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 613 records · Page 34Linked to original sources
PubMed2001

Evidence for redox regulation of cytochrome C release during programmed neuronal death: antioxidant effects of protein synthesis and caspase inhibition.

Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitochondria is central to this death. In this report we present evidence that cellular redox state regulates cytochrome c redistribution in these neurons. An increase of mitochondrial-produced reactive oxygen species (ROS) occurred in rat sympathetic neurons in cell culture within 3 hr of NGF withdrawal. Caspase inhibitors blocked this ROS burst. By 6 hr after NGF deprivation, glutathione (GSH) levels had increased, neutralizing elevated hydrogen peroxide levels and returning cellular redox state to basal levels. By 12 hr after deprivation, ROS levels had again increased and remained elevated during the rest of the apoptotic process. The later ROS burst appeared to have both caspase-dependent and caspase-independent components and was coincident with the period of cytochrome c release. Inhibition of protein synthesis with cycloheximide (CHX) and treatment with the antioxidant compound, N-acetyl-l-cysteine (l-NAC), blocked both the early and late ROS bursts by increasing cellular GSH levels (Ratan et al., 1994; Tan et al., 1998). Both compounds, and a membrane-permeant form of GSH, also inhibited cytochrome c release and death. Treatment of NGF, CHX-, l-NAC-, and GSH-saved cells with hydrogen peroxide caused rapid cytochrome c release. These data suggest a role for cellular redox state in regulating cytochrome c release during apoptosis induced by NGF withdrawal.

Animals↗
PubMed2003

BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome.

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.

Apoptosis↗
PubMed2003

Activation of two caspase cascades, caspase 8/3/6 and caspase 9/3/6, during photodynamic therapy using a novel photosensitizer, ATX-S10(Na), in normal human keratinocytes.

BACKGROUND/PURPOSE: Photodynamic therapy (PDT) is a potent treatment for skin tumors. Although the therapeutic effect of PDT is supposed to be due to cellular cytotoxicity, the precise mechanism is still unknown. ATX-S10(Na) [13,17-bis(1-carboxypropionyl)carbamoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt], a novel hydrophilic chlorin photosensitizer, shows good accumulation in tumors and is suitable for use in PDT. In this study, we investigated the mechanism of PDT-induced cell death using ATX-S10(Na). METHODS: . Following ATX-S10(Na) treatment for 12 h, normal human keratinocytes (NHK) were irradiated using a diode laser. PDT-induced cell death and the activity of various caspases were measured. Activation of Fas antigen was also determined by immunoprecipitation analysis. The expression of Bax, cytochrome c, and apoptosis-inducing factor (AIF) was determined by Western blotting. RESULTS: ATX-S10(Na)-PDT had induced apoptosis of NHK by 2 h and the maximal effect was observed at 6 h following irradiation. The effect was suppressed by pretreatment of NHK with inhibitors of caspases 3, 6, 8 and 9. A caspase activity assay revealed the sequential activation of caspases 8, 3 and 6, and caspases 9, 3 and 6, respectively. Immunoprecipitation analysis indicated multimerization of Fas antigen without Fas ligand binding in ATX-S10(Na)-PDT-treated NHK. Western blotting revealed cytosolic release of cytochrome c and AIF accompanied by decreased Bax expression in the cytosol. CONCLUSIONS: ATX-S10(Na)-PDT induces apoptosis of NHK, and this was mediated by sequential activation of two caspase cascades, caspases 8, 3 and 6, and caspases 9, 3 and 6. This was accompanied by multimerization of Fas antigen and cytosolic release of cytochrome c and AIF.

Apoptosis↗
PubMed2001

Apoptotic activity in gestational trophoblastic disease correlates with clinical outcome: assessment by the caspase-related M30 CytoDeath antibody.

