Daxx overexpression in T-lymphoblastic Jurkat cells enhances caspase-dependent death receptor- and drug-induced apoptosis in distinct ways

The role of Daxx, in particular, its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in apoptosis signaling of malignant lymphocytes, Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. We thus demonstrate that ectopic expression of Daxx substantially increases the rate of apoptosis upon incubation with death receptor agonists such as Fas and TRAIL as well as upon incubation with the cytotoxic drug doxorubicin (DOX). Analysis of the molecular changes induced in the extrinsic and intrinsic apoptosis pathways reveals that augmentation of apoptosis by Daxx overexpression is conveyed by distinctly different mechanisms. Although enforced apoptosis caused by ectopic Daxx expression is caspase-dependent in both cases, major differences between Fas/TRAIL-induced apoptosis and doxorubicin-induced apoptosis are observed in expression patterns of X-linked inhibitor of apoptosis (XIAP), p53, Bid, ZIP kinase, and prostate apoptosis response gene 4 (Par-4). Moreover, we could show that addition of a CD95 blocking antibody to the clones treated with doxorubicin was able to increase apoptosis as compared to doxorubicin treatment alone and was accompanied by an enhancement of the mitochondrial branch of apoptosis. In conclusion, we here outline the major molecular mechanisms underlying the apoptosis-promoting effect of Daxx in neoplastic lymphocytes and demonstrate fundamental molecular differences elicited by the overexpression of Daxx in the extrinsic and intrinsic signaling pathways.     

Fas-Mediated apoptosis is inhibited by TSH and iodine in moderate concentrations in primary human thyrocytes in vitro.

Programmed cell death (apoptosis) can be found in normal thyroid tissue and in various diseases affecting the thyroid gland. The Fas/Fas ligand (FasL) system is involved in the induction of apoptosis in human thyrocytes. Cross-linking the Fas receptor with its own ligand or with an antibody capable of oligomerizing with the receptor induces programmed cell death. We investigated the role of Fas-induced apoptosis in primary human thyrocytes in vitro. Cell cultures of normal human thyrocytes were prepared from specimens obtained during surgery for uninodular goiter. Apoptosis was induced by incubation of the cells with a monoclonal IgM anti-Fas antibody. The presence of apoptosis was determined by FACS analysis of FITC-labelled annexin V binding combined with dye exclusion of propidium iodide. We found a significant rate of Fas-induced apoptosis in normal thyrocytes after activation with a monoclonal anti-Fas antibody. TSH was able to inhibit Fas-mediated apoptosis in a dose-dependent manner. This effect was more pronounced when thyrocytes were incubated in the presence of interferon-gamma. Low concentrations of iodine were able to inhibit apoptosis, while high concentrations of iodine increased the rate of Fas-induced apoptosis. Our results show that Fas-mediated apoptosis is inducible in normal human thyrocytes in vitro and is influenced by TSH and iodine. The Fas/FasL system may play an important role in the regulation of cell number within the thyroid gland, and may be involved in the processes leading to goiter in iodine deficiency.     

哺乳动物卵母细胞凋亡的研究进展

细胞凋亡是发育过程中的基本生命现象,除各种体细胞凋亡外,生殖细胞的发生过程中也发生细胞凋亡。就雌性生殖系而言,细胞凋亡是其发育过程中的一个重要组成部分。在哺乳动物中,超过99．9％的雌性生殖细胞都会在卵子发生的不同阶段发生凋亡。有三种学说解释这一现象：1)被忽视死亡;2)因缺陷死亡;3)自我牺牲死亡。本文主要综述了哺乳动物卵母细胞凋亡的现象、卵母细胞凋亡学说、线粒体遗传与卵母细胞凋亡的关系以及凋亡的分子机理,同时还探讨了卵母细胞凋亡的生物学意义。     

