MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20 ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca²⁺concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca²⁺-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca²⁺regulator or antioxidant, preventing lipid peroxidation in the process.     

p38丝裂原活化蛋白激酶在不同细胞内定位的研究

用共聚焦显微镜对不同原代培养细胞的ｐ３８丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ,ＭＡＰＫ）进行定位,以探讨ｐ３８激活后入核在不同的细胞中是否具有普遍性。发现与单核细胞相似,未受刺激静止的心肌细胞、平滑肌细胞和内皮细胞的ｐ３８荧光强度呈弥散性分布;脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）刺激后,细胞核区荧光强度均明显增加,胞浆荧光强度均降低     

ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR-α-INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS

Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor-α (TNF-α) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF-α-induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF-α in a dose-dependent manner. UVB but not TNF-α induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF-α-induced apoptosis through the induction of TNFRs, Fas, and Fas-L in murine fibroblasts.     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

P38和ERK1/2在肝细胞癌中的表达及意义

目的探讨p38（mitogen-activated protein kinase p38,p38）和细胞外信号调节激酶1/2（extracellular reg-ulated kinase 1/2,ERK1/2）在不同分化程度肝细胞癌（human hepatocellular carcinoma,HCC）中的表达情况,以及两者的相关性。方法通过免疫组化（Envision）法,检测52例肝癌组织及l0例癌旁正常肝脏组织中p38和ERK1/2的表达。结果：p38和ERK1/2在肝癌组织中均有表达,与正常组比较差异有显著性,且阳性率的高低与其分化程度有关,p38在肝癌组织中的表达随分化程度的增高阳性率降低（P〈0.05）;ERK1/2在中、低分化的肝癌组织分别与高分化相比差异具有显著性（P〈0.05）。但低分化与中分化的肝癌组织比较虽也有差异但无统计学意义（P〉0.05）。癌组织中p38和ERK1/2的表达呈中度正相关（r=0.703,P〈0.05）。结论HCC中,p38和ERK1/2表达和活性增加,且存在一定的相关性。     

NECROSIS OF LUNG EPITHELIAL CELLS BY FILARIAL PARASITIC PROTEIN VIA AN EARLY INDUCTION OF c-H-rasAND TNFα EXPRESSION

The direct interaction of filarial proteins with lung epithelial cells was examined to determine the possible mechanism of inducing cell death, an event that is observed in patients with tropical pulmonary eosinophilia. Exposure of lung epithelial cells to filarial parasitic proteins,Brugia malayi(BmA),Setaria digitata(Sd), and recombinant filarial protein (pGT 7)in vitrofor more than 2 days, causes the appearance of DNA fragments both in the cytoplasm and culture supernatants, while no fragmentation was observed in the untreated controls. The release of DNA fragments both in the cytoplasm and the culture supernatants simultaneously, indicates that cell death is induced by a necrotic event rather than apoptosis. Fluorescent-labelled studies also indicate the fragmentation of DNA increasing in a time-dependent manner. Normal cellular function is controlled through several oncogenes. The modulation of specific proto-oncogenes like myc, ras and TNFα during exposure to filarial parasitic proteins reveal elevated levels of expression of ras and TNFα as early as 2 hours, implicating their involvement prior to DNA fragmentation leading to pathogenesis.     

Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

二甲基苯并蒽对肺肿瘤细胞P1-450基因表达的调节

人肺肿瘤细胞(ChaGo)经二甲基苯并蒽(DMBA)处理,刺激了p 1-450基因的高水平表达,用亚致死剂量内的DMBA 处理细胞,检出了p 1-450基因的mRNA 水平随DMBA浓度和处理时间而增加;p 1-450基因的主要编码区和3’末端区“—CCGG—”序列的甲基化型式不受DMBA 处理的影响,但DMBA 处理细胞影响到基因5’末端和侧翼区的“—CC-GG—”中“—C—”残基的位点特异的低甲基化效应,这种低甲基化效应,可能关系到p 1-450基因的异常高水平表达,也可能同时存在着别的分子调节机制。     

胞内钙与蛋白激酶C对人脐静脉内皮细胞组织因子途径抑制物及组织因子表达的影响

为探讨细胞内钙及蛋白激酶C在血管内皮细胞释放组织因子（TF）,组织因子途径抑制物（TFPI）中的作用,本文采用钙离子载入剂A23187与蛋白激酶C激动剂佛波脂（PMA）刺激原代培养人脐静脉内皮细胞（HUVEC）,观察各实验组内皮细胞冻融液中TF活性及条件培养液中TFPI活性的变化,以一期凝固法测TF活性,二期生色法测定TFPI活性。结果显示：A23187,PMA及A23187＋PMA组TF活性较对照明显升高（P＜0.001）,三组中A23187和A23187＋PMA组活性明显低于PMA组（P＜0．05）,但前两组间无显著性差异（P＞0．05）;A23187组TFPI活性与对照无明显差别,但PMA与A23187＋PMA组TFPI活性较对照及A23187组明显升高（P＜0.01）。结果提示：细胞内钙升高及PKC激活促进血管内皮细胞TF的合成,以PKC为强;PKC促进血管内皮细胞释放TFPI,而钙对此无明显作用。     