AIMS: The objective of this study was to assess apoptotic activity in gestational trophoblastic disease (GTD) and its prognostic value in hydatidiform mole (HM). METHODS AND RESULTS: Expression of the specific caspase cleavage site within cytokeratin 18 was assessed immunohistochemically using the monoclonal antibody M30 CytoDeath in 12 spontaneous abortions, 22 partial and 57 complete HM, eight choriocarcinoma (CCA) and 28 normal placentas. The M30 immunoreactivity occurred predominantly in the syncytiotrophoblasts. A significantly higher M30 index in HM and CCA was found when compared with normal placentas and spontaneous abortions (P < 0.001). The M30 index of those HM which spontaneously regressed was significantly higher than those HM which developed persistent disease requiring chemotherapy (P < 0.001). The M30 index correlated with another apoptotic index previously detected by TdT-mediated dUTP nick-end labelling (TUNEL) (P = 0.007) and the proliferation index assessed by the Ki67 antigen (P = 0.034). CONCLUSIONS: We conclude that apoptosis is important in the pathogenesis of GTD. Assessment of apoptotic activity in HM by the M30 index may be considered as an alternative prognostic indicator for predicting the clinical behaviour.

Abortion, Spontaneous↗
PubMed2004

Progression of hair cell ejection and molecular markers of apoptosis in the avian cochlea following gentamicin treatment.

Aminoglycoside treatment induces caspase-dependent apoptotic death in inner ear sensory hair cells. The timing of apoptotic signaling in sensory hair cells following systemic aminoglycoside treatment has not been characterized in vivo. We administered a single subcutaneous injection of the aminoglycoside gentamicin (300 mg/kg) to 12-16-day-old chicks and used immunocytochemical techniques to document the following responses in affected hair cells: T-cell restricted intracellular antigen-related protein (TIAR) translocation from the nucleus to the cytoplasm, cytochrome c release from the mitochondria, caspase-3 activation, nuclear condensation, and an orderly progression of hair cell ejection from the proximal end of the basilar papilla. Hair cells in the proximal tip exhibited TIAR translocation from the nucleus and aggregation into punctate granules in the cytoplasm 12 hours after injection and the response progressed distally. Cytochrome c release from the mitochondria into the cytoplasm and caspase-3 activation were observed in affected hair cells immediately prior to and during ejection. Hair cell ejection occurred between 30 and 54 hours after injection, beginning in the proximal tip and progressing distally. Nuclear condensation accompanied ejection while the loss of: 1) membrane integrity; 2) phalloidin labeling of F-actin; and 3) TO-PRO-1 labeling of nuclear contents occurred within 48 hours following ejection. Our results present a timeline of aminoglycoside-induced inner ear sensory hair cell apoptotic death that includes an 18-hour window between the initial apoptotic response and the later stages of programmed death signaling that accompany ejection and a gradual breakdown of hair cells following ejection.

Animals↗
PubMed2005

Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells.

Berberine, a main component of Coptidis Rhizoma, is a plant alkaloid with a long history of medicinal use in Chinese medicine. Berberine has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi. The mechanism by which berberine initiates apoptosis remains poorly understood. In the present study, we demonstrated that berberine exhibited significant cytotoxicity in hepatoma HepG2 cells but is ineffective in Chang liver cells. Herein we investigated cytotoxicity mechanism of berberine in HepG2 cells. The results showed that HepG2 cells underwent internucleosomal DNA fragmentation after 24-h treatment with berberine (50 microM). Moreover, berberine induced the activation of caspase-8 and -3, and caused the cleavage of poly ADP-ribose polymerase (PARP) and the cytochrome c release, whereas the expression of Bid and anti-apoptosis factor Bcl-XL were decreased markedly. The loss of mitochondrial membrane potential (Delta psim) at 24 h and activation of Fas at 12 h were also seen in the berberine-treated HepG2 cells. These findings supported the fact that the inhibitors of caspases, DEVD-FMK, IETD-FMK and VAD-FMK, prevented apoptosis and restored the expression of Bcl-XL, Bcl-2 and Bid. These results indicated that the potential of anti-hepatoma activity of berberine may be mediated through a caspases-mitochondria-dependent pathway.

Apoptosis↗
PubMed2006

Fluorinated Cpd 5, a pure arylating K-vitamin derivative, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating MAPK.

