高等植物细胞周期调控研究进展

高等植物的细胞周期（cell cycle)在其生长发育过程中受严格调控的,细胞周期的运转是基因有序表达的结果,并受的因素的影响,植物细胞周期研究近年来已取得的较大的进展,本文综述了近几年与植物细胞周期调控相关的细胞周期蛋白（cyclins),细胞周期蛋白依赖性激酶（CDKs)等内部调控因子及外源影响因素的研究进展。     

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.     

植物细胞周期调控因子及其功能的研究进展

细胞周期调控因子能通过影响细胞周期对植物细胞的生长、分裂和分化产生作用,进而调节植物的生长发育。本文综述了近几年来植物细胞周期调控因子中细胞周期蛋白（cyclin,CYC）、周期蛋白依赖激酶（cyclin-dependent kinase,CDK）等的作用机理及研究进展,阐述了各调控因子在植物生长发育过程中的作用。     

ZFP36L1通过下调细胞周期蛋白D1抑制舌癌细胞增殖

C3H1型的锌指蛋白36 (zinc finger protein 36,C3H type-like 1,ZFP36L1)是一种高度保守且具有CCCH型RNA结合结构域的蛋白质。近年来,ZFP36L1在多种肿瘤中的作用被报道,但是在舌癌中的表达型和作用机制尚不清楚。Western印记结合荧光定量PCR检测发现,ZFP36L1在舌癌细胞中的表达明显低于人永生化表皮细胞Hacat。在相对低表达ZFP36L1的舌癌细胞SCC15和SCC25中,稳定过表达ZFP36L1,细胞计数实验发现,SCC15细胞的数目由(4.768±0.09225)×10~3个降低到(3.089±0.09745)×10~3个,SCC25细胞的数目由(6.274±0.01311)×10~3个降低到(4.037±0.01173)×10~3个;平板克隆实验提示,SCC15和SCC25细胞克隆数目是对照组的0.67倍,0.68倍,0.7倍和0.59倍,0.57倍,0.59倍;过表达ZFP36L1组G_1期的SCC15和SCC25细胞分别由61.82±0.8933%增加到88.72%±0.8378,由56.31%±1.029增加到71.7%±0.9303;而S期的细胞由25.21%±0.9865减少到11.31%±0.6567,由28.58%±0.8182减少到18.61%±0.6798。过表达ZFP36L1能明显下调SCC15和SCC25细胞中细胞周期蛋白D1(cyclinD1)的蛋白质水平。过表达ZFP36L1组的SCC15和SCC25细胞中,细胞周期蛋白D1 mRNA的表达量分别是对照组的0.217倍和0.175倍。在舌癌细胞中,上调细胞周期蛋白D1的表达水平可消除由过表达ZFP36L1引起的细胞增殖能力降低。总之,ZFP36L1在舌癌中呈低表达;可通过下调细胞周期蛋白D1的表达,抑制舌癌细胞增殖。     

细胞周期蛋白D1特异性核酶表达载体的构建及其活性

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf( )中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。     

通过共表达转铁蛋白和细胞周期蛋白D1改造CHO细胞株

目的:对现有的CHO-DG44细胞株进行改造,得到在无血清培养基中更具生长优势的CHO宿主细胞株。方法:分别克隆转铁蛋白和细胞周期蛋白D1基因,构建共表达2种基因的pIRES质粒,转染CHO-DG44细胞株,筛选G418抗性克隆。结果与结论:得到3株G418阳性克隆株,其中转铁蛋白和细胞周期蛋白D1表达水平最高的S6与CHO-DG44相比,在无血清培养基中生长更快、密度更高。     

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。     

细胞周期蛋白（cyclin）D在鼻咽癌中的表达及功能初？…

探讨了细胞周期蛋白（ｃｙｃｌｉｎ）Ｄ在ＥＢ病毒（Ｅｐｓｔｅｉｎ－Ｂａｒｒ ｖｉｒｕｓ）潜伏膜蛋白１（ｌａｔｅｎｔ ｍｅｍｂｒａｎｅ ｐｒｏｔｅｉｎ,ＬＭＰ１）阳性和阴性表达的鼻咽癌细胞５系中的表达特征,利用流式细胞仪同时从ＤＮＡ及蛋白质水平初步分析了ｃｙｃｌｉｎ Ｄ１在鼻咽癌细胞系中的功能。Ｗｅｓｔｅｒｎ印迹的结果发现ｃｙｃｌｉｎｈ Ｄ１在ＣＮＥ－ＬＭＰ１中过表达,在ＨＮＥ１中表达较弱;Ｃｙｃｌｉ     

靶向细胞周期蛋白B1的抗肿瘤研究

细胞周期蛋白B1(cyclin B1,CCNB1)是有丝分裂期(M期)周期蛋白,与细胞周期蛋白依赖性激酶1(cyclin-dependent kinase 1,CDK1)结合形成成熟促进因子(maturation promotingfactor,MPF),MPF的激活为真核细胞启动有丝分裂所必需.CCNB1在肿瘤组织中普遍高表达,该蛋白质作为一种细胞周期进程调节剂在肿瘤细胞内的表达量是判断肿瘤恶性程度高低的指标之一,被视为肿瘤抗原,故靶向CCNB1进行抗肿瘤治疗是肿瘤基因治疗中一个极有潜力的策略.首先分析了CCNB1与肿瘤的相关性,进而从CCNB1活性抑制因子、药物或化合物对CCNB1的抑制作用及CCNB1与抗肿瘤免疫等多方面对靶向CCNB1的抗肿瘤研究进行了综述.     

人细胞周期蛋白D1/CDK4基因的真核表达及生物活性鉴定

通过生物工程获得人重组细胞周期蛋白 (cyclinD1 )及细胞周期蛋白激酶CDK4蛋白 ,作为抗癌药物筛选的分子靶点 .从人HL 6 0细胞中获得细胞周期蛋白D1 CDK4基因的cDNA ,先克隆至pGEMT Easy载体上 ,再经重组构建供体质粒pFastBac D1和pFastBac CDK4 .重组供体质粒转化感受态DH1 0Bac细胞 ,挑取确证为白色克隆的菌落振荡培养 ,分离制备高纯度杆粒DNA .以重组病毒适量感染昆虫细胞Tn 5B1 4 ,利用Bac to Bac杆状病毒表达系统在昆虫细胞Tn 5B1 4 (Hi5 )中表达相应的重组蛋白 .应用昆虫杆状病毒表达系统 (Bac to Bac)在昆虫细胞Tn 5B1 4中分别高效表达了人细胞周期蛋白D1和CDK4蛋白 .SDS PAGE分析表明 ,表达量占细胞可溶性蛋白质的 2 0 %左右 ,表达产物经Ni2 + NTA亲和层析纯化后纯度达 85 %以上 .研究表明 ,昆虫细胞表达的细胞周期蛋白D1和CDK4蛋白能促进Rb蛋白的磷酸化 ,具有生物活性 .成功构建了细胞周期蛋白D1及CDK4真核杆状病毒表达载体 ,并且在昆虫细胞中正确表达了具有生物活性的细胞周期蛋白D1及CDK4融合蛋白 .     

Progression through the G1-phase of the on-going cell cycle

Cell cycle progression is dependent upon the action of cyclins and their partners the cyclin dependent kinases (CDKs). Each cell cycle phase has its own characteristic cyclin-CDK combination, cyclin D-CDK4,6 and cyclin E-CDK2 being responsible for progression through G(1)-phase into S-phase. Progression through G(1)-phase is regulated by signal transduction cascades activated by polypeptide growth factors and by extracellular matrix (ECM) components. Studies aiming to unravel the molecular mechanism by which these extracellular components activate the cyclin-CDK complexes in the G(1)-phase, are usually performed using serum-starved cells (G(0) cells). These cells are activated by addition of growth factors, or the cells are detached from the substratum by trypsinization and subsequently allowed to re-attach. An alternative approach, however, is to study the effects of growth factors and attachment in the ongoing cell cycle by synchronization of the cells by the mitotic shake-off method. These cells are not serum starved and not actively detached from the substratum. In this contribution it is shown that both methods yield significant different results. These observations demonstrate that data obtained with model systems should be interpreted with care, especially if the findings are used to explain cell cycle progression in cells in an intact organism.     

Networks and circuits in cell regulation

Large-scale “omics” data are often represented as networks of interacting components, but such representation is inherently static and, as such, cannot provide a realistic picture of the temporal dynamics of complex cellular functions. These difficulties suggest moving to a modeling strategy that explicitly takes into account both the wiring of the components and the task they perform. From an engineering perspective, this problem resembles that of “circuit analysis”. In this paper, we focus on a limited but relevant biological circuit, the G1 to S transition in yeast cell cycle, and investigate both the network representation and the corresponding circuit described by a mathematical model, by means of a wide range of numerical simulation analysis. Reliable predictions of system-level properties are achieved and the parameters that mostly affect these properties are found out.     

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.     

DDA1, a novel oncogene,promotes lung cancer progression through regulation of cell cycle

Lung cancer is globally widespread and associated with high morbidity and mortality. DDA1 (DET1 and DDB1 associated 1) was first discovered and registered in the GenBank database by our colleagues. DDA1, an evolutionarily conserved gene, might have significant functions. Recent reports have demonstrated that DDA1 is linked to the ubiquitin–proteasome pathway and facilitates the degradation of target proteins. However, the function of DDA1 in lung cancer was previously unknown. This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of lung cancer. We found that the expression of DDA1 in normal lung cells and tissue was significantly lower than that in lung cancer and was associated with poor prognosis. DDA1 overexpression promoted proliferation of lung tumour cells and facilitated cell cycle progression in vitro and subcutaneous xenograft tumour progression in vivo. Mechanistically, this was associated with the regulation of S phase and cyclins including cyclin D1/D3/E1. These results indicate that DDA1 promotes lung cancer progression, potentially through promoting cyclins and cell cycle progression. Therefore, DDA1 may be a potential novel target for lung cancer treatment, and a biomarker for tumour prognosis.     

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K⁺) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K⁺ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K⁺ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21^WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27^Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8 h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21^WAF1/Cip1 and p27^Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K⁺ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E.     

The Role of the Anaphase-Promoting Complex/Cyclosome in Plant Growth

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that plays a major role in the progression of the eukaryotic cell cycle. This unusual protein complex targets key cell cycle regulators, such as mitotic cyclins and securins, for degradation via the 26S proteasome by ubiquitination, triggering the metaphase-to-anaphase transition and exit from mitosis. Because of its essential role in cell cycle regulation, the APC/C has been extensively studied in mammals and yeasts, but relatively less in plants. Evidence shows that, besides its well-known role in cell cycle regulation, the APC/C also has functions beyond the cell cycle. In metazoans, the APC/C has been implicated in cell differentiation, disease control, basic metabolism and neuronal survival. Recent studies also have shed light on specific functions of the APC/C during plant development. Plant APC/C subunits and activators have been reported to play a role in cellular differentiation, vascular development, shoot branching, female and male gametophyte development and embryogenesis. Here, we discuss our current understanding of the APC/C controlling plant growth.