Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.     

Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms.

In an initial efforts to characterize the virological basis of neoplasia in the Shope papilloma-carcinoma system, the extent to which the viral genome is present in non-virus-producing benign and malignant tumors in domestic rabbits was established. Employing nick-translated radioactive viral DNA purified from productively infected papillomas on cotton tail rabbits as a probe, it was found that (i) papillomas, primary carcinomas, and metastatic carcinomas contain 10 to about 100 copies of the viral genome per diploid cell equivalent of DNA and (ii) viral DNA is present in detectable amounts in essentially all neoplastic cells. These results are consistent with the suggestion that continued presence of the viral genome is necessary for induction and maintenance of malignant as well as benign neoplasms.     

Mechanism-based inactivation of caspases by the apoptotic suppressor p35.

Caspases play a crucial role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, one of which--p35--has so far only been found in baculoviruses. P35 has previously been shown to rapidly form essentially irreversible complexes with its target caspases in a process that is accompanied by peptide bond cleavage. To determine the protease-inhibitory pathway utilized by this very selective protease inhibitor, we have analyzed the thermodynamic and kinetic stability of the protein. We show that the conformation of p35 is stabilized following cleavage within its reactive site loop. An inactive catalytic mutant of caspase 3 is bound by p35, but much less avidly than the wild-type enzyme, indicating that the protease catalytic nucleophile is required for stable complex formation. The inhibited protease is trapped as a covalent adduct, most likely with its catalytic Cys esterified to the carbonyl carbon of the scissile peptide bond. Together these data reveal that p35 is a mechanism-based inactivator that has adopted an inhibitory device reminiscent of the widely distributed serpin family, despite a complete lack of sequence or structural homology.     

ATP and adenosine prevent via different pathways the activation of caspases in apoptotic AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly disintegrate after serum deprivation.27 ATP or adenosine added immediately after serum removal afforded substantial protection against cell death even for a long period of 24 h. ED50 values were 14 and 110 microM for ATP and adenosine, respectively. In the presence of 5 microg/ml cycloheximide the protective effect of both substances was suppressed, indicating that protein synthesis is required. The protective effect of ATP was highly specific since among numerous tested derivatives only ATP-[gamma-S] exhibited a substantial protective effect.The ability of ATP and adenosine to modulate cell division was analyzed. Both substances did not exhibit any mitogenic effect. Adenosine completely blocked PDGF-BB induced cell division, whereas ATP had no effect. Unlike adenosine, ATP strongly stimulated Ca2+-release from intracellular stores. On the other hand, adenosine stimulated an increase in the intracellular concentration of cAMP from 0.4 - 1.5 microM, whereas ATP decreased the content below 0.1 microM. ATP stimulated the phosphorylation of MAP-kinase, RSK and p70S6-kinase; adenosine was inactive. After complexation of [Ca2+]i the protective effect of ATP was greatly lost while adenosine was still active. Surprisingly neither ATP nor adenosine caused an activation of PKC-isoforms. After incubation with pertussis toxin, the protection by ATP was reduced indicating an involvement of Gi-proteins in the signal transduction induced by ATP. Our results indicate that ATP as well as adenosine are potent inhibitors of cell death caused by serum deprivation and that this protective effect apparently occurs via distinct pathways. However, both pathways must converge at the point of caspase activation, since the stimulation of DEVDase- and VEIDase-activities, respectively, are suppressed by either ATP or adenosine.     

Quantification of active caspase 3 in apoptotic cells

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes.     

Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Ubiquitin-protein conjugates and alpha B crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos.

Ubiquitin-protein conjugates and alpha B crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and alpha B crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and alpha B crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo. Some form of functional association between protein ubiquitination and alpha B crystallin in cells may explain why alpha B crystallin is found with ubiquitin-protein deposits in some neurodegenerative diseases.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

GroEL-related molecular chaperones are present in the cytosol of oat cells.

In eukaryotic cells GroEL-related molecular chaperones (cpn 60) are considered to be restricted to plastids and mitochondria. Re-evaluation of the intracellular localization of chaperonins by electron microscopy, using two different anti-chaperonin antisera, revealed additionally their presence in the cytosol of oat primary leaf and coleoptile cells. The distribution of cpn 60 is not influenced by heat or light treatments.     

The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.     

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

CPP32 cDNA的克隆及其表达和活性检测

CPP32在细胞编程死亡调控中起重要的作用.利用CPP32专一性引物,用RT-PCR方法从人鼻咽癌CNE细胞中扩增出约830 bp的片段,经序列分析证明与已发表的CPP32序列完全一致.将扩增出的编码人全长CPP32的cDNA片段克隆入pGEX-2T中,转化大肠杆菌DH5α.转化菌经诱导表达出较高含量的GST-CPP32融合蛋白.进一步研究显示,细菌中表达的CPP32蛋白能自我切割,而且能裂解体外翻译的PARP,从而证明其具有生物活性.     

Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death

The homeostatic control of beta-cell mass in normal and pathological conditions is based on the balance of proliferation, differentiation, and death of the insulin-secreting cells. A considerable body of evidence, accumulated during the last decade, has emphasized the significance of the disregulation of the mechanisms regulating the apoptosis of beta-cells in the sequence of events that lead to the development of diabetes. The identification of agents capable of interfering with this process needs to be based on a better understanding of the beta-cell specific pathways that are activated during apoptosis. The aim of this article is fivefold: (1) a review of the evidence for beta-cell apoptosis in Type I diabetes, Type II diabetes, and islet transplantation, (2) to review the common stimuli and their mechanisms in pancreatic beta-cell apoptosis, (3) to review the role of caspases and their activation pathway in beta-cell apoptosis, (4) to review the caspase cascade and morphological cellular changes in apoptotic beta-cells, and (5) to highlight the putative strategies for preventing pancreatic beta-cells from apoptosis.     

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.     

Early stages of the apoptotic pathway in plant cells are reversible

Chromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.