APOPTOSIS-INDUCED BY TRAIL AND TNF-α IN HUMAN MULTIPLE MYELOMA CELLS IS NOT BLOCKED BY BCL-2

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor is a member of the tumour necrosis factor (TNF) family of death signal tranduction proteins with a mechanism of cell death, similar to the Fas and Fas ligand (Fas-L) system. Here, we provide first time evidence that TRAIL and TNF-α are potent inducers of apoptosis in multiple myeloma (MM) cell lines and freshly isolated myeloma cells. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner reaching 80% within 48 h of treatment with a dose of 160 ng/ml. Bcl-2 transfected 8226 and ARP-1 cells were equally sensitive to apoptosis by TRAIL. Apoptosis with TNFα, reached >60% within 48 h of treatment with a dose of 160 ng/ml. In addition to MM cell lines, freshly isolated, flow-sorted myeloma cells from 8 different MM patients expressing variable levels of bcl-2 were equally sensitive to both TRAIL and TNF-α. We have previously shown that anti-Fas-induced apoptosis is not blocked by endogenous or ectopic bcl-2 in MM cell lines. Here we extend our observation with Fas to include TNF-α and TRAIL to the apoptotic signals that are not be blocked by bcl-2, in MM cells.     

MODULATION OF INTERFERON γ INDUCED INCREASES IN CATHEPSIN B IN THP-1 CELLS BY ADRENERGIC AGONISTS AND ANTAGONISTS

In order to investigate the possible modulation of macrophage function by the autonomic nervous system, the effect of adrenergic agonists and antagonists on interferon (IFN)-γ-induced increases in cathepsin B (CB) in a macrophage-like cell line was studied. It has been shown previously that IFN-γ induces increased CB activity in phorbol myristate acetate (PMA)-primed THP-1 cells. Isoproterenol (ISO) (10μm ), a mixed β-receptor agonist, increased the induction of CB activity in the cells but norepinephrine (10μm ) and epinephrine (10μm ), the α and β receptor agonists, had little effect. The addition of the mixed α-receptor antagonist phentolamine (10μm ) had no effect on ISO induced increases but the mixed β-receptor antagonist propranolol (10μm ) and the selective β₁-receptor antagonist atenolol produced significant inhibition. These results suggest that the activation of β-receptors could be involved in the induction of CB activity in macrophages and provide a possible mechanism for the regulation of macrophage effector function by the autonomic nervous system. Dibutyryl cAMP (1mm ) alone also induced increases in CB in THP-1 cells, and H-89 or HA1004 abrogated the effect of dibutyryl cAMP, suggesting that the effect of ISO on CB could be through the elevation of cAMP and the activation of cAMP-dependent protein kinases.     

P15INK4B高表达对人黑色素瘤细胞G1/S进程的阻滞及与MAPK信号相关性之初探

利用TdR-N_2O同步法分别获得P15高表达的MLIK6和表达空载体的MLC2的M期细胞和G_1期细胞,~3H-TdR掺入结果显示,与对照组细胞MLC2相比,实验组细胞MLIK6从G_1期进入S期时间延长2h,并且掺入强度明显减弱,DNA合成被抑制。进一步观察了P15~(INK4B)对G_1/S相关调控蛋白的影响,在M期细胞释放8h(晚G_1期细胞)后,与对照组MLC2细胞相比,实验组MLIK6细胞中CyclinD1,CyclinE,Cdk4,C-Myc蛋白水平均降低。相反,P27~(KIP1)的表达却上升。同时探讨了MAPK信号在P15~(INK4B)阻抑A375细胞G_1/S转换中的作用与相关性,结果显示晚G_1期的MLIK6细胞中ERK1,ERK2水平变化不大,而具有活性的P-ERK1和P-ERK2均表现出下降。上述实验表明,P15~(INK4B)可能通过作用于G_1期相关的周期调节蛋白和抑制ERK1和ERK2活性,阻滞G_1/S转换与抑制DNA合成。     

Induction of Heme Oxygenase-1 by Na+-H+ Exchanger 1 Protein Plays a Crucial Role in Imatinib-resistant Chronic Myeloid Leukemia Cells

Resistance toward imatinib (IM) and other BCR/ABL tyrosine kinase inhibitors remains troublesome in the treatment of advanced stage chronic myeloid leukemia (CML). The aim of this study was to estimate the reversal effects of down-regulation of Na⁺/H⁺ exchanger 1 (NHE1) on the chemoresistance of BCR-ABL-positive leukemia patients'' cells and cell lines. After treatment with the specific NHE1 inhibitor cariporide to decrease intracellular pH (pH_i), the heme oxygenase-1 (HO-1) levels of the K562R cell line and cells from IM-insensitive CML patients decreased. HO-1, as a Bcr/Abl-dependent survival molecule in CML cells, is important for the resistance to tyrosine kinase inhibitors in patients with newly diagnosed CML or IM-resistant CML. Silencing PKC-β and Nrf-2 or treatment with inhibitors of p38 pathways obviously blocked NHE1-induced HO-1 expression. Furthermore, treatment with HO-1 or p38 inhibitor plus IM increased the apoptosis of the K562R cell line and IM-insensitive CML patients'' cells. Inhibiting HO-1 enhanced the activation of caspase-3 and poly(ADP-ribose) polymerase-1. Hence, the results support the anti-apoptotic role of HO-1 induced by NHE1 in the K562R cell line and IM-insensitive CML patients and provide a mechanism by which inducing HO-1 expression via the PKC-β/p38-MAPK pathway may promote tumor resistance to oxidative stress.     

Okadaic acid,sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase Fa/GSK-3α in A431 cells

Exposure of A431 cells to a rapid and sudden increase from 37°C to 46°C for 30 min could induce an increase in protein level and cellular activity of protein (kinase Fa /GSK-3α) up to ∼200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37°C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37°C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46°C for another 30 min, the heat induction on kinase Fa /GSK-3α was found to be completed blocked. In sharp contrast, when cells were first treated with 1 μM TPA at 37°C for 24 h or with 5 μM sphingosine at 37°C for 30 min to down-regulate cellular PKC, the heat induction on kinase Fa /GSK-3α was found to be reversely promoted up to ∼ 250% of control level, demonstrating that kinase Fa /GSK-3α may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase Fa /GSK-3α in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase Fa /GSK-3α, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process. © 1996 Wiley-Liss, Inc.     

p38α- and DYRK1A-dependent phosphorylation of caspase-9 at an inhibitory site in response to hyperosmotic stress

The cysteine aspartyl protease caspase-9 is a critical component of the intrinsic apoptotic pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125, which is catalysed by the mitogen-activated protein kinases (MAPKs) ERK1/2 in response to growth factors, by the cyclin-dependent protein kinase CDK1-cyclin B1 during mitosis, and at a basal level by the dual-specificity tyrosine-phosphorylation regulated protein kinase DYRK1A. Here we show that inhibitory phosphorylation of caspase-9 at Thr125 is induced in mammalian cells by hyperosmotic stress. This response does not require ERK1/2 or ERK5, but it is diminished by ablation of DYRK1A expression by siRNA or chemical inhibition of DYRK1A by harmine. Phosphorylation of Thr125 in response to hyperosmotic stress is also reduced by chemical inhibition of p38 MAPK and is abolished in p38α^−/− mouse embryonic fibroblasts. These results show that both DYRK1A and p38α play roles in the inhibitory phosphorylation of caspase-9 following hyperosmotic stress and suggest a functional interaction between these protein kinases. Phosphorylation of caspase-9 at Thr125 may restrain apoptosis during the acute response to hyperosmotic stress.     

Swiprosin‐1 is expressed in mast cells and up‐regulated through the protein kinase CβI/η pathway

Swiprosin‐1 exhibits the highest expression in CD8⁺ T cells and immature B cells and has been thought to play a role in lymphocyte physiology. Here we report that swiprosin‐1 is also expressed in mast cells and up‐regulated in both in vitro cultured mast cells by phorbol ester and in vivo model tissues of passive cutaneous anaphylaxis and atopic dermatitis. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC‐βI/η are involved in the expression of swiprosin‐1 in the human mast cell line HMC‐1. In contrast, down‐regulation of swiprosin‐1 by A23187 or ionomycin suggests that calcium‐signaling plays a negative role. The ectopic expression of swiprosin‐1 augmented PMA/A23187‐induced NF‐κB promoter activity, and resulted in increased expression of cytokines. Moreover, knock‐down of swiprosin‐1 attenuated PMA/A23187‐induced cytokine expression. Collectively, these results suggest that swiprosin‐1 is a PKC‐βI/η‐inducible gene and it modulates mast cell activation through NF‐κB‐dependent pathway. J. Cell. Biochem. 108: 705–715, 2009. © 2009 Wiley‐Liss, Inc.     

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.