芥子酸对高糖诱导下大鼠血管平滑肌细胞增殖和凋亡的影响

目的:探讨芥子酸对高糖诱导下大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和凋亡的影响及机制。方法:将培养的A7r5细胞随机分组处理,MTT法检测细胞活力,Brd U法检测细胞DNA合成,流式细胞术检测细胞周期进程和细胞凋亡,ELISA检测细胞活性氧簇(reactive oxygen species,ROS)生成,Western blot检测cyclin D1、P21和P27等蛋白的表达,以及蛋白激酶C(PKC)和P38的磷酸化水平。结果:与正常组比较,高糖组细胞活力显著升高,DNA合成加快,细胞周期加快,P21和P27表达降低,cyclin D1表达增加,ROS水平增加,细胞凋亡率降低,p-PKC和p-P38蛋白水平增加(P0.05)。而芥子酸(0.1、1和10μmol/L)处理引起细胞增殖活性降低,DNA合成减弱,细胞周期受阻,P21和P27表达增加,cyclin D1表达降低,ROS水平降低,细胞凋亡率升高,p-PKC和p-P38蛋白水平降低,且呈一定浓度依赖性(P0.05)。P38抑制剂SB203580和PKC抑制剂chelerythrine均显著抑制高糖诱导的PKC/P38活化和细胞活力(P0.05)。结论:芥子酸可通过抑制PKC/P38激活降低高糖诱导的VSMCs增殖,并促进细胞凋亡。     

肌肽对高糖诱导的心肌成纤维细胞增殖的影响

目的 研究肌肽(carnosine)对高糖环境下大鼠心脏成纤维细胞(cardiac flbroblasts,CFs)增殖的影响及机制.方法 以CFs为研究对象,使用Brdu-酶联免疫吸附试验(enzyme-linked immu-nosorbent assays,ELISA)检测法观察肌肽抑制高糖环境下心肌成纤维细胞DNA的合成;细胞周期的测定采用流式细胞仪法;Western blot法观察不同条件下CFs中CDK2、CyclinE、PCNA、ERK1/2、p-ERK1/2及p21和p27的蛋白表达情况.结果 肌肽能够抑制高糖环境下CFs的DNA合成,通过调节细胞周期和细胞周期的相关蛋白表达来实现这种作用.与高糖组比较,20 mmol/L肌肽组明显抑制了高糖刺激的大鼠CFs中CDK2、CyclinE、PCNA蛋白的表达增加(t CDK2=6.19、t CyclinE=5.7、t PCNA=8.56,P<0.05),差异具有统计学意义;与高糖组比较20 mmol/L肌肽则使p21、p27表达水平增加了(t p21=5.16、t p27=8.558,P<0.05),具有统计学意义;与高糖组比较20 mmol/L的肌肽或20 mol/L的PD-98059(ERK1/2抑制剂)明显抑制了高糖刺激的大CFs中p-ERK1/2的表达增加(t肌肽=5.64、t PD=8.68,P<0.05),差异具有统计学意义.结论 肌肽能够通过ERK1/2 MAPK信号通路抑制高糖环境下心肌成纤维细胞的DNA合成.     

p27~(Kip1)调控永生化人神经前体细胞分化的机制

目的研究p27Kip1在永生化人神经前体细胞分化中的作用,探讨神经前体细胞的分化机制。方法取本课题组已建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)进行培养,在细胞进入对数生长期时给予1μmol/L全反式视黄酸(RA)诱导,重复诱导3次,每次均在诱导的第3、5、7天收集细胞,用流式细胞仪分析RA诱导第3天细胞周期的变化,用免疫印迹法检测RA诱导第3、5、7天p27Kip1、p21cip1细胞周期蛋白依赖激酶2(cdk2)及S期激酶相关蛋白2(skp2)的变化。结果流式细胞术结果显示,hSN12W-TERT细胞经1μmol/LRA诱导3d后,G0/G1期细胞的比例由77.25%上升至85.68%,而S期的比例由9.38%下降到8.57%。免疫印迹法结果显示,RA诱导第3天,hSN12W-TERT细胞p27Kip1蛋白表达比未经RA诱导的细胞增加,并在RA诱导第5天达到高峰(P0.05)。未经RA诱导的正常hSN12W-TERT细胞p21Cip1蛋白表达弱,RA诱导后p21Cip1蛋白的表达略呈下降趋势。RA诱导前后hSN12W-TERT细胞cdk2蛋白的表达变化不明显,但反映cdk2活性的磷酸化cdk2(p-cdk2)的表达在RA诱导后明显下降,同时,参与p27Kip1泛素降解途径的重要因子skp2的表达在RA诱导后明显下降。结论在RA诱导hSN12W-TERT细胞分化的过程中,p27Kip1通过抑制cdk2的活性而发挥促细胞分化的作用,且RA诱导后p27Kip1蛋白含量增加与其泛素降解途径被抑制密切相关。     

