06-735 Sigma-AldrichAnti-Caspase 3 Antibody

The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

Modulation of the cellular response by the administration of probiotic bacteria may be an effective strategy for preventing or inhibiting tumour growth. We orally pre-inoculated mice with probiotics Lactobacillus acidophilus NCFM (La) for 14 d. Subcutaneous dorsal-flank tumours and segmental orthotopic colon cancers were implanted into mice using CT-26 murine colon adenocarcinoma cells. On day 28 after tumour initiation, the lamina propria of the colon, mesenteric lymph nodes (MLN) and spleen were harvested and purified for flow cytometry and mRNA analyses. We demonstrated that La pre-inoculation reduced tumour volume growth by 50·3 %, compared with untreated mice at 28 d after tumour implants (2465·5 (SEM 1290·4) v. 4950·9 (SEM 1689·3) mm³, Pless than 0·001). Inoculation with La reduced the severity of colonic carcinogenesis caused by CT-26 cells, such as level of colonic involvement and structural abnormality of epithelial/crypt damage. Moreover, La enhanced apoptosis of CT-26 cells both in dorsal-flank tumour and segmental orthotopic colon cancer, and the mean counts of apoptotic body were higher in mice pre-inoculated with La (Pless than 0·05) compared with untreated mice. La pre-inoculation down-regulated the CXCR4 mRNA expressions in the colon, MLN and extra-intestinal tissue, compared with untreated mice (Pless than 0·05). In addition, La pre-inoculation reduced the mean fluorescence index of MHC class I (H-2Dd, -Kd and -Ld) in flow cytometry analysis. Taken together, these findings suggest that probiotics La may play a role in attenuating tumour growth during CT-26 cell carcinogenesis. The down-regulated expression of CXCR4 mRNA and MHC class I, as well as increasing apoptosis in tumour tissue, indicated that La may be associated with modulating the cellular response triggered by colon carcinogenesis.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.

Necrosis and apoptosis are well established as two primary cell death pathways. Mixed neuroglial cultures are commonly used to study cell death mechanisms in neural cells. However, the ages of these cultures vary across studies and little attention has been paid to how cell death processes may change as the cultures mature. To clarify whether neuroglial culture age affects cell death mechanisms, we treated 1- and 3-week-old neuroglial cultures with either the excitotoxic stimulus, N-methyl-D-aspartate (NMDA), or with the oxidative stressor, hydrogen peroxide (H2O2). Although NMDA is known to be toxic only in cultures that are at least 2 weeks old, H2O2 is toxic in cultures of all ages. Here, we confirm that, in 1-week-old neuroglial cultures, NMDA does not induce toxicity, whereas H2O2 induces both calpain-mediated and caspase-mediated neuronal death. In 3-week-old cultures, both NMDA and H2O2 trigger calpain-mediated, but not caspase-mediated, neuronal death. Further, we observed a decrease in caspase-3 levels and an increase in calpain levels in untreated neuroglial cultures as they aged. The findings presented here show that neuronal cell death mechanisms vary with culture age and highlight the necessity of considering culture age when interpreting neural cell culture data.

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene NTRK3 yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of NTRK3 are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system.We identify one microRNA (miR-151-3p) that represses the full-length isoform of NTRK3 and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated NTRK3, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor BCL2.Our results show that the regulation of NTRK3 by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.

The ability of the central nervous system (CNS) to generate innate immune responses was investigated in an in vitro model of CNS infection. Cultures containing CNS cells were infected with mouse hepatitis virus-JHM, which causes fatal encephalitis in mice. Immunostaining indicated that viral infection had a limited effect on culture characteristics, overall cell survival, or cell morphology at the early postinfection times studied. Results from Affymetrix gene array analysis, assessed on RNA isolated from virally and sham-infected cultures, were compared with parallel protein assays for cytokine, chemokine, and cell surface markers. Of the 126 transcripts found to be differentially expressed between viral and sham infections, the majority were related to immunological responses. Virally induced increases in interleukin-6 and tumor necrosis factor alpha mRNA and protein expression correlated with the genomic induction of acute-phase proteins. Genomic and protein analysis indicated that viral infection resulted in prominent expression of neutrophil and macrophage chemotactic proteins. In addition, mRNA expression of nonclassical class I molecules H2-T10, -T17, -M2, and -Q10, were enhanced three- to fivefold in virus-infected cells compared to sham-infected cells. Thus, upon infection, resident brain cells induced a breadth of innate immune responses that could be vital in directing the outcome of the infection and, in vivo, would provide signals which would summon the peripheral immune system to respond to the infection. Further understanding of how these innate responses participate in immune protection or immunopathology in the CNS will be critical in efforts to intervene in severe encephalitis.

Antiandrogens such as Casodex (Bicalutamide) are designed to treat advance stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. Treatment of androgen sensitive, non-metastatic LNCaP human prostate cancer cells with 0-100 microM Casodex or 0-10 ng/ml TNF-alpha induces cell death in 20-60% of the cells by 48 h in a dose-dependent manner. In cells treated with TNF-alpha, this is accompanied by the loss of mitochondrial membrane potential (DeltaPsim) and cell adhesion. In contrast, cells treated with Casodex display loss of cell adhesion, but sustained mitochondrial dehydrogenase activity. Overexpression of Bcl-2 in LNCaP cells attenuates the induction of cell death by TNF-alpha but not Casodex, suggesting that mitochondria depolarization is not required for the induction of cell death by Casodex. While both TNF-alpha and Casodex-induced release of cytochrome c in LNCaP cell is predominantely associated with the translocation and cleavage of Bax, our data also suggest that Casodex induces cell death by acting on components downstream of decline of DeltaPsim and upstream of cytochrome c release. Furthermore, while induction of both caspase-3 and caspase-8 activities are observed in TNF-alpha and Casodex-treated cells, a novel cleavage product of procaspase-8 is seen in Casodex-treated cells. Taken together, these data support the hypothesis that Casodex induces cell death by a pathway that is independent of changes in DeltaPsim and Bcl-2 actions and results in an extended lag phase of cell survival that may promote the induction of an invasive phenotype after treatment.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) induces cell death through a laser light-activated photosensitizer and is a treatment option for tumors resistant to radio- and chemo-therapy. STUDY DESIGN/MATERIALS AND METHODS: We investigated whether m-THPC-PDT induces cell death by necrosis and/or apoptosis, and whether these responses are modulated by p53 and/or ATM, two cancer-associated genes. Sensitivity of atm(+/+)p53(+/+), atm(+/+)p53(-/-), and atm(-/-)p53(-/-) mouse embryonic fibroblasts to m-THPC-PDT performed at a wavelength of 652 nm was determined by the MTT assay, trypan blue-exclusion, and the TUNEL and caspase3-cleavage apoptosis assays. c-Abl protein level was determined by immunoblotting. RESULTS: m-THPC-PDT rapidly induced cell death in a substantial fraction of cells by p53- and Ataxia telangiectasia mutated (ATM)-independent non-apoptotic processes. However, in the subset of apoptotic cells, apoptosis was reduced by loss of p53 and was even more reduced by the additional loss of ATM. Apoptosis correlated inversely with c-Abl level. CONCLUSIONS: p53 and ATM are not required for necrosis, but may be required for PDT-mediated apoptosis.