Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro

Summary We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.     

Overexpression of E2F-1 inhibits progression of gastric cancer in vitro

E2F-1 plays a critical role in cell cycle regulation and other biological processes in cells. E2F-1 mediates apoptosis and suppresses tumorigenesis in many tissue types, but there are few data available on E2F-1 expression and its relationship to tumor kinetics in gastric cancer. To gain better insight into the involvement of E2F-1 in the biological characteristics of gastric tumors, we investigated the effect of E2F-1 overexpression on the progression of gastric carcinoma cells. A gastric cancer cell line stably overexpressing E2F-1 (MGC-803/E2F-1) was established. The influence of E2F-1 overexpression on in vitro cell growth was assessed by measuring cell survival, colony formation, and cell cycle progression. The results clearly show that overexpression of E2F-1 significantly inhibits cell growth and proliferation, blocking entry into the S-phase of the cell cycle. MGC-803/E2F-1 cells also had a higher apoptotic rate than control cells. In addition, E2F-1 reduced the motility and invasion of gastric cancer cells.     

PDT effects of m-THPC and ALA, phototoxicity and apoptosis

The purpose of this study was to estimate the efficacy of an endogenous sensitizer (-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD₅₀ with m-THPC was 2.8 g/ml and 4.7 g/ml under irradiation of 25 J/cm² respectively for PROb and B16 cells. With ALA, LD₅₀ was 150 g/ml and 175 g/ml under 25 J/cm² respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 g/ml m-THPC at 10 J/cm² for PROb and B16 cells and for 50 g/ml ALA at 25 J/cm² for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD₇₈ for PROb cells and LD₃₀ for B16 cells and after ALA-PDT with LD₂₂ for PROb cells and LD₁₈ for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm². On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm² as compared to 50 J/cm² (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.     

siRNA干扰PDGFRβ促进胃癌MGC-803/VCR细胞的凋亡

为研究血小板衍生生长因子受体β(PDGFRβ)在胃癌组织和长春新碱(vincristine, VCR)耐药胃癌细胞MGC-803/VCR中的表达,并探讨PDGFRβ沉默对MGC-803/VCR细胞增殖和凋亡的影响,本研究分别通过免疫组化和蛋白质印迹法(Western blotting)检测PDGFRβ蛋白在胃癌组织和耐药细胞株中的表达水平。通过Lipofectamine 2000将PDGFRβ小干扰RNA (si-PDGFRβ)转染到MGC-803/VCR细胞,Western blotting检测转染后PDGFRβ蛋白表达水平,Cell Counting Kit-8 (CCK-8)和流式细胞术分别检测si-PDGFRβ对VCR诱导人胃癌MGC-803细胞增殖和凋亡的影响。研究结果显示:PDGFRβ蛋白在胃癌组织中的表达显著高于正常胃组织;PDGFRβ蛋白在MGC-803/VCR细胞中的表达极显著高于MGC-803细胞,并且转染si-PDGFRβ后MGC-803/VCR细胞的PDGFRβ蛋白表达水平显著降低;1μmol/L、2μmol/L、4μmol/L、8μmol/L的VCR诱导MGC-803/VCR细胞后,si-PDGFRβ组细胞增殖抑制率分别为(21.97±0.84)%、(37.63±1.32)%、(55.77±1.39)%和(72.17±1.16)%,与对照组的(13.60±0.49)%、(22.33±1.01)%、(38.30±1.56)%和(52.90±1.08)%分别比较有极显著的差异(p<0.01);流式检测结果显示,与对照组的细胞凋亡率(13.61±0.49)%比较,发现si-PDGFRβ组胃癌MGC-803/VCR细胞凋亡率为(29.80±0.64)%,说明两者差异极显著(p<0.01)。本研究初步结论表明,si RNA干扰PDGFRβ能够促进VCR诱导的胃癌MGC-803/VCR细胞凋亡。     

组蛋白乙酰化在二烯丙基二硫诱导MGC803细胞分化中的作用

探讨二烯丙基二硫(DADS)诱导人胃癌MGC803细胞分化过程中组蛋白乙酰化状态的改变情况。运用形态学方法及成瘤实验观察DADS诱导MGC803细胞分化,应用Western印迹观察DADS诱导MGC803细胞分化与其调控细胞组蛋白乙酰化水平和相关p21WAF1的关系。形态学观察结果显示,30mg/LDADS处理MGC803细胞24h后,细胞异型性明显减少,且经裸鼠成瘤实验证实,处理后的细胞均未在裸鼠体内形成肿瘤;Western印迹显示,30mg/LDADS处理细胞12h后,其组蛋白H3乙酰化程度明显升高,与未处理组比较增加了38%(P<0.05);H4乙酰化程度无明显改变。用15、30、60mg/LDADS处理细胞12、24h后,p21WAF1均较对照组升高,以30mg/LDADS处理24h升高最显著。研究结果表明,DADS可诱导MGC803细胞分化,其作用可能与增加核组蛋白乙酰化水平及p21WAF1表达有关。     

