细胞程序式死亡途径的新进展

关于细胞程序式死亡(programmed cell death,PCD)的分类目前有多种不同的观点,如有凋亡、副凋亡、胀亡和自噬等.但随着对PCD的深入研究,发现过去的分类是不合理的.出现了彼此重叠的机制.这是由于研究者认识的局限性造成的.因此,很有必要对细胞程序式死亡途径进行重新认识.根据最新研究成果将PCD主要划分为7种,它们分别为经典凋亡、自体吞噬、凋亡样程序性死亡、坏死样程序性死亡、类凋亡、丝裂灾变和衰老.     

细胞程序性死亡的非Caspase依赖型途径

程序性细胞死亡是一种程序化的主动性细胞死亡,半胱胺酸天冬氨酸特异性蛋白酶家族(在该过程中起着不可忽视的作用.基于Caspase在程序性细胞死亡过程中所起的作用,将程序性细胞死亡分为两大类:Caspase依赖型和Caspase非依赖型.前者即典型的凋亡,后者包括自体吞噬、副凋亡、有丝分裂灾变、凋亡样程序性死亡、坏死样程序性死亡等.这些Caspase非依赖型的细胞程序性死亡途径与生理及病理现象密切相关.     

胱冬肽酶非依赖性的细胞程序性死亡

在多细胞有机体的组织内稳态维持和正常发育过程中,细胞程序性死亡发挥着重要的作用。细胞程序性死亡有多种形式(如细胞凋亡、类细胞凋亡和类坏死等),其中了解较清楚的是细胞凋亡。一直以来,胱冬肽酶(caspase)被认为是细胞凋亡发生中关键的一种蛋白酶。但是最近的研究表明,包括细胞凋亡在内的一些细胞程序性死亡可以以一种不依赖胱冬肽酶的方式发生。细胞程序性死亡与胱冬肽酶之间存在非依赖性关系。     

神经酰胺以Caspase依赖和非依赖方式诱导线粒体凋亡蛋白释放

本研究探讨了外源性C2-神经酰胺诱导入结肠癌HT-29细胞凋亡中,线粒体膜间隙凋亡蛋白的释放机制．不同浓度C2-神经酰胺作用HT-29细胞,流式细胞仪检测线粒体膜电位（△ψm）,线粒体／细胞液分离试剂盒分离亚细胞成分,聚丙烯酰胺凝胶电泳检测细胞色素C（Cytc）、高温必需蛋白A2（HtrA2）、线粒体源性半胱天冬氨酸蛋白酶第二活化因子（Smac）、凋亡抑制蛋白（XtAP）和半胱天冬氨酸蛋白酶-3（Caspase-3）蛋白表达水平．实验结果显示25和50μmol／L C2-神经酰胺作用细胞6h,△ψm即开始下降（P〈0．05）,且环孢霉素能通过调节线粒体膜通透性转换孔抑制△ψm的下降．C2-神经酰胺对Cyt c,HtrA2和Smac总蛋白表达没有明显影响,但能诱导Cyt c,HtrA2和Smac从线粒体释放入细胞液中,并下调XIAP蛋白的表达及活化Caspase-3．在Caspase抑制剂存在下,C2-神经酰胺仍能诱导Cyt c和HtrA2从线粒体释放,但不能诱导Smac释放．因此认为C2-神经酰胺能通过线粒体凋亡通路诱导HT-29细胞凋亡,C2-神经酰胺诱导Cytc和HtrA2从线粒体的释放是Caspase非依赖性的,而Smac释放是Caspase依赖性的．     

一种高效抑制Jurkat细胞表达RhoGDI2的方法

目的:RhoGDI2是RhoGTP酶的解离抑制因子,在白血病细胞内表达量很高。构建Rho GDI2短发夹RNA(sh RNA)的慢病毒载体,并鉴定该慢病毒在急性T细胞白血病细胞系Jurkat内的抑制效率。方法:针对人rho GDI2基因序列合成sh RNA序列,通过限制性内切酶Bam HⅠ和XhoⅠ、T4DNA连接酶,将该sh RNA序列插入慢病毒载体p EN_h H1c,测序正确后通过LR重组酶与p DSL_hp UGIP重组,转化感受态细菌,筛选阳性克隆,测序鉴定正确后用脂质体法将质粒与ps PAX2、p MD2共转染293T细胞、包装病毒并侵染Jurkat细胞,采用荧光显微镜观察侵染效率、West-ern印迹鉴定Rho GDI2 sh RNA在Jurkat细胞内的表达。结果:构建的Rho GDI2 sh RNA慢病毒成功侵染Jurkat细胞并抑制Rho GDI2的表达。结论:构建并鉴定了RhoGDI2 shRNA的慢病毒表达质粒,为研究RhoGDI2在白血病中的作用奠定了基础。     