人组织激肽释放酶基因重组腺病毒转染对血管平滑肌细胞增殖和迁移的影响

目的:探讨重组腺病毒介导的人组织激肽释放酶(hKLK1)基因转移对血小板源性生长因子-BB(PDGF-BB)诱导下的自发性高血压大鼠(SHR)血管平滑肌细胞(VSMCSHR)增殖和迁移的影响。方法:自行构建双顺反子重组腺病毒载体,携带强绿色荧光蛋白(EGFP)标志基因和目的基因hKLK1;用细胞计数法和四甲基偶氮唑盐(MTT)比色法检测细胞增殖,流式细胞仪检测细胞生长周期;蛋白免疫印迹法(Western blotting)测定细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1的表达。采用改良Boyden微孔膜双槽法测定VSMCSHR迁移。结果:(1)hKLK1基因转移呈感染复数依赖性(20-100MOI)抑制PDGF-BB诱导的VSMCSHR生长,100MOI时抑制率为39.3%;呈时间依赖性抑制VSMCSHR生长,第5d时达高峰,抑制率为35.2%。(2)hKLK1基因转移可显著抑制PDGF-BB诱导的VSMCSHR增殖,峰值抑制率为30.2%(P0.01);细胞周期阻滞于G0/G1期的VSMCSHR明显增多,最大阻滞率为36.4%(P0.01),而缓激肽B2受体特异性阻断剂Hoe140逆转了hKLK的抑制作用。(3)hKLK1基因转移明显上调PDGF-BB诱导VSMCSHR的p27Kip1、p21Cip1表达,Hoe140明显降低p27Kip1、p21Cip1表达。(4)hKLK1基因转移可明显抑制PDGF-BB诱导的VSMCSHR细胞迁移,抑制率为34.6%,且Hoe140不影响该抑制作用。结论:hKLK1基因转移可抑制PDGF-BB诱导的VSMCSHR增殖,主要由缓激肽B2受体介导的,通过上调细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1表达的途径。而hKLK1基因转移抑制VSMCSHR迁移效应可能不通过B2受体。     

p27~(Kip1)在永生化人神经前体细胞分化中的作用研究

目的:利用全反式视黄酸(all-trans retinoic acid,RA)诱导永生化的人神经前体细胞(immortalized human neural progenitor cells,hSN12W-TERTcells)分化,研究分化过程中细胞周期调节蛋白p27Kip1,p21Cip1,细胞周期蛋白激酶2(cyclin-dependent kinase2,cdk2)及细胞周期蛋白E(cyclinE)的变化,探讨永生化人神经前体细胞分化的相关分子机制。方法:取本课题组已经建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)培养并给予1μmol/L RA诱导。在RA诱导的第3,7d观察细胞形态变化,用RT-PCR方法检测RA诱导前后hSN12W-TERT细胞p27Kip1,p21Cip1,cdk2及cyclinE mRNA的变化,用免疫细胞化学染色方法比较RA诱导前后p27Kip1蛋白表达的变化。结果:RA诱导第3d,hSN12W-TERT细胞比未经RA诱导的正常hSN12W-TERT细胞生长缓慢且形态发生改变,表现为胞体变小,突起延长增多。至RA诱导第7d时,hSN12W-TERT细胞形态变化更加明显,接近成熟神经元形态。RT-PCR结果表明,hSN12W-TERT细胞中p27Kip1mRNA的表达在RA诱导后明显增加,而p21Cip1mRNA的表达在RA诱导后略呈下降趋势。hSN12W-TERT细胞中cdk2、cyclinE的mRNA水平在RA诱导前后没有明显变化。免疫细胞化学染色结果显示,RA诱导第3d,hSN12W-TERT细胞p27Kip1的表达比未经RA诱导的正常hSN12W-TERT细胞明显增加(P0.05),RA诱导第7d,p27Kip1的表达进一步增加(P0.05)。结论:p27Kip1参与RA诱导的永生化人神经前体细胞生长阻滞和分化过程,p27Kip1可能在永生化人神经前体细胞的神经元分化过程中发挥重要作用;且RA诱导后p27Kip1蛋白含量的增加是通过转录水平调节的。     

