Caspase Inhibitor Z-VAD Research Articles

Expressed isolated integrin β1 subunit cytodomain induces endothelial cell death secondary to detachment

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Reactive oxygen species mediation of Baizhu-induced apoptosis in human leukemia cells

Baizhu ( Atractylodes macrocephala Koidz) has traditionally been used as an important ingredient of several Chinese herbal medicines, which have been used for abdominal pain and gastroenterology diseases for thousands of years. Despite its popularity in herbal therapies, little is known about the anticancer effect of Baizhu. In this study, the anticancer potential of Baizhu on human hepatoma and leukemia cell lines was evaluated. Baizhu methanol extract induced apoptosis in human lymphoma Jurkat T cells, leukemia U937, and HL-60 cells. This was confirmed by several methods, including hypodiploid cells detection using flow cytometry, the examination of apoptotic bodies containing cells using confocal laser scanning microscopy, and hypodiploid cell population inhibition using the broad spectrum caspase inhibitor z-VAD. Finally, the intracellular reactive oxygen species (ROS), especially hydrogen peroxide (H 2O 2) and superoxide anion (O 2 −), were found to be elevated after treatment of these cells with Baizhu extracts. Antioxidant N-acetyl cysteine (NAC) pretreatment almost completely inhibited Baizhu-induced apoptosis, suggesting that ROS are the key mediators for Baizhu-induced apoptosis. All these data indicate that Baizhu is a possible anti-tumor agent that induces apoptosis of human leukemia cells through ROS generation.

Caspase Inhibition Attenuates Contractile Dysfunction Following Cardioplegic Arrest and Rewarming in the Setting of Left Ventricular Failure

Hyperkalemic cardioplegic arrest (HCA) and rewarming evokes postoperative myocyte contractile dysfunction, a phenomenon of particular importance in settings of preexisting left ventricular (LV) failure. Caspases are intracellular proteolytic enzymes recently demonstrated to degrade myocardial contractile proteins. This study tested the hypothesis that myocyte contractile dysfunction induced by HCA could be ameliorated with caspase inhibition in the setting of compromised myocardial function. LV myocytes were isolated from control pigs (n = 9, 30 kg) or pigs with LV failure induced by rapid pacing (n = 6, 240 bpm for 21 days) and were randomized to the following: (1) normothermia (2003 myocytes), incubation in cell culture medium for 2 hours at 37 degrees C; (2) HCA only (506 myocytes), incubation for 2 hours in hypothermic HCA solution (4 degrees C, 24 mEq K); or (3) HCA + z-VAD, incubation in hypothermic HCA solution supplemented with 10 microM of the caspase inhibitor z-VAD (z-Val-Ala-Asp-fluoromethyl-ketone, 415 myocytes). Inotropic responsiveness was examined using beta-adrenergic stimulation (25 nM isoproterenol). Ambient normothermic myocyte shortening velocity (microm/s) was reduced with LV failure compared with control values (54 +/- 2 versus 75 +/- 2, respectively, P < 0.05). Following HCA, shortening velocity decreased in the LV failure and control groups (27 +/- 5 and 45 +/- 3, P < 0.05). Institution of z-VAD increased myocyte shortening velocity following HCA in both the LV failure and control groups (49 +/- 5 and 65 +/- 5, P < 0.05). Moreover, HCA supplementation with z-VAD increased beta-adrenergic responsiveness in both groups compared with HCA-only values. This study provides proof of concept that caspase activity contributes to myocyte contractile dysfunction following simulated HCA. Pharmacologic caspase inhibition may hold particular relevance in the execution of cardiac surgical procedures requiring HCA in the context of preexisting LV failure.