Streptococcus pneumoniae: the role of apoptosis in host defense and pathogenesis

Programmed cell death or apoptosis is a recognised feature of infection with Streptococcus pneumoniae, and is observed during pneumococcal meningitis and pneumonia. The cholesterol-dependent cytolysin, pneumolysin, is a major trigger of apoptosis in the brain in association with pneumococcal production of hydrogen peroxide. Pneumococcal cell wall is also an important stimulus for apoptosis. Microbial factors and host factors combine in causing apoptosis in the brain, with hippocampal neurons being particularly susceptible. In pulmonary infection epithelial cell apoptosis contributes to tissue injury but macrophage apoptosis may benefit the host, aiding microbial killing and downregulating the inflammatory response. During sepsis lymphocyte apoptosis may be harmful to the host while dendritic cell apoptosis may limit the generation of an adaptive immune response during infection. Apoptosis induction may be harmful or potentially beneficial during pneumococcal infection and understanding its function in each setting is essential to allow specific therapeutic intervention.     

Involvement of Apoptosis in Host-Parasite Interactions in the Zebra Mussel

The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism.     

Regulation of FasL/Fas in human trophoblasts: possible implications for chorioamnionitis

Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor alpha-induced and interferon gamma-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.     

Novel role for RbAp48 in tissue-specific, estrogen deficiency-dependent apoptosis in the exocrine glands

Although tissue-specific apoptosis in the exocrine glands in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy, the molecular mechanism responsible for tissue-specific apoptosis remains obscure. Here we show that RbAp48 overexpression induces p53-mediated apoptosis in the exocrine glands caused by estrogen deficiency. RbAp48-inducible transfectant results in rapid apoptosis with p53 phosphorylation (Ser9) and alpha-fodrin cleavage. Reducing the expression of RbAp48 through small interfering RNA inhibits the apoptosis. Prominent RbAp48 expression with apoptosis was observed in the exocrine glands of C57BL/6 ovariectomized (OVX) mice but not in OVX estrogen receptor alpha(-/-), p53(-/-), and E2F-1(-/-) mice. Indeed, transgenic expression of the RbAp48 gene induced apoptosis in the exocrine glands but not in other organs. These findings indicate that estrogen deficiency initiates p53-mediated apoptosis in the exocrine gland cells through RbAp48 overexpression and exerts a possible gender-based risk of autoimmune exocrinopathy in postmenopausal women.     

The role of autophagy in corpus luteum regression in the rat

Autophagy is associated with luteal cells death during regression of the corpus luteum (CL) in some species. However, the involvement of autophagy or the association between autophagy and apoptosis in CL regression are largely unknown. Therefore, we investigated the role of autophagy in CL regression and its association with apoptosis. Ovaries were obtained from pseudopregnant rats at Days 2 (early), 7 (mid-), and 14 and 20 (late-luteal stage) of the pseudopregnancy; autophagy-associated protein (microtuble-associated protein light chain 3 [LC3]) was immunolocalized and its expression level was measured. Luteal cell apoptosis was evaluated by measuring cleaved caspase 3 expression. LC3 expression increased slightly from early to mid-luteal stage, with maximal levels detected at the late-luteal stage in steroidogenic luteal cells. The expression level of the membrane form of LC3 (LC3-II) also increased during luteal stage progression, and reached a maximum at the end point of late-luteal stage (Day 20). This pattern coincided with cleaved caspase 3 expression. Furthermore, LC3-II expression increased, as did levels of cleaved caspase 3 in luteal cells cultured with prostaglandin F(2alpha) known to induce CL regression. These findings suggest that luteal cell autophagy is directly involved in CL regression, and is correlated with increased apoptosis. In addition, autophagic processes were inhibited using 3-methyladenine or bafilomycin A1 to evaluate the role of autophagy in apoptosis induction. Inhibition of autophagosome degradation by fusion with lysosomes (bafilomycin A1) increased apoptosis and cell death. Furthermore, inhibition of autophagosome formation (3-methyladenine) decreased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces luteal cell apoptosis. In conclusion, these results indicate that autophagy is involved in rat luteal cell death through apoptosis, and is most prominent during CL regression.     

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.     

线粒体在细胞凋亡中的变化与作用

要在各种凋亡信号的诱导下,线粒体会发生显著的结构与功能性的变化,包括各种促凋亡蛋白(如细胞色素c,凋亡诱导因子等）的释放,线粒体膜电位的丢失,电子传递链的变化,以及细胞内氧化还原状态的变化;核转录因子以线粒体为中介也参与了细胞凋亡的调控。线粒体在哺乳动物细胞凋亡中具有核心地位和作用,昆虫细胞凋亡的研究表明,线粒体与昆虫细胞凋亡也有密切的关系。线粒体在细胞凋亡中的作用可能具有普遍意义。     

Functional role of caspases in sphingosine-induced apoptosis in human hepatoma cells

We have previously shown that sphingosine increased caspase-3 activity and induced apoptosis in human hepatoma cells. Our data also suggest that other caspases may be involved in sphingosine-triggered apoptosis. In order to clarify this issue, we used different approaches to study the functional role of several initiator or executioner caspases in apoptosis induced by sphingosine. Activation of procaspases-2, -7, and -8, was clearly demonstrated during sphingosine-triggered apoptosis. Pretreatment with chemical inhibitors for caspase-7 and -8, attenuated apoptotic cell death induced by sphingosine. Conversely, pretreatment with specific caspase-2 inhibitor Z-VDVAD-FMK did not show any protective effect. In addition, enforced expression of constitutively activated AKT kinase which is known to inhibit apoptosis induced by sphingosine, potently suppressed activation of procaspases-7 and -8. In summary, these data suggest that in addition to caspases-3, caspase-7 and -8 are involved in the apoptosis induced by sphingosine.     

Apoptosis and autophagy in polycystic kidney disease (PKD)

Apoptosis in the cystic epithelium is observed in most rodent models of polycystic kidney disease (PKD) and in human autosomal dominant PKD (ADPKD). Apoptosis inhibition decreases cyst growth, whereas induction of apoptosis in the kidney of Bcl-2 deficient mice increases proliferation of the tubular epithelium and subsequent cyst formation. However, alternative evidence indicates that both induction of apoptosis as well as increased overall rates of apoptosis are associated with decreased cyst growth. Autophagic flux is suppressed in cell, zebra fish and mouse models of PKD and suppressed autophagy is known to be associated with increased apoptosis. There may be a link between apoptosis and autophagy in PKD. The mammalian target of rapamycin (mTOR), B-cell lymphoma 2 (Bcl-2) and caspase pathways that are known to be dysregulated in PKD, are also known to regulate both autophagy and apoptosis. Induction of autophagy in cell and zebrafish models of PKD results in suppression of apoptosis and reduced cyst growth supporting the hypothesis autophagy induction may have a therapeutic role in decreasing cyst growth, perhaps by decreasing apoptosis and proliferation in PKD. Future research is needed to evaluate the effects of direct autophagy inducers on apoptosis in rodent PKD models, as well as the cause and effect relationship between autophagy, apoptosis and cyst growth in PKD.     

Tamoxifen modulates apoptosis in multiple modes of action in CreER mice

Tamoxifen‐inducible Cre (CreER) has become a powerful tool for in vivo manipulation of the genome. Here, we investigated opposing effects of tamoxifen on apoptosis during embryogenesis using Olig2–CreER knock‐in mice, namely, tamoxifen‐induced apoptosis through CreER‐mediated toxicity and cytoprotective activity of tamoxifen independent of CreER. First, we examined tamoxifen‐induced apoptosis; in the homozygous mice, we observed region‐specific apoptosis in the ventral neural tube, with no obvious increase in the heterozygotes. Next, we detected a cytoprotective effect on apoptosis in the homozygous dorsal root ganglia (DRG). This apoptosis is a secondary phenotype of Olig2‐null mice, as Olig2/CreER is not expressed in the DRG. The cytoprotective effect is DRG‐specific, because tamoxifen did not rescue apoptosis in the interdigital mesenchyme. These data indicate that tamoxifen has multiple effects on apoptosis during development and caution that careful examination is necessary when interpreting results obtained from tamoxifen‐induced recombination: in Olig2‐CreER mice, heterozygotes are usable for lineage‐tracing experiment without obvious toxicity, while homozygotes show efficient recombination, despite enhanced apoptosis. genesis 46:775–781, 2008. © 2008 Wiley‐Liss, Inc.     

Phenoptosis in yeasts

The current view on phenoptosis and apoptosis as genetic programs aimed at eliminating potentially dangerous organisms and cells, respectively, is given. Special emphasis is placed on apoptosis (phenoptosis) in yeasts: intracellular defects and a plethora of external stimuli inducing apoptosis in yeasts; distinctive morphological and biochemical hallmarks accompanying apoptosis in yeasts; pro- and antiapoptotic factors involved in yeast apoptosis signaling; consecutive stages of apoptosis from external stimulus to the cell death; a prominent role of mitochondria and other organelles in yeast apoptosis; possible pathways for release of apoptotic factors from the intermembrane mitochondrial space into the cytosol are described. Using some concrete examples, the obvious physiological importance and expediency of altruistic death of yeast cells is shown. Poorly known aspects of yeast apoptosis and prospects for yeast apoptosis study are defined.     

Role of elevated pressure in TRAIL-induced apoptosis in human lung carcinoma cells

TNF-related apoptosis-inducing ligand (TRAIL, Apo2L) is a promising anticancer agent with high specificity for cancer cells. Many strategies have been proposed to enhance the sensitivity of cancer cells to TRAIL-mediated apoptosis, including the use of combination treatment with conventional cancer therapies. However, few reports have evaluated the effects of TRAIL in combination with mechanical stress, which can also cause apoptosis of cancer cells. In the present study, we describe a custom-designed culture system that delivers two atmospheres of elevated pressure (EP) by using compressed air, and which enhances the sensitivity of cancer cells to TRAIL-mediated apoptosis. The combination of TRAIL and EP significantly increased apoptosis of human H460 lung cancer cells more than hyperbaric normoxia or normobaric mild hyperoxia. EP-potentiating TRAIL-mediated apoptosis of H460 cells was accompanied by up-regulated death receptor 5 (DR5), activation of caspases, decreased mitochondrial membrane potential, and reactive oxygen species production. We also observed EP-induced sensitization of TRAIL-mediated apoptosis in other cancer cell types. In contrast, human normal cells showed no DNA damage or cell death when exposed to the combined treatment. In a chicken chorioallantoic membrane model, EP enhanced TRAIL-mediated apoptosis of tumors that developed from transplanted H460 cells. Collectively, EP enhanced TRAIL-induced apoptosis of human lung carcinoma cells in vitro and in vivo. These findings suggest that EP is a mechanical and physiological stimulus that might have utility as a sensitizing tool for cancer therapy.     

Apoptotic pathways in adipose tissue

To treat the ever growing number of obese patients, reduction of adipocyte number by apoptosis may complement other therapeutic options. On the other hand in free fat grafts, apoptosis along with necrosis is responsible for long term volume reduction. To ensure successful soft tissue reconstruction it is mandatory to keep apoptosis on a low level in adipocytes, adipose-derived stromal cells and others cells of the fat graft. Apoptotic pathways have been sufficiently studied in various tissues, but the knowledge about apoptotic pathways in adipocytes is surprisingly scarce. Current knowledge about apoptotic pathways in adipose tissue is elaborately reflected in this review as well as the association of cancer with obesity. Possibilities to induce and reduce adipose tissue apoptosis in animal models are discussed as well as clinical implications of fat cell apoptosis. Mechanisms of apoptosis induction have been studied in animal models and suggest that a tight control of apoptosis induction is necessary because otherwise detrimental metabolic effects of fat mass loss will occur that may mimic lipodystrophic diseases. At present, targeted induction of adipocyte apoptosis appears to be of some concern related to increased blood lipid concentrations, ectopic lipid storage and other detrimental metabolic effects. Treatment of autologous adipocytes used for lipofilling procedures with appropriate substances may result in more satisfactory long-term outcomes as well as stimulation of stem cell differentiation in a strictly local manner.