碱性成纤维细胞生长因子对人肝癌Bel-7402细胞Skp2和p27^Kip表达的影响

目的:研究碱性成纤维细胞生长因子(bFGF)对肝癌Bel-7402细胞Skp2、p27Kip表达的影响,探讨bFGF调控肝癌细胞增殖的信号转导机制.方法:分为对照组、bFGF组、bFGF+Wortmannin组,MTT法分析细胞增殖程度;流式细胞术分析细胞周期;RT-PCR法检测Skp2的mRNA表达;免疫印迹法检测Skp2、p27Kip蛋白的表达.结果:bFGF组细胞增殖比显著增加;bFGF可诱导细胞由G1期进入S期,而且上调Skp2的mRNA和蛋白表达、下调p27Kip蛋白表达;PI3K的抑制剂Wortmannin可部分抑制bFGF的上述作用.结论:bFGF可能通过激活PI3K/PKB途径调节Skp2、p27Kip表达,促进肝癌Bel-7402细胞增殖.     

Probucol抑制氧化低密度脂蛋白诱导的大鼠主动脉平滑肌细胞增殖

目的:研究Probucol抑制ox-LDL诱导RASMCs增殖与信号蛋白分子ERK1/2、MKP-1、HO-1和Trx-1表达之间的关系。方法:采用MTT、流式细胞术和Western blotting观察ox-LDL刺激条件下probucol对细胞周期、细胞增殖和凋亡、ERK1/2、MKP-1、HO-1和Trx-1表达的影响。结果:(1)Probucol抑制ox-LDL刺激RASMCs增殖:100μmol/Lprobucol+35mg/Lox-LDL组与35mg/Lox-LDL组比较,A值下降了34.9%(P0.01);(2)Probucol通过使RASMCs停滞在G0/G1期和诱导细胞凋亡2种方式抑制ox-LDL刺激细胞增殖。(3)ox-LDL显著抑制MKP-1的蛋白表达,与对照组比较下降了60.0%(P0.01),同时使p-ERK1/2表达增加了34.7%;Probucol使MKP-1蛋白表达显著增加2倍,p-ERK1/2表达降低了15.7%(P0.01);(4)35mg/Lox-LDL使细胞内Trx-1蛋白表达下降28.9%(P0.05),HO-1蛋白表达轻度增加(P0.05)。与ox-LDL组比较,probucol使Trx-1蛋白表达增加了91.6%(P0.01),HO-1表达增加31.9%(P0.01)。结论:Probucol通过增强MKP-1和HO-1蛋白表达、抑制细胞周期运转和诱导细胞凋亡的机制抑制RASMCs增殖。     

p27Kip1反义寡核苷酸对B淋巴瘤细胞WEHI-231细胞周期的影响

目的　探讨p2 7Kip1在抗原受体介导的B淋巴瘤细胞WEHI- 2 31细胞周期停止信号中的作用。方法　用抗IgM抗体诱导WEHI- 2 31细胞细胞周期停止。用合成的p2 7Kip1反义寡核苷酸抑制p2 7Kip1基因的表达。采用流式细胞仪 ,分析细胞核的DNA含量和细胞周期的变化。用体外激酶实验检测Cdk2的活性 ,Westernblot检测Rb蛋白的磷酸化水平。结果　合成的p2 7Kip1反义寡核苷酸能阻断抗IgM抗体诱导的p2 7Kip1基因表达的上调。用p2 7Kip1反义寡核苷酸处理WEHI-2 31细胞 ,可恢复抗原受体交联引起的周期素依赖性激酶Cdk2活性的降低 ,以及Rb蛋白磷酸化水平的下降 ,并使细胞周期恢复运转。结论　p2 7Kip1可能在抗原受体信号介导的WEHI-2 31细胞的细胞周期停止中发挥主要作用     

