Caspase Inhibitor Z-VAD Research Articles

IL-24 Induces Apoptosis of Chronic Lymphocytic Leukemia B Cells Engaged into the Cell Cycle through Dephosphorylation of STAT3 and Stabilization of p53 Expression

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived monoclonal B cells mostly arrested at the G(0)/G(1) phase of the cell cycle. CLL cells strongly express intracellular melanoma differentiation-associated gene-7 (MDA7)/IL-24. However, adenovirus-delivered MDA7 was reported to be cytotoxic in several tumor cell lines. We report herein that rIL-24 alone had no effect; however, sequential incubation with rIL-2 and rIL-24 reduced thymidine incorporation by 50% and induced apoptosis of CLL cells in S and G(2)/M phases of the cell cycle, but not of normal adult blood or tonsil B cells. IL-24 stimulated STAT3 phosphorylation in IL-24R1-transfected cells but not in normal or CLL B cells. In contrast, IL-24 reversed the IL-2-induced phosphorylation of STAT3 in CLL, and this effect was neutralized by anti-IL-24 Ab. Phospho- (P)STAT3 inhibition induced by IL-24 was reversed by pervanadate, an inhibitor of tyrosine phosphatases. The addition of rIL-24 to IL-2-activated CLL B cells resulted in increases of transcription, protein synthesis. and phosphorylation of p53. The biological effects of IL-24 were reversed by the p53 inhibitor pifithrin-alpha and partly by the caspase inhibitor zvad. Troglitazone (a protein tyrosine phosphatase, PTP1B activator) phosphatase inhibited PSTAT3 and augmented p53 expression. PSTAT3 is a transcriptional repressor of p53, and therefore IL-24 induction of p53 secondary to PSTAT3 dephosphorylation may be sensed as a stress signal and promote apoptosis in cycling cells. This model explains why IL-24 can protect some resting/differentiated cells and be deleterious to proliferating cells.

Characterization of β-N-Acetylglucosaminidase Cleavage by Caspase-3 during Apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.

Involvement of Protective Autophagy in TRAIL Resistance of Apoptosis-defective Tumor Cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.

Caspase-related apoptosis in chronic ischaemic microangiopathy following experimental vein occlusion in mini-pigs.

Acute brain ischaemia (stroke) causes a central area of coagulation necrosis. Peripheral to it and after a few hours, apoptosis causes neurons throughout the entire area to die progressively. However, this sequence of events is related to the reperfusion of regenerated capillaries or collateral circulation, and is considered to be potentially salvageable. Similar findings have been reported in the retina after ischaemia-reperfusion injury in rats. In the present study, we intended to investigate whether delayed cell death is involved in neuronal injuries to the inner retina during chronic retinal ischaemia. Experimental branch retinal vein occlusion (BRVO) was induced in miniature pigs using indirect argon laser. The eyes were prelevated at 4, 24 and 48 hours and at 1 and 3 weeks following BRVO. The caspase inhibitor Z-VAD was injected intravitreally 24 hours after BRVO. Affected retinas were examined 24 hours later for any protective effect from apoptotic cell death. Histological examination with cresyl violet staining and TUNEL (TdT-mediated dUTP-biotin nick-end labelling) was performed on the samples. A progressive oedema of the nerve fibre, ganglion cell and inner plexiform layers, related to a widely diffused cell necrosis, was observed in the affected territory within 4-24 hours after BRVO. This was followed by a wave of apoptosis localized at the periphery of the affected territory, which peaked approximately 48 hours after BRVO and was associated with a diffuse oedema of the inner nuclear layer. A progressive atrophy of the inner retina was observed 1-3 weeks after BRVO. Injection of the caspase inhibitor Z-VAD (24 hours after BRVO) decreased the amount of apoptotic cell bodies 48 hours after BRVO. This study shows that although necrosis is the predominant form of neuronal death in the early phase, massive delayed neuronal cell death caused by apoptosis occurs on a widespread basis as a result of chronic ischaemia after BRVO in the retina. Further studies are needed to evaluate the possibility of rescuing retinal neurons from death by neuroprotective treatments.

