蛋白激酶B在佛波酯诱导人胃癌细胞系凋亡中的作用

目的探讨佛波酯(TPA)诱导胃癌细胞系凋亡过程中蛋白激酶B(PKB)的作用。方法通过BrdU处理,流式细胞术检测以及DAPI染色,荧光显微镜观察分析TPA对胃癌BGC-823细胞的影响;免疫印迹法检测TPA对PKB蛋白表达水平、磷酸化的影响;核浆分离获得核浆蛋白,用于免疫印迹法检测TPA对胃癌细胞内PKB和其Ser 473位点磷酸化的核浆表达影响,以及激光扫描共焦显微镜观察TPA是否改变胃癌细胞内PKB的分布。结果TPA诱导BGC-823细胞凋亡;TPA下调BGC-823细胞内PKB蛋白表达,并且与TPA作用时间和TPA浓度呈正相关,与PP2A降解作用无关;TPA抑制PKB的Ser 473位点磷酸化,而对其Thr 308位点磷酸化没有明显影响;TPA下调细胞核内PKB的表达以及Ser 473位点的磷酸化;TPA并不改变PKB在胃癌细胞内的分布。结论PKB的抑制作用可能参与TPA诱导胃癌细胞凋亡过程;由TPA诱导的胃癌细胞凋亡可能部分是由于细胞核内PKB蛋白表达和Ser 473位点磷酸化下降所致。     

佛波酯处理人胃癌MGC80-3细胞过程中磷酯酶C-γ2的意义

目的 探讨佛波酯(TPA)处理人胃癌MGC80-3细胞过程中磷酯酶C-γ2(PLC-γ2)的意义.方法 通过DAPI染色,荧光显微镜观察分析TPA对胃癌MGC80-3细胞的影响;借助核浆分离手段获得胃癌细胞核浆蛋白,并通过免疫印迹(Western blotting)检测TPA对PLC-γ2蛋白表达水平影响;通过免疫荧光技术处理,激光扫描共焦显微镜观察胃癌细胞内PLC-γ2蛋白的定位和转运;用PLC-γ2的抑制剂(U73122)预处理细胞,免疫印迹和激光扫描共焦显微镜分别检测其对TPA作用胃癌细胞内PLC-γ2的影响;以及荧光显微镜下观察分析U73122是否影响TPA对胃癌细胞的作用.结果 TPA诱导胃癌MGC80-3细胞凋亡;同时TPA提高PLC-γ2蛋白表达水平,并诱导其发生核浆转运;其抑制剂(U73122)可以降低TPA对PLC-γ2蛋白表达水平的作用,但并不能影响TPA诱导胃癌细胞凋亡;而TPA诱导的PLC-γ2核浆转运却没有被PLC-γ2抑制剂(U73122)阻止.结论 尽管TPA提高了胃癌MGC80-3细胞中PLC-γ2表达水平,但PLC-γ2表达水平和TPA诱导的胃癌MGC80-3凋亡没有直接关系,而其核浆转运可能与TPA诱导的胃癌细胞凋亡相关.     

皮质酮快速抑制C6细胞摄取甘氨酸的信号转导

目的:探讨皮质酮快速作用的信号转导机制。方法:应用液体闪烁技术检测了皮质酮对C6细胞摄取甘氨酸的快速抑制作用,以及多种信号转导蛋白的激动剂或抑制剂对其作用的影响。结果:牛血清白蛋白偶联皮质酮和硫酸皮质酮快速抑制C6细胞摄取甘氨酸的效果相同;新霉素能部分阻断皮质酮的快速抑制C6细胞摄取甘氨酸的作用;GDP-β-S及chelery部分阻断皮质酮快速作用,而H₈₉对皮质酮的快速作用无明显影响。皮质酮对C6细胞和SK-N-SH细胞作用的Km和Vmax不同。结论:上述结果提示蛋白激酶C参与了硫酸皮质酮快速抑制C6细胞摄取甘氨酸的信号转导过程。皮质酮对C6细胞和SK-N-SH细胞作用的差异可能是由于二种细胞甘氨酸转运系统不同所致。     

佛波酯对细胞间隙连接通讯的影响及其作用机制

佛波酯对细胞间隙连接通讯的抑制有其自身的作用特点。抑制作用的主要机制是经蛋白激酶C介导的致连接蛋白的过磷酸化。另外已发现佛波酯可通过影响钙依赖性细胞粘附分子的功能及调控连接蛋白基因的表达而抑制细胞的间隙连接通讯。     