AIF在细胞程序性死亡中的研究进展

凋亡诱导因子(apoptosis inducing factor, AIF)是参与细胞程序性死亡的重要因子,最早在线粒体凋亡途径中被发现。AIF通过非半胱天冬酶(caspase)依赖的凋亡途径参与细胞凋亡过程,在凋亡信号刺激下,AIF从线粒体释放,引起染色质凝聚和DNA断裂。有研究发现,AIF参与了多聚ADP核糖聚合酶-1(poly ADP-ribose polymerase-1, PARP-1)依赖性细胞死亡(parthanatos)过程。Parthanatos是由PARP-1过度活化后诱导PAR聚合物合成和积累并与AIF结合,随后诱导AIF从线粒体释放来引起大规模的DNA断裂的过程。AIF参与的细胞凋亡和parthanatos在细胞程序性死亡的研究中具有同等重要的意义。本文以AIF为线索,对细胞凋亡和parthanatos两种细胞死亡的机制和相关疾病的研究进展进行总结。     

神经细胞死亡的细胞和分子机制

神经细胞死亡的细胞和分子机制朱国璋,张永莲,龚岳亭（中国科学院上海生物化学研究所,上海２０００３１）关键词神经细胞死亡,细胞和分子机制无论是在脊椎动物或无脊椎动物的神经系统发育和神经网络构建过程中,在外周神经和中枢神经细胞增殖、分化、成熟的同时,伴随...     

白藜芦醇通过诱导细胞自噬性死亡抑制宫颈癌的研究

白藜芦醇作为一种广泛存在于药食同源植物中的非黄酮类多酚化合物,其抗肿瘤效果受到广泛关注,但在抑制宫颈癌方面仍缺乏体内效应的实验依据.本研究通过体内实验发现白藜芦醇具有明显的抗肿瘤生长作用,组织水平LC3B、P62和Beclin-1表达改变,推测白藜芦醇可能通过促进癌细胞的过度自噬抑制宫颈癌的进展;进一步通过体外细胞实验...     

应用原子力显微镜分析正常淋巴细胞和Jurkat细胞的形态和机械性质

淋巴细胞形态和机械性质的变化与人的健康、疾病的治疗和诊断有着密切关系。本研究利用原子力显微镜研究淋巴细胞和Jurkat细胞形态和机械性质。结果显示,这2种细胞的形态较为相似,但通过对力曲线的分析得出这2种细胞的机械性质明显不同。正常淋巴细胞粘弹力范围大致为(796.7±248.5)pN,而Jurkat细胞分布于(158.5±37.5)pN;正常淋巴细胞的杨氏模量(0.471kPa±0.081kPa)近4倍于Jurkat细胞(0.0964kPa±0.0229kPa);而Jurkat细胞(4.322mN/m±0.382mN/m)的硬度近2倍于正常淋巴细胞(2.278mN/m±0.488mN/m)。结果表明原子力显微镜能可在临床诊断上区分正常细胞与肿瘤细胞,即使两者形态区别不明显。     

p53转录非依赖活性介导细胞凋亡

p53主要通过两条途径诱导细胞凋亡：p53作为转录因子,促进细胞凋亡的靶基因的表达上调,如PUMA、NOXA、PIDD、p53AIP1、COP1等,并通过这些蛋白参与内源和外源凋亡途径;另一方面,胞浆中的p53能转位到线粒体,激活内源性的线粒体途径,促进凋亡。后者已成为研究p53促凋亡机制的热点。本文就p53对转录非依赖活性诱导细胞凋亡途径的研究进展作一概述。     

Caspase-3-mediated degradation of condensin Cap-H regulates mitotic cell death

Mitotic death is a major form of cell death in cancer cells that have been treated with chemotherapeutic drugs. However, the mechanisms underlying this form of cell death is poorly understood. Here, we report that the loss of chromosome integrity is an important determinant of mitotic death. During prolonged mitotic arrest, caspase-3 is activated and it cleaves Cap-H, a subunit of condensin I. The depletion of Cap-H results in the loss of condensin I complex at the chromosomes, thus affecting the integrity of the chromosomes. Consequently, DNA fragmentation by caspase-activated DNase is facilitated, thus driving the cell towards mitotic death. By expressing a caspase-resistant form of Cap-H, mitotic death is abrogated and the cells are able to reenter interphase after a long mitotic delay. Taken together, we provide new insights into the molecular events that occur during mitotic death.     