MicroRNA-140-5p通过蛋白激酶C亚型ε促进肾癌细胞增殖（英文）

目的探讨microRNA-140-5p(miR-140-5p)在肾癌发生中的作用。方法 Real-time PCR方法检测肾癌细胞(Caki-1、ACHN、OS-RC-2和786-O)中miR-140-5p的表达。MTS,流式细胞术和Western blotting检测细胞增殖,细胞周期和相关蛋白的改变。结果降低Caki-1细胞中miR-140-5p的水平后,细胞增殖被抑制,G0/G1期细胞数量显着增加,cyclin D、cyclin E和CDK2、CDK4也随之减少;此外,增加786-O细胞中miR-140-5p的水平后,促进体外细胞增殖和体内肿瘤发生。基因芯片结果发现,蛋白激酶C亚型ε(PKCε)基因在改变Caki-1和786-O的细胞中显著性升高。si PKCε在降低肾癌细胞(RCC)中PKCε蛋白表达的同时,还能显著性反转抑制物/模拟物诱导的细胞增殖和细胞周期的改变。结论 miR-140-5p通过PKCε促进肾癌细胞增殖。     

细胞周期调控研究进展

细胞周期是细胞生命活动的基本过程 ,由细胞周期素 (Cyclin)依次激活相应的细胞周期蛋白依赖激酶 (CDK)所推动。Cyclin的周期性积累与分解对细胞周期进展起着正调控作用 ,而细胞周期蛋白依赖性激酶抑制因子 (CKI)通过在细胞周期适当的时间点上抑制 CDK的活性 ,从而对细胞周期进展起负调控作用。 Cyclin、CDK、CKI多因素相互协同作用 ,通过 p Rb途径和 p5 3途径共同影响细胞周期进程。对细胞周期调控的研究有助于阐明肿瘤的发生以及终末细胞的分化 ,将为治疗肿瘤和诱导终末分化细胞的增殖提供有益的思路     

High glucose-induced hypertrophy of mesangial cells requires p27(Kip1), an inhibitor of cyclin-dependent kinases

Hypertrophy of mesangial cells is one of the earliest morphological alterations in the kidney after the onset of diabetes mellitus. We have previously shown that cultured mesangial cells exposed to high ambient glucose arrest in the G1 phase of the cell cycle and that this is associated with an increased expression of inhibitors of the cyclin-dependent kinase (CDK)-inhibitors p21(Cip) and p27(Kip1). To further investigate a potential role of p27Kip1 in the development of glucose-induced hypertrophy, mesangial cells from p27Kip1 wild-type (+/+) and knockout (-/-) mice were established. High glucose medium (450 mg/dl) increased p21(Cip1) protein in p27Kip1+/+ and -/- mesangial cells, and increased p27Kip1 protein levels in p27Kip1+/+ cells. In contrast to high glucose increasing de novo protein synthesis in p27Kip1+/+ cells, high glucose did not increase protein synthesis in p27Kip1-/- cells. High glucose also reduced DNA synthesis and caused cell cycle arrest in p27Kip1+/+ cells. In contrast, despite an increase in transforming growth factor (TGF)-beta mRNA and protein expression, DNA synthesis and cell cycle progression were increased by high glucose in p27Kip1-/- cells. Exogenous TGF-beta comparably induced fibronectin mRNA in p27Kip1+/+ and -/- cells suggesting intact TGF-beta receptor transduction. In addition, high glucose failed to increase the total protein/cell number ratio in p27Kip1-/- cells. However, in the presence of high glucose, reconstituting p27Kip1 expression by transient or stable transfection in p27Kip1-/- cells, using an inducible expression system, increased the de novo protein synthesis and restored G1-phase arrest. These results show that p27Kip1 is required for glucose-induced mesangial cell hypertrophy and cell cycle arrest.     