川楝素诱导人胃癌MGC-803细胞凋亡及其机制

目的: 本研究旨在探讨川楝素诱导人胃癌MGC-803细胞凋亡及其机制。方法: 将人胃癌MGC-803细胞分为5组,每组3个复孔,采用氟尿嘧啶(5-FU)和0 nmol/L川楝素(TSN)分别作为阳性对照和阴性对照。其余3组分别加入终浓度为30 nmol/L、50 nmol/L、70 nmol/L的川楝素。川楝素处理细胞48 h后,利用激光共聚焦显微镜观察细胞形态结构变化;流式细胞术检测线粒体膜电位变化;酶标法检测Caspase-3和Caspase-9活性;利用qRT-PCR和Western blot检测凋亡相关基因Bcl-2、Bax、Cyt c和APAF-1 mRNA和蛋白水平。结果: 与0 nmol/L TSN组相比,30 nmol/L、50 nmol/L、70 nmol/L的川楝素作用于人胃癌MGC-803细胞48 h,可见细胞体积缩小,细胞核裂解,部分染色质凝集等形态学变化;Caspase-3和Caspase-9活性升高(P＜0.05);而线粒体膜电位明显下降(P＜ 0.05);Bax、Cyt c和APAF-1 基因mRNA及蛋白表达量显著升高(P＜0.05),Bcl-2 基因mRNA及蛋白表达量显著降低(P＜0.05)。结论: 川楝素通过上调Bax、Cyt c和APAF-1的表达,下调Bcl-2基因表达,增强Caspase-3、Caspase-9活性诱导人胃癌MGC-803细胞凋亡。     

青钱柳多糖对人胃癌MGC_803细胞生长的影响

本文研究青钱柳多糖(polysaccharides isolated from Cyclocarya paliurus(Batal.)Iljinskjk,PCP)对人胃癌MGC_803细胞生长的影响.采用噻唑蓝(MTT)法检测PCP对MGC_803细胞生长的影响;Annexin法检测PCP对MGC_803细胞诱导凋亡的作用.实验结果表明PCP在50、100、200、400 μg/mL浓度条件下,均可极显著性抑制人胃癌MGC_803细胞生长(P＜0.01),抑制率可达65.07%,细胞凋亡率可达25,44%.说明青钱柳多糖具有抑制人胃癌MGC_803细胞生长的生物活性.     

Design,synthesis and biological mechanisms research on 1,2,3-triazole derivatives of Jiyuan Oridonin A

Two series of derivatives with 1,2,3-triazole as heterocyclic moiety of Jiyuan Oridonin A, a new ent-kaurene diterpenoid which was isolated from genus Isodon rubescens, were synthesized and biologically evaluated. All the derivatives possessed good anti-proliferative activities. Among them, compound 8g was found to significantly induce cell apoptosis and cell cycle arrest in MGC-803 via a series of signals activated by the increased intracellular ROS levels.     

桦木酸对人胃癌MGC-803细胞增殖的影响

目的:探究不同浓度桦木酸对人胃癌MGC-803细胞增殖的影响.方法:将人胃癌MGC-803细胞分成4组,每组设置3个复孔,对照组细胞为加入浓度为0 μg/ml的桦木酸实验组细胞分别加入终浓度为10、20、30 μg/ml的桦木酸,各组细胞在含5％的CO2培养箱中孵育48 h后,使用吉姆萨染色法和台盼蓝拒染法检测桦木酸对...     

不同浓度桦木酸对人胃癌MGC-803细胞凋亡的影响

目的: 以人胃癌MCG-803细胞为实验材料,探讨不同浓度桦木酸(BA)对人胃癌MGC-803细胞凋亡的影响,为其临床应用提供依据。方法: 将人胃癌MGC-803细胞分成 4 组,每组设置 3 个复孔,对照组为未加入桦木酸的 MGC-803 细胞,3 组实验组分别加入终浓度为10、20、30 μg/ml桦木酸处理细胞48 h后,通过激光共聚焦显微镜观察各组细胞形态变化;检测桦木酸对细胞Caspase-3和Caspase-9活性的影响;利用流式细胞术检测细胞线粒体膜电位变化;qRT-PCR和Western blot检测凋亡相关基因Caspase-3、Caspase-9和Cytochrome C(Cyt c)mRNA及蛋白水平的表达变化。结果: 与对照组比较, 各处理组Caspase-3和Caspase-9活性显著升高(P＜0.01),线粒体膜电位显著下降(P＜0.01),Caspase-3、Caspase-9和Cyt c mRNA及蛋白表达均显著上调(P＜0.01)。结论: 在终浓度为10 ~30 μg/ml浓度范围内,桦木酸通过调节Caspase-3、Caspase-9和Cyt c的表达诱导人胃癌MGC-803细胞凋亡。