The Inhalation Anesthetic Desflurane Induces Caspase Activation and Increases Amyloid β-Protein Levels under Hypoxic Conditions

Perioperative factors including hypoxia, hypocapnia, and certain anesthetics have been suggested to contribute to Alzheimer disease (AD) neuropathogenesis. Desflurane is one of the most commonly used inhalation anesthetics. However, the effects of desflurane on AD neuropathogenesis have not been previously determined. Here, we set out to assess the effects of desflurane and hypoxia on caspase activation, amyloid precursor protein (APP) processing, and amyloid beta-protein (Abeta) generation in H4 human neuroglioma cells (H4 naïve cells) as well as those overexpressing APP (H4-APP cells). Neither 12% desflurane nor hypoxia (18% O(2)) alone affected caspase-3 activation, APP processing, and Abeta generation. However, treatment with a combination of 12% desflurane and hypoxia (18% O(2)) (desflurane/hypoxia) for 6 h induced caspase-3 activation, altered APP processing, and increased Abeta generation in H4-APP cells. Desflurane/hypoxia also increased levels of beta-site APP-cleaving enzyme in H4-APP cells. In addition, desflurane/hypoxia-induced Abeta generation could be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the Abeta aggregation inhibitor clioquinol and gamma-secretase inhibitor L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In summary, desflurane can induce Abeta production and caspase activation, but only in the presence of hypoxia. Pending in vivo confirmation, these data may have profound implications for anesthesia care in elderly patients, and especially those with AD.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Sevoflurane-Mediated Activation of p38-Mitogen-Activated Stresskinase is Independent of Apoptosis in Jurkat T-Cells

Modulation of the inflammatory stress response by anesthesia may be responsible for an increased susceptibility to infectious complications, such as wound infection or pneumonia. Sevoflurane, a specific inhibitor of activator protein-1, an immediate early transcription factor, induces apoptosis in T-cells. Because p38 can be involved either in pro- or antiapoptotic processes, we examined whether the sevoflurane-induced apoptosis is mediated by p38 activation in Jurkat T-cells. Jurkat T-cells were exposed to different concentrations of sevoflurane, isoflurane, or desflurane in vitro. Phosphorylation of mitogen-activated protein (MAP) kinases, upstream kinases, downstream activating transcription factor 2 ATF-2, and caspase-3 processing were evaluated by Western blot. p38 kinase activity was evaluated after immunoprecipitation and phosphorylation of the substrate ATF-2 using Western blot. Apoptosis was assessed using flow cytometry after staining with green fluorescent protein-annexin V. While desflurane had no effect, sevoflurane and isoflurane induced p38 phosphorylation with sevoflurane inducing p38 kinase activity. Sevoflurane did not affect the MAP kinases ERK and JNK. Sevoflurane exposure also induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1), MAP kinase kinases 3 and 6 (MKK3/MKK6), and ATF-2. Pretreatment of cells with the general caspase inhibitor Z-VAD.fmk did not prevent the sevoflurane-induced phosphorylation of p38. Isoflurane- and sevoflurane-mediated caspase-3 processing and apoptosis could not be abolished by pretreatment with the specific p38 inhibitors SB202190 and SB203580. Sevoflurane is a specific activator of the apoptosis signal-regulating kinase-1-, MKK3/MKK6-p38 MAP kinase cascade in Jurkat T-cells. Our data suggest that sevoflurane-induced p38 activation is not affected by caspase activation. Furthermore, sevoflurane-induced apoptosis is not dependent on p38 MAP kinase activation.

Apoptosis induces expression of sphingosine kinase 1 to release sphingosine‐1‐phosphate as a “come‐and‐get‐me” signal

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen

Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.

