Genetic variants and haplotypes of the caspase-8 and caspase-10 genes contribute to susceptibility to cutaneous melanoma

Caspase-8 (CASP8) and caspase-10 (CASP10) play key roles in regulating apoptosis, and their functional polymorphisms may alter apoptosis and cancer risk. However, no reported studies have investigated the association between such polymorphisms and the risk of cutaneous melanoma (CM). In a hospital-based study of 805 non-Hispanic white patients with CM and 835 cancer-free age-, sex-, and ethnicity-matched controls, we genotyped three reported putatively functional polymorphisms of CASP8 and CASP10-CASP8 D302 H (rs1045485:G>C), CASP8 -652 6N del (rs3834129:-/CTTACT), and CASP10 I522L (rs13006529:A>T)-and assessed their associations with risk of CM and interactions with known risk factors for CM. We also calculated the false-positive report probability (FPRP) for significant findings. CASP8 302 H variant genotypes (DH: adjusted odds ratio [OR], 0.70; 95% confidence interval [CI], 0.50-0.98; DH+HH: unadjusted OR, 0.78; 95% CI, 0.62-0.98; FPRP, 0.79) and CASP8 -652 6N del variant genotypes (ins/del: OR, 0.74; 95% CI, 0.57-0.97; ins/del+del/del: OR, 0.76; 95% CI, 0.61-0.95; FPRP, 0.61) were associated with significantly lower CM risk than were the DD and ins/ins genotypes, respectively. However, the CASP10 522L variant genotypes were not associated with significantly altered CM risk. Also, the D-del-I haplotype was associated with a significantly lower CM risk (OR, 0.52; 95% CI, 0.37-0.74; FPRP, 0.04) than was the most common haplotype, D-ins-I. Furthermore, multivariate logistic regression analysis revealed that CASP8 D302 H, CASP8 -652 6N del, and CASP10 I522L were independent risk factors for CM. Therefore, these CASP8 and CASP10 polymorphisms may be biomarkers for susceptibility to CM.     

Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site

The sequence of the spike (also called peplomer or E2) protein gene of the Mebus strain of bovine coronavirus (BCV) was obtained from cDNA clones of genomic RNA. The gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an N-terminal hydrophobic signal sequence of 17 amino acids, 19 potential N-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the C terminus, and a hydrophilic cysteine-rich C terminus of 35 amino acids. An internal Lys-Arg-Arg-Ser-Arg-Arg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the S apoprotein into S1 and S2 segments of 85690 and 65153 mol wt, respectively. Amino terminal amino acid sequencing of the virion-derived gp 100 spike subunit confirmed the location of the predicted cleavage site, and established that gp 120 and gp 100 are the glycosylated virion forms of the S1 and S2 subunits, respectively. Sequence comparisons between BCV and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (S1) than in the stem region (S2).     

Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity

The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.     

浙江地区汉族人群caspase-8、-10基因四个单核苷酸多态性位点的单倍型研究

目的分析汉族人群caspase-8、-10基因的单核苷酸多态性（single nucleotide polymorphisins，SNPs）位点及其构成的单倍型，为研究caspase-8、-10基因与多基因复杂疾病的关联分析奠定基础。方法采用PCR、变性高效液相色谱技术和DNA测序技术检测caspase-10基因的第2．5外显子，caspase-8基因的第8-10外显子及其部分侧翼序列的多态性位点；分析配对位点的连锁不平衡关系，最大期望值法估算它们构成的单倍型。结果（1）caspase-10基因的第2、5外显子分别检出一个SNP位点A2823G和A12799G，其中A12799G是新发现的低信息度的SNP；caspase-8基因中检测到3个SNP位点A43466G、G51484A、G52951A，分别位于第8、9外显子和第9内含子；它们均未改变所编码蛋白的一级结构；（2）caspase-10基因中A2823G位点与caspase-8基因中3个位点间已达到连锁平衡，caspase-8基因中A43466G与G52951A、G51484A与G52951A也达到连锁平衡，连锁不平衡系数分别接近于0；只有A43466G与G51484A存在强的连锁不平衡，连锁不平衡系数接近于1；（3）caspase-10基因的A2823G位点与caspase-8基因的3个位点预计产生11种单倍型，其中A-2823／A-43466／G-51484／G-52951是主要单倍型，频率为0．381l，其次是A．2823／A-43466／G-51484／A-52951，频率为0．2536；这4个SNP位点联用，多态信息含量可达到0．7106。结论浙江地区汉族人群caspase-10、-8基因中的SNP位点至少处于3个不同的单倍型块；联合多个相邻的位点构成单倍型，可以弥补单个SNP信息度较低的不足。     