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

Xanthomonas caspase displays an inherent PARP-like activity

In an earlier study, intracellular accumulation of metabolites such as pyruvate and citrate in Xanthomonas campestris pv. glycines (Xcg) was found to result in a caspase dependent stationary phase rapid cell death (RCD). In the present study, the presence of poly ADP-ribose polymerase (PARP)-like activity associated with caspase-3-like protein of Xcg is reported. This activity was found to be responsible for depletion of cellular NAD(+) levels in RCD-promoting media such as Luria-Bertani medium and starch medium fortified with citrate. Addition of PARP-specific inhibitors such as 3-aminobenzamide to RCD-promoting media restored the intracellular NAD(+) levels and thereby prevented RCD. The inherent association of PARP-like activity with the caspase protein was demonstrated by PARP cellular assay, immuno-precipitation and Western analysis. A truncated polysaccharide deacetylase gene having a caspase-like domain was cloned. The expressed protein though found to be inactive, cross-reacted with human caspase and PARP antibodies. This is the first report demonstrating the presence of a PARP-like activity in a prokaryote and its involvement in cell death.     

Characterization of 4-O-methyl-ascochlorin-induced apoptosis in comparison with typical apoptotic inducers in human leukemia cell lines

Apoptosis can be induced by various stimuli such as the ligands of death receptors, chemotherapeutic drugs and irradiation. It is generally believed that chemotherapeutic drugs induce mitochondrial damage, cytochrome c release and activation of caspase-9, leading to apoptosis. Here, we found that an isoprenoid antibiotic, 4-O-methyl ascochlorin, significantly induces typical apoptotic events in Jurkat cells including the degradation of poly (ADP-ribose) polymerase, DNA fragmentation, activation of caspase-3, -9 and -8, and cytochrome c release from mitochondria. Similar to Fas stimulation, 4-O-methyl ascochlorin but not staurosporine, cycloheximide and actinomycin D, induced apoptosis in SKW6.4 cells, in which apoptosis is strongly dependent on death-inducing signaling-complex. Bcl-2 overexpression in Jurkat cells completely suppressed the apoptosis, but procaspase-9 processing was partially induced. A caspase-8 inhibitor, IETD-fmk, effectively suppressed poly (ADP-ribose) polymerase cleavage and cytochrome c release. However, 4-O-methyl ascochlorin induced apoptosis in Jurkat cells deficient of caspase-8 or Fas-associated death domain protein. These results suggest that 4-O-methyl ascochlorin induces apoptosis through the mechanism distinct from conventional apoptosis inducers.     

A possible intermediate step during apoptotic execution

Many proteases are known to be involved in apoptosis. Among them, interleukin-1beta converting enzyme (ICE) and its family proteases, which are called caspases, play critical roles in the execution stage of apoptosis. We previously reported that a proteasome-inhibitor, benzyloxycarbonyl Leu-Leu-leucinal (ZLLLal), induced apoptosis in MOLT-4 cells. In the present study, in order to analyze the detailed mechanism of ZLLLal-induced apoptosis, we examined the effect of a caspase-inhibitor, acetyl(Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AcYVADcmk), on ZLLLal-induced apoptosis in the cells. Agarose gel electrophoresis revealed that low concentrations of AcYVADcmk efficiently suppressed apoptotic DNA fragmentation. However, the cells presented morphology different from normal, apoptotic or necrotic cells, although DNA fragmentation was suppressed. The same examination was performed on the cells with anti-Fas antibody-induced apoptosis, and the same results were obtained. Some cells with a similar morphology were found even without the caspase-inhibitor in the early stage of anti-Fas antibody-induced physiological apoptosis. In addition, apoptotic cascade was reactivated by washing out the caspase inhibitor from the DNA degradation-suppressed cells. Therefore, this newly found morphological feature shows the presence of a step prior to caspase activation in the cells, and this is the first report presenting the pre-caspase-activated step in the apoptotic cascade.     

Leishmania mexicana metacaspase is a negative regulator of amastigote proliferation in mammalian cells

Metacaspases (MCAs) are caspase family cysteine peptidases that have been implicated in cell death processes in plants, fungi and protozoa. MCAs have also been suggested to be involved in cell cycle control, differentiation and clearance of aggregates; they are virulence factors. Dissecting the function of MCAs has been complicated by the presence in many organisms of multiple MCA genes or limitations on genetic manipulation. We describe here the creation of a MCA gene-deletion mutant (Δmca) in the protozoan parasite Leishmania mexicana, which has allowed us to dissect the role of the parasite''s single MCA gene in cell growth and cell death. Δmca parasites are viable as promastigotes, and differentiate normally to the amastigote form both in in vitro macrophages infection and in mice. Δmca promastigotes respond to cell death inducers such as the drug miltefosine and H₂O₂ similarly to wild-type (WT) promastigotes, suggesting that MCAs do not have a caspase-like role in execution of L. mexicana cell death. Δmca amastigotes replicated significantly faster than WT amastigotes in macrophages and in mice, but not as axenic culture in vitro. We propose that the Leishmania MCA acts as a negative regulator of amastigote proliferation, thereby acting to balance cell growth and cell death.