of cyclins D1 and D3 is dependent on the ERK- and CSK-dependent pathways, respectively; moreover, a decrease in the levels of the cyclin-dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell-cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D-type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D-type cyclin-associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET-1, are regulated by distinct signaling pathways.     

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C elegans

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.     

A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis

The Arabidopsis genome encodes 10 D-type cyclins (CYCD); however, their differential role in cell cycle control is not well known. Among them, CYCD4;2 is unique in the amino acid sequence; namely, it lacks the Rb-binding motif and the PEST sequence that are conserved in CYCDs. Here, we have shown that CYCD4;2 suppressed G1 cyclin mutations in yeast and formed a kinase complex with CDKA;1, an ortholog of yeast Cdc28, in insect cells. Hypocotyl explants of CYCD4;2 over-expressing plants showed faster induction of calli than wild-type explants on a medium containing lower concentration of auxin. These results suggest that CYCD4;2 has a promotive function in cell division by interacting with CDKA;1 regardless of the unusual primary sequence.     

Advanced glycation end-product-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 are dependent on glycogen synthase kinase 3beta in LLC-PK1 cells

Glycogen synthase kinase 3β (GSK3β) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3β in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 μg/ml) time-dependently (8-48 h) increased phospho-GSK3β-Tyr216 (active GSK3β) and time-dependently (4-24 h) decreased phospho-GSK3β-Ser21/9 (inactive GSK3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK3β kinase at 8-48 h. AGE (100 μg/ml) dose-dependently (75-100 μg/ml) decreased β-catenin protein expression but AGE did not decrease β-catenin protein expression until 48 h. SB216763 (a GSK3β inhibitor) and GSK3β shRNA attenuated AGE (100 μg/ml)-inhibited cell proliferation and protein expression of β-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3β in LLC-PK1 cells. AGE-inhibited β-catenin and cyclin D1 protein expression are dependent on GSK3β. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3β.     

Paradoxical roles of cyclin D1 in DNA stability

Maintenance of DNA integrity is vital for all of the living organisms. Consequence of DNA damaging ranges from, introducing harmless synonymous mutations, to causing disease-associated mutations, genome instability, and cell death. A cell cycle protein cyclin D1 is an established cancer-driving protein. However, contribution of cyclin D1 to cancer formation and cancer survival is not entirely known. In cancer tissues, overexpression of cyclin D1 is associated with both cancer genome instability, and resistance to DNA-damaging cancer drugs. Emerging evidence indicated that cyclin D1 may play novel direct roles in regulating DNA repair. Here we provide an insight how cyclin D1 expression may contribute to DNA repair and chromosome instability, and how these functions may facilitate cancer formation, and drug resistance.     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

糖原合酶激酶3β以细胞周期蛋白D1依赖性方式引发人肺腺癌细胞A549细胞周期阻滞

对糖原合酶激酶3β（glycogen synthase kinase 3β,6SK3β）在细胞增殖中的作用研究,在不同细胞系和不同刺激因素作用下得出了不同结论,本文旨在探讨GSK3β在人肺腺癌细胞系A549细胞生长中的直接作用。A549细胞瞬时转染持续激活型S9A-GSK3β以及显性负突变型KM-GSK3β两种GSK3β突变型质粒,改变GSK3β活性。24 h后,分别进行细胞计数,流式细胞术及Western blot检测。结果显示,增强GSK3β活性可导致细胞数量下降,G．期细胞百分比升高。细胞周期蛋白D1表达水平被GSK3β下调。结果提示,GSK3β可能以细胞周期蛋白D1依赖性方式引发A549细胞的G,期阻滞,从而发挥生长抑制效应。     

Apoptosis related protein 3, an ATRA-upregulated membrane protein arrests the cell cycle at G1/S phase by decreasing the expression of cyclin D1

Human Apr3 was first cloned from HL-60 cells treated by ATRA. In this study, we further demonstrated that Apr3 could be obviously upregulated by ATRA in many other ATRA sensitive cells, suggesting a common role of Apr3 in ATRA effects. Indirect immunofluorescence assay indicates that Apr3 is a membrane protein, while its truncated form without the predicted transmembrane and intracellular domain, was likely a secreted one. Furthermore, FACS analysis showed that Apr3 overexpression could cause an obvious G1/S phase arrest which might be induced by dramatic reduction of cyclin D1 expression. Strikingly, the truncated Apr3 antagonized the negative role of Apr3 on cell cycle and cyclin D1. Taken together, our data suggest that Apr3 should play an important role in ATRA signal pathway and the predicted transmembrane and/or the intracellular domain mediates Apr3 membrane localization and is vital for the negative regulation on cell cycle and cyclin D1.     

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.