Effect of PKC pathway on G1/S progression control in HeLa cells

The effect of PKC activity on G1/S progression in HeLa cells has been studied.The result shows that (ⅰ) PKC activity alteration in G1 phase affects G1/S progression in HeLa cells.It has been observed that G1/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X.(ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G1 phase.(ⅲ) During G1/S progression,the expression of CyclinD1 is stimulated by TPA treatment and inhibited by GF-109203X treatment.There is no effect on the expression of CDK4.It is likely that PKC pathway regulates G1/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.     

PKA和PKC对HeLa细胞G2/M/G1进程的影响

为了研究ＰＫＡ和ＰＫＣ对ＨｅＬａ细胞Ｇ２→用Ｍ→Ｇ１期进程的影响,用ＰＫＡ和ＰＫＣ抑制剂分别处理同步的Ｇ２期和Ｍ期的ＨｅＬａ细胞后,测定了细胞有丝分裂指数和ＰＫＡ,ＰＫＣ与ＣＤＣ２激酶的活性。     

PKA和PKC对HeLa细胞G_2/M/G_1进程的影响

为了研究PKA和PKC对HeLa细胞G2 →M期和M→G1期进程的影响 ,用PKA和PKC抑制剂分别处理同步的G2 期和M期的HeLa细胞后 ,测定了细胞有丝分裂指数和PKA ,PKC与CDC2激酶的活性 .结果表明 ,PKAⅢ型抑制剂在促进HeLa细胞G2 /M /G1进程的同时 ,刺激了PKC的活性 ,反之 ,PKC的抑制剂GF 10 92 0 3X在阻抑HeLa细胞G2 /M /G1进程的同时 ,激活了PKA的活性 ,与其相关的CDC2激酶活性也发生了相应的变化 .实验表明 ,PKA和PKC分别负调和正调HeLa细胞G2 /M/G1进程 ,两者表现出拮抗关系 .     

Effect of P15INK4b expression on the cell cycle and G1 phase related regulatory genes in human melanoma cells

Using the transfeetion teehnique. P15INK4b was introduced into P15INk4b gene deleted human melanoma A375 cells,and a cell model MLED6 overexpressing P15INK4b WAS CONSTRUCTED.Comparing with the control cells MLC2,MLEK6cells in G1phase increased by 11%,but those in Sphase decreased by 15%by FCM.By the method of thymidine(TdR)and N2O arresting,the proportions of synchronized Mphase cells of MLEK6 ana MLC23 were measured and found to be 89.1% and 76.8%respectively ,and the cells in G1phase were 74.3% for MLID6 AND 76. 4% forMLC2.The result of3 H-TdR incorporation indicated that the transition of G1/Sof MLEK6 cell was delayed 2h as compared with that of MLC2 cells,and incorporation rate also decreased.The observation on exprissions of some G1/ S-resates relatory rigusating genes showed that in MLIK6 cells the protein leves of P27KIPI increased with the decreasing expressions of cyclinD1,cyclinE and c-myc,especially cyclinD1 in late G1phade.The expression of cyclinE obviously decreased at G1/S transition ,and c-myc wad inhibited throughout all the process of G1 S phase.All the risults suggest that P15INK4b can delayG1/S transition of MLEK6 cells by inhibiting the cell cycle engine ,and by increasing the expression of Cdk ingibitor P27KIPI in different stages of G1 phase.     

Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate （MMS）. The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS＋UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.     

含5-氟尿嘧啶稀土磷钨酸盐诱导细胞凋亡作用研究

 多金属氧酸盐作为一种无机金属-氧簇化合物,在抗肿瘤、抗病毒等药物化学领域引起广泛关注。研究了以下5种含5-氟尿嘧啶稀土磷钨酸盐K₉（C₄H₄FN₂O₂）₂La（PW₁₁O₃₉）₂·18H₂O,K₉（C₄H₄FN₂O₂）₂Ce（PW₁₁O₃₉）₂·23H₂O,K₉（C₄H₄FN₂O₂）₂Nd（PW₁₁O₃₉）₂·25H₂O,K₉（C₄H₄FN₂O₂）₂Sm（PW₁₁O₃₉）₂·11H₂O和K₉H（C₄H₄FN₂O₂）Eu（PW₁₁O₃₉）₂·11H₂O（FLnPW,Ln=La、Ce、Nd、Sm、Eu）对HeLa细胞凋亡和周期的影响,以5-氟尿嘧啶为阴性对照,同时比较了含5-氟尿嘧啶磷钨酸盐K11C₄H₄FN₂O₂（PW₁₁O₃₉）·7H₂O（FPW）及磷钨酸H₃PW₁₂O₄₀（PW）的生物活性。细胞形态检测表明,化合物作用于HeLa细胞后均出现明显凋亡形态特征,细胞核染色质呈高度浓缩和边缘化现象（PW除外）。流式细胞周期检测表明,化合物作用后HeLa细胞均出现S期阻滞,与5-氟尿嘧啶相比,FPW作用后S期阻滞增强,而FCePW、FNdPW和FEuPW组同时出现S期和G₂/M期阻滞。流式细胞检测表明,化合物诱导HeLa细胞发生凋亡（PW除外）,且诱导凋亡活性顺序为FLnPW > FPW > 5-氟尿嘧啶。Caspase 3检测表明,化合物作用后Caspase 3活性增强（PW除外）,活性顺序与凋亡活性顺序相同,其中FCePW组和FEuPW组Caspase 3相对活性显著增强。实验结果表明,所考查化合物（PW除外）能诱导细胞周期阻滞、诱导凋亡活性以及激活Caspase 3,且FLnPW的活性均高于FPW和5-氟尿嘧啶,而PW只能使肿瘤细胞发生坏死,说明5-氟尿嘧啶和稀土元素对化合物的抗肿瘤活性发挥关键作用,FLnPW可能是通过诱导细胞周期阻滞以及激活Cas-pase 3细胞凋亡通路,实现显著抑制HeLa细胞增殖。     

细胞外信号调节激酶1/2信号通路在小鼠T细胞增殖、周期和活化中的作用

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在小鼠T淋巴细胞增殖、周期和活化中的作用.方法:分离小鼠淋巴结细胞,藉多克隆刺激剂刀豆蛋白(ConA)或佛波醇酯(PDB)加离子霉素(Ion)刺激,流式细胞术分析ERK1/2信号通路的特异性阻断剂PD98059对小鼠T淋巴细胞增殖、周期和活化的影响.结果:活体染料羧基荧光素乙酰乙酸染色分析显示,不同浓度(5、10、20、30、40 μmol/L)的PD98059对ConA诱导的T淋巴细胞增殖具有明显的抑制作用,呈现剂量依赖关系(r=0.985,P＜0.01).碘化丙锭染色分析表明,PD98059可阻止ConA刺激的T淋巴细胞进入S期和G2/M期,PD98059对PDB Ion刺激的T淋巴细胞细胞周期的影响与ConA刺激相似,不同的是S期和G2/M期的变化较ConA作用更显著.荧光标记单克隆抗体染色显示,不同浓度的PD98059仅能轻微影响T淋巴细胞表面活化标志CD69和CD25的表达.结论:PD98059对小鼠T淋巴细胞的增殖有明显抑制作用,并阻止其进入S期和G2/M期,但不能明显抑制小鼠T淋巴细胞的早期和中期活化.     

microRNA-16 expression in HeLa cells is upregulated in cell-cycle and drug dependent manner

microRNAs are single-stranded, non-coding RNAs that regulate gene expression. The microRNA-16 family has been reported to be involved in cell-cycle regulation, which could also downregulate expression of multiple pro-proliferation genes. The present results demonstrated that miR-16 expression in HeLa cells increased when the cells were arrested during S-phase under methyl methanesulfate (MMS) treatment. This further resulted in downregulation of a target protein CDC25A, whereas miR-16 expression did not increase when HeLa cells were arrested during the MMS-treated G₀/G₁ or G₂/M phase. Furthermore, when HeLa cells were arrested during S-phase with hydroxyurea treatment, miR-16 expression did not increase. These results suggest that expression levels of microRNAs in mammalian cells are delicately regulated under variable cellular conditions.     

水飞蓟宾通过上调P15~(INK4B)和P21~(WAF1/CIP1)活化JNK诱导人胰腺癌细胞G1期阻滞及细胞凋亡

目的:探讨水飞蓟宾对胰腺癌As PC-1细胞的增殖抑制作用及其作用机制.方法:MTT法和克隆形成抑制实验观察水飞蓟宾对人胰腺癌As PC-1细胞的增殖抑制作用,碘化丙锭(PI)单染色检测细胞周期改变,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平,Western blotting检测细胞周期及细胞凋亡相关蛋白的表达.结果:不同浓度的水飞蓟宾对胰腺癌As PC-1细胞的生长均有抑制作用,且呈剂量-效应和时间-效应关系(P0.05),水飞蓟宾作用于As PC-1细胞48、72 h的IC50浓度分别为224.20、87.25μmol/L;克隆形成抑制实验显示,随着水飞蓟宾浓度增加,As PC-1细胞克隆形成逐渐减少.细胞周期检测结果显示,随着水飞蓟宾浓度的增加,胰腺癌As PC-1细胞出现明显G1期阻滞;水飞蓟宾处理组细胞的周期蛋白Cyclin D1、Cyclin E2、Cyclin A、Cyclin B1表达下降,细胞周期蛋白激酶CDK4、CDK6表达不变,细胞周期素依赖性蛋白激酶抑制蛋白P15INK4B、P21WAF1/CIP1表达升高,与流式检测的结果相一致.不同浓度水飞蓟宾作用48 h后,出现明显的凋亡细胞群;同时发现Caspase-9、Caspase-3活化降解,Caspase3下游效应蛋白PARP出现切割条带.JNK蛋白表达增加并磷酸化活化,Bcl-2蛋白家族中抗凋亡蛋白Bcl-2、Bcl-x L、Mcl-1表达明显降低,促凋亡蛋白Bax表达基本不变,BH3-only蛋白Bclxs、Bid、Bim表达增加.结论:水飞蓟宾明显抑制胰腺癌细胞增殖,通过诱导P15INK4B、P21WAF1/CIP1表达阻滞细胞周期在G1期,并通过诱导JNK活化激活线粒体细胞凋亡途径,进而诱导胰腺癌As PC-1细胞凋亡.     