Dominance of cytokine- over FasL-induced impairment of the mitochondrial transmembrane potential (ΔΨm) in the pancreatic β-cell line NIT-1

Mitochondria of pancreatic beta-cells are potential targets of intrinsic and extrinsic apoptotic pathways in the autoimmune pathogenesis of type 1 diabetes. We aimed to investigate whether cytokine- and FasLigand (FasL)-induced apoptosis is associated with impaired mitochondrial transmembrane potential (Deltapsim) in the pancreatic beta-cell line NIT-1. NIT-1 cells were exposed to the interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma) cytokine combination to induce apoptosis in vitro. Low concentrations of cytokines resulted in Deltapsim impairment, and increasing concentrations had only a minor additional effect. Treatment with the inducible nitric oxide synthase (iNOS) inhibitor Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME) prevented cytokine-mediated Deltapsim impairment, implying that cytokines affect Deltapsim via nitric oxide. The broad-spectrum caspase inhibitor Z-VAD(Ome)-FMK (ZVAD) revealed dichotomic actions. In the presence of ZVAD, cytokine-induced nitrite generation was increased but cell death and Deltapsim impairment were reduced. Deltapsim impairment was also reduced by inhibitors of caspases 1, 6 and 8. Induction of Fas by IL-1beta/IFN-gamma coupled with activation by Super-FasL augmented cytokine-induced cell death. We observed a clear dominance of cytokine- over FasL-induced effects on Deltapsim. Our findings show that IL-1beta/IFN-gamma cytokines have a strong effect to impair Deltaym and prime beta-cells for apoptosis via the intrinsic pathway mediated by iNOS and caspases. Furthermore, at least in NIT-1 cells, the extrinsic FasL/Fas pathway has only a minor additive effect on cytokine-induced Deltapsim impairment.

Smac’d to Death

Inhibitor of apoptosis (IAP) proteins bind, through their baculovirus IAP repeat (BIR) domains, to caspases, the proteases that mediate apoptosis, thus preventing apoptotic cell death. IAPs also contain RING domains and so act as E3 ubiquitin ligases that target proteins (and themselves) for proteasomal degradation. IAPs are more abundant in cancer cells than in normal cells, making IAPs attractive therapeutic targets. IAP activity is blocked by Smac (second mitochondrial activator of caspases), whose release from mitochondria is triggered by proapoptotic stimuli. Two groups have now designed and characterized mimetics of Smac. Varfolomeev et al . treated various cancer cell lines with their compounds, MV1 and BV6, and saw the rapid degradation of the IAP family members, c-IAP1 and c-IAP2, as assessed by Western blotting, which was blocked by an inhibitor of the proteasome. Both compounds also led to a dose-dependent increase in cell death, which was blocked by the caspase inhibitor z-VAD. Blockade of tumor necrosis factor-α (TNF-α) inhibited BV6-mediated apoptosis, as did a small interfering (si) RNA against the TNF-α receptor (TNFR1). Vince et al . found similar results when characterizing their IAP antagonist compounds. TNF-α production is characteristic of nuclear factor κB (NF-κB) activation. Both groups showed that the canonical and noncanonical NF-κB pathways were activated in cells in which IAPs were antagonized. NIK (NF-κB-inducing kinase), an activator of the noncanonical pathway, was constitutively bound to IAPs and then degraded, which was prevented by treatment with the Smac mimetics. Apoptosis mediated by these compounds was blocked by inhibitors of NF-κB. IAP antagonism also increased the recruitment to the TNFR1 of the kinase receptor-interacting protein 1 (RIP1), which is important for canonical NF-κB activation. Together these studies show that rather than simply interfering with the ability of IAPs to bind to caspases, Smac mimetics trigger IAP degradation, which has a twofold outcome. First, caspases are now uninhibited and responsive to a proapoptotic signal. Second, IAP degradation removes a block to noncanonical NF-κB activation, leading to the production of TNF-α that acts in an autocrine fashion to trigger cell death. Wu et al . discuss the potential use of these compounds in anticancer therapies. E. Varfolomeev, J. W. Blankenship, S. M. Wayson, A. V. Fedorova, N. Kayagaki, P. Garg, K. Zobel, J. N. Dynek, L. O. Elliott, H. J. A. Wallweber, J. A. Flygare, W. J. Fairbrother, K. Deshayes, V. M. Dixit, D. Vucic, IAP antagonists induce autoubiquitination of c-IAPs, NF-κB activation, and TNFα-dependent apoptosis. Cell 131 , 669-681 (2007). [Online Journal] J. E. Vince, W. W.-L. Wong, N. Khan, R. Feltham, D. Chau, A. U. Ahmed, C. A. Benetatos, S. K. Chunduru, S. M. Condon, M. McKinlay, R. Brink, M. Leverkus, V. Tergaonkar, P. Schneider, B. A. Callus, F. Koentgen, D. L. Vaux, J. Silke, IAP antagonists target cIAP1 to induce TNFα-dependent apoptosis. Cell 131 , 682-693 (2007). [Online Journal] H. Wu, J. Tschopp, S.-C. Lin, Smac mimetics and TNFα: A dangerous liaison? Cell 131 , 655-658 (2007). [Online Journal]

