The N-terminal internal region of BLM is required for the formation of dots/rod-like structures which are associated with SUMO-1

Chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than caspases 3 or 8 (Glaser et al., Oncogene 18, 5044-5053, 1999). Here, we report that caspases 1, 2, 3, 7, 8, and 9 are constitutively expressed in most human malignant glioma cell lines. Cytotoxic drug-induced apoptosisinvolves delayed activation of caspases 2, 7, and 9, but not 8 and 3, and is blocked by a broad spectrum caspase inhibitor, zVAD-fmk. Cytochrome c release from mitochondria precedes caspase activation during drug-induced apoptosis and is unaffected by zVAD-fmk or ectopic expression of the viral caspase inhibitor, crm-A. In contrast, ectopic expression of BCL-X(L) prevents drug-induced cytochrome c release, caspase activation and cell death. Thus, cancer chemotherapy targets the mitochondrial, caspase-dependent death pathway in human malignant glioma cells.     

Role of caspase‐9 in the effector caspases and genome expressions,and growth of bovine skeletal myoblasts

Caspase‐9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase‐9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase‐9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase‐9 mRNA expression significantly. Particularly, the mRNA expression level of caspase‐9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase‐9 led to the significant increases in the mRNA and protein expressions of effector caspase‐3, whereas the reduction in mRNA and protein expressions of caspase‐7. The microarray results showed that the suppression of caspase‐9 resulted in significant upregulations of cell proliferation‐, adhesion‐, growth‐, development‐ and division‐regulating genes, whereas the reduction in the expressions of cell death program‐ and stress response‐regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase‐9 could play an important role in the control of cell growth, and knockdown of caspase‐9 may have genuine potential in the treatment of skeletal muscle atrophy.     

2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases

2-Methoxyoestradiol (2-MeOE2) is an endogenous oestrogen metabolite which inhibits tubulin polymerisation and has anti-tumour and anti-angiogenic activity. 2-MeOE2 induces apoptosis in a wide range of cancer cell types and has recently been demonstrated to cooperate with TRAIL to induce apoptosis in breast cancer cells. 2-Methoxyoestradiol-3,17-bis-O,O-sulphamate (2-MeOE2bisMATE) is a sulfamoylated derivative of 2-MeOE2 with enhanced activity and improved pharmacokinetic properties, and 2-MeOE2bisMATE is a promising candidate for early clinical trials. It is important, therefore, to understand the mechanisms by which 2-MeOE2bisMATE acts, and whether it retains the ability to cooperate with TRAIL. We demonstrate that 2-MeOE2bisMATE-induced apoptosis of CAL51 breast cancer cells was associated with rapid activation of caspase 3 and 9, but not caspase 8 (as measured by BID cleavage) and was completely prevented by the caspase inhibitor zVADfmk. Interfering with Fas- or TRAIL-receptor function did not prevent 2-MeOE2bisMATE-induced apoptosis. Whereas CAL51 cells were resistant to TRAIL-induced apoptosis, 2-MeOE2bisMATE and TRAIL cooperated to induce cell death. This apoptosis was associated with enhanced activation of caspases, but not increased expression of the DR5 TRAIL receptor, previously demonstrated to be induced by 2-MeOE2. Therefore, 2-MeOE2bisMATE-induced apoptosis is dependent on caspases and like 2-MeOE2, 2-MeOE2bisMATE can overcome resistance to TRAIL by stimulating activation of downstream caspases. Our results suggest that 2-MeOE2bisMATE and TRAIL might be a particularly effective combination of anti-cancer agents.     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.     

Expression of selected apoptosis related genes,MIF, IGIF and TNF alpha,during retinoic acid-induced neural differentiation in murine embryonic stem cells

Apoptosis plays an important role during embryonic development. Apoptotic cell death is executed by caspases and can be regulated by the Bcl-2 family of genes. Ribonuclease protection assay was used to investigate the expression of selected apoptosis-related genes of the Bcl-2 family, pro-apoptotic Bax, Bad and anti-apoptotic Bcl-2, during differentiation of murine embryonic stem cells (ES) mediated by all-trans-retinoic acid. The mRNA expression of caspase 3, caspase 6 and certain pro-inflammatory cytokines was also investigated simultaneously. ES cells exposed to 1 microM all-trans-retinoic acid on day 8, 9 and 10 of differentiation revealed increased expression of Bax and Bad compared to the vehicle-treated cells. No effect on Bcl-2 mRNA was noted after all-trans-retinoic acid treatment. Increased mRNA expression of caspase 3 and caspase 6 in all-trans-retinoic acid-exposed ES cells suggested that caspases play an important role in retinoic acid-mediated apoptosis during ES differentiation. Increase in the expression of TNF alpha and macrophage migration inhibitory factor (MIF) was noted in retinoic acid-treated cells on day 14. Significant increase observed in interferon gamma inducing factor (IGIF/IL-18) mRNA expression in all-trans-retinoic acid-treated cells on day 14 and 17 did not translate to increased INF gamma expression. No change in the expression of other pro-inflammatory cytokines was noted with all-trans-retinoic acid treatment. The function of TNF alpha, IGIF/IL-18 and MIF in all-trans-retinoic acid-treated cells during ES differentiation and apoptosis is still speculatory. Results suggested that RA-mediated apoptosis during neural differentiation of ES cells involves up-regulation of caspase 3, caspase 6, Bad, and Bax.     

Targeting apoptotic caspases in cancer

Members of the caspase family of proteases play essential roles in the initiation and execution of apoptosis. These caspases are divided into two groups: the initiator caspases (caspase-2, -8, -9 and -10), which are the first to be activated in response to a signal, and the executioner caspases (caspase-3, -6, and -7) that carry out the demolition phase of apoptosis. Many conventional cancer therapies induce apoptosis to remove the cancer cell by engaging these caspases indirectly. Newer therapeutic applications have been designed, including those that specifically activate individual caspases using gene therapy approaches and small molecules that repress natural inhibitors of caspases already present in the cell. For such approaches to have maximal clinical efficacy, emerging insights into non-apoptotic roles of these caspases need to be considered. This review will discuss the roles of caspases as safeguards against cancer in the context of the advantages and potential limitations of targeting apoptotic caspases for the treatment of cancer.     

Targeted suppression of μ‐calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.     

Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis

Background and Aims. H. pylori infection results in an increased epithelial apoptosis in gastritis and duodenal ulcer patients. We investigated the role and type of activation of caspases in H. pylori‐induced apoptosis in gastric epithelial cells. Methods. Differentiated human gastric cancer cells (AGS) and human gastric mucous cell primary cultures were incubated with H. pylori for 0.5–24 hours in RPMI 1640 medium, and the effects on cell viability, epithelial apoptosis, and activity of caspases were monitored. Apoptosis was analyzed by detection of DNA‐fragments by Hoechst stain®, DNA‐laddering, and Histone‐ELISA. Activities of caspases were determined in fluorogenic assays and by Western blotting. Cleavage of BID and release of cytochrome c were analyzed by Western blot. Significance of caspase activation was investigated by preincubation of gastric epithelial cells with cell permeable specific caspase inhibitors. Results. Incubation of gastric epithelial cells with H. pylori caused a time and concentration dependent induction of DNA fragmentation (3‐fold increase), cleavage of BID, release of cytochrome c and a concomittant sequential activation of caspase‐9 (4‐fold), caspase‐8 (2‐fold), caspase‐6 (2‐fold), and caspase‐3 (6‐fold). No effects on caspase‐1 and ‐7 were observed. Activation of caspases preceded the induction of DNA fragmentation. Apoptosis could be inhibited by prior incubation with the inhibitors of caspase‐3, ‐8, and ‐9, but not with that of caspase‐1. Conclusions. Activation of certain caspases and activation of the mitochondrial apoptotic pathway are essential for H. pylori induced apoptosis in gastric epithelial cells.     

Cycloxygenase-2 is Essential for the Survival and Proliferation of Gastric Cancer Cells

Cycloxygenase-2 catalyzes the synthesis of prostaglandins from arachidonic acid and this enzyme has been implicated in the metastasis of gastric cancer. In order to examine the significance of cycloxygenase-2 (Cox-2) in the survival and proliferation of gastric cancer cells, we have stably overexpressed an antisense Cox-2 in two gastric cancer cell lines, SGC7901 and AGS, in order to reduce the expression of this protein. The sense and antisense Cox-2 expression vectors were created by cloning COX-2 cDNA, in pIRES2-EGFP plasmid. Cox-2 gene expression was monitored by RT-PCR and Western blotting and the results indicated that cells with antisense Cox-2 construct had significantly reduced Cox-2 expression in comparison to the cells that received sense-Cox-2 plasmid. Reduction of Cox-2 expression in SGC7901 and AGS gastric cancer cells led to markedly decreased proliferation. The metastatic capability of the two cell lines, as assessed by in vitro colony formation assay, is also significantly compromised by lowered Cox-2 expression. Thus, this study demonstrates that Cox-2 activity is necessary for the proliferation and metastasis of gastric cancer cells.     

Regulation of interferon and retinoic acid-induced cell death activation through thioredoxin reductase

Interferons (IFNs) and retinoids are potent biological response modifiers. The IFN-beta and all-trans-retinoic acid combination, but not these single agents individually, induces death in several tumor cell lines. To elucidate the molecular basis for these actions, we have employed an antisense knockout approach to identify the gene products that mediate cell death and isolated several genes associated with retinoid-IFN-induced mortality (GRIMs). One of the GRIM cDNAs, GRIM-12, was identical to human thioredoxin reductase (TR). To define the functional relevance of TR to cell death and to define its mechanism of death-modulating functions, we generated mutants of TR and studied their influence on the IFN/RA-induced death regulatory functions of caspases. Wild-type TR activates cell death that was inhibited in the presence of caspase inhibitors or catalytically inactive caspases. A mutant TR, lacking the active site cysteines, inhibits the cell death induced by caspase 8. IFN/all-trans-retinoic acid-induced cytochrome c release from the mitochondrion was promoted in the presence of wild type and was inhibited in the presence of mutant TR. We find that TR modulates the activity of caspase 8 to promote death. This effect is in part caused by the stimulation of death receptor gene expression. These studies identify a new mechanism of cell death regulation by the IFN/all-trans-retinoic acid combination involving redox enzymes.     

1,25 dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells.

Commitment of members of the monocyte/macrophage family to the bone resorptive phenotype, in vitro, requires contact, of these osteoclast precursors, with osteoblasts or related stromal cells. The osteoclast-inductive properties of these stromal cells are typically expressed, however, only in the presence of steroid hormones such as 1,25 dihydroxyvitamin D (1,25D3) and dexamethasone (DEX). To gain insight into the means by which steroid treated accessory cells induce osteoclast differentiation we asked, using differential RNA display (DRD), if gene expression by this stromal cell population differs from that of their untreated, non-osteoclastogenic counterpart. We identified four known genes specifically expressed by 1,25D3/DEX-treated ST2 stromal cells: 1) a family of rat organic anion transporters, 2) Na/K ATPase ss-subunit, 3) tazarotene-induced gene 2 (TIG2), and 4) prostaglandin G/H synthase I, or cyclooxygenase 1 (Cox-1). The regulation of these genes in 1,25D3/DEX-treated ST2 cells was demonstrated by Northern blot analysis of treated (osteoclast-supporting) and untreated (non-osteoclast-supporting) ST2 cells; the genes have a limited and specific tissue mRNA expression pattern. Northern blot analysis of treated and untreated ST2 cell total RNA using either a DRD-derived Cox-1 cDNA or a Cox-1 specific oligonucleotide confirmed the steroid regulation of Cox-1 mRNA. Surprisingly, there is no detectable expression by untreated or steroid exposed ST2 cells, of Cox-2, the classical regulated cyclooxygenase isoform. In contrast to 1, 25D3/DEX, serum treatment rapidly induces Cox-2 mRNA, substantiating the capacity of ST2 cells to express the gene. These data establish that steroid induction of the osteoclastogenic properties of stromal cells is attended by Cox gene expression, a phenomenon consistent with the capacity of eicosinoids to impact the resorptive process. The response of osteoclast-supporting ST2 cells to 1,25D3/DEX treatment may be one prostaglandin-mediated event which specifically involves Cox-1 regulation.     

Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent manner.

Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4-1BB, which is known to initiate a signal cascade activating NF-kappaB, was significantly up-regulated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF-kappaB reporter luciferase, even in the presence of the 4-1BB-Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4-1BB. Furthermore, up-regulation of 4-1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4-1BB serves as the mediator of signals from T3 to activate caspases.     

Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.     

Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia

Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemia-reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.     

Proteases for cell suicide: functions and regulation of caspases.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.