We previously synthesized several K-vitamin derivatives, which are potent growth inhibitors of human tumor cells, including Hep3B human hepatoma cells. Among these, Cpd 5 was the most potent. However, being a quinone derivative, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a fluorinated derivative of Cpd 5, F-Cpd 5. The calculated reduction potential of F-Cpd 5 was much higher than that for Cpd 5 and it was not predicted to generate ROS. This was supported by our observation that F-Cpd 5 generated significantly lower ROS than Cpd 5. F-Cpd 5 was three times more potent than Cpd 5 in inhibiting Hep3B cell growth. Interestingly, under identical culture conditions, F-Cpd 5 inhibited mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold less potently than Hep3B cells. F-Cpd 5 was found to induce caspase-3 cleavage and nuclear DNA laddering, evidences for apoptosis. It preferentially inhibited the activities of the cell cycle controlling phosphatases Cdc25A and Cdc25B, by binding to their catalytic cysteines. Consequently, inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 were induced. F-Cpd 5 also induced phosphorylation of the MAPK proteins ERK1/2, JNK1/2 and p38 in Hep3B cells and the MAPK inhibitors (U0126, JNKI-II, and SB 203580) antagonized its growth inhibition. F-Cpd 5 inhibited the action of cytosolic ERK phosphatase activity, which likely caused the ERK phosphorylation. F-Cpd 5 thus differentially inhibited growth of normal and tumor cells by preferentially inhibiting the actions of Cdc25A and Cdc25B phosphatases and inducing MAPK phosphorylation.

Animals↗
PubMed2002

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A(2) and delta(12)-PGJ(2).

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A(2) and delta(12)-PGJ(2) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(2) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Apoptosis↗
PubMed2006

Semaphorin 3C regulates endothelial cell function by increasing integrin activity.

Class 3 semaphorins (sema 3) are secreted guidance proteins. Sema 3A expressed by endothelial cells controls vascular morphogenesis through integrin inhibition. Sema 3C is required for normal cardiovascular patterning. Here we examined the potential role of sema 3C as regulator of endothelial cell function in vitro using mouse glomerular endothelial cells (MGEC). We determined that MGEC express sema 3C mRNA and protein and its receptors mRNA. Recombinant sema 3C induced MGEC proliferation 18 +/- 2% above control, as assessed by bromodeoxyuridine (BrdU) incorporation, and reduced starvation-induced apoptosis by 46 +/- 3%, as indicated by an in situ marker of activated caspase 3. Sema 3C increased MGEC adhesion to fibronectin 79 +/- 13% and to collagen 55 +/- 12% as compared with control. Sema 3C-induced MGEC adhesion was prevented by integrin blocking antibodies and involved beta1 integrin serine phosphorylation. Sema 3C-induced MGEC adhesion and proliferation were similar to those induced by vascular endothelial growth factor (VEGF)-A. Sema 3C induced a 44 +/- 11% increase in MGEC directional migration and stimulated MGEC capillary-like network formation on collagen I gels. Collectively, our data indicate that sema 3C promotes glomerular endothelial cell proliferation, adhesion, directional migration, and tube formation in vitro by stimulating integrin phosphorylation and VEGF120 secretion, functions that are similar to VEGF-A and opposite to sema 3A.

Animals↗
PubMed2004

Decreased proliferation precedes growth factor changes after physeal irradiation.

The effects of irradiation on growth plate chondrocytes and mediators of chondrocytic differentiation are poorly understood. In earlier work on rat growth plate changes 1/2 to 4 weeks after irradiation, a nadir was identified at 1 week in proliferation and growth factor expression coincident with maximal histomorphometric derangement. The purpose of this study was to determine the earlier sequential relationship of proliferative, growth factor, and histomorphometric changes after irradiation leading to the 1-week nadir. Twenty-four weanling 5-week-old male Sprague-Dawley rats had right knee irradiation with single fraction 17.5 Gy whereas the left leg served as an internal control. The earliest change identified was a significant decrease in BrdU evidence of proliferative activity between 6 and 12 hours after irradiation, which persisted through 48 hours. Twelve to 24 hours after irradiation, caspase-3 staining for apoptosis was higher than that in growth plates not having received radiotherapy. Histomorphometric changes after irradiation were observed as early as 24 hours. Growth factors and their downstream antiapoptotic and proapoptotic mediators did not differ significantly between limbs through 48 hours. The current study suggests that decreased proliferation and apoptosis precede any change in histomorphometric features of the growth plate after irradiation and that decreased growth factor expression occurs later.

Analysis of Variance↗
PubMed2006

Suicide cancer gene therapy using pore-forming toxin, streptolysin O.

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.

Adenoviridae↗
PubMed2002

PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis.

Treatment with the proteasome inhibitor, PS-341 resulted in concentration- and time-dependent effects on Bcl-2 phosphorylation and cleavage in H460 cells that coincided with the PS-341-induced G2-M phase arrest. The observed Bcl-2 cleavage paralleled the degree of PS-341-induced apoptosis but was detected to a similar extent with comparable concentrations of two other proteasome inhibitors (MG-132 and PSI). Calpain inhibitors, ALLM and ALLN, and the caspase inhibitors, Z-VAD and AC-YVAD did not induce BcI-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional Mr 25,000 cleavage fragment of Bcl-2, whereas only a Mr 23,000 fragment was observed with other anticancer agents. The formation of the Mr 25,000 fragment was not prevented by caspase inhibitors unlike the Mr 23,000 fragment, which suggests mediation by a caspase-independent pathway. Cell fractionation studies revealed that the Bcl-2 cleaved fragments localize within membrane structures and was an early event (at approximately 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), beta-catenin, and DNA fragmentation (at approximately 36 h posttreatment). The Mr 23,000 Bcl-2 cleavage product was inhibited by the pan-caspase inhibitor and the inhibitors of capase-3, -8, -9; but the PARP cleavage was prevented only by the pan-caspase and caspase-3 inhibitors, which suggests that the Mr 23,000 Bcl-2 cleavage occurred at both the initiation and execution stages of apoptosis. The inhibition of the ubiquitin/proteasome pathway by PS-341 leads, at an early stage of apoptosis, to Bcl-2 phosphorylation and a unique proteolytic cleavage product, which are associated with G2-M phase arrest and the induction of apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2002

[Killing and proapoptotic effects of topoisomerase I inhibitor on K562 cells].

BACKGROUND AND OBJECTIVE: Recently, it has been demonstrated that topoisomerase I(Topo I) inhibitor is a useful alternative for treatment of the patients with chronic myelogenous leukemia(CML) in accelerated or blast crisis. In order to get insight into pharmalogic activity of Topo I inhibitor, this study was designed to investigate the killing and proapoptotic activity of Topo I inhibitor(Topotecan, TPT) using K562 cell line, which carry abnormal chromosome[t(9;22)] of characteristics of CML. METHODS: The cytotoxic effect of TPT on K562 cells was determined using MTT assay; TPT-induced apoptosis in K562 cells was identified by morphological analysis and Annexin V FITC staining; correlation between TPT-mediated killing or apoptosis and caspase-8 activation was investigated using caspase-8 inhibitor IETD-fmk. RESULTS: After incubation of K562 cells with 0.15 mumol/L TPT for 12, 24, 48, and 72 h, the survival rates, compared with control, progressively reduced to (92 +/- 36)% [P > 0.05 vs (94 +/- 27)%], (68 +/- 21)% [P < 0.05 vs (119 +/- 13)%], (54 +/- 15)% [P < 0.05 vs (132 +/- 31)%], and (21 +/- 10)% [P < 0.01 vs (114 +/- 19)%]. Targeted cells meanwhile demonstrated phosphatidylserine externalization, cell shrinkage, chromatin margination, karyorrehxis and eventually disintegrate into numerous apoptotic bodies. After exposure to TPT in combination with IETD-fmk for 24 h and 48 h, K562 cells were still survival at viability of (95 +/- 29)% and (87 +/- 11)%, the latter was significantly higher than that of to TPT only [P < 0.05 vs (54 +/- 15)%]. CONCLUSION: Topoisomerase I inhibitor TPT possess killing activity and was induce apoptosis to K562 cells, which could be dependent on activation of caspase-8.

Apoptosis↗
PubMed2004

Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive.

