Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Caspase 12”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed2004

A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage.

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent.

Antineoplastic Agents, Phytogenic↗
PubMed2004

Alleviating ischemia-reperfusion injury in aged rat liver by induction of heme oxygenase-1.

BACKGROUND: Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic ischemia-reperfusion (I/R) injury, a critical factor in the dysfunction of the aged liver after transplantation. METHODS: We used hemin to overexpress HO-1 and analyze its effects in a model of I/R in aged livers used for orthotopic transplantation. RESULTS: The SGOT levels in the hemin group were significantly lower than those of the saline treatment group. Hemin liver grafts showed markedly fewer apoptotic (TUNEL+) liver cells after reperfusion compared with the controls. The plasma nitric oxide levels in the hemin group were significantly lower than those in the control group. Unlike untreated or hemin + Znpp-treated orthotopic liver transplant controls, iNOS expression in the hemin group was almost absent at 12 and 24 hours, after reperfusion. In contrast, eNOS was comparable in hemin and saline orthotopic liver transplants. The increased levels of Bcl-2 expression compared with saline controls were most pronounced at 12 hours after transplantation. In contrast, caspase 3 was lower at 24 hours among the hemin-pretreated group compared with saline-treated liver transplant controls. CONCLUSIONS: HO-1 alleviated the I/R injury in the aged liver by suppressing local expression of inducible nitric oxide synthase and by modulating pro- and antiapoptotic pathways.

Aging↗
PubMed2006

Apoptosis is required during early stages of tail regeneration in Xenopus laevis.

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.

Animals↗
PubMed2003

Immunomodulation of hepatic ischemic injury via increased Bcl-X(L) and decreased Bcl-X(S).

BACKGROUND: Although classic ischemia-reperfusion injury is mediated by reactive oxygen intermediaries, increasing evidence implicates a role for immune-mediated apoptosis during ischemic injury in transplantation. Herein, we report the effects of polyclonal rabbit anti-thymocyte globulin (rATG) on mediators of hepatic apoptosis during cold storage. METHODS: Three-month-old male Lewis rats were placed under halothane anesthesia and the portal vein cannulated. University of Wisconsin (UW) solution (35 ml) with (n = 5) and without (n = 5) 20 mg/kg anti-rat rATG was infused before hepatectomy. The liver was stored in UW solution +/- rATG (143 ng/ml) at 4 degrees C for various times up to 24 h. Specimens were terminal deoxyuridine nick end labeling-stained for apoptosis. Tissue lysates were analyzed by Western blotting and densitometry. RESULTS: Compared to UW alone, significantly fewer apoptotic cells were present in UW + rATG perfused and stored livers. There were early and sustained significant increases in Bcl-X(L) and decreases in Bcl-X(S) with rATG. There was an initial, but not sustained, significant decrease in Bax with rATG. Moreover, there was a significant one-third decrease in caspase-9 production with rATG at 0, 6, 12, and 18 h. CONCLUSION: Decreased proapoptotic Bcl-X(S) and increased antiapoptotic Bcl-X(L), as well as decreased downstream proapoptotic caspase-9 expression, during liver ischemia after treatment with rATG, all favor cell survival. Because apoptotic ischemic injury results in allograft dysfunction, preservation strategies that ameliorate such immunological effects may improve organ function.

Adenosine↗
PubMed2004

Selective efficacy of depsipeptide in a xenograft model of Epstein-Barr virus-positive lymphoproliferative disorder.