胡桃醌对卵巢癌SKOV3细胞氧化应激和凋亡的影响

 目的: 探讨胡桃醌对人卵巢癌SKOV3细胞的毒性作用及机制。方法: 采用MTT法检测胡桃醌对卵巢癌SKOV3细胞生长的作用;流式细胞术检测胡桃醌对卵巢癌SKOV3细胞凋亡的影响;采用DCF-DA染色荧光显微镜观察细胞内的活性氧簇(ROS)水平;采用Western blot检测卵巢癌SKOV3细胞细胞色素C(Cyt C)和活化caspase-3的水平。结果: 与对照组比较,胡桃醌对SKOV3细胞的生长有明显抑制作用,且抑制作用随药物浓度和时间的增加而增强(P<0.05)。流式细胞术检测50 μmol/L胡桃醌作用细胞24 h和48 h后诱导细胞凋亡,早期凋亡率和晚期凋亡率均高于对照组(P<0.01)。胡桃醌显著提高细胞内的ROS水平,上调细胞Cyt C和活化caspase-3的蛋白水平。结论: 胡桃醌通过促进SKOV3细胞ROS的积累诱导细胞凋亡和抑制细胞生长。     

人胚胸腺细胞对多种刺激剂反应性的研究

本文研究结果表明:PHA、SEB(葡萄球菌肠毒素B)、A23187、TPA均有不同程度诱导人胚胸腺细胞活化和增殖作用;TPA单独诱导作用较弱,但加入低浓度rHuIL-2可显著地促进其诱导增殖作用。而加入rHuIL-1则无促进作用;PHA和TPA间在诱导人胚胸腺细胞增殖中有很强的协同效应,而A23187对PHA诱导的增殖有明显的抑制作用,对TPA诱导的增殖有显著的促进作用。不同(20～36)周龄人胚胸腺细胞对上述刺激剂的反应结果基本一致。此结果对于了解人胚胸腺发育与胸腺细胞功能以及对于完善人胚胸腺细胞活化途径均有一定的意义。     

CD226和9.1C3特异性抗体对NK-92细胞杀伤作用的影响

目的 :探讨NK细胞活化型受体CD2 2 6和NK细胞抑制型受体 9 1C3分子特异性抗体对活化NK细胞系NK 92杀伤的调节作用。方法 :通过间接免疫荧光染色和FCM分析 ,鉴定CD2 2 6和 9 1C3分子在NK 92细胞的表达 ,采用51 Cr释放实验和重导向杀伤实验观察CD2 2 6mAb和 9 1C3mAb对NK 92杀伤作用的影响。结果 :NK 92细胞为CD2 2 6阳性 ,9 1C3弱阳性。CD2 2 6mAb和 9 1C3mAb对NK 92自然杀伤作用没有明显的调节作用。在重导向杀伤实验中 ,CD2 2 6mAb能增强NK 92对P815细胞的杀伤作用 ,而 9 1C3mAb对NK 92的自然杀伤以及CD2 2 6mAb诱导的杀伤均没有抑制作用。结论 :NK细胞活化型受体CD2 2 6分子可能参与NK 92细胞杀伤作用的正向调节 ,而NK细胞抑制受体 9 1C3分子对NK 92细胞杀伤功能的影响不大。     

黏液瘤病毒作用PI3K/AKT通路诱导子宫内膜癌细胞凋亡

目的 探讨黏液瘤病毒（MV）对子宫内膜癌HEC-IB细胞凋亡和增殖的作用.方法 通过荧光染色等观察细胞核固缩和染色体碎片等形态学变化,MTT法测定MV对HEC-IB细胞的生长抑制作用,采用流式细胞仪和免疫印迹实验检测HEC-IB细胞的生长凋亡情况及其相关蛋白的表达变化.结果 MV对子宫内膜癌HEC-IB细胞具有明显的生长抑制作用,并能诱导细胞发生凋亡,随着作用时间的延长,细胞的生长抑制率及细胞凋亡率均明显升高.MV抑制细胞生长及诱导细胞发生凋亡的过程中,细胞磷脂酰肌醇-3激酶（PI3K）、蛋白激酶B（AKT）、糖原合酶激酶3（GSK3）表达水平及活性显著降低.结论 MV能够通过多条信号途径促进人子宫内膜癌HEC-IB细胞发生凋亡,通过抑制PI3K/AKT的活性是其体外诱导人子宫内膜癌HEC-IB细胞发生凋亡和抑制增值的重要作用机制.     

C225单用及与DDP联用对HeLa细胞的抑制作用与机制研究

探讨C225单用及与DDP联用对人子宫颈癌HeLa细胞的体外抑制作用及其机制。分别用C225(100、300μg/ml)与DDP(0.1、0.5、1μg/ml)以单用和联用处理HeLa细胞72 h后,MTT法检测对HeLa细胞生长的抑制作用及协同效应;FCM技术检测C225(300μg/ml)与不同工作浓度DDP单用及联用72 h后对细胞周期及凋亡的影响;并用HOE-CHEST33258染色技术进行细胞凋亡的形态学检测。结果C225能明显抑制HeLa细胞增殖,与DDP联合具有协同作用或相加作用;C225能使细胞周期阻滞于G1期,并诱导细胞凋亡;与DDP联用后凋亡率更高,使细胞周期进一步阻滞于G2期,同时降低S期细胞比例。荧光显微镜下可见实验对照组所发荧光较弱,无凋亡小体出现,而各药物干预组均出现一定数量的凋亡细胞,并随药物浓度的增加而增多,其中尤以联合用药组最为明显。两药单用及联用均对HeLa细胞的体外生长具有显著抑制作用,C225可通过将细胞周期阻滞于G0-G1期及诱导细胞凋亡发挥其抑瘤效应;两药间存在协同作用,机制可能与C225增加HeLa细胞对DDP的敏感性有关。     

大蒜提取液对细胞生长,细胞周期进展及蛋白激酶C活性的影响

实验证明大蒜提取液对人羊膜FL细胞株的细胞增殖有抑制作用,其作用环节在细胞周期的G_2/M期;适当浓度的大蒜提取液对促癌剂12-0-十四酰基-佛波醇-13-乙酸酯(TPA)诱发的蛋白激酶-C(PK-C)活性变化有拮抗作用,但大蒜提取液在较高浓度时本身能引起类似于TPA诱发的PK-C活性变化。     

Regulation of the 12-o-tetradecanoyl-phorbol-13-acetate-induced inhibition of intercellular communication

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase C (PKC) inhibitors on cell-cell communication were studied in a normal rat liver cell line, clone 9. Communication was observed and quantitated with microspectrofluorometric and image analysis techniques following scrape-loading of the cells with lucifer yellow. Lucifer yellow migrated as far as ten cells away from the scraped edge in control populations. Two minute TPA (25–50 μg/ml) treatment inhibited dye movement such that the dye remained mainly in the cells at the cut edge. The TPA-induced inhibition of cell-cell communication could be partially blocked by 15 min treatment of the cell populations with the PKC inhibitors trifluoperazine (30 μg/ml), staurosporine (2 × 10^?8 or 2 × 10^?6 M), sangivamycin (15 or 200 μM), or a PKC inhibitor peptide (20 μg/ml) scraped in at the same time as lucifer yellow. Normal communication was observed in cultures treated only with PKC inhibitors. Lower concentrations of TPA (50 ng/ml-1 μg/ml) used for 2 min did not inhibit dye communication. Our results demonstrate the phorbol ester-induced interruption of cell-cell communication. The inhibition of PKC by inhibitors eliminates the effect of TPA on communication. Our data are consistent with a role of PKC in the control of junctional communication. © 1993 Wiley-Liss, Inc.     

三磷酸腺苷对人横纹肌肉瘤细胞系诱导分化作用的研究

为了探讨三磷酸腺苷（ＡＴＰ０对人横纹肌肉瘤细胞增殖和分化的影响，用ＡＴＰ作用于人横纹肌肉瘤细胞亚系（ＲＤＬ６）细胞，观察到ＡＴＰ可抑制ＲＤＬ６细胞的增殖，使其生长速度明显减慢，作用第５ｄ时增殖抑制率为８１％，流式光度术检测；观察到ＡＴＰ ＲＤＬ６细胞Ｓ期的细胞数明显增多，说明细胞停滞在Ｓ期，用罗氏黄荧光染料传法实，ＡＴ家恢复ＲＤＬ６细胞间隙加接通讯功能的作用。用 光细胞化学方法观察到经ＡＴＰ处理后     

Effects of phorbol ester and related compounds on acetylcholine-evoked [3H]leucine-labelled protein secretion in permeabilized rat pancreatic acini

The effects of the phorbol ester, 12-tetradecanoyl-phorbol-13-acetate (TPA) and related compounds on acetylcholine (ACh)-evoked [3H]leucine-labelled protein release (secretion) were tested in isolated permeabilized rat pancreatic acini. The aim was to determine whether the diacylglycerol-like compounds can still potentiate the actions of ACh during unfluctuating supramaximal elevation of cytosolic free calcium (Ca2+) levels. TPA and R59022, an inhibitor of diacylglycerol kinase, evoked marked biphasic dose-dependent increases in 3H-labelled protein secretion from permeabilized rat pancreatic acini. Synthetic diacylglycerol (OAG) and 8-bromo cyclic GMP elicited small increases in 3H-labelled protein release while an inactive phorbol ester (4 alpha-phorbol-12,13-didecanoate; 4 alpha-PDD) and polymyxin B, an inhibitor of protein kinase C, were unable to stimulate secretion. Combining polymyxin B with TPA resulted in an inhibition of 3H-labelled protein secretion. Acetylcholine also induced a dose-dependent increase in 3H-labelled protein output, but when TPA or R59022 was combined with ACh, there was a marked potentiation of the ACh-evoked secretory response, particularly at higher concentrations of ACh. This synergism was unaffected by the protein kinase C inhibitor, polymyxin B. The results show that cytosolic free Ca2+ and protein kinase C may not be the only mediators of ACh-evoked secretion. Moreover, they indicate that protein kinase C may not be involved in the potentiation by TPA of ACh-evoked 3H-labelled protein release.     

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.     

Protein kinase C, a coupling element between stimulus and secretion of basophils

Protein kinase C plays a crucial role in the transmission and control of secretory cell membranal signals. This Ca2+ and phospholipid dependent kinase have been isolated and partially purified from histamine secreting rat basophilic leukemia cells (RBL-2H3 line). The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate ester (TPA) directly activated this isolated enzyme. In the intact RBL-2H3 cells, TPA did not significantly affect free intracellular Ca2+ ions concentration or induce secretion. However, at low concentrations it synergistically enhanced secretion induced either by antigen or ionophore. Significantly, at TPA concentrations exceeding 25 ng/ml both the increase in cytosolic free Ca2+ and the ensuing degranulation were inhibited. The synergism between TPA and the ionophore reaches saturation. These findings suggest that free cytosolic Ca2+ and kinase C-mediated protein phosphorylation are synergistically involved in the mediation of the cellular response. Moreover, kinase C appears to play a dual role both in the activation and termination of secretion. The latter is most probably achieved by closure of the Ca2+ channels in the cells.     

Induction of chemotaxis in mouse peritoneal macrophages by activators of protein kinase C

Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 microM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by diC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that chemotaxis in macrophages may be mediated by activation of protein kinase C.     

The effects of the protein kinase C inhibitors staurosporine and H7 on the IgE dependent mediator release from RBL 2H3 cells

RBL 2H3 cells, a model for mast cell function, sensitized with rat IgE, released histamine and peptidoleukotrienes (LT) in response to rabbit anti-rat IgE in a concentration-dependent manner. The calcium ionophore, A23187 also stimulated the release of both mediators but to a greater extent. The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) failed to influence mediator release when added alone, but when added with either A23187 or anti-IgE, TPA significantly enhanced the release of both histamine and LT. The effects of anti-IgE, TPA and A23187 were completely inhibited by prior addition of the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) but not by N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide dihydrochloride (HA1004), a compound which has similar potency to H7 as an inhibitor of some protein kinases but is less potent as a protein kinase C inhibitor. Although other explanations are possible, these results support the hypothesis that the release of histamine and leukotrienes from RBL 2H3 cells resulting from the cross bridging of the IgE receptors, is dependent on activation of protein kinase C.     

Ultraviolet A exposure induces reversible disruption of gap junction intercellular communication in lens epithelial cells

Gap junction intercellular communication (GJIC) is essential for the proper function of many organs including the lens. Disruption of GJIC can cause lens metabolic disorder and can induce cataracts. The purpose of this study was to investigate the signal transduction pathways involved in GJIC disruption following ultraviolet A (UVA) exposure in lens epithelial cells. Following exposure of human lens epithelial cells to UVA, connexin 43 (Cx43), the main component of gap junctions, was down-regulated at both the mRNA and protein levels. Furthermore, we observed that UVA exposure can increase protein kinase C activity and stimulate reactive oxygen species generation and lipid peroxidation. Using scrape load dye transfer technique, we found that the GJIC is compromised by UVA exposure. In addition, we demonstrated that UVA-induced modulation of GJIC was associated with p38 mitogen-activated protein kinase activation. More importantly, at non-lethal doses (10 J/cm2), the UVA-induced GJIC disruption and the consequent alterations were reversible. Collectively, our data revealed a new signaling pathway in GJIC disruption following UVA exposure, suggesting that UVA-compromised gap junction activity may sensitize human lens to photoaging and cataract formation.