Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (DeltaPsim) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and DeltaPsim disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and DeltaPsim disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or DeltaPsim disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.     

Modulation of tumor necrosis factor apoptosis-inducing ligand- induced NF-kappa B activation by inhibition of apical caspases.

Tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), a member of the TNF family, induces apoptosis in many transformed cells. We report TRAIL-induced NF-kappaB activation, concomitant with production of the pro-inflammatory cytokine Interleukin-8 in the relatively TRAIL-insensitive cell line, HEK293. In contrast, TRAIL-induced NF-kappaB activation occurred in HeLa cells only upon pretreatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.fmk), indicating that this was due to a caspase-sensitive component of TRAIL-induced NF-kappaB activation. NF-kappaB activation was mediated by the death receptors, TRAIL-R1 and -R2, but not by TRAIL-R3 or -R4 and was only observed in HeLa cells in the presence of z-VAD.fmk. Receptor-interacting protein, an obligatory component of TNF-alpha-induced NF-kappaB activation, was cleaved during TRAIL-induced apoptosis. We show that receptor-interacting protein is recruited to the native TRAIL death-inducing signaling complex (DISC) and that recruitment is enhanced in the presence of z-VAD.fmk, thus providing an explanation for the potentiation of TRAIL-induced NF-kappaB activation by z-VAD.fmk in TRAIL-sensitive cell lines. Examination of the TRAIL DISC in sensitive and resistant cells suggests that a high ratio of c-FLIP to caspase-8 may partially explain cellular resistance to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis was also modulated by inhibition or activation of NF-kappaB. Thus, in some contexts, modulation of NF-kappaB activation possibly at the level of apical caspase activation at the DISC may be a key determinant of sensitivity to TRAIL-induced apoptosis.     

Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.     

Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis

Endotoxin is one of the major factors causing myocardial depression and death during sepsis in humans. Recently, it was reported that endotoxin may induce cardiomyocyte apoptosis. Also, multiple caspase activation has been implicated in endotoxin-induced apoptosis in several organ systems. In this study, we investigated whether endotoxin would increase myocardial caspase activities and evaluated the effects of in vivo administration (3 mg/kg) of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone(z-VAD.fmk), the caspase-3-like inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone (z-DEVD.cmk), and the caspase-1-like inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD. fmk), on endotoxin-induced myocardial dysfunction and apoptosis. Endotoxin administration (10 mg/kg iv) induced myocardial contractile dysfunction that was associated with caspase activity increases and nuclear apoptosis. Broad-spectrum z-VAD.fmk and z-DEVD.cmk improved endotoxin-induced myocardial dysfunction and reduced caspase activation and nuclear apoptosis when given immediately and 2 h after endotoxin. In contrast, no effects of Ac-YVAD.fmk were observed on myocardial function and caspase-induced apoptosis. Administration of caspase inhibitors 4 h after endotoxin treatment was not able to protect the rat heart from myocardial dysfunction and nuclear apoptosis. These observations provide evidence that in our model, caspase activation plays a role in endotoxin-induced myocardial apoptosis. Caspase inhibition strategy may represent a therapeutic approach to endotoxin-induced myocardial dysfunction.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.     

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.     

Apoptosis triggered redistribution of caspase-9 from cytoplasm to mitochondria

Caspase-9 is an apoptosis initiator protease activated as a response to the mitochondrial damage in the cytoplasmic complex apoptosome. By fluorescence labelling of proteins, confocal microscopy and subcellular fractionations we demonstrate that caspase-9 is in the cytoplasm of non-apoptotic pituitary cells. The activation of apoptosis with rotenone triggers the redistribution of caspase-9 to mitochondria. Experiments using the general caspase inhibitor z-VAD.fmk and the specific caspase-9 inhibitor z-LEHD.fmk show that the caspase-9 redistribution is a regulated process and requires the activity of a caspase other than the caspase-9. We propose that this spatial regulation is required to control the activity of caspase-9.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway

Caspase-3 activity has been described to be essential for drug-induced apoptosis. Recent results suggest that in addition to its downstream executor function, caspase-3 is also involved in the processing of upstream caspase-8 and -9. To test the absolute requirement for caspase-3, we examined mitomycin C (MMC)-induced apoptosis in the caspase-3 deficient human breast cancer cell line MCF-7. MMC was used as anticancer drug since this agent was preferentially active compared to chemotherapeutic compounds with differing mechanisms of action such as cisplatin, docetaxel, or lovastatin. MMC treatment led to pronounced caspase-8, -9, and -7 processing and early morphological features of apoptosis within 48 h. This could be inhibited by the broad-spectrum caspase inhibitor z-VAD.fmk and to a lesser extent by z-IETD.fmk and z-LEHD.fmk, which have a certain preference for inhibiting caspase-8 and -9, respectively. MMC induced apoptosis in MCF-7 cells was not mediated by the death receptor pathway as demonstrated by experiments using the inhibiting anti-Fas antibody ZB4 and transfections with CrmA, a viral serpin inhibitor of caspase-8, and the dominant negative Fas-associated death domain (FADD-DN). Stable expression with Bcl-2 significantly prevented the processing of caspase-9 but also of caspase-8 and blocked the induction of apoptosis. Thus, we provide evidence that caspase-3 activity is dispensable for MMC-induced apoptosis and for caspase-8 and -9 processing in MCF-7 cells.     

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.     

c-IAP1 blocks TNFalpha-mediated cytotoxicity upstream of caspase-dependent and -independent mitochondrial events in human leukemic cells

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.     

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.     

Serum deprivation induced autophagy and predominantly an AIF-dependent apoptosis in hippocampal HT22 neurons

Neuronal death induced by serum deprivation (SD) in HT22-cells was accompanied by a moderate activation of caspase-3, a prominent upregulation of AIF and its translocation into the nucleus. In addition protein levels of autophagy markers such as LC3 and beclin-1 were affected by SD. The ratio of LC3-II/LC3-I was significantly increased in serum deprived cultures. Furthermore, the addition of the pan-caspase inhibitor z-VAD(OMe)-FMK (zVAD) does not protect HT22-cells from SD-induced neurodegeneration. However, addition of the autophagy inhibitors such as 3-methyladenine (3-MA) or bafilomycin A1 (BafA1) provided a potentiation of cell death induced by SD. z-VAD and 3-MA in combination were not only ineffective in rescuing cells from the damaging effects of SD, but seem likely to act in synergy to potentiate slightly SD-induced cell death. The results of the current study suggest that SD induced predominantly caspase-independent apoptosis in hippocampal HT22 cells and that inhibition of autophagy is rather deleterious than protective.     

Taurine monochloramine activates a cell death pathway involving Bax and Caspase-9

Taurine is an abundant free amino acid that interacts with the potent oxidant hypochlorous acid to form the less toxic and more stable oxidant taurine monochloramine (TauNHCl). TauNHCl has diverse cellular effects ranging from inhibiting the production of proinflammatory mediators to inhibiting cell proliferation and inducing cell death. We hypothesized that TauNHCl could activate a cell death pathway involving Bcl-2 members and the activation of caspase proteases. FL5.12 cells are lymphocytic cells that undergo apoptosis following interleukin-3 (IL-3) withdrawal. Therefore, cell death following TauNHCl treatment of FL5.12 cells was compared and contrasted with IL-3 withdrawal. We found that TauNHCl treatment activates a cell death pathway with kinetics very similar to IL-3 withdrawal. TauNHCl-treated cells undergo an annexin V-positive/propidium iodide-negative phase of death consistent with apoptosis. TauNHCl treatment results in a conformational change in BAX that is associated with its activation. Both Bcl-2 and, to a lesser degree, the dominant negative form of caspase-9 inhibit cell death following TauNHCl treatment. In contrast with IL-3 withdrawal, TauNHCl treatment of FL5.12 cells results in a rapid cell cycle arrest that is cell cycle phase-independent. These results demonstrate that TauNHCl treatment induces a rapid, cell cycle-independent proliferative arrest followed by the activation of a cell death pathway involving Bcl-2 family members and caspase activation.     

Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos)

Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.     

Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.     

Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.