BACKGROUND: Immune-compromised individuals are at increased risk for developing aggressive Epstein-Barr virus (EBV)-associated lymphoproliferative disorders after primary EBV infection or for reactivation of a preexisting latent EBV infection. We evaluated the effect of depsipeptide, a histone deacetylase inhibitor, on EBV-positive lymphoblastoid cell lines (LCLs) and Burkitt lymphoma cell lines in a mouse model and explored its mechanism of action in vitro. METHODS: We studied EBV-transformed LCLs, which express a latent III (Lat-III) viral gene profile, as do some EBV-positive lymphoproliferative malignancies, and Burkitt lymphoma cell lines, which express a Lat-I viral gene profile. Cell lines were used to characterize depsipeptide-induced apoptosis, which was evaluated by flow cytometry. Flow cytometry, western blot analyses, and histone deacetylase inhibitors were used to investigate components of prodeath and survival pathways in vitro. We studied depsipeptide's effects on survival with a mouse xenograft model of EBV-positive human B-cell tumors (groups of 10 mice). All statistical tests were two-sided. RESULTS: Depsipeptide (5 mg/m2 of body surface area) treatment was associated with statistically significantly improved survival of mice carrying Lat-III EBV-positive LCL tumors, compared with that of control-treated mice (day 30: for depsipeptide-treated mice, 90% survival, 95% confidence interval [CI] = 73.2% to 100%; for control-treated mice, 20% survival, 95% CI = 5.79% to 69.1%; P<.001), but it was not associated with survival of mice carrying Lat-I EBV-positive Burkitt lymphoma tumors. Depsipeptide induced apoptosis in 64% of LCLs and in 14% of EBV-positive Burkitt lymphoma cells in vitro. Depsipeptide-treated LCL cultures had two distinct cell populations--one sensitive and one resistant to depsipeptide. Depsipeptide-mediated apoptosis was associated with a 12-fold increased level of active caspase 3, but some apoptosis persisted despite z-VAD-fmk treatment to inhibit caspase activity. Depsipeptide-resistant LCLs expressed higher levels of latent membrane protein 1 (LMP1; P = .017), BCL2 (P = .032), and nuclear factor kappaB (NF-kappaB) (P<.001) than depsipeptide-sensitive LCLs; this resistance was circumvented by treatment with PS-1145, an inhibitor of NF-kappaB activation (P<.001). CONCLUSIONS: Apoptosis is induced by depsipeptide via caspase-dependent and -independent pathways in Lat-III EBV-positive LCLs and is enhanced by inhibiting NF-kappaB activity. Depsipeptide as a treatment for Lat-III EBV-associated lymphoproliferative disorders should be explored further in clinical trials.

Animals↗
PubMed2003

Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression.

Calorie restriction, followed by ad libitum refeeding, results, respectively, in loss and regeneration of pulmonary alveoli. We now show 35% of alveoli are lost within 72 h of onset of calorie restriction ((2/3) decreased daily chow intake), and an additional 12% of alveoli are lost over a subsequent 12 days of calorie restriction. Tissue necrosis was not seen. Within 72 h of refeeding, after 15 days of calorie restriction, the number of alveoli returns to precalorie restriction values. Microarray lung gene profiling, in conjunction with Western and RNase protection assay, demonstrate an increase of granzyme and caspase gene expression 2-3 h after onset of calorie restriction. By 12 h, granzyme and caspase expression is no longer increased, but tumor necrosis factor death receptor expression is elevated. At 336 h, Fas death receptor expression is increased. Because granzymes are found only in cytotoxic lymphocytes (CTLs) and natural killer (NK) cells, we suggest calorie restriction activates these cells, initiating a series of molecular events that results in alveolar destruction. The evidence of involvement of CTLs and NK cells and the absence of necrosis are similar to alveolar destruction in chronic obstructive pulmonary disease.

Animals↗
PubMed2005

[The effect and mechanism of arsenic trioxide on hepatocellular carcinoma].

OBJECTIVE: To investigate the effect and mechanism of arsenic trioxide on hepatoma cell line BEL-7402 and observe the effect and best administration method of arsenic trioxide on hepatocellular carcinoma patients who were not suitable for operation. METHODS: The cell activity and morphologic changes were studied after being treated with arsenic trioxide in different concentrations. The apoptosis was detected by flow cytometry assay and DNA fragmentation assay. The caspase-3 level of mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and fluoro-spectrophotometer. The growth inhibition of implant tumor was observed in nude mice treated with arsenic trioxide in different concentrations. Arsenic trioxide was used in hepatocellular carcinoma patients by intravenous dropping and continuous regional infusion through hepatic artery. RESULTS: The effect of arsenic trioxide on hepatoma cell lines was dependent on the time and concentration obviously. The decrease in cell number was preceded by morphological changes in the treated BEL-7402 cells that were characteristic of apoptosis, including membrane blebbing, shrunken cytoplasm, nuclear condensation and loss of adhesion. Flow cytometry assay showed an arrestment at G(2)/M phase and sub-G(1) cell peak. DNA fragmentation assay showed a marked DNA ladder. The mRNA level of caspase-3 was no change in RT-PCR whereas the protein of caspase-3 was increased after added As(2)O(3) 1 - 36 h. Caspase-3 activity began to increase after 2 h and reached a maximal level after 12 h in a linear fashion. Then, the level of caspase-3 was decreased, but still in a high level. The growth inhibition of implant tumors was obviously in nude mice. The intravenous usage of arsenic trioxide could improve the quality of life with low toxicity in hepatocellular carcinoma patients not suitable for operation. The tumor size decreased in 30 patients and AFP value decreased in 19 patients by continuous regional infusion through hepatic artery. CONCLUSIONS: Arsenic trioxide can obviously inhibit the growth of hepatoma cell line BEL-7402 through inducing hepatoma cell apoptosis. The activation and increase of Caspase-3 is the possible mechanism of apoptosis, and the acting point is in pro-enzyme level. The best result of arsenic trioxide on non-operative patients should be gotten in continuous infusion through hepatic artery.

Adult↗
PubMed2005

Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by paclitaxel.

BACKGROUND: Paclitaxel, an antineoplastic drug, inhibits cell growth and cell cycle progression and induces apoptosis in human leukemia HL-60 cells. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. METHODS: We examined the cell morphology and apoptosis in HL-60 cells after exposure to paclitaxel and measured caspase-3 activities with or without z-VAD-fmk (a broad-spectrum caspase inhibitor) pretreatment by flow cytometric analysis and Western blotting. RESULTS: Together, our results were (1) paclitaxel mainly induced G2/M cell cycle arrest in HL-60 cells (p<0.001); (2) time (p<0.001)- and dose-dependent (p<0.001) apoptosis of HL-60 cells was induced by paclitaxel; (3) in HL-60 cells, z-VAD-fmk blocked paclitaxel-induced apoptosis (12 h: p<0.001; 24 h: p<0.01; 48 h: p<0.01; 72 h: p<0.001) and caspase-3 activation (12 h: p<0.05; 24 h: p<0.01; 48 h: p<0.01; 72 h: p<0.01). CONCLUSIONS: These results suggest that paclitaxel can induce G2/M cell cycle transition and apoptosis via caspase-3 activity in HL-60 cells.

Amino Acid Chloromethyl Ketones↗
PubMedDate not supplied

Cis 9, trans 11- and trans 10, cis 12-conjugated linoleic acid isomers induce apoptosis in cultured SW480 cells.

Dietary conjugated linoleic acid (CLA) has been shown to reduce colon tumor incidence in rodents by mechanisms probably involving apoptosis. The aim of this study was to evaluate the effects of three commercial CLA preparations (pure c9, t11-CLA, pure t10, c12-CLA and a CLA mixture, containing 29.5% c9, t11 and 29% t10, c12-CLA) on caspase-dependent apoptosis in colon SW480 tumor cells. After 4 days incubation, all CLA-treated cells displayed an increase in caspase 3 (27-34%) and caspase 9 activities (37-47%), cleavage of pro-caspase 3 (32 kDa) to 17 and 12 kDa subunits, increased membrane annexin V levels and reduced expression of bcl-2 compared with untreated controls. Cytosolic cytochrome c was increased (p < 0.05) by all CLA preparations, with the t10, c12-CLA isomer being the most potent. The data indicate that t10, c12-CLA may be the more biologically active isomer for inhibition of colon tumor cell proliferation in vitro.

Annexin A5↗
PubMed2001

The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces monocytic differentiation of human myeloid leukemia cells and inhibits proliferation of diverse human tumor cell lines. The present studies on human osteosarcoma cells demonstrate that CDDO induces mitochondrial cytochrome c release, caspase-3 activation, and internucleosomal DNA fragmentation. Overexpression of the caspase-8 inhibitor CrmA blocked CDDO-induced cytochrome c release and apoptosis. By contrast, overexpression of the antiapoptotic Bcl-x(L) protein blocked CDDO-induced cytochrome c release, but only partly inhibited caspase-3 activation and apoptosis. In concert with these findings, we demonstrate that CDDO: 1) activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism and 2) induces cytochrome c release by caspase-8-dependent cleavage of Bid. The results also demonstrate that treatment of osteosarcoma cells with CDDO induces differentiation, as assessed by alkaline phosphatase activity and osteocalcin production, and that this response is abrogated in cells that overexpress CrmA. These findings demonstrate that CDDO induces both osteoblastic differentiation and apoptosis by caspase-8-dependent mechanisms.

Apoptosis↗
PubMed2002

Alterations of Fas-pathway genes associated with nodal metastasis in non-small cell lung cancer.

Many types of cancer cells are resistant to Fas-mediated apoptosis by several mechanisms, including the mutations of the genes involved in Fas-mediated apoptosis. In this study, to explore the possibility that the mutations of the genes involved in the proximal pathway of Fas-mediated apoptosis (Fas, FADD, caspase 8 and caspase 10) are involved in cancer metastasis, we have analysed somatic mutation and deletion of these genes in 80 non-small cell lung cancers (NSCLCs) with (n=43) and without (n=37) metastasis to the regional lymph nodes. We found 12 mutations (four Fas, four FADD, and four caspase 10 mutations) in 11 of 80 NSCLCs (13.8%). Interestingly, of these mutations, most mutations (10 out of 12) were detected in the NSCLCs with metastasis, and the frequency in the metastasis lesions (23%) was higher than that in the primary lesions of the NSCLCs without metastasis (5.4%). Furthermore, transfection study revealed that the tumor-derived mutants have decreased apoptosis inductions compared to the wild types. These data suggest that the inactivating mutations of the genes in the proximal pathway of Fas-mediated apoptosis may lead to a decreased cancer cell death and play a role in the metastasis of NSCLC.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Basic fibroblast growth factor suppresses retinal neuronal apoptosis in form-deprivation myopia in chicks.

PURPOSE: Chorioretinal atrophy including retinal neuronal apoptosis occurred in chronic form-deprivation myopia induced by lid suture in chicks. We investigated whether exogenous basic fibroblast growth factor (bFGF) could simultaneously inhibit the excessive axial elongation and retinal neuronal loss in the myopic chick eyes. METHODS: Unilateral form deprivation was produced in neonatal male Hyline chicks by lid suture 24 hr after hatching. Ten microliters of solution containing 5 ng bFGF or PBS (vehicle) was injected into the vitreal chamber at 10 weeks of age, once every 3 days until week 12, when the animals were sacrificed. Ocular refraction and axial length were assessed by retinoscopy and calipers at the age of 12 weeks. Retinal apoptotic neurons and their caspase-3-like protease activity were analyzed by transmission electron microscopy, the terminal-deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) staining, and a colorimetric method using Ac-DEVD-pNA as a substrate. The number of TUNEL-positive cells was counted to analyize the survival activity of bFGF on retina. RESULTS: After 12 weeks of lid suture, the control eyes were either emmetropic or hyperopic, whereas the deprived eyes became myopic with axial length increased. TUNEL-positive nuclei, condensed nuclear chromatin, and apoptotic bodies were observed in posterior retinal outer nuclear layer (ONL) and inner nuclear layer (INL) in the myopic chick eyes. Activity of retinal caspase-3-like protease in these eyes was elevated. In addition to ameliorating myopic ocular growth, intravitreal bFGF therapy significantly reduced the number of retinal apoptotic neurons and downregulated caspase-3 activity. CONCLUSIONS: Exogenous bFGF effectively ameliorates the excessive axial elongation and retinal neuron apoptosis in chronic form-deprivation myopia in chicks. It is possible that bFGF will be a promising therapeutic agent for high human myopia.

Animals↗
PubMed2000

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis.

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.

Annexin A5↗
PubMed2001

Apoptosis induced in HepG2 cells by short exposure to millimolar concentrations of ethanol involves the Fas-receptor pathway.

PURPOSE: We previously found that ethanol-induced apoptosis is associated with an activation of caspase-3. However, the initial triggering of this process is yet unknown. Therefore, the present study was designed to determine whether the Fas-receptor pathway plays a role in the initiation by ethanol of human hepatocellular carcinoma (HepG2) cell apoptosis. METHODS: HepG2 cells were incubated with or without 1 mM ethanol for 24 h. Apoptosis was assessed by DNA fragmentation and caspase-8 activity. Selective inhibitors of caspase-8 and caspase-9 were used to analyze the role of both caspases on apoptosis. Soluble human Fas ligand (Fas-L) was determined by enzyme-linked immunosorbent assay (ELISA). A fluorescent dye was used to investigate the permeability of the mitochondrial outer membrane. A recombinant Fas fusion protein was used to inhibit the activation of Fas receptors. Human anti-Fas-L antibody was employed to neutralize Fas-L released from the cells. RESULTS: Caspase-8 activity increased significantly threefold (P < 0.005) after 12 h incubation of HepG2 cells with 1 mM ethanol whereas no change was observed in control cells. Incubation with caspase-8 inhibitor completely prevented apoptosis induced by ethanol (P < 0.001). In contrast, a caspase-9 inhibitor did not significantly reduce apoptosis. The permeability of the outer mitochondrial membrane was not altered. Neutralization of Fas-receptors by Fas fusion proteins completely attenuated ethanol-induced apoptosis in HepG2 cells treated with ethanol. CONCLUSIONS: These findings show that apoptosis induced by low concentrations of ethanol in human HepG2 cells is associated with Fas-receptor activation and subsequent caspase-8 activation. Triggering of apoptosis through Fas-receptors represents a mechanism of action different from that observed with high concentrations of ethanol.

Antineoplastic Agents↗
PubMed2001

Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity.

Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.

Animals↗
PubMed2000

Expression and activation of caspase-3/CPP32 in CD34(+) cord blood cells is linked to apoptosis after growth factor withdrawal.

Caspase-3/CPP32, a member of the interleukin-1 converting enzyme (ICE) family, is considered an executioner protease in mammalian cells during apoptosis. Although expression and activation of caspase-3/CPP32 protein have been studied in many tissues and leukemia cell lines, this has not been explored in primitive hematopoietic CD34(+) cells. In this study, we evaluated expression and activation of caspase-3/CPP32 protein in CD34(+) cells from cord blood (CB) during apoptosis induced by growth factor deprivation. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and flow cytometry analysis were used in this study to determine the expression of caspase-3/CPP32 in CD34(+) CB cells during apoptosis. Our results demonstrated that caspase-3/CPP32 mRNA was constitutively expressed at a very low level in freshly isolated CD34(+) cells. Expression of caspase-3/CPP32 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3/CPP32 proenzyme was detected in the freshly isolated CD34(+) cells and after 3 days expansion with cytokines. Within 12 hours after growth factor withdrawal from expanded cells caspase-3/CPP32 was activated and a cleavage 20 kDa protein was detected; a poly(ADP-ribose) polymerase (PARP) was cleaved by activated caspase-3/CPP32. Activation of caspase-3/CPP32 and apoptosis upon growth factor withdrawal were inhibited/reduced by the caspase inhibitors, z-VAD-fmk and DEVD-CHO. These results demonstrate that caspase-3/CPP32 is involved in apoptosis of primitive CB CD34(+) cells but may not be the only mechanism involved.

Antigens, CD34↗
PubMed2001

Differential role of I kappa B kinase 1 and 2 in primary rat hepatocytes.

Nuclear factor-kappa B (NF-kappa B) protects hepatocytes from undergoing apoptosis during embryonic development and during liver regeneration. Activation of NF-kappa B is mediated through phosphorylation of its inhibitor, I kappa B, by a kinase complex that contains 2 I kappa B kinases. We analyzed the differential role of I kappa B kinase 1 (IKK1) and I kappa B kinase 2 (IKK2) in tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 beta (IL-1 beta)-mediated NF-kappa B activation in primary rat hepatocytes. Maximal induction of IKK activity was observed 5 minutes after TNF-alpha and 15 minutes after IL-1 beta treatment, and activated IKK was able to phosphorylate GST-I kappa B (1-54) and GST-p65 (354-551), but not a GST-p65 (354-551) substrate with a serine-to-alanine substitution at position 536. Infection with an adenovirus containing catalytically inactive IKK2K44M (Ad5IKK2dn) completely blocked both TNF-alpha- and IL-1 beta-induced GST-I kappa B and GST-p65 phosphorylation, I kappa B degradation, and NF-kappa B DNA binding. Adenovirally transduced, catalytically inactive IKK1K44M (Ad5IKK1dn) reduced IKK activity and NF-kappa B DNA binding only slightly. Accordingly, Ad5IKK2dn induced apoptosis in 75% (+/-6%) of hepatocytes after 12 hours of TNF-alpha, which was accompanied by activation of caspases 3 and 8, nuclear fragmentation, and DNA laddering. In contrast, Ad5IKK1dn led to 21% (+/-2%) apoptosis in TNF-alpha-treated hepatocytes after 12 hours and comparatively low activity of caspases 3 and 8. Furthermore, Ad5IKK2dn completely blocked the induction of inducible nitric oxide synthase (iNOS), whereas Ad5IKK1dn had no influence on the expression of iNOS. Thus, IKK2 is the main mediator for cytokine-induced NF-kappa B activation in primary hepatocytes and protects against TNF-alpha-induced apoptosis, whereas IKK1 kinase activity is not required for NF-kappa B activation.

Adenoviridae↗