硒化麒麟菜多糖对宫颈癌细胞株生长和淍亡的影响

目的：探讨硒化麒麟菜多糖在体外对宫颈癌Hela细胞株生长和凋亡的作用及机制。方法：以宫颈癌Hela细胞株为研究对象，用MTT法检测硒化麒麟菜多糖对肿瘤细胞增殖的影响，流式细胞仪检测细胞周期、细胞调亡以及凋亡基因Fas的表达情况，以顺铂为对照组，比较硒化麒麟菜多糖与顺铂的疗效。结果：硒化麒麟菜多糖可阻滞宫颈癌Hela细胞的生长使之停留在S和G2-M期，从而抑制了肿瘤细胞的增殖，同时通过促进Fas表达，诱导Hela细胞凋亡。结论：硒化麒麟菜多糖通过阻滞肿瘤细胞的生长及诱导细胞凋亡等机制，对宫颈癌细胞的增殖有一定的抑制作用。     

MDV中meq基因对CEF细胞chTERT,chTR表达影响的研究

利用TaqMan探针技术,采用实时荧光相对定量RT-PCR方法检测了MDV中meq基因对鸡胚成纤维细胞(CEF)的端粒酶催化亚单位基因(chTERT)、端粒酶RNA亚基(chTR)表达水平的影响,并用TRAP法测定了端粒酶活性,用流式细胞仪检测了细胞周期的变化.实验结果表明,转染48 h后chTERT表达水平为转染后72 h的16倍,chTR表达水平在转染前后基本不变;转染48 h后CEF细胞的相对端粒酶活性是转染72 h后的12倍;在转染后72 h S期细胞的百分比较未转染细胞显著增加.     

Effect of P15INK4b/MTS2 on the proliferation of human hepatoma cells SMMC-7721

The full length cDNA coding for P15 INK4b, which is a cyclin-dependent kinase inhibitor, was cloned to plasmid PXJ41-neo (Eco RⅠ/XhoⅠ site) and the new constructed plasmid pXJp15 was obtained. pXJp15 was transferred into the human hepatoma SMMC-7721 cells by lipofectine reagent. After G418 selection, a series of cell lines stably expressing high levels of P15 (named SHT) and the clone containing vector PXJ41-neo only (named SVXJ) were obtained by Northern and Western analysis. The results showed that the proliferation of SHT cells is inhibited compared with that of SVXJ cells. Cell cycle analysis indicated that overexpressing of P15 inhibited the growth of SHT cells by decreasing progrssion of cells from G1 to S and G2 to M phases. The levels of c-Myc and c-Fos were obviously decreased in SHT cells compared with control cells by Western blotting. The decreased expression of oncogene may be one of the molecular mechanisms of the effect of P15 on the proliferation of in SHT cells.     

白藜芦醇抑制HeLa细胞肿瘤活性的自由基机理

白藜芦醇是一种植物抗毒素，属多元酚类。虽然迄今的研究表明，白藜芦醇能有效抑制许多癌细胞的肿瘤活性，但均未从自由基生物角度来阐明其抑制肿瘤活性的机理。该研究以离体HeLa细胞为材料，利用细胞培养法研究了白藜芦醇抗肿瘤活性的功效，以及产生这种功效的自由基途径，旨在从自由基生物学的角度进一步揭示白藜芦醇抗肿瘤活性的机量。噻唑蓝法及流式细胞仪分析结果表明，白藜芦醇能强烈抑制HeLa细胞增殖阻断HeLa细胞由S期向G2期转变。HeLa细胞内源性活性氧（ROS）及HeLa细胞内部氧化还原态的实验结果表明，白藜芦醇对细胞自分泌的总ROS有一定的抑制作用，它抑制HeLa细胞自分泌的O^-2浓度与抑制HeLa细胞增殖之间有明显的相关关系；同时，白藜芦醇可明显增高HeLa细胞内源SOD的活性以及GSH的含量，但可使CAT的活性显著下降，提示白藜芦醇能显著改变细胞内部的氧化还原态。以上结果为白藜芦醇抗癌机制提供了自由基生物学方面的实验依据。     

核糖体蛋白RPL34-PS1对B细胞淋巴瘤的生长促进作用

一种核糖体蛋白RPL34具有促进B细胞淋巴瘤生长的作用,通过分子克隆将RPL34基因的序列克隆到逆转录病毒载体pBABE-Puro上后,包装逆转录病毒并感染Balb/c小鼠来源的B淋巴瘤细胞38B9,经嘌呤霉素筛选后构建稳定过表达RPL34的B淋巴瘤细胞系RPL34-38B9。体外培养计数比较细胞增殖变化情况;流式检测细胞凋亡和细胞周期水平;小鼠皮下荷瘤并测量肿瘤体积。结果表明:过表达的核糖体蛋白RPL34能够显著促进B淋巴瘤细胞的生长速率,减少细胞凋亡,同时能够显著增加B淋巴瘤细胞的成瘤速率。     

HCAP1基因对Raji细胞凋亡及凋亡相关蛋白Bax/Bcl - 2的作用

目的：研究外源HCAP1基因产物对Burkitt淋巴瘤细胞系Raji细胞凋亡及凋亡相关蛋白Bax／Bcl-2的作用。方法：用脂质体介导的基因转移方法，把外源HCAP1基因转染到Raji细胞中，用流式细胞术和Hochest 33258荧光染色检测细胞凋亡；用Western blot检测外源HCAP1基因对凋亡相关蛋白Bax和Bcl-2蛋白表达的调节。结果：外源HCAPl基因导入Raji细胞24、48和96h后，细胞凋亡率增加。Western blot显示Bax／Bel-2蛋白表达比值明显增高。结论：转染外源性HCAP1基因可诱导Raji细胞凋亡，使Bax／Bcl-2蛋白表达比例上调可能是HCAP1诱导凋亡的机制之一。     

车用燃料电池发动机控制系统与协调控制研究

介绍了燃料电池发动机的基本结构和特点,设计了实用的控制系统,并阐述了各个子系统的工作原理和控制策略,根据其自身特性和运行工况,提出了燃料电池发动机各子系统的协调控制方法,获得了较好的控制效果。     

核糖体蛋白S3表达减少对果蝇发育的影响

为探讨核糖体蛋白S3在果蝇（Drosophila）发育中的作用,采用RNAi法,分别在果蝇和果蝇s2细胞中干扰核糖体蛋白S3表达,观察对果蝇表型的影响。结果显示,减少核糖体蛋白S3表达引起果蝇幼虫发育延迟,眼睛、翅膀和刚毛生长缺陷等。进一步实验表明,在翅原基中有明显的凋亡信号;S2细胞数目减少;细胞凋亡和细胞周期相关基因表达异常。上述结果暗示核糖体蛋白s3丧达减少导致的果蝇发育改变可能通过细胞凋亡的方式来实现。     

Multifunctions of histone H1 proteins

Among various histones, histone H1 proteins have been appreciated for their multiple functions in diverse biological processes. In addition to being a structural protein in chromatin, H1 proteins also play critical roles in cell cycle, gene expression, and development. Recent studies reveal the possible effects of H1 in some diseases, such as cancer and neurodegenerative diseases. Here, we review different variants of HI, the functions, and post translational modifications of ill variants are also discussed.     

CXCR7对HeLa细胞增殖、粘附和侵袭能力的影响

基质细胞衍生因子-1(SDF-1)在肿瘤侵袭、转移过程中起着重要的调控作用.此前认为SDF-1是通过其唯一受体CXCR4来起作用.近年来发现SDF-1还有另一作用受体——CXCR7,SDF-1/CXCR7在部分肿瘤侵袭转移过程中起重要作用,但其在宫颈癌HeLa细胞中的作用目前尚未明确.通过Western blotting检测HeLa细胞中CXCR4和CXCR7的表达,阻断CXCR4或CXCR7后,通过MTT法评价细胞增殖能力,细胞粘附实验评价细胞粘附能力,Transwell实验评价细胞侵袭能力.结果表明,CXCR4和CXCR7在HeLa细胞中表达.阻断CXCR4或CXCR7后,SDF-1诱导的HeLa细胞增殖、侵袭和与内皮细胞的粘附能力均被阻断.结果提示CXCR7在SDF-1诱导HeLa细胞增殖、侵袭和与内皮细胞的粘附过程中起着重要作用,将有望成为治疗宫颈癌转移的新靶点.