Different apoptotic mechanisms are involved in the antiproliferative effects of 7β-hydroxysitosterol and 7β-hydroxycholesterol in human colon cancer cells

Plant sterols are found in fruits and vegetables. Their cholesterol-lowering effect is well documented. Our study aimed at comparing antiproliferative effects of 7beta-hydroxysitosterol (7beta-OHsito) versus 7beta-hydroxycholesterol (7beta-OHchol) on the human colon cancer cell line Caco-2. When cells were exposed for 32 h to 60 microM 7beta-OHsito or to 30 microM 7beta-OHchol, both compounds caused 50% growth inhibition. Cells treated with 7beta-OHsito showed enhanced caspase-9 and -3 activities followed by DNA fragmentation. In contrast, 7beta-OHchol did not activate caspase-3 and activation of caspase-9 and DNA fragmentation were delayed. The treatment of cells with the caspase inhibitor Z-VAD.fmk retarded the 7beta-OHsito-induced apoptotic process but not that triggered by 7beta-OHchol. Our data suggest that the two compounds in spite of their structural similarities target different cellular pathways, which lead to cell death.

NO-donating aspirin inhibits the growth of leukemic Jurkat cells and modulates β-catenin expression

β-Catenin has been implicated in leukemic cell proliferation. We compared the effects of aspirin (ASA) and the ortho, meta, and para positional isomers of NO-donating aspirin (NO-ASA) on cell growth and β-catenin expression in human Jurkat T leukemic cells. Cell growth inhibition was strong: IC 50 for p-, o-, and m- were 20 ± 1.6 (mean ± SEM), 15 ± 1.5, and 200 ± 12 μM, respectively, in contrast to that of ASA (3200 ± 375 μM). The para isomer of NO-ASA degraded β-catenin in a dose- and time-dependent manner coinciding with increasing expression of activated caspase-3. The caspase inhibitor ZVAD blocked β-catenin cleavage by p-NO-ASA and partially reversed cell growth inhibition by p-NO-ASA but not that by ASA. A denitrated analog of p-NO-ASA did not degrade β-catenin indicating the importance of the NO-donating moiety. Our findings suggest that NO-ASA merits further study as an agent against leukemia.

Bortezomib Sensitizes Non-Hodgkin's Lymphoma Cells to Apoptosis Induced by Antibodies to TRAIL Receptors R1 and R2.

TRAIL-R1 and -R2 signaling induces apoptosis via a pathway that activates caspase 8. The proteosome inhibitor bortezomib may act via several pathways. Agonistic antibodies to TRAIL-R1 and -R2 and bortezomib are in clinical trials in solid tumors and hematologic malignancies. To develop rational combinations for future clinical studies, we investigated the actions of these agents on non-Hodgkin s lymphoma (NHL) cell lines. The t(14;18)+, EBV- NHL cell lines DoHH2 and WSU-FSCCL were treated with agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR1) and -R2 (HGS-ETR2) (Human Genome Sciences, Rockville, MD) and/or bortezomib. While HGS-ETR 1 and HGS-ETR 2 are effective inducers of apoptosis in FSCCL, DoHH2, which expresses dim TRAIL-R1 (DR4, HGS-ETR1 target) and TRAIL-R2 (DR5, HGS-ETR2 target), shows minimal growth inhibition or apoptosis induction by HGS-ETR1 or HGS-ETR2. Bortezomib has modest effects on DoHH2 cells in growth inhibition and apoptosis assays. HGS-ETR1 and HGS-ETR2 induction of apoptosis in WSU-FSCCL is efficiently blocked by the caspase inhibitor ZVAD. In contrast, bortezomib effects are not blocked by ZVAD, indicating an independent mechanism of action. To determine if these separate pathways would provide enhanced combination activity, DoHH2 cells were pre-treated with bortezomib for 30 min, followed by incubation with HGS-ETR1 or HGS-ETR2. This led to supra-additive induction of apoptosis (annexin V staining). We conclude that bortezomib sensitizes DoHH2 cells to the action of HGS-ETR1 and HGS-ETR2. Further, bortezomib induces apoptosis in DoHH2 cells by an independent mechanism, and the combination of TRAIL-receptor signaling and bortezomib may be a useful combination to explore.

Inhibition of XIAP Renders Newly CD40-Activated Chronic Lymphocytic Leukemia Cells Sensitive to Fas (CD95) Mediated Apoptosis.

CD40 ligation induces CLL cells to express high levels of Fas (CD95) and DR5, a death receptor for the tumor necrosis factor (TNF) receptor apoptosis-inducing ligand (TRAIL). Despite expression of these death receptors, CD40-activated CLL cells initially are resistant to CD95- or TRAIL-mediated apoptosis, even under conditions that repress CD40-induced expression of both isoforms of the flice-inhibitory protein (FLIP) that can inhibit recruitment of pro-caspase 8 to the death-inducing signaling complex (Proc Natl Acad Sci U S A 100:3854–9, 2002). One potential explanation for how newly CD40-activated CLL cells can resist CD95/DR5-mediated apoptosis is through the high-level expression of XIAP, an important downstream inhibitor of caspase activation that is expressed by CLL cells and many other types of cancer. The anti-apoptotic activity of XIAP can be circumvented by SMAC (Diablo), a natural inhibitor to the inhibitors of apoptosis that is released from mitochondria following activation of the intrinsic apoptotic pathway. SMAC inhibits XIAP by blocking its BIR domain(s), thereby precluding XIAP from inhibiting active caspases, such as caspase 9. Using mixture-based combinatorial libraries, we identified a series of polyphenylureas that selectively target the BIR2 domain of XIAP and that do not compete with SMAC for binding to XIAP (Cancer Cell 5:25–35, 2004). Recently, structural activity studies resulted in the identification of analogs with improved drug-like characteristics. We used an active (TPI 1540–14) and inactive structural analog (TPI 1540–20) in this study to investigate whether inhibition of XIAP could enhance the sensitivity of CD40-activated CLL cells to CD95-mediated apoptosis. Consistent with prior studies, CLL cells following CD40-ligation initially were resistant to apoptosis induced by the anti-CD95 IgM mAb CH11, despite having high-level, de novo expression of CD95. However, when these CH11-resistant cells were treated with TPI 1540-14, but not with the control compound TPI 1540-20, the CD40-activated CLL cells became sensitive to CH11-mediated apoptosis. Immunoblot analyses of lysates made from treated cells demonstrated that CD40-activated CLL cells treated with TPI1540-14 experienced activation of caspases 8 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP) upon treatment with CH11. The non-specific caspase inhibitor zVAD could inhibit the apoptosis induced by CH11. We also observed similar activity with TPI 1540-14 on blood mononuclear cells collected from patients one day after they were treated with an infusion of autologous CD154-transfected CLL cells. This study provides the first evidence that distal apoptosis regulators play a role in the resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that such inhibitors may work synergistically with immune-therapy strategies that target CD40, such as CD40-ligand (CD154) gene therapy.

The cowpox virus host range gene, CP77, affects phosphorylation of eIF2 α and vaccinia viral translation in apoptotic HeLa cells

Host restriction of vaccinia virus has been previously described in CHO and RK13 cells in which a cowpox virus CP77 gene rescues vaccinia virus growth at the viral protein translation level. Here we investigate the restrictive stage of vaccinia virus in HeLa cells using a vaccinia mutant virus (VV-hr) that contains a deletion of 18-kb genome sequences resulting in no growth in HeLa cells. Insertion of CP77 gene into VV-hr generated a recombinant virus (VV-36hr) that multiplied well in HeLa cells. Both viruses could enter cells, initiate viral DNA replication and intermediate gene transcription. However, translation of viral intermediate gene was only detected in cells infected with VV-36hr, indicating that CP77 relieves host restriction at the intermediate gene translation stage in HeLa cells. Caspase-2 and -3 activation was observed in HeLa cells infected with VV-hr coupled with dramatic morphological alterations and cleavage of the translation initiation factor eIF4G. Caspase activation was reduced in HeLa cells infected with VV-36hr, indicating that CP77 acts upstream of caspase activation. Enhanced phosphorylation of PKR and eIF2α was also observed in cells infected with VV-hr and was suppressed by CP77. Suppression of eIF4G cleavage with the caspase inhibitor ZVAD did not rescue virus translation, whereas expression of a mutant eIF2α protein with an alanine substitution of serine at amino acid position 51 (eIF2αS51A) partially restored viral translation and moderately increased virus growth in HeLa cells.

Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores.

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.

Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas.

Non-small-cell lung carcinomas (NSCLCs) are resistant to the induction of apoptosis by conventional anticancer treatment. However, NSCLC cell lines are sensitive to the action of the broad protein kinase inhibitor, staurosporine (STS). In the NSCLC cell line U1810, STS induced the mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) followed by activation of caspases, nuclear condensation, DNA fragmentation and finally cell death. Although preincubation of U1810 cells with the broad-spectrum caspase inhibitor z-VAD.fmk delayed the occurrence of nuclear apoptosis induced by STS, it did not impede mitochondrial alterations (such as the release of Cyt c and AIF) and cell death to occur. Moreover, the microinjection of neither Cyt c nor recombinant active caspase-3 into the cytoplasm promoted nuclear apoptosis-related changes in U1810 cells. Evaluation of the role of the caspase-independent factor AIF in STS-mediated death revealed that, upon immunodepletion of AIF, cytosols from STS-treated U1810 lost their capacity to induce nuclear condensation when incubated with isolated nuclei. In addition, microinjection of an anti-AIF antibody prevented AIF from translocating to the nuclei of STS-treated U1810 cells and reduced STS-induced cell death. Finally, although the transfection-enforced overexpression of AIF was not sufficient to induce cell death, it did enhance STS-mediated cell killing. Altogether, these results indicate that activation of caspases is not sufficient to kill U1810 cells and rather suggests an important role for the AIF-mediated mitochondrial-mediated death pathway.

Differentiation-induced HL-60 cell apoptosis: a mechanism independent of mitochondrial disruption?

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.

AAV-encoded expression of TRAIL in experimental human colorectal cancer leads to tumor regression.

Gene transfer vectors based on the adeno-associated virus (AAV) are used for various experimental and clinical therapeutic approaches. In the present study, we demonstrate the utility of rAAV as a tumoricidal agent in human colorectal cancer. We constructed an rAAV vector that expresses tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) and used it to transduce human colorectal cancer cells. TRAIL belongs to the TNF superfamily of cytokines that are involved in various immune responses and apoptotic processes. It has been shown to induce cell death specifically in cancer cells. Transduction with AAV.TRAIL gave rise to rapid expression of TRAIL, followed by induction of apoptosis, which could be inhibited by the caspase inhibitor z-VAD.fmk, in several human colon cancer cell lines. The apoptotic mechanism included activation of caspase-3, as well as cytochrome c release from mitochondria. The outgrowth of human colorectal tumors grown in mice was completely blocked by transduction with AAV.TRAIL in vitro, while in vivo transduction significantly inhibited the growth of established tumors. AAV vectors could provide a safe method of gene delivery and offer a novel method of using TRAIL as a therapeutic protein.

Effect of Ginkgo biloba (EGb 761) on staurosporine-induced neuronal death and caspase activity in cortical cultured neurons

Previous studies suggest the protective potentiality of Ginkgo biloba (EGb 761) against apoptotic cell death induced by hydroxyl radicals, staurosporine, serum deprivation and β-amyloid (βA) peptide. We have extended these observations to cultured cortical neurons and studied the effect of EGb 761 on neuronal survival (evaluated as MTT reduction), the presence of condensed nuclei (monitored as Hoechst staining), the time-course of caspase-1, caspase-3 and caspase-9 activation (measured by cleavage of specific fluorescent substrates) and superoxide anion production (evaluated by hydroethidine staining) after the exposure to staurosporine. Results show that 200 μg/ml of EGb 761 increased cell survival and reduced the number of condensed nuclei after the exposure to 200 nM staurosporine. Vitamin E and the spin trapper α-phenyl- N- tert-butylnitrone (PBN) also significantly increased cell survival. In contrast, the broad-spectrum caspase inhibitors ZVAD and ZBIOT showed no protection. Similarly, selective inhibitors of caspase-1 (YVAD-CHO), caspase-2 (VDVAD-CHO), caspase-3 (DEVD-CHO) and caspase-8 (IETD-CHO) did not protect against cell damage induced by staurosporine. The protective effect of EGb 761 was not enhanced when coincubated with vitamin E or DEVD-CHO. Caspase-3 activity was maximally induced 5–8 h after staurosporine exposure. Both EGb 761 and vitamin E showed a tendency to decrease caspase-3 activity. In contrast, activation of caspase-1 and caspase-9 was not observed at any of the times studied after STS exposure. Exposure to staurosporine resulted in increased superoxide production that was maximal at 5 h. EGb 761 significantly inhibited superoxide production at short times after staurosporine exposure. Vitamin E and PBN also significantly reduced superoxide production. Results suggest that EGb 761 neuroprotective effect might be mediated by its well-known antioxidant activity, which might also influence caspase-3 activation. Inhibition of capase-3 induced by EGb 761 and vitamin E does not seem to contribute to their observed protective action.

Degradation of poly(A)-binding protein in apoptotic cells and linkage to translation regulation.

We have recently shown that poly(A)-binding protein (PABP) is cleaved during poliovirus and Coxsackievirus infection by viral 3Cprotease and that 3Cprotease modification of a subset of PABP can result in significant translation inhibition. During apoptosis, translation undergoes significant down-regulation that correlates with caspase-3 mediated cleavage of several translation factors, including eIF4G, 4EBP1 and eIF2alpha. The fate of PABP in apoptotic cells has not yet been examined. Here we show that PABP levels decline significantly via proteolytic degradation in apoptotic HeLa, Jurkat and MCF7 cells. The degradation of PABP correlated with translation inhibition but lagged behind cleavage of eIF4GI. In apoptotic MCF7 cells translation inhibition occurred without modification of most translation factors and correlated with PABP degradation. PABP was not cleaved during incubation with several caspases, yet caspase 3 induced weak PABP degradative activity in cells lysates. Both the caspase inhibitor zVAD and calpain inhibitors blocked PABP cleavage in vivo, while the proteosome inhibitor MG132 induced PABP degradation. Protease(s) activated during apoptosis preferentially degraded PABP associated with ribosomes and translation factors, but not PABP in other cellular compartments. The data suggest that targeted degradation of PABP contributes to translation inhibition in apoptotic cells.

Adriamycin Induced Apoptosis of H9c2 Cardiomyocytes via a Caspase-Independent Pathway

Background: The cardiotoxicity of adriamycin limits its clinical usefulness as a powerful drug for solid tumors and malignant hematological disease. Although the exact mechanism by which it causes cardiac damage is not yet known, several reports have suggested apoptosis to be the principal process in adriamycin-induced cardiomyopathy, which exhibits DNA fragmentation, cytochrome C release, and caspase activation. However, no direct evidence has linked the critical involvement of caspase-3 in adriamycin-induced apoptosis. Methods: To determine the requirements for the activation of caspase-3 in adriamycin-treated cardiac cells, we examined the effect of caspase inhibitor on the cell survival and apoptotic changes, using MTT assay, microscopy and Western blotting. Results: Exposure of H9c2 cells to adriamycin resulted in time- and dose-dependent cell death and cleavage of pro-caspase-3 and the nuclear protein poly (ADP-ribose) polymerase (PARP). However, neither the reduction of cell viability nor the characteristic morphological changes induced by adriamycin was prevented by pretreatment with the general caspase inhibitor z-VAD.FMK. In contrast, caspase inhibition effectively blocked the apoptosis induced by H2O2 in H9c2 cells, but was not essential for adriamycin-induced apoptosis in H9c2 cells. We also observed that p53 expression was increased by adriamycin, and this increase was not affected by the inhibition of caspase activity, suggesting a role for p53 in adriamycin-induced caspase-independent apoptosis in cardiac toxicity. Conclusions: Our results demonstrate that adriamycin specifically activates an apoptotic pathway that is not

Administration of nitric oxide with caspase inhibitors minimizes bacterial translocation in experimental intestinal transplantation.

Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after small-bowel transplantation (Trp). Nitric oxide (NO) and apoptosis could affect cell demise. The aim of this study was to asses whether supplementation of University of Wisconsin (UW) solution with NO donors and apoptosis inhibitors can abolish BT in Trp. The following experimental groups were studied: sham, Trp, intestinal transplantation, Trp+spermine NONOate (NONOs), and Trp+NONOs+caspase inhibitor Z-Val-Ala-Asp(Ome)-fluoromethylketone(Z-VAD-fmk). Histologic analysis, caspase-3 activity, DNA fragmentation, and BT from graft to mesenteric lymph nodes, liver, and spleen were measured in tissue samples after transplantation. During intestinal transplantation, apoptosis and necrosis were increased, showing graft injury and high levels of BT. The rats treated with NONOs showed a histologic protection of transplanted graft and a decrease in BT despite caspase-3 and DNA fragmentation-inducing effects. Administration of caspase inhibitor Z-VAD to NONOs-treated rats reversed the NO apoptosis-inducing effects and showed the lowest levels of BT in all tissues. Exogenous administration of NO associated with the inhibition of apoptosis maintains the graft in optimal conditions in terms of BT and improves the histology of the graft after intestinal transplantation in rats.

In vitro neurodegeneration model: dopaminergic toxin-induced apoptosis in human SH-SY5Y cells

The brains of patients with Parkinson's disease (PD) show evidence that mitochondrial complex I is suppressed. Therefore, it is considered that rotenone, a specific inhibitor of mitochondrial complex I, is a useful tool in animal models of PD. However, the mechanism of rotenone-induced neuronal death is not fully understood. In human SH-SY5Y cells, rotenone induced the degradation of caspases-9 and -3, followed by cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and cell death. At that time, we detected a caspase-12-like protein (casp-12LP) that has a molecular mass (about 60 kDa) similar to that of mouse caspase-12, and it was degraded by rotenone, like an endoplasmic reticulum (ER) stress. Pretreatment with phorbol-12-myristate-13-acetate inhibited the rotenone-induced decrease in caspases-9 and -3, but not that in casp-12LP. In contrast, nonselective caspase inhibitor z-VAD.fmk inhibited the decrease in casp-12LP, but not those in caspases-9 and -3 in this study. These results suggest that rotenone-induced apoptosis may be mediated through both mitochondria- and ER-dependent pathways in human SH-SY5Y cells.

N-(4-hydroxyphenyl)retinamide induces growth arrest and apoptosis in HTLV-I-transformed cells.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth and induces apoptosis in many human cell lines. We explored the effects of HPR on human T-cell lymphotropic virus type I (HTLV-I)-positive and HTLV-I-negative malignant T-cell lines, most of which are resistant to all-trans retinoic acid. Clinically achievable concentrations of HPR caused a dramatic inhibition of cell proliferation, G(0)/G(1) arrest, and massive apoptosis in all tested malignant T cells, while no effect was observed on resting or activated normal lymphocytes. Interestingly, HTLV-I-negative cell lines were significantly more sensitive to HPR compared to HTLV-I-positive and Tax-transfected cells. In HTLV-I-negative cells only, HPR-induced apoptosis was associated with ceramide accumulation, sharp decrease in mitochondrial membrane potential, and activation of caspases 8, 9 and 3, and could be partially reverted by the caspase inhibitor z-VAD suggesting that Tax protects infected cells from ceramide accumulation and caspase-mediated apoptosis. In HTLV-I-positive cells, HPR treatment rapidly induced proteasomal-mediated degradation of p21, downregulated cyclin D(1), and upregulated bax protein levels. These findings support a potential therapeutic role for HPR in both HTLV-I-associated adult T-cell leukemia/lymphoma (ATL) and HTLV-I-negative peripheral T-cell lymphomas.

TNFalpha impairs glucose stimulated insulin release in human islets by iCOX-independent mechanisms

