Pan-caspase Inhibitor zVAD-fmk Research Articles

MEK1/2 Inhibitors Promote Ara-C-Induced Apoptosis but Not Loss of ΔΨm in HL-60 Cells

The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 μM), PD98059 (40 μM), or PD184352 (10 μM) with 10–100 μM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (ΔΨm), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of ΔΨm, implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.

Heat Shock Protein 70 Protects from Caspase-Independent Programmed Cell Death via Suppression of Stress Kinase JNK.

METHODS. To increase levels of Hsp70 we used adenovirus expressing human Hsp72 under control of tet-promoter, and adenovirus expressing dominant-negative mutant of SEK1 was used to inhibit JNK activation. Cell death was assessed by acridine orange/ethidium bromide staining under fluorescent microscope, or by poly-ADP ribose (PARP) cleavage and caspase-3 activation by immunoblotting. Activities of MAP kinases (ERK1/2, p38, and JNK) were measured by immunoblotting with antibodies to phosphorylated (activated) form of the kinases. RESULTS. Although the protective effect of Hsp70 against caspase-dependent apoptosis is well established, little is known about its role in caspase-independent cell death. We studied the effect of Hsp70 in two models of caspase-independent cell death: heat-induced killing of normal human fibroblasts and death of myogenic cells after transient ATP depletion. Morphology of heat-treated IMR90 diploid fibroblasts resembled that of apoptotic cells (e.g., after TNF or staurosporine treatment), and the plasma membrane remained intact. Morphology of H9c2 myogenic cells after transient ATP depletion was also similar to that of apoptotic cells, but some cells lost plasma membrane integrity. Both forms of cell death were not accompanied either by caspase-3 activation or PARP cleavage. Moreover, pancaspase inhibitor zVAD-fmk did not protect either from heat-induced killing of fibroblasts or ischemic death of H9c2 cells. Both heating and transient energy deprivation activated several MAP kinases, Erk1/2, p38, and JNK in these cells. As with classical apoptosis, inhibition of kinase, Erk1/2, decreased cell survival in both models. In contrast, inhibition of JNK (but not p38) almost completely protected from heat-induced killing of fibroblasts and markedly reduced death of H9c2 after energy deprivation. These types of caspase-independent cell death were effectively prevented by increased levels of Hsp70. Hsp70 overexpression greatly diminished activation of JNK after heat shock or transient energy deprivation; on the other hand, it did not increase activity of Erk1/2. Therefore, the protection of cells from the caspase

Temperature-dependent Arrest of Neutrophil Apoptosis: FAILURE OF Bax INSERTION INTO MITOCHONDRIA AT 15 °C PREVENTS THE RELEASE OF CYTOCHROME c

Apoptosis is essential for the resolution of neutrophilic inflammation. To define the mechanisms triggering the execution phase of apoptosis we developed and utilized a model in which culture of human neutrophils at 15 degrees C for 20 h arrested apoptosis and subsequent warming to 37 degrees C triggered a synchronous burst of apoptosis. Treatment of 15 degrees C cultured neutrophils with the pan-caspase inhibitor zVAD-fmk just before warming to 37 degrees C inhibited the morphological changes associated with apoptosis, but did not prevent the insertion of the proapoptotic protein Bax into mitochondria nor the inhibition of secretion and the externalization of phosphatidylserine, indices of neutrophil apoptosis. In both intact neutrophils and a cell-free extract, cytochrome c released from mitochondria induced proteolytic cleavage of procaspase-3. At 15 degrees C the binding of Bax to mitochondria was uncoupled from Bax insertion into the mitochondrial membrane required for the release of cytochrome c. Apoptosis was also inhibited by low pH during warming to 37 degrees C, suggesting that changes to the conformation of Bax, necessary for membrane insertion, were being inhibited. Bax insertion was only sensitive to zVAD-fmk when added at the start of the 15 degrees C culture period, suggesting that a cytoplasmic substrate of the effector caspases may mediate in the mechanism of Bax insertion into mitochondria.

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development.

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.

Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms.

Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.

Ceramide Induces Cell Death in the Human Prostatic Carcinoma Cell Lines PC3 and DU145 but Does Not Seem to Be Involved in Fas-Mediated Apoptosis

Treatment of cells with synthetic C2-ceramide has been reported to induce apoptosis in several cell systems, and endogenously formed ceramide has been proposed to act as a second messenger, activating signaling pathways which contribute to the execution of apoptotic cell death after Fas ligation or tumor necrosis factor receptor-1 ligation. In this study, we examined the effect of exogenously administered C2-ceramide on the human prostatic carcinoma cell lines PC3 (Fas-sensitive) and DU145 (Fas-resistant). In both cell lines, C2-ceramide induced cell death in a dose-dependent manner, whereas a structural analog, C2-dihydroceramide, did not. The pan-caspase inhibitor zVAD-fmk did not prevent C2-ceramide–induced cell death but did prevent C2-ceramide–induced DNA fragmentation, indicating that apoptotic and non-apoptotic mechanisms are involved in C2-ceramide–induced death. Interestingly, cycloheximide prevented C2-ceramide–induced DNA fragmentation, indicating that ceramide-induced apoptosis in PC3 and DU145 requires new protein synthesis. In addition, because cycloheximide converts Fas-resistant DU145 to Fas-sensitive as assessed by DNA fragmentation, ceramide does not seem to play a major role in the Fas-mediated pathway in this cell line. We also determined the levels of endogenous sphingomyelin after Fas ligation in PC3. No decrease of sphingomyelin levels could be detected after Fas activation. We conclude that sphingomyelinase-generated ceramide does not play a role in Fas-mediated apoptosis in PC3, and that there are fundamental differences in the mechanisms of cell death induced by C2-ceramide and Fas ligation.

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotease-mediated shedding

. Purpose : L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. Materials and methods : Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITClabelled annexin-V in the presence of propidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. Results : PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. Conclusion : UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte tra Ýcking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.

Retinoic acid induces apoptosis-associated neural differentiation of a murine teratocarcinoma cell line.

Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspase inhibitor zVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10(-7) M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron-specific protein kinase C substrate GAP-43. RA-induced apoptosis increased with the plating density of PCC7-Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA-damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle-arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis-inhibiting factors by the cultured cells is indicated. Neither ApoI/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl-2 proto-oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13-dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long-term treatment with phorbol 12,13-dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apoptosis of PCC7-Mz1 cells. We conclude that the apoptotic activity following RA treatment of cultured PCC7-Mz1 cells probably is controlled by the same cascade of gene regulatory events that govern the early cell lineage determinations in this in vitro model system of neural development.