IAP Antagonists Induce Autoubiquitination of c-IAPs, NF-κB Activation, and TNFα-Dependent Apoptosis

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

EF24 Induces G2/M Arrest and Apoptosis in Cisplatin-resistant Human Ovarian Cancer Cells by Increasing PTEN Expression

We report that EF24, a synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one, greatly inhibits cisplatin-resistant (CR) human ovarian cancer cell proliferation. The inhibitory effect of EF24 on cell proliferation is associated with G(2)/M phase cell cycle arrest and increased G(2)/M checkpoint protein (pp53, p53, and p21) levels. Within 24 h following treatment, EF24 induced apoptosis in CR cells. The apoptosis was partially blocked by the general caspase inhibitor z-VAD. Within 12 h, EF24 induced a membranous FasL expression, consistent with a substantial decrease in the Ser(473) and Thr(308) phosphorylation of Akt, a known negative regulator of FasL transcription. Also, EF24 activated the phosphorylated PTEN and marginally up-regulated total PTEN expression through the inhibition of ubiquitin-mediated PTEN degradation. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt phosphorylation at Ser(473) and Thr(308), resulting in decreased apoptosis and increased cell survival. On the other hand, overexpression of PTEN markedly induced apoptosis. Our results clearly suggested that EF24 induced significant increase in PTEN expression. The up-regulation of PTEN inhibited Akt and MDM2, which enhanced the level of p53, thereby inducing G(2)/M arrest and apoptosis. Therefore, EF24 appears to have a potential therapeutic role in human ovarian cancer through the activation of PTEN.

PROTECTIVE AND APOPTOTIC CASPASES CONTROL THE SURVIVAL OF THE RAT INITIAL SEGMENT THROUGH CLEAVAGE OF THE REACTIVE OXYGEN SPECIES GENERATOR PHOSPHOLIPASE A2

The role of caspases is no longer restricted to cell death pathways. Caspases have been implicated in development, proper functioning of the immune system, and even in the protection of cells. The initial segment of the epididymis is a tissue that undergoes cell death within 12–18 hours after withdrawal of luminal testicular factors by efferent duct ligation (EDL). The signaling proteins controlling this region-specific cell death remain unknown. Attempts to identify caspases involved through Western Blotting have demonstrated that the initiator caspase 9 and the executioner caspase 3 are cleaved and activated in both the normal and EDL tissue and that the presence of these active caspases is confined to the most proximal region of the initial segment. Western Blotting of nuclear and cytoplasmic extracts has confirmed that cleaved caspase 3 is located in both the cytoplasm and nucleus of normal and EDL treated initial segment tissue. Immunohistochemistry has confirmed that upon 18 hour EDL, in addition to cytoplasmic cleaved caspase 3, there is an an increase in nuclear-localized cleaved caspase 3 staining suggesting that caspase 3 plays a role in initial segment cell death. To determine if cleaved caspase 3 plays a role in the protection of the initial segment in normal tissue, the caspase inhibitor Z-VAD FMK was injected into the initial segment of EDL and normal initial segment tissue. A trend was visible that seemed to suggest an increase in cell death in Z-VAD FMK treated initial segments following a 12 hour EDL. In addition, many cells within normal initial segment tissue injected with Z-VAD FMK appeared to undergo cell death and sloughing into the lumen, suggesting that inhibition of caspases could promote cell death. Lastly, Western Blotting confirmed that phospholipase A2, an enzyme that metabolizes phospholipids and generates reactive oxygen species through the generation of oxidative biproducts, is inactivated by caspases in the normal initial segment tissue through cleavage, and that this cleavage is inhibited in initial segments that have undergone EDL. These data suggest that prodeath caspases contribute to cell death in the initial segment and that a potentially separate population of caspases performs a protective function in the initial segment that involves cleavage and inhibition of the reactive oxygen species generator, phospholipase A2. Supported by NIH-NICHD HD045890 (poster)