Pro-inflammatory cytokines such as IL-1β, TNFα and IFNγ impair glucose stimulated insulin release (GSIR) and induce apoptosis of human islets in vitro. Previous work has suggested that the inducible cyclooxygenase (iCOX)/prostaglandin E2 (PGE2) pathway mediates inhibition of insulin secretion by IL-1β. We tested the hypothesis that iCOX mediates TNFα-induced injury in human islets. Human islets isolated from cadaveric donors were treated with recombinant human TNFα (1000 U/ml) alone or with a cytokine mixture (CM) containing TNFα, IL-1β (50 U/ml) and IFNγ (750 U/ml) +/− iNOS inhibitor L-NMMA (1 mM), iCOX inhibitor NS-398 (0.01 mM) or caspase inhibitor z-VAD (0.1 mM). At 40 hours, nitrite and PGE2 released to the medium were determined. Islets were subjected to either glucose challenge or RNA extraction. RNase protection assay was used to determine cytokine-regulated gene expression for iCOX and iNOS. At 72 hours, islets were dissociated and flow cytometry used to determine rates of apoptosis. Control islets demonstrated a 15.2 ± 0.5 fold insulin secretion index (ISI, insulin released in high glucose vs. that in low glucose containing solutions). TNFα and CM treated islets demonstrated impaired ISI ( 6.2 ± 0 and 4.1 ± 0.5, respectively). Control islets in culture for 72 hours demonstrated an apoptosis rate of 6.2 ± 0.2%. TNFα induced the rate to 8.0 ± 0.3%, CM treatment 11.1 ± 0.5%. TNFα stimulated iCOX but not iNOS gene expression, while CM stimulated the expression of both genes. PGE2 produced by control islets was 4.4 ± 0.1 pg/ml. TNFα stimulated PGE2 production to 25.2 ± 0.3 pg/ml and CM 100 ± 1.1 pg/ml. Control and TNFα-treated islets had a nitrite production level of 1.6 ± 0.1 μM, CM 3.1 ± 0.2 μM. Co-treatment with NS-398 completely blocked cytokine-induced PGE2 production by the islets. Co-treatment with L-NMMA completely blocked CM-induced nitrite production. z-VAD protected the islets from cytokine-induced apoptosis. NS-398, L-NMMA, and z-VAD all failed, however, to protect the islets from TNFα- or CM-impaired GSIS. Proinflammatory cytokines inhibit islet insulin secretion in response to glucose challenge. iCOX- and iNOS-independent mechanisms are involved in the process.

A flow cytometric method to detect protein-protein interaction in living cells by directly visualizing donor fluorophore quenching during CFP-->YFP fluorescence resonance energy transfer (FRET).

Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency. A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by comparing CFP intensity between FRET-positive and -negative populations. To accomplish this, we compared the CFP intensity in FRET-positive and FRET-negative cells within the same population transfected with a CFP/YFP fusion protein, in which the molar ratio of CFP:YFP was one. By using separate lasers to excite CFP and YFP, the detection of FRET was separated from that of YFP. Therefore, after direct excitation, the YFP emission spectrum remained constant in all transfected cells, whereas the emission spectrum of CFP varied with the extent of FRET in individual cells. Specific CFP/YFP fusion constructs were prepared to evaluate this approach. The first one consisted of CFP and YFP separated by two caspase cleavage sites (CFP-LEVD-YFP). A second construct consisted of CFP and YFP separated by a structurally restricted 232-amino acid (aa) spacer. No FRET was observed by transfectants expressing this construct. Transfection of CFP-LEVD-YFP into Hela cells resulted in a FRET-positive population and a FRET-negative one. The appearance of the FRET-negative population was inhibited by the caspase inhibitor z-VAD. Moreover, substituting D for A in the caspase cleavage sites of this probe abolished the FRET-negative population, demonstrating the probe's specificity for caspase activity. Comparison of the CFP emission in the FRET-positive and FRET-negative population was used to document the relationship of FRET to donor quenching and permit the calculation of FRET efficiency and relative molecular distance between CFP and YFP. Similar results were noted when cells transfected with the caspase-sensitive probe (in the presence of z-VAD) were mixed with cells expressing the CFP-YFP construct with the 232-aa spacer and therefore were FRET negative. This demonstrated the validity of calculating CFP donor quenching and FRET efficiency by comparing emission spectra of an unknown construct with that of a known positive control, both expressed by the same population of cells. Using this approach, we confirmed that members of the TNF receptor-associated factor (TRAF) family engaged in both homotypic and heterotypic interactions. We have established a novel flow cytometric approach to assess donor CFP quenching during CFP-->YFP FRET, which can be used for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties. This method can be used not only to analyze cells that express a CFP and YFP fusion protein, but also independent CFP-coupled and YFP-coupled interacting proteins.