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.     

SIGNALLING TO TRANSLATIONAL ACTIVATION OF TUMOUR NECROSIS FACTOR-α EXPRESSION IN HUMAN THP-1 CELLS

Monocytic THP-1 cells expressed tumour necrosis factor-α (TNF-α) mRNA, but hardly any detectable TNF-α protein and a partially activated MAP kinase ERK-2 in the unstimulated state. Stimulation with phorbol ester led to expression of TNF-α protein without significant changes in mRNA, a response that was sensitive to the MEK-1/2 inhibitors PD98059 and U0126. A calcium signal also led to expression of TNF-α protein, but now accompanied by a rapid increase in mRNA. A synergistic effect between phorbol ester and calcium ionophore was evident at the level of TNF-α protein, but not its mRNA. Stimulation with anisomycin led to a TNF-α expression that was sensitive to the p38 inhibitor SB203580. Actinomycin D lowered TNF-α mRNA in a similar way as PD98059 but was less inhibitory on PMA- or anisomycin-induced formation of TNF-α, thus confirming that these agents acted by causing translational derepression. Thus, in THP-1 cells MAP kinase pathways involving MEK-1/2 and possibly ERK-2 as well as the human p38 analogue were essential for basal TNF-α mRNA expression and translational activation.     

APOPTOSIS,REDIFFERENTIATION AND ARRESTING PROLIFERATION SIMULTANEOUSLY TRIGGERED BY OXIDATIVE STRESS IN HUMAN HEPATOMA CELLS

The effects of oxidative stress (ascorbic acid—ferrous system) on the proliferation, differentiation and apoptosis of the human hepatoma cell SMMC-7721 were studied. Oxidative stress significantly inhibited cell proliferation and induced morphological differentiation. Whatever the indices related with cell malignancy, such as α-fetoprotein and c-glutamyltranspeptidase or the index related with cell differentiation, such as tyrosine-α-ketoglutarate transaminase, all inclined evidently to normalization. The tumour's clonogenic potential decreased significantly. Moreover, together with differentiation, the phenomenon of apoptosis was found by the appearance of apoptotic bodies, detached cells, and apoptotic morphological feature. Although, their DNA was not degraded into oligonucleosomal fragmentation, the DNA was cut into larger fragments (about 21.2kbp) of a size associated with chromatin loops. These findings indicated that oxidative stress can induce both differentiation and apoptosis simultaneously in tumour cells. All the results showed that oxidative stress may initiate the tumour cells reverse transformation. The possible mechanism of the differentiation and apoptosis induced by oxidative stress may be related to the lipid peroxidation of cell membrane.     

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.     

IL-4 and TNF-α-MEDIATED PROLIFERATION OF THE HUMAN MEGAKARYOCYTIC LINE M-O7E IS REGULATED BY INDUCED AUTOCRINE PRODUCTION OF GM-CSF

In this study, the authors examined the effects of recombinant human interleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alpha (rhTNF-α) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e. While rhIL-4 or rhTNF-α alone induced only a weak proliferative response, a synergistic proliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-α is mediated predominantly by the 55-kDa TNF receptor because the agonistic monoclonal antibody Htr-9 and the Trp₃₂Thr₈₆TNF-α mutant protein specific for this receptor type produced similar results to rhTNF-α. In contrast, the Asn₁₄₃Arg₁₄₅TNF-α mutant protein specific for the 75-kDa TNF receptor produced only minimal proliferation of M-O7e cells. Using RT-PCR, we found that rhTNF-α rapidly and strongly induced granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient inducer of GM-CSF mRNA. However, there was little evidence of the TNF-α/IL-4 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with IL-4 or TNF-α alone. Thus, the observed synergistic effect of TNF-α/IL-4 costimulation of M-O7e cells seems to be mediated via induction of GM-CSF secretion rather than an enhanced production of GM-CSF mRNA. Higher levels of GM-CSF were detectable in supernatants of cells treated with both rhIL-4 and rhTNF-α than in cells stimulated with either cytokine alone. Furthermore, addition of a neutralising antibody against GM-CSF abrogated the observed synergistic effect of rhIL?4 and rhTNF-α treatment, indicating that the rhIL-4/THF-α combination acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.     

流感病毒感染诱导MDCK细胞凋亡的研究

用荧光染色、DNA凝胶电泳等方法检测了A型流感病毒株A1／京防86－1和B型流感病毒株B/沪防93－1诱导狗肾体代细胞(MDCKcells)的凋亡情况,并采用MTT法和流式细胞仪比较了这2株病毒对MMCK细胞的毒力和凋亡诱导能力水平。结果显示：病毒感染6h后,细胞DNA发生断裂,病毒感染12h后,可见明显的染色质凝聚;在一定范围内,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.     

氧胁迫对人肝癌细胞生长分化和凋亡的影响

采用不同浓度的抗坏血酸(50—800μmol/L)和硫酸亚铁(2.5—40μmol/L)系统生成以羟自由基为主的各种程度氧胁迫,使之作用于人肝癌细胞。本文所采用的不同程度的氧胁迫均能抑制癌细胞的生长。低水平氧胁迫可使肝癌细胞失去某些恶性特征,趋向分化,表现为细胞表面对Con-A的凝集力、甲胎蛋白含量、γ-谷氨酰转肽酶和酪氨酸-α-酮戊二酸转氨基酶活性都朝着分化方向变化,差异显著。分化后的细胞克隆形成能力显著降低。在分化过程中,出现一定量的凋亡细胞。随着氧胁迫程度的增高,凋亡细胞增多,表现为非贴壁细胞增多,细胞体积变小,染色质凝缩在核膜边缘,呈新月形,核碎裂,但质膜完整。细胞核中DNA降解成大约21.2kbp大小的大片段DNA。有望通过严格控制氧胁迫程度来减慢肝癌细胞增殖,促进分化和凋亡,使恶性细胞逆转成良性细胞。     

POTENTIATION OF TNF-α TOXICITY BY CONJUGATION WITH RICIN A-CHAIN

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor α (TNF-α) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-α can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-α cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-α and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC₅₀=4 pM) not sensitive to native TNF-α, and sensitive WEHI cells with substantially increased lethality (LD₅₀=0.01 fM). This increased TNF-α cytotoxic activity suggests that hybrid toxins containing TNF-α may have therapeutic applications in the treatment of cancer.     

Inhibition by Bacterial Lipopolysaccharide of Spontaneous and TNF-α-Induced Human Neutrophil Apoptosis In Vitro

In the previous paper (Takeda et al, Int. Immunol., 5, 691-694, 1993), we demonstrated that tumor necrosis factor-α (TNF-α) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF-α-induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.     

Nicotine blocks TNF-α-mediated neuroprotection to NMDA by an α-bungarotoxin-sensitive pathway

Excitotoxic neuronal death mediated by N-methyl-D -aspartate (NMDA) glutamate receptors can contribute to the extended brain damage that often accompanies trauma or disease. Both the inflammatory cytokine tumor necrosis factor-α (TNF-α) and nicotine have been identified as possible neuroprotective agents to NMDA assault. We find that TNF-α protection of a subpopulation of cultured cortical neurons to chronic NMDA-mediated excitotoxic death requires both the activation of the p55/TNFRI, but not p75/TNFRII, and the release of endogenous TNF-α. Nicotine protection to NMDA was mediated through an α-bungarotoxin-sensitive receptor. When coapplied, neuroprotection to NMDA by either TNF-α or nicotine was abolished but could be recovered with α-bungarotoxin. These results suggest that the cytokine TNF-α and α-bungarotoxin-sensitive nicotinic neurotransmitter receptors confer neuroprotection through potentially antagonistic pathways. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 29–36, 1998     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

冷休克诱导肿瘤细胞凋亡的动力学特征

应用扫描电镜观察、末端DNA转移酶介导的脱氧UTP缺口末端标记技术（TUNEL）和流式细胞术研究低温冷休克诱导人乳癌细胞系（ZR－75－30）细胞凋亡,并对其细胞凋亡的动力学特征测定、分析。结果显示：－5℃、0℃、4℃冷休克均可诱导ZR－75－30细胞凋亡,但细胞凋亡进程受实验细胞预培养时间;冷休克温度、时间;细胞冷休克后恢复培养时间的影响,这些影响因素具有明显的动力学特征。经过对冷休克诱导ZR－75－30细胞凋亡的动力学特征分析、比较,优选出研究冷休克诱导肿瘤细胞凋亡的实验体系：即细胞经37℃细胞凋亡的动力学特征分析、比较, 优选出冷休克诱导肿瘤细胞凋亡的实验体系：即细胞经37℃预培养36h→冷休克（-5℃24h,0℃36h,4℃48h）→细胞冷休克后于37℃恢复培养18－24h→细胞凋亡检测→检测结果综合分析。应用该实验体系要使细胞凋亡率达50－60％,且重复性尚佳。优化体系的深入研究冷休克诱导细胞凋亡的信号传递及其分子机制提供实验模型。