Neuronal death in the lateral geniculate nucleus of young ferrets following a cortical lesion: Time-course, age dependence and involvement of caspases

In humans and many other mammalian species, the behavioural consequences of a cortical lesion tend to be milder when it occurs early in life, and there is evidence that an important factor contributing to the behavioural sparing in the young is the formation of new thalamo-cortical connections by thalamic neurons initially connected with the lesioned area. However, this plasticity may be hindered by the secondary death of many of these neurons owing to the elimination by the primary lesion of their trophic support from the cortex. With the long-term aim of preventing this neuronal death, we have here characterised its timing in the lateral geniculate nucleus of ferrets following lesions of the visual cortex on postnatal days 5, 10, 20 or 35. After the earliest lesions (P5 or P10), this cell death began rapidly and occurred synchronously, being maximal at 48 h and declining to zero over the next few days. Following later lesions the cell death began more slowly and continued for longer. The dying neurons contained activated caspase-3 and fragmented DNA and their number 2 days after a P5 lesion was reduced by the broad-band caspase inhibitor z-VAD.fmk. These experiments open the way for a concerted effort to enhance adaptive plasticity by neuroprotection in the hours or days following a cortical lesion.

Sensitization of vascular smooth muscle cell to TNF-α-mediated death in the presence of palmitate

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-α-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-α-mediated nuclear factor kappa B (NF-κB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-κB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-κB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-α-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.

The Inhalation Anesthetic Isoflurane Induces a Vicious Cycle of Apoptosis and Amyloid β-Protein Accumulation

The anesthetic isoflurane has been reported to induce apoptosis and increase Abeta generation and aggregation. However, the molecular mechanism underlying these effects remains unknown. We therefore set out to assess whether the effects of isoflurane on apoptosis are linked to amyloid beta-protein (Abeta) generation and aggregation. For this purpose, we assessed the effects of isoflurane on beta-site amyloid beta precursor protein (APP)-cleaving enzyme (BACE) and gamma-secretase, the proteases responsible for Abeta generation. We also tested the effects of inhibitors of Abeta aggregation (iAbeta5, a beta-sheet breaker peptide; clioquinol, a copper-zinc chelator) on the ability of isoflurane to induce apoptosis. All of these studies were performed on naive human H4 neuroglioma cells as well as those overexpressing APP (H4-APP cells). Isoflurane increased the levels of BACE and gamma-secretase and secreted Abeta in the H4-APP cells. Isoflurane-induced Abeta generation could be blocked by the broad-based caspase inhibitor Z-VAD. The Abeta aggregation inhibitors, iAbeta5 and clioquinol, selectively attenuated caspase-3 activation induced by isoflurane. However, isoflurane was able to induce caspase-3 activation in the absence of any detectable alterations of Abeta generation in naive H4 cells. Finally, Abeta potentiated the isoflurane-induced caspase-3 activation in naive H4 cells. Collectively, these findings suggest that isoflurane can induce apoptosis, which, in turn, increases BACE and gamma-secretase levels and Abeta secretion. Isoflurane also promotes Abeta aggregation. Accumulation of aggregated Abeta in the media can then promote apoptosis. The result is a vicious cycle of isoflurane-induced apoptosis, Abeta generation and aggregation, and additional rounds of apoptosis, leading to cell death.

Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.

Apoptosis in sulfur mustard treated A549 cell cultures

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30–1000μM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100–500 μM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

Study on the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by tumor necrosis factor related apoptosis inducing ligand

To explore the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic. Apoptosis, integration of mitochondria (including DeltaPsim, cardiolipin), activity of Caspase-9 and release of cytochrome c in colon carcinoma cells SW1116 treated with TRAIL, were detected by means of flowcytometry, fluorometer method and western-blot at the different time point. After treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with DeltaPsim, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was (48.12+/-2.21)micromol.L(-1).h(-1).mg(-1) protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk. Mitochondrial pathways involved in the apoptosis of colon carcinoma cell induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondrial.