RASSF1A基因对肝癌细胞化疗药物敏感性的影响

目的探讨RASSF1A基因的表达对肝细胞肝癌化疗药物敏感性的影响。方法利用已构建成功的稳定表达野生型和突变型RASSF1A基因的肝癌细胞株QGY-7703,化疗药物Mitomycin、Adriamycin、Etoposide、5-Fluorouracilur、Cisplatin分别作用各细胞株后,比较生长抑制率、细胞周期及P53、P21、Bax、Caspase-3蛋白的表达水平。结果野生型RASSF1A的表达可提高Mitomycin诱导的肝癌细胞生长抑制率、凋亡发生率（P<0.05）和Caspase-3的活性,对P53、P21、Bax基因的蛋白表达水平没有影响。结论野生型RASSF1A基因可提高肝癌细胞对Mitomycin的敏感性。     

外源性SHIP基因对K562细胞周期及其相关蛋白表达的影响

目的:探讨SHIP基因对人白血病细胞系K562细胞周期的调控作用.方法:用携带野生型SHIP基因及绿色荧光蛋白 (green fluorescent protein,GFP)基因的慢病毒及空载体慢病毒转染人白血病K562细胞.FCM检测转染效率、细胞凋亡率及细胞周期变化,实时荧光定量PCR(real-time fluorescent quantitative PCR,RFQ-PCR)检测SHIP mRNA水平的变化,Western 印迹法检测SHIP、cyclin D1、p21WAF1/CIPI、p27KIP1蛋白表达水平的变化.结果:与转染空载体组和未转染组相比,转染野生型SHIP基因的K562细胞增殖减慢,凋亡率明显上升(P＜0.05);细胞周期显示,G0/G1期延长,G1期前出现亚二倍体的凋亡峰,S期和G2/M期比例降低;cyclin D1表达降低,p21WAF1/CIPI和p27KIP1表达增加.结论:转染野生型SHIP基因可使细胞周期阻滞在G0/G1期,抑制白血病细胞增殖,促进白血病细胞凋亡.     

p53基因对人胚肺成纤维细胞周期影响的研究

背景与目的:研究野生型和179位残基突变型p53基因在HELF细胞周期调控中的作用.探讨p53基因的179位残基突变对细胞生长的影响.材料与方法:用野生型p53(pcDNA3-wtp53)和179位残基突变的突变型p53(pcDNA3-mtp53)转染HELF细胞,观察细胞生长情况,绘制细胞生长曲线;用流式细胞仪分析细胞周期;用RT-PCR和Western blotting方法检测p53基因转染后HELF细胞周期相关基因mRNA和蛋白的表达.结果:野生型p53表达的上调使HELF细胞周期阻滞于G1期,细胞体积减小,并下调cyclin D3、cyclin E、Cdk2和Cdk4的表达,同时上调p21的表达.而179位残基突变的突变型p53表达的上调则促进细胞周期从G1期到S期的转换,同时细胞体积增大,上调cyclin A和Cdk4的表达.结论:p53的179位残基突变对于HELF细胞cyclin A和Cdk4的表达有诱导作用,并可能借此促进细胞周期进程.     

贲门腺癌中RASSF1A基因甲基化与cyclin D1表达的关系

目的:探讨贲门腺癌(gastric cardiac adenocarcinoma,GCA)中RASSF1A基因的甲基化状态及其与cyclin D1蛋白表达之间的相关性.方法:分别应用甲基化特异性PCR(mothylation-specific PCR,MSP)方法和免疫组织化学SP法检测贲门腺癌组织及相应癌旁组织的RASSF1A基因甲基化情况和cyclin D1蛋白表达情况.结果:92例贲门腺癌组织中有54例RASSF1A发生了甲基化,甲基化率为58.7%,显著高于癌旁正常组织(P<0.001).Ⅲ期和Ⅳ期贲门腺癌患者中RASSF1A基因发生甲基化的比率显著高于Ⅰ期和Ⅱ期患者(P<0.05).92例贲门腺癌组织中有72例(78.3%)cyclin D1蛋白表达阳性,与相应癌旁正常组织比较,cyclin D1在贲门癌组织中的表达显著升高(P<0.001);且随肿瘤分化程度的降低,cyclin D1的阳性表达率明显升高(P<0.05).RASSF1A基因在贲门腺癌中的高甲基化与cyclin D1蛋白表达之间有明显的相关性(P<0.05).结论:RASSF1A基因启动子区的高甲基化以及cyclin D1蛋白的过表达可能参与了贲门腺癌的发生发展过程.     

5-氮杂-2′-脱氧胞苷对MCF-7乳腺癌细胞生物学行为影响的研究

目的:研究5-氮杂2′-脱氧胞苷(5-Aza-CdR)对MCF-7乳腺癌细胞株生长周期及凋亡的影响,探讨其临床治疗乳腺癌的可能性.方法:分别使用浓度为0.4、1.6、6.4、25.6和102.4 μmol/L的甲基化抑制剂5-Aza-CdR处理MCF-7乳腺癌细胞株.通过MTT检测5-Aza-CdR对MCF-7乳腺癌细胞株存活率的影响.应用流式细胞仪检测5-Aza-CdR对MCF-7乳腺癌细胞株生长周期及凋亡的影响.RT-PCR检测处理前后抑癌基因RASSF1A mRNA在MCF-7乳腺癌细胞株表达的变化.结果:5-Aza-CdR 6.4 μmol/L作用1 d或0.4 μmol/L作用3 d就可以显著抑制MCF-7乳腺癌细胞的增殖,P<0.05.作用3 d后,在6.4 μmol/L时细胞周期中处于G0/G1期的细胞的显著增加(P<0.05),细胞周期阻滞于G0/G1期;在0.4 μmol/L时细胞的凋亡率为(11.80±0.30)%,与对照组比较有显著升高,P<0.01.以上作用呈时间-剂量依赖性关系.5-Aza-CdR处理后,无RASSF1A表达的MCF-7乳腺癌细胞株可检测出基因RASSF1A的重新表达.结论:5-Aza-CdR可消除RASSF1A启动子甲基化状态,使其重新表达而抑制MCF-7乳腺癌细胞株的生长,使细胞周期阻滞于G0/G1期,并促进其凋亡.     

膜结合型前列腺素E2合酶 1抑制剂MK886对白血病HL-60／A细胞周期的影响

 【摘要】　目的　探讨膜结合型前列腺素E2合酶 1（mPGES-1）抑制剂MK886对人类急性髓系白血病耐药细胞HL-60／A细胞周期的作用。方法　采用流式细胞术、Western blot、酶联免疫吸附（ELISA）等方法,检测HL-60／A及健康人单个核细胞（MNC）、敏感细胞株HL-60的细胞周期、 mPGES-1蛋白、细胞周期素D1（cyclin D1）的差异,检测不同浓度的MK886对HL-60／A细胞周期的作用及对cyclin D1、mPGES-1、PGE2、P-Akt、c-myc蛋白的影响。结果　与健康人MNC及敏感细胞株HL-60比较,HL-60／A细胞cyclin D1、mPGES-1表达最高,G0～G1期细胞比例为（30.53±2.15）％,较正常MNC[（62.63±6.58）%]及HL-60细胞[（38.86±2.25）%]减少（均P＜0.05）;S期比例为（57.56±1.54）％,较正常MNC[（12.18±4.43）%]及HL-60细胞[（47.70±1.88）%]明显增多（均P＜0.05）。MK886作用后,被阻滞于G0～G1期的细胞增多,同时mPGES-1表达下调,合成PGE2减少,cyclin D1、P-Akt、c-myc蛋白表达下降。结论　MK886对白血病HL-60／A细胞有细胞周期阻滞作用,其机制与抑制mPGES-1表达,减少PGE2的合成,抑制cyclin D1、P-Akt、c-myc表达有关。     

白桦脂酸对Jurkat白血病细胞增殖和凋亡的影响

目的 观察白桦脂酸对人T淋巴细胞白血病Jurkat细胞株细胞增殖、凋亡和细胞周期的影响,并探讨其分子机制.方法 以不同浓度的白桦脂酸处理Jurkat细胞,采用二苯基溴化四氮唑蓝(MTY)法检测细胞的增殖活性,Hoechst 33258染色和Annexin-V/PI双标法检测细胞凋亡,流式细胞仪检测细胞周期分布,逆转录聚合酶链反应(RT-PCR)法检测Jurkat细胞中cyclin D3和bcl-xlmRNA含量的变化,Western blot法检测cyclin D3和bcl-xl蛋白的表达.结果 白桦脂酸对Jurkat细胞有明显的增殖抑制作用,并呈时间和浓度依赖性.白桦脂酸可诱导Jurkat细胞凋亡,Hoechst 33258染色可见典型的凋亡小体;Annxin-V/PI双标法显示,20、60和100 μmol/L白桦脂酸作用于Jurkat细胞24 h时,细胞的早期凋亡率分别为8.7%±0.3%、28.0%±1.3%和33.6%±2.0%.白桦脂酸可使Jurkat细胞的细胞周期阻滞于G0/G1期.白桦脂酸可使Jurkat细胞中凋亡相关基因cyclin D3和bcl-xl mRNA及蛋白的含量均明显减少,并呈浓度依赖性.结论 白桦脂酸可抑制Jurkat细胞增殖并诱导其凋亡,可使细胞阻滞于G0/G1期;其可能通过下调cyclin D3和bcl-xl mRNA及蛋白的表达而发挥抗肿瘤作用.     

转染外源CC10对肺腺癌细胞系A549的CyclinD1蛋白及mRNA的影响(英文)

目的研究转染外源性CC10基因对肺腺癌A549细胞株的细胞周期及周期蛋白CyclinD1蛋白和mRNA表达影响。方法用脂质体法分别将外源性抑癌基因CC10转染到人肺腺癌细胞株A549中,8h、16h、24h、36h、48h后,分别用Western blot法检测转染CC10基因的A549细胞CC10蛋白表达,用流式细胞仪观察细胞周期的变化,用免疫细胞化学及RT-PCR检测转染CC10基因对周期蛋白CyclinD1蛋白、mRNA的影响。结果转染外源CC10基因对A549细胞生长具有抑制作用,细胞生长阻止于G0／G1,细胞周期S期+G2／M期比例下降。转染外源 CC10基因的A549细胞CC10蛋白表达增高,而CyclinD1的蛋白及mRNA表达明显降低。结论转染外源CC10基因对肺癌细胞G0／G1周期的抑制作用可能与CyclinD1基因表达下调有关。     

稳定表达RFX6基因肝癌细胞株的建立

目的:建立稳定表达RFX6基因的肝癌细胞株。方法:构建人源RFX6基因的重组质粒(pLNCX2-RFX6),pVSV-G质粒分别与pLNCX2-RFX6和空载体pLNCX2共转染至GP2-293细胞中进行包装,并用包装的逆转录病毒感染人肝癌QGY-7703细胞株,G418筛选出阳性克隆。RT-PCR和蛋白质印迹法验证稳定细胞株RFX6基因表达情况。采用Image J软件进行灰度值分析比较两株细胞系的差异。结果:在QGY-7703-pLNCX2-RFX6和QGY-7703-pLNCX2细胞中,RFX6/18SmRNA电泳条带灰度比值分别为5.044 0±1.384 0和0.357 2±0.407 9,QGY-7703-pLNCX2-RFX6细胞RFX6/18S是QGY-7703-pLNCX2细胞的14.1倍,差异有统计学意义,t=4.143,P<0.05;而在两细胞株中,RFX6/GAPDH的蛋白质印迹条带灰度值分别为1.391 4±0.141 3和0.127 8±0.037 3,QGY-7703-pLNCX2-RFX6细胞RFX6/GAPDH是QGY-7703-pLNCX2细胞的10.9倍,差异有统计学意义,t=14.966,P<0.05。结论:成功构建稳定表达RFX6基因的肝癌细胞株,为研究RFX6基因在肝癌细胞中的功能奠定了基础。     

5-氮杂-2'-脱氧胞苷对肺癌细胞H460生物学行为及抑癌基因RASSF1A表达的影响

 目的　观察5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对肺癌细胞H460的生物学行为及RASSF1A mRNA表达的影响。方法　5-Aza-CdR处理H460细胞,通过MTT方法、平板克隆试验观察细胞生长活性的变化,PCR检测RASSF1A甲基化状态,Western blotting法检测RASSF1A的蛋白表达,流式细胞术进行细胞周期分析。结果　H460细胞经5-Aza-CdR处理后,与未处理组相比,生长速度出现不同程度减慢,克隆形成率明显降低,RASSF1A甲基化程度降低,延缓H460细胞周期G1／S进程,使细胞阻滞于G1期。结论　在肺癌细胞系中,RASSF1A基因甲基化可能导致其表达缺失,而5-Aza-CdR能够恢复RASSF1A基因的表达,为肺癌的去甲基化治疗提供理论依据。     

雌激素受体β亚型对人乳腺癌细胞株生物学特性的影响

目的探讨雌激素受体β亚型（ER8）对人乳腺癌细胞株生物学特性的影响和作用机制。方法构建ERβ稳定高表达的MDA—MB-435细胞株。运用噻唑蓝（MTT）法、流式细胞术和Tramwell等方法,观察不同雌激素浓度下ERβ对该细胞株增殖、侵袭和转移特性的影响。采用RRPCR和（或）Western blot、明胶酶谱技术检测相关基因的表达水平。结果ERβ可显著提高MDA-MB-435细胞的增殖速度和侵袭迁徙能力,且呈非雌激素依赖性。与对照细胞相比,ERβ高表达的细胞S期比例显著增多（P=0．01）;p21 mRNA表达水平降低33．3％（P=0．03）,蛋白表达水平降低47．4％（P=0．02）;基质金属蛋白酶（MMP）-9 mRNA表达水平增高91．3％（P〈0．01）,活性增高67．3％（P=0．02）;Ets-1 mRNA表达水平增高62．2％（P=0．01）,蛋白表达水平增高51．0％（P=0．01）;cyclin A、cyclin E、cyclin D1、MMP-1、MMP-2、MMP-7、血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）在mRNA水平未见明显差异。结论ERβ有促进乳腺癌细胞增殖、侵袭和转移的作用。降低p21的表达、增加Ets-1和MMP-9的表达并增强MMP-9的活性是其可能的作用机制之一。     

A plant oxylipin, 12-oxo-phytodienoic acid,inhibits proliferation of human breast cancer cells by targeting cyclin D1

Cyclin D1 overexpression has been associated with poor prognosis and resistance to therapy in human breast cancer. Thus, the development of therapeutic agents that selectively target cyclin D1 activity is of clinical interest. This study demonstrates that 12-oxo-phytodienoic acid (OPDA), a phytohormone with critical functions in growth and development in plants, induces growth arrest in MDA-MB-231 and T47D breast cancer cells. In response to OPDA treatment, the human breast cancer cell lines exhibit a progressive decline in cyclin D1 expression, which is tightly associated with the accumulation of hypophosphorylated form of the retinoblastoma protein (Rb) and G1 arrest. The decrease in cyclin D1 protein expression accompanies a dramatic decline in nuclear but not membranous beta-catenin expression and activation of glycogen synthase kinase-3-beta (GSK3beta) caused by inhibition of its serine-9 phosphorylation. The proteasome inhibitor MG132 blocks OPDA-mediated decrease in cyclin D1. In addition, the overexpression of T286A, a cyclin D1 mutant which is refractory to phosphorylation by GSK3beta and proteosomal degradation, is resistant to OPDA-mediated Rb dephosphorylation as well as G(1) cell cycle arrest. Thus, our results demonstrate that degradation of cyclin D1 protein is a key event in OPDA induced growth inhibition in breast cancer cells. These data provide the basic foundation for future efforts to develop OPDA-based approaches in the prevention and treatment of breast cancer and other types of cancer.     

大蒜素对人胃癌细胞BGC823 cyclin D1和p27Kip1表达的影响

背景与目的：在研究大蒜素抑制人胃癌细胞BGC823恶性增殖,将其阻滞于G1期并诱导BGC823细胞凋亡的分子机制及其靶基因过程中,我们发现cvclin D1和p27^Kip1。蛋白在G1期阻滞中起重要作用。本实验从mRNA和蛋白水平探讨大蒜素对人胃癌细胞BGC823 cvclin D1和p27^Kip1表达水平的影响。方法：用25μg／ml大蒜素处理BGC823细胞,并分别提取对照组和大蒜素处理组的总RNA和总蛋白,然后采用RT-PCR和Western blot法检测大蒜素处理前后cvclin D1和p27^Kip1 mRNA和蛋白的变化。结果：经25μg／ml大蒜素处理BGC823细胞24h,cyclin D1蛋白表达下调,p27^Kip1蛋白表达上调,处理48h,上述变化更明显。而mRNA水平未见明显改变。结论：大蒜素可影响cyclin D1和p27^Kip1蛋白的表达,而不影响mRNA的转录。     

Indomethacin induces differential expression of beta-catenin, gamma-catenin and T-cell factor target genes in human colorectal cancer cells.

Indomethacin-induced G(1) arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in beta-catenin protein levels. Beta-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. Beta-catenin, gamma-catenin and T-cell factor (TCF) target gene (cyclin D1, c-MYC and PPARdelta) expression was studied following indomethacin treatment of SW480 and HCT116 cells. Cyclin D1 was used as a model TCF target gene for analysis of beta-catenin-TCF-4 DNA binding and trans-activation. Indomethacin treatment was associated with a specific decrease in beta-catenin (but not gamma-catenin) expression. Resulting TCF target gene expression was gene specific (cyclin D1, decreased; c-MYC, increased; PPARdelta, no significant change). Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a beta-catenin-TCF-4-DNA complex. Indomethacin-induced G(1) arrest and apoptosis is associated with specific beta-catenin down-regulation in human CRC cells in vitro. Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.     

Biphasic effect of medroxyprogesterone-acetate (MPA) treatment on proliferation and cyclin D1 gene transcription in T47D breast cancer cells

While progesterone is a known differentiation-inducing factor in the human endometrium, for the breast epithelium both proliferation-inducing and -inhibiting effects have been described. Cyclin D1, which is required for cell cycle progression in G1 and has been shown to play an important role in the pathogenesis of breast cancer has been implicated as a possible mediator of such effects. In the present study we thus investigated the effects of the progestin agonist MPA (medroxy-progesterone acetate) on proliferation of T47D breast cancer cells. In parallel experiments, the regulation of the human cyclin D1 promoter as well as cyclin D1 protein levels under the influence of MPA were studied. Our results show an increase of proliferative activity in T47D cells after 24 and 48 h of MPA treatment followed by inhibition of proliferation after 72 h. In Western blot analysis an increased expression level of cyclin D1 protein can be observed after 24h of MPA stimulation, while at 72h the protein levels are barely detectable. Transient transfection experiments with a luciferase reporter plasmid containing the human cyclin D1 promoter showed an induction of the promoter after 24 and 36h of MPA treatment followed by a reduction in promoter activity. In conclusion, our results confirm the existence of a biphasic response of T47D cell proliferation in response to MPA treatment, consisting of stimulation of proliferation followed by inhibition, and further implicate cyclin D1 as a mediator of these effects, since the cyclin D1 promoter shows a similar biphasic response in this context.