Argyrophilic dark neurons (DNs) reflect the early histopathological state of neurons following various brain insults. We examined the fate of DNs, about to either die or recover, following two types (heavy and light damage) of brain insult. Wistar rats were injected ibotenic acid unilaterally into the hippocampal CA1 region (ibotenic acid [IA] injection) or were forced to swim (SWIM). Argyrophil III (DNs)-, activated caspase-3 immuno-, TUNEL- and hematoxylin-eosin (H-E)-staining and ultrastructural examinations were then performed. One to three hours after IA injection, typical DNs (argyrophilic both in somata and dendrites) with corkscrew-like dendrites were densely packed in the pyramidal cell layer of hippocampal CA1 around the injection site. After 12-24 h, DNs were argyrophilic only in the somata and proximal dendrites but absent in distal dendrites in the CA1 region. However, at this time typical DNs were found in remote areas. At 3 h, caspase-3 activation was detected at the injection site, which increased to a peak level after 12 h. Three to 7 days after injection, TUNEL positive cells were detected in the CA1 pyramidal cell layer. Immediately following SWIM, "brown" rather than "dark" neurons were detected in the various areas and most frequently in the CA1 pyramidal cell layer. No typical DNs were detected over the first 3 days. Some activation of caspase-3 was detected in a few CA3 pyramidal cells but no TUNEL-positive cells were detected. Ultrastructural examination revealed a diffuse distribution of aggregated silver particles in the dendrites and cytoplasm of pyramidal cells at the sites of IA injection. After SWIM, silver particles were detected mainly on mitochondria of affected cells. These data suggest that DNs provide a measure of neuronal damage: typically dark neurons with broad damage to the cytoskeleton of dendrites would die, while non-typical brown neurons, that may have a disturbance in mitochondria, predominantly survive.

Animals↗
PubMed2005

Aggressive vestibular schwannomas showing postoperative rapid growth - their association with decreased p27 expression.

Vestibular schwannomas (VSs) are relatively slow growing tumors. However, some rapidly regrow or recur after surgical resection. The objective of this study was to identify those molecular characteristics predicting rapid recurrence after surgical resection. Immunohistochemically determined expressions of several cell cycle regulators and apoptosis-associated proteins in 12 cases of aggressive VS (AVS) and in 15 control cases of usual VS (UVS) cases were compared. The expressions of p53 and Bax (pro-apoptotic protein), Bcl-2 (anti-apoptotic protein), Fas, and Fas-L (apoptotic death receptor and ligand), caspase 3 (apoptotic effector caspase proteins), and p27 and p21 (cyclin-dependent kinase inhibitors) were analyzed using tissue array blocks. Loss of p27 expression was observed in 8 of 12 AVS cases (67%) and in 3 UVS cases (20%); p21 was expressed in all cases. Loss of Bax was observed in 3 AVS and 3 UVS cases. The anti-apoptotic protein, Bcl-2, was expressed in 9 AVS (75%) and 11 UVS (73%), and p53, Fas-L, and caspase 3 were negative and Fas was positive in all AVS and UVS cases. Of these, only the loss of p27 was statistically significant (P = 0.02). The loss of p27 in AVS may explain the unusually high proliferative potential of AVS versus UVS, and p27 may be a predictor of VS aggressiveness. The expressions of other apoptosis associated proteins were not significantly different in the two groups. This may be the first report to identify a molecular entity associated with aggressive VS. However, further studies are required.

Cell Proliferation↗
PubMed2001

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis.

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2005

Phorbol myristate acetate induces neutrophil death through activation of p38 mitogen-activated protein kinase that requires endogenous reactive oxygen species other than HOCl.

Stimulation of normal mouse neutrophils with phorbol 12-myristate 13-acetate resulted in an acceleration of chromatin condensation and phosphatidylserine externalization that was not associated with caspase-3 activation. Caspase-independent death was completely inhibited by GF109203X and SB202190, specific inhibitors for protein kinase C and p38 mitogen-activated protein kinase respectively. Activation of p38 mitogen-activated protein kinase was completely suppressed by GF109203X, indicating that this enzyme is regulated by protein kinase C. On the other hand, cell death was abolished in NADPH oxidase-deficient neutrophils lacking superoxide production. Of note, p38 mitogen-activated protein kinase was activated by phorbol 12-myristate 13-acetate in normal and myeloperoxidase-deficient neutrophils lacking production of HOCl, whereas no activation was observed in NADPH oxidase-deficient neutrophils. These results strongly suggest that activation of p38 mitogen-activated protein kinase is regulated by endogenously generated superoxide or its metabolites other than HOCl, a critical regulator of inducer-stimulated death of neutrophils.

Animals↗
PubMed2003

Mechanisms of glucocorticoid-induced Leydig cell apoptosis.

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.

Animals↗