Caspase-8 and caspase-10 activate NF-kappaB through RIP,NIK and IKKalpha kinases

NF-kappaB regulates the expression of various genes involved in cell growth and differentiation, immune response and inhibition of apoptosis. Recently, some death effector domain (DED)-containing proteins, such as FADD and c-FLIP were reported to activate NF-kappaB. We previously reported that the prodomain-only isoforms of caspase-8 and -10 (PDCasp8/10), containing two DED motifs, could inhibit Fas-mediated apoptosis. Here, we demonstrate that these isoforms also activate NF-kappaB, implying this to be one of the mechanisms by which these polypeptides inhibit apoptosis. The GST pull-down assay revealed that, among upstream kinases that activate NF-kappaB, only NIK and RIP, but not RICK or IKKalpha/beta, could directly bind to PDCasp8/10. In addition, both modules ofDED in PDCasp8/10 were required for these interactions as well as NF-kappaB activation. Experiments using a kinase-dead mutant of IKKalpha and an RIP mutant lacking a kinase domain, both of which function as dominant-negative mutants for their wild-type counterparts, blocked PDCasp8/10-mediated NF-kappaB activation. Using small interfering RNA technology, we further demonstrate that the down-regulation of IKKalpha but not IKKbeta significantly inhibits PDCasp8-mediated NF-kappaB activation. Taken together, these results suggest that caspase-8 and -10 have roles in a non- or anti-apoptotic signaling pathway leading to NF-kappaB activation through RIP, NIK and IKKalpha.     

Virus infection induces proteolytic processing of IL-18 in human macrophages via caspase-1 and caspase-3 activation

There is increasing evidence that IL-18 is a key pro-inflammatory cytokine and an important mediator of Th1 immune response. The main source of IL-18 is macrophage-like cells. In the present study we have investigated IL-18 protein expression in primary human macrophages in response to influenza A and Sendai virus infections. Macrophages constitutively expressed proIL-18 but produced biologically active IL-18 only after virus infection. The IL-18 release was due to virus infection-induced proteolytic processing of 24-kDa proIL-18 into its mature 18-kDa form. ProIL-18 processing required active caspase-1 enzyme and the release of mature IL-18 was blocked with a caspase-1-specific inhibitor. Caspase-3 inhibitor also reduced IL-18 production in response to virus infection. Inactive proforms of caspase-1 and caspase-3 were basally expressed in macrophages, and virus infection induced the cleavage of procaspases into their mature forms. Besides increasing the expression of caspase proteins, virus infection enhanced caspase mRNA expression in macrophages. The enhancement of caspase gene expression was abrogated by anti-IFN-alpha antibody. Furthermore, IFN-alpha and IFN-gamma could induce caspase gene expression. These results imply that interferons are involved in virus-induced caspase activation that leads to proIL-18 processing and subsequent release of mature IL-18.     

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.     

Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration

We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly activates caspase-3 in PC12 cells. Finally, overexpression of the kinase-inactive PKCdelta(K376R) mutant in rat mesencephalic dopaminergic neuronal cells attenuates dieldrin-induced caspase-3 activity and DNA fragmentation, further confirming the pro-apoptotic function of PKCdelta in dopaminergic cells. Together, we conclude that caspase-3-dependent proteolytic activation of PKCdelta is a critical event in dieldrin-induced apoptotic cell death in dopaminergic cells.     

Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells

Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.     

Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues

Gel-forming mucins are major contributors to the viscoelastic properties of mucus secretion. Currently, four gel-forming mucin genes have been identified: MUC2, MUC5AC, MUC5B, and MUC6. All these genes have five major cysteine-rich domains (four von Willebrand factor [vWF] C or D domains and one Cystine-knot [CT] domain) as their distinctive features, in contrast to other non-gel-forming type of mucins. The CT domain is believed to be involved in the initial mucin dimer formation and have very succinct relationship between different gel-forming mucins across different species. Because of gene duplication and evolutional modification, it is very likely that other gel-forming mucin genes exist. To search for new gel-forming mucin candidate genes, a "Hidden Markov Model"(HMM) was built from the common features of the CT domains of those gel-forming mucins. By using this model to screen all protein databases as well as the six-frame translated expression sequence tag and translated human genomic databases, we identified a locus located at the peri-centromere region of human chromosome 12 and the corresponding homologous region of mouse chromosome 15. We cloned the 3' end of this gene and its mouse homolog. We found one vWF C domain, one CT domain, and various mucin-like threonine/serine-rich repeats. Phylogenetic analysis indicated the close relationship between this gene and the submaxillary mucin from porcine and bovine. A polydispersed signal was observed on the Northern blot, which indicates very large mRNA size. Further analysis of the upstream genomic sequences generated from human and mouse genome projects revealed three additional vWF D domains and many mucin-like threonine/serine-rich repeats. The expression of this gene is restricted to the mucous cells of various glandular tissues, including sublingual gland, submandibular gland, and submucosal gland of the trachea. Based on the chronological convention, we have given the name MUC19 to the human ortholog and Muc19 to the mouse.     

Murine coronavirus-induced apoptosis in 17Cl-1 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and bid cleavage

Mouse hepatitis virus (MHV) infection in murine 17Cl-1 cells results in apoptotic cell death. Inhibition of MHV-induced apoptosis by the pancaspase inhibitor Z-VAD-FMK promoted virus production late in infection, indicating that apoptosis could be a host response to limit the production of viral progeny. Activation of the mitochondria-mediated apoptotic pathway was indicated by the activation of caspase-9 and delay of apoptosis by Bcl-2 overexpression. Analyses of the subcellular distribution of cytochrome c, procaspase-9, and Apaf-1 suggested an aberrant apoptosome formation in the vicinity of the mitochondria, which could be a cell type-specific event. An increase in the amount of Fas (APO-1/CD95), caspase-8 activation, caspase-8-mediated Bid cleavage, and subsequent translocation of truncated Bid to mitochondria, all of which relate to the Fas-mediated pathway, also occurred in MHV-infected 17Cl-1 cells, whereas the formation of the death-inducing signaling complex, a direct indication of the activation of Fas-mediated pathway, was undetectable. Caspase-8 and Bid activation appeared to be downstream of mitochondria, because Bcl-2 overexpression suppressed both events, suggesting that infected 17Cl-1 cells might have activated a receptor-mediated "type II" signaling pathway, in which primary and low levels of receptor-mediated pathway activation lead to the activation of the mitochondria-mediated pathway. All our data indicate that a mitochondria-mediated pathway played a major regulatory role in apoptosis in MHV-infected 17Cl-1 cells.     

姜黄素对Aβ_(25-35)诱导的PC12细胞caspase-3、caspase-8和caspase-9表达的影响

目的:探讨姜黄素对β淀粉样蛋白25-35(Aβ_(25-35))诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞存活率、乳酸脱氢酶(LDH)释放率及凋亡的影响。方法:以Aβ_(25-35)作用于PC12细胞,分别采用MTT法和LDH试剂盒检测细胞的存活率和LDH释放率;实验随机分为空白对照组、模型组、姜黄素10μmol/L组和姜黄素20μmol/L组,采用流式细胞术及Annexin V-FITC/PI双染法检测凋亡情况,采用比色法和Western blot法检测凋亡相关蛋白胱天蛋白酶(caspase)-3、caspase-8和caspase-9的活性和表达。结果:与模型组比较,姜黄素可显著升高Aβ_(25-35)诱导的PC12细胞存活率,降低LDH释放率和凋亡率(P0.01);同时可显著降低caspase-3、caspase-8和caspase-9的活性和表达(P0.05或P0.01)。结论:姜黄素可显著抑制caspase-3、caspase-8和caspase-9的表达,从而抑制Aβ_(25-35)诱导的PC12细胞凋亡。     

Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors

Caspases are essential initiators and executioners of apoptosis. Changes in their expression may contribute to the development of proliferative disorders such as cancer, by altering the death-proliferation homeostatic balance. The aim of this work was to analyze the expression of a broad panel of caspases at the epithelial level in human prostate tissues to assess possible prostatic disease-related alterations. We comparatively analyzed by immunohistochemistry the expression of pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved caspase-3, cleaved caspase-8, and caspase-7, in normal and pathologic (benign hyperplasic, premalignant [high-grade intraepithelial neoplasia], and cancerous [prostate cancer]) human prostate epithelium. Expression of caspases was correlated with clinicopathologic features, including preoperative prostate-specific antigen levels, Gleason scores, and biochemical progression. Percentage of positive samples for all the analyzed caspases decreased in prostate cancer versus normal prostate epithelium. The values obtained for benign prostatic hyperplasia and high-grade intraepithelial neoplasia more qualitatively resembled those of the prostate cancer group. Our results indicate that caspase expression in prostate malignant cells is reduced in a substantial number of patients and that such an alteration occurs in the premalignant stage. Loss of caspase expression could constitute a useful marker for prostate cancer diagnosis. Therapeutic approaches aimed to recover or enhance caspase expression might be effective against prostate cancer.     

超声对软骨细胞凋亡、caspase-8和caspase-3表达的影响

背景：超声治疗能够缓解疼痛,改善膝骨关节炎患者的运动功能,但目前关于超声治疗的文献缺乏一致性。 目的：进一步验证超声治疗的膝骨关节炎有效性。 方法：24 只兔子随机分为3组,正常组不干预;模型组兔行前交叉韧带离断诱导膝骨关节炎,不接受任何治疗;超声组兔建模后接受超声波治疗10 min/次,1次/d,0.3 W/cm2,1 MHz,共治疗10次。采用苏木精-伊红染色进行兔关节软骨组织学观察,运用免疫印迹和 RT-PCR技术检测兔软骨中的caspases3和caspases8 的表达,TUNEL技术检测兔膝骨关节软骨细胞凋亡率。 结果与结论：正常兔软骨组织软骨细胞呈柱状整齐的排列,模型中软骨层变薄,软骨细胞排列无序而少。超声治疗后软骨细胞重新排列,趋于整齐,细胞数增加。模型组为膝骨关节炎模型;与正常组比较,模型组和超声组改良Mankin 评分较高,模型组和超声组软骨细胞凋亡指数更高,模型组高于超声组。与正常组比较,模型组和超声组caspase-3 和caspase-8表达较高,超声治疗后两项指标表达下降。结果表明,超声可以改善软骨组织结构、降低caspase-3、caspase -8的表达,降低软骨细胞凋亡。提示超声治疗膝骨关节炎是有效的。     

Frequent nuclear localization of ICAD and cytoplasmic co-expression of caspase-8 and caspase-3 in human lymphomas

Lymphoma cells often display in vitro resistance to FAS-induced apoptosis, in which caspases act as crucial cell death effectors. Following FAS stimulation, caspase-8 activates caspase-3, which in turn activates the caspase-activated DNAse (CAD) by proteolysis of its inhibitor (ICAD). To investigate the mechanism of FAS resistance, the expression of caspase-8 was analysed by immunohistochemistry, together with that of the substrates caspase-3 and ICAD, in 52 representative samples from non Hodgkin's lymphoma (NHL), 12 from Hodgkin's disease (HD), and eight benign lymphoid tissues. In benign tissues, caspase-8 was co-expressed with caspase-3 in the cytoplasm in germinal centre (GC) cells and was co-expressed with ICAD in the nuclei of the mantle and marginal zone cells. ICAD expression was weak or absent in GC cells. Cytoplasmic staining for both caspase-8 and caspase-3 was present in 11/12 cases of diffuse large cell B-NHL. Caspase-8 positivity was nuclear and cytoplasmic in 9/9 follicular NHLs, in 5/5 mantle cell NHLs and in 6/6 marginal zone NHLs. Five out of six peripheral T-cell NHLs expressed cytoplasmic caspase-8. Ten out of the 12 HD cases lacked significant cytoplasmic staining for caspase-3 and caspase-8 in the majority of Reed-Sternberg cells. All lymphoma cases exhibited predominant nuclear ICAD positivity. Subcellular fractionation analysis of three lymphoma samples and normal mantle zone cells confirmed that ICAD and caspase-8 were at least partly localized in the nucleus. These results show that the profile of caspase-8 expression is correlated with histological lymphoma subtypes; that caspase-8 is co-expressed with caspase-3 in GC cells and their neoplastic counterparts; that ICAD has an immunohistochemical nuclear localization in vivo; and that caspase-8 and ICAD can be co-expressed in the nuclei of mantle zone and marginal zone cells; their unexpected nuclear localization allows a reappraisal of the biochemical cascade of caspase activation.