Role of p27Kip1 protein in the cell cycle and its appearance in lymphoid tissues, particularly non-Hodgkin's B-cell lymphomas. Review

Cell cycle progression is governed by cyclin dependent kinases (CDK) that are activated by cyclin binding and inhibited by CDK inhibitors. Protein p27Kip1 functions as a CDK inhibitor, which controls the progression from G1 to S phase. Further, p27Kip1 may have a positive regulative influence. In nonneoplastic tissues and in the majority of tumors investigated so far, the immunohistochemical positivity of p27Kip1 showed an inversely proportional relationship to the proliferation index. Among B-cell non-Hodgkin lymphomas, the exceptions to this rule are represented by mantle cell lymphoma, hairy cell leukemia, and the immunoblastic Epstein-Barr virus latent membrane antigen positive diffuse large B-cell lymphoma in AIDS patients. The loss of p27Kip1 expression is a negative prognostic factor in numerous tumors, including the majority of B-cell lymphomas.     

细胞周期素依赖性激酶2及p27Kipl与血管瘤

目的:进一步探讨细胞周期素依赖性激酶2(CDK2)及p27Kipl.在血管瘤发生、发展及退化过程中的作用机制.方法:采用免疫组织化学SP法检测49例皮肤毛细血管瘤增生期、退化期及正常皮肤组织中CDK2和p27Kipl的表达水平;采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对CDK2和p27Kipl表达的平均光密度和阳性面积率进行图像分析.结果:增生期组CDK2的表达明显高于退化期组和正常皮肤组织组;增生期血管瘤内皮细胞p27Kipl的表达显著低于退化期血管瘤内皮细胞.结论:p27Kipl能抑制血管瘤内皮细胞的增殖,在血管瘤的退化过程中起了重要作用;而CDK2能促进血管瘤内皮细胞增殖,存血管瘤的增生过程巾起了重要作用.     

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development

The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.     

High-glucose-induced prostaglandin E(2) and peroxisome proliferator-activated receptor delta promote mouse embryonic stem cell proliferation

Peroxisome proliferator-activated receptor is a nuclear receptor that has been implicated in blastocyst implantation, cell cycle, and pathogenesis of diabetes. However, the signal cascades underlying this effect are largely unknown in embryo stem cells. This study examined whether or not there is an association between the reactive oxygen species-mediated prostaglandin E(2) (PGE(2))/peroxisome proliferator-activated receptor (PPAR) delta and the growth response to high glucose levels in mouse ESCs. A high concentration of glucose (25 mM) significantly increased the level of [3H]thymidine incorporation, the level of 5-bromo-2'-deoxyuridine incorporation, and the number of cells. Moreover, 25 mM glucose increased the intracellular reactive oxygen species, phosphorylation of the cytosolic phospholipase A(2) (cPLA(2)), and the release of [3H]arachidonic acid ([3H]AA). In addition, 25 mM glucose also increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of PGE(2). Subsequently, high glucose-induced PGE(2) stimulated PPARdelta expression directly or through Akt phosphorylation indirectly through the E type prostaglandin receptor receptors. The PPARdelta antagonist inhibited the 25 mM glucose-induced DNA synthesis. Moreover, transfection with a pool of PPARdelta-specific small interfering RNA inhibited the 25 mM glucose-induced DNA synthesis and G1/S phase progression. Twenty-five millimolar glucose also increased the level of the cell cycle regulatory proteins (cyclin E/cyclin-dependent kinase [CDK] 2 and cyclin D1/CDK 4) and decreased p21(WAF1/Cip1) and p27(Kip1), which were blocked by the inhibition of the cPLA(2), COX-2, or PPARdelta pathways. In conclusion, high glucose promotes mouse ESC growth in part through the cPLA(2)-mediated PGE(2) synthesis and in part through PPARdelta pathways.     

Inhibition of cell cycle progression via p27Kip1 upregulation and apoptosis induction by an ethanol extract of Rhus verniciflua Stokes in AGS gastric cancer cells

Botanical preparations are widely used by patient with cancer in Korea, Japan and China. Rhus verniciflua Stokes (RVS) has traditionally been used as a medicinal ingredient for the therapy of stomach and uterine cancer. In this study, we showed that exposure to an ethanol extract of RVS (50 microg/ml) resulted in a synergistic inhibitory effect on cell growth in AGS cells. Growth inhibition was related with the inhibition of proliferation and induction of apoptosis. The extract induces G1-cell cycle arrest through the regulation of cyclins, the induction of p27Kip1, and decrease the CDK2 kinase activity. The upregulated p27Kip1 level is caused by protein stability increment by the reduction of Skp2, a key molecule related with p27Kip1 ubiquitination and degradation, and de novo protein synthesis. RVS extract induces apoptosis through the expression of Bax, poly(ADP-ribose) polymerase (PARP) and activation of caspase-3. RVS extract induces G1-cell cycle arrest via accumulation of p27Kip1 controlled by Skp2 reduction and apoptosis passing through an intrinsic pathway in human gastric cancer cells but not in normal cells, therefore we suggest that this extract could be a candidate medicine or compound for the development of novel class of anti-cancer drugs.     

Modulation of liver X receptor signaling as a prevention and therapy for colon cancer

Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor family and are important regulators of cholesterol, fatty acid, and glucose homeostasis. LXR agonists are effective for treatment of murine models of atherosclerosis, diabetes, and Alzheimer's disease. Recently we and other groups observed that LXR agonists suppressed proliferation of multiple human cancer cell lines in vitro as well as suppressed the growth and progression of prostate tumor xenografts in nude mice. LXR agonists appear to cause G1 cell cycle arrest in cancer cells by reducing the protein expression level of Skp2, cyclin A2, cyclin D1, and the phosphorylation of Rb, while increasing the protein expression level of cell cycle inhibitor p27(Kip1) and p53. LXR agonist also suppressed the oncogenic activity of β-catenin, an important regulator in Wnt signaling, as well as the proliferation in human colon cancer cells. Phytosterols, the plant equivalent of mammalian cholesterol, have been shown to be agonists for LXRs. Intake of phytosterol-rich diets reduced the incidence of colon cancer. We therefore propose that activation of LXR signaling via treatment with LXR agonists or intake of phytosterols-rich diets can reduce the incidence and suppress the tumor growth of colon cancer.     

The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27

E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27^Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27^Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27^Kip1 expression at protein level.     

Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E-cadherin expression in gastric cancer

BACKGROUND: Gastric carcinoma is characterised by numerous genetic and epigenetic alterations that influence cell cycle progression, apoptosis and DNA repair. These alterations include down-regulation of the cyclin-dependent kinase (CDK) inhibitors p21(WAF1/CIP1) and p27(Kip1), and mutations of the tumour suppressor protein p53 and the cell adhesion molecule E-cadherin. Combined evaluation of the prognostic significance of these alterations has not been reported in Mexican Mestizo patients. AIMS: To evaluate p21(WAF1/CIP1), p27(Kip1), p53 and E-cadherin protein expression, including mutant E-cadherin variants with deletion of exon 8 (del 8) or 9 (del 9), in gastric cancer from Mexican patients. METHODS: Immunohistochemistry for the above-mentioned markers, including mutation-specific E-cadherin antibodies, was carried out in 69 gastric carcinomas; expression levels were correlated with histotype, tumour stage and prognosis. RESULTS: Expression of p21(WAF1/CIP1) alone or in combination with p27(Kip1) or in the absence of p53 was associated with favourable prognosis. Staining of del 8 and del 9 E-cadherin was found exclusively in patients negative for p53 and positive for p21(WAF1/CIP1), suggesting that the p21(WAF1/CIP1) regulatory function of p53 was intact. CONCLUSION: Combined evaluation of the prognostic significance of cell cycle regulators and E-cadherin should be performed. Even though patients negative for p53 and positive for p21(WAF1/CIP1) have a favourable prognosis, it may have a negative influence on prognosis if they acquire in addition E-cadherin mutations which have been shown previously to be associated with poor survival.