Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.     

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.     

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.     

Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2

Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was characterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastructural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of the cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl-2 and these all showed a markedly decreased sensitivity towards injected cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. They also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, but can also interfere with caspase activation induced by cytochrome c microinjected directly into the cytoplasm.     

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.     

Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD. cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibtor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD. cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.     

Apoptosis of human neutrophils induced by protein phosphatase 1/2A inhibition is caspase-independent and serine protease-dependent

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V-positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro-apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl-1. zVAD-fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase-3 activity, procaspase-3 processing, and the apoptotic rate of neutrophils. However, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA-induced mobility shift in procaspase-3, caspase-3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase-3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD-fmk. These results suggest that OA induces procaspase-3 processing but that OA-induced apoptosis is caspase-independent and serine protease-dependent.     

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.     

Inhibition of Caspases Rescues Brown Adipocytes from Apoptosis Downregulating BCL-XS and Upregulating BCL-2 Gene Expression

Serum deprivation of the immortalized brown adipocyte cell line resulted in growth arrest inG₀/G₁phases of the cell cycle and apoptosis, as detected by DNA laddering, nuclei condensation and fragmentation, and an increase in the percentage of hypodiploid cells. In addition, apoptosis in these cells is accompanied by an induction of the expression of the apoptotic form of the Bcl-x gene, the isoform Bcl-xS, and by a decrease of Bcl-2 expression, Bcl-xL remaining almost undetectable. The loss of mitochondrial membrane potential was associated with apoptosis. Z-VAD, a cell-permeable inhibitor of caspases, but not cycloheximide, precludes DNA laddering under serum deprivation. Moreover, Z-VAD rescues serum-deprived brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the percentage of apoptotