盘基网柄菌突变型细胞allC的细胞周期异常

研究盘基网柄菌(Dictyostelium discoideum)细胞周期的相关问题可以为真核生物细胞周期调控研究提供理论基础。细胞计数和细胞倍增时间计算的结果表明,突变allC细胞的倍增时间为2.36 h,仅为KAx-3细胞倍增时间的1/3。进一步利用流式细胞术测定两种细胞的细胞周期,并结合实时荧光定量PCR技术测定cycB1和cdk1基因的相对表达量的比值,我们发现,培养16 h的allC细胞处于G2期的数目(1.51%)显著少于KAx-3细胞(16.61%)(P0.05)。allC细胞和KAx-3细胞的细胞周期素B1(cyclinB1,cycB1)基因相对表达量分别是2.5和0.24(P0.05),两者相差10倍。两种类型细胞中处于G2期的细胞数目差异十分明显,cycB1的相对表达量也存在显著差异,表明cycB1的过表达可能在一定程度上影响allC细胞的细胞周期正常的调控机制,与突变细胞的G2期异常有一定关系,但具体机制仍需进一步探究。     

人类T型钙通道α1H亚单位基因在细胞增殖中的功能研究

将人类T型钙通道α1H 亚单位基因 (CACNA1H)cDNA转染到HEK 2 93(humanembroyonickidney)细胞系得到稳定过量表达的细胞株 ,以此为体外模型来研究T型钙通道在细胞增殖中的直接作用。RT -PCR和标准全细胞膜片钳记录分别从mRNA转录水平和T型钙通道蛋白功能水平验证了α1H 亚单位的过量表达。生长曲线结果表明 ,T型钙通道α1H 亚单位基因的过量表达能显著促进HEK - 2 93细胞增殖 ,将细胞群体倍增时间从对照细胞的 2 2 1± 1.1h缩短到稳定转染细胞的 14 0± 0 .4h ,细胞群体倍增时间缩短了约 8h ;流式细胞分析结果表明 ,在稳定转染细胞处于S期的细胞百分率比对照细胞高 ,相反地处于G1 期的百分率比对照细胞低 ,以上结果证明了过量表达T型钙通道亚单位α1H 基因能促进细胞增殖。Western印迹结果提示 ,T型钙通道α1H 亚单位基因表达产物是通过某种信号途径 ,提高了与细胞周期有关基因 (CDK2、cyclinA和cyclinE)的蛋白质表达水平 ,从而刺激了细胞周期的进程。此研究结果有助于理解细胞增殖的机制并为开发治疗与细胞增殖异常有关疾病的新药提供了理论依据     

PC-1基因在前列腺癌细胞系LNCaP和C4-2不同细胞周期的表达

目的:检测PC-1基因在前列腺癌细胞周期中各时间点的表达变化。方法:用200 ng/mL诺可唑(nocoda-zole)处理前列腺癌细胞系LNCaP和C4-2,16 h后使细胞处于G2/M期,在不同时间点收获细胞,分别进行流式分析和Western印迹,检测PC-1基因的表达。结果:流式分析和Western印迹结果显示,在G2/M期,LNCaP和C4-2前列腺癌细胞系中PC-1基因高表达。结论:PC-1基因的表达与前列腺癌细胞的细胞周期有关,提示PC-1可能在细胞周期调控中发挥作用。     

NGX6基因对人结肠癌细胞HT-29细胞周期的影响

NGX6基因是新克隆的候选抑瘤基因,研究表明NGX6重表达可抑制结肠癌细胞的增殖.为进一步研究NGX6对细胞周期的影响,采用流式细胞仪检测NGX6重表达对结肠癌细胞HT-29细胞周期的影响,发现NGX6重表达可增加HT-29细胞在G0/G1期的分布比例,减少了S,G2,M期细胞数.利用蛋白质印迹和流式细胞术分析NGX6转染前后HT-29细胞周期素(cyclins)和细胞周期素依赖性蛋白激酶抑制物(cyclin-dependentkinaseinhibitor,CKI)的表达变化,发现NGX6可下调HT-29细胞中cyclinE、cyclinD1的表达及上调p27的表达,对cyclinA和cyclinB的表达无明显影响,p16在三组结肠癌细胞中均无表达.研究结果表明,NGX6在HT-29细胞中通过下调cyclinE、cyclinD1和上调p27的表达,阻滞细胞周期于G0/G1期,从而发挥其在结肠癌中的抑瘤作用.     

RNA干扰Chk1/2调控二烯丙基二硫诱导的G2/M期阻滞作用及相关周期蛋白表达

在细胞周期检测点信号传导通路中,Chkl和Chk2起着重要作用,主要参与G2/M期细胞周期检测点信号传导.首先采用RNAi技术在BGC823细胞中将Chk1、Chk2基因沉默,Chk1、Chk2 siRNA转染BGC823细胞后24h加入15mg/L二烯丙基二硫(diallyl disulfide,DADS),接着通过Real-timePCR、Western blot分析Chk1、Chk2基因在转染前后的表达差异,然后运用流式细胞术和Western blot分析Chk1、Chk2基因沉默后对DADS诱导的细胞周期G2/M期阻滞作用及相关周期蛋白CDC25C和cyclinB1表达的影响.实验结果表明,与未转染对照组相比,转染Chk1、Chk2siRNA后BGC823细胞中Chk1、Chk2表达明显被抑制,二者的mRNA表达分别下降84.7%和69.0%,蛋白质水平分别下降73.4%和78.5%.流式细胞术分析结果发现,ChklsiRNA转染的BGC823细胞在15mg/LDADS处理24h后,G2/M期比例由单纯DADS处理组的58.1%降至10.4%(P<0.05).但Chk2 siRNA转染后加入15mg/...     

层粘连蛋白对晚G1期人胃癌BGC-823细胞生长的促进作用

为研究层粘连蛋白（laminin,LN）促进肿瘤细胞生长作用,采用脉冲标记计数有丝分裂百分率(percentage labeling mitosis,PLM)法测得体外培养人胃癌 (BGC 823) 细胞周期时间为41 h,其中G1期时间为24.5 h. 分裂细胞脱离法获取分裂期细胞,继续培养23 h,在细胞运行进入G1晚期时,将其置于LN 0、0.11、0.55、1.10 μmol/L基质上孵育4 h; 细胞荧光光度计检测晚G1期细胞内Ca2+浓度、钙调蛋白、DNA含量. 结果显示,LN与其膜上受体结合后引起细胞内Ca2+浓度、钙调蛋白、DNA含量增加,尤以在0.55 μmol/L LN作用显著（P<0. 001）.蛋白质免疫印迹分析证明,cPKC α呈现表达,提示 LN与其受体结合可增强其细胞cPKC-α的活性;分析G1期细胞周期蛋白E(cyclinE)、细胞周期蛋白依赖性激酶CDK2表达水平,呈现逐渐增强的趋势; LN可诱导c-Myc蛋白呈现高表达,提示 LN与其受体结合增强与细胞增殖密切相关的基因表达;在LN作用前后的BGC 823细胞均未检测到Bax蛋白表达.结果提示,在人胃癌 (BGC 823)细胞G1/S期交界处,层粘连蛋白与其膜上受体结合引起细胞内Ca2+浓度升高,诱导钙调蛋白的释放,其含量增加,增强蛋白激酶C的活化,导致细胞内DNA含量增加、G1/S期细胞周期蛋白与CDK表达增强、诱导原癌基因c-Myc呈持续表达状态,而凋亡基因Bax不表达.     

T型钙通道α1G亚单位在细胞增殖中的功能研究

利用稳定过表达人类T型钙通道α_(1G)亚单位的HEK-293细胞研究了T型钙通道在细胞增殖中的作用。RT-PCR和标准全细胞膜片钳记录分别从mRNA转录水平和T型钙通道蛋白功能水平验证了α_(1G)单位的过表达的实现。生长曲线表明,T型钙通道α_(1G)亚单位的过表达能显著促进细胞增殖,HEKα_(1G)~+细胞的细胞群体倍增时间(13.7±0.3h)比对照HEK-293细胞的细胞群体倍增时间(22.1±1.1h)缩短了约8h;流式细胞分析结果也与此吻合,在稳定过表达了人类T型钙通道α_(1G)亚单位的细胞处于S期的细胞百分率比对照HEK-293细胞高,相反地处于G_0/G_1期的百分率比对照HEK-293细胞低,以上结果证明过表达T型钙通道α_(1G)亚单位能促进细胞增殖;T型钙通道特异性阻断剂mibefradil抑制HEKα_(1G)~+细胞增殖,IC_(50)为3.5/μmol/L,表明在HEK-293细胞过表达T型钙通道对细胞增殖的促进作用可以被T型钙通道特异性阻断剂mibefradil抑制,这就进一步证明了T型钙通道在促进细胞增殖方面的直接作用。Western杂交结果提示了T型钙通道α_(1G)亚单位的表达是通过某种信号途径提高了与细胞周期有关的蛋白质,cyclin A、cyclin E和CDK2的表达水平,从而刺激了细胞周期的进程。本研究有助于理解细胞增殖的机制并为开发治疗与细胞增殖异常有关疾病的新药提供了理论依据。     

癌基因D52家族新基因PC-1在前列腺癌细胞系LNCaP和C4-2B的G2／M期高表达

目的：检测癌基因D52家族新基因船一,在细胞周期各时相的表达变化。方法：用1mmol／L羟基脲处理前列腺癌细胞系LNCaP和C4-2B 40h,使细胞同步化于G1／S期,在药物撤除后0-16h不同时间点收获细胞,分别进行流式分析和Westernblot检测。结果：流式分析和Westernblot检测在LNCaP和C4-2B细胞系中得到了趋势一致的结果,印PC-1基因在GVM期高表达。结论：PC-1基因的表达与前列腺癌细胞的细胞周期有关,表明PC-1蛋白可能在G2／M期发挥作用。     

塞来昔布诱导HCT-116结肠癌细胞G2/M阻滞

目的:研究选择性COX-2抑制剂塞来昔布诱导结肠癌细胞株HCT-116细胞周期阻滞的作用及其可能的机制。方法:应用流式细胞仪检测塞来昔布对HCT-116细胞周期的影响,定量PCR检测细胞周期素cyclinB1及COX-2 mRNA表达水平,Western-Blot检测细胞周期素cyclinB1的蛋白水平。结果:塞来昔布诱导HCT-116细胞G2/M阻滞的作用呈剂量依赖性,塞来昔布在mRNA及蛋白水平下调HCT-116细胞的cyclinB1。结论:塞来昔布能在体外抑制HCT-116细胞的增殖,诱导G2/M的阻滞,其作用与下调细胞周期素cyclinB1有关。     

植物激素处理和环境胁迫对‘金魁’猕猴桃AdRAVs基因表达特性的影响

以‘金魁’猕猴桃(Actinidia deliciosa‘Jinkui’)组培苗及2年生扦插苗为实验材料,采用qRT-PCR技术对0.1 mmol·L-1水杨酸(SA)、0.05 mmol·L-1茉莉酸甲酯(MeJA)、0.01 mmol·L-11-氨基环丙烷-1-羧酸(ACC)和0.01 mmol·L-1脱落酸(ABA)4种植物激素处理后0、4、12和48 h,低温(4℃)和0.2 mol·L-1 NaCl胁迫0、4、12和48 h,高温(48℃)胁迫0、2和4 h及恢复培养6 h,以及干旱胁迫14 d后叶片中AdRAV1、AdRAV2和AdRAV3基因的相对表达量进行了测定。结果显示：不同处理条件下3个AdRAVs基因相对表达量的变化存在一定差异。 SA、MeJA和ACC处理后4和12 h,AdRAV1基因的相对表达量显著(P<0.05)升高,但ABA处理后该基因的相对表达量无明显变化;SA、MeJA、ACC和ABA处理后48 h,AdRAV2基因的相对表达量显著降低;4种植物激素处理后4、12和48 h,AdRAV3基因的相对表达量总体上显著降低。低温胁迫下,AdRAV1和AdRAV2基因的相对表达量无明显变化,但胁迫48 h时AdRAV3基因的相对表达量却显著升高。 NaCl胁迫12 h时,AdRAV1和AdRAV2基因的相对表达量均显著升高,而AdRAV3基因的相对表达量则显著降低。高温胁迫4 h时, AdRAV1基因的相对表达量显著降低, AdRAV2基因的相对表达量显著升高;胁迫2 h时,AdRAV3基因的相对表达量显著降低;恢复培养6 h时,3个基因的相对表达量均无法恢复至起始水平。干旱胁迫14 d后,AdRAV1基因的相对表达量显著高于对照(正常浇水);AdRAV2的相对表达量高于对照,而AdRAV3基因的相对表达量则低于对照,且均与对照无显著差异。研究结果表明：不同胁迫条件对‘金魁’猕猴桃AdRAVs基因的表达特性有不同诱导效应。根据实验结果,推测AdRAV1、AdRAV2和AdRAV3基因可能参与SA、MeJA、ACC和ABA信号转导途径以及耐盐和耐高温过程;并且,AdRAV1基因还可能参与耐旱过程,而AdRAV3基因则可能参与耐寒过程。     

Shortening of the cell cycle and developmental interruption in a Dictyostelium discoideum cell line due to RNAi-silenced expression of allantoicase

The signaling molecules NH(3) (unprotonated volatile ammonia), as well as cyclic adenosine monophosphate and differentiation-inducing factor, play important roles in the multicellular development of the slime mould Dictyostelium discoideum. One of the downstream metabolic products catalyzed by allantoicase (allC) is ammonia. We observed the role of allC by RNAi-mediated manipulation of its expression. The allC gene of D. discoideum was silenced by RNAi. We found significant downregulation of allC mRNA and protein expression levels. Recombinant allC RNAi mutant cell lines had a shortened cell cycle, a reduction in cell size relative to wild-type cells and interrupted development. We conclude that the normal functions of allC include retarding cell division until a specific cell size is reached and coordinating the progression of development.     

Characterisation and Gene Expression Profiling of a Stable Cell Line Expressing a Cell Cycle GFP Sensor

The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has increased significantly in recent years. In this study we have useda range of complimentary analytical techniques to examine the characteristicsof a cell line stably expressing a EGFP cell cycle sensor relative to parentalU2OS cells. Analysis of cell cycle duration and cell cycle phase distribution bycell growth assays and flow cytometry revealed that the two cell lines hadidentical doubling times and cell cycle distributions. Measurement of EGFPfusion protein mRNA by quantitative RT-PCR indicated a EGFP sensorexpression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2).Microarray analysis showed a 0.9% (>2 fold at p     

LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression

Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Regulation of the Cell Cycle by Focal Adhesion Kinase

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant ΔC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in ΔC14. Biochemical analyses indicated that FAK mutant ΔC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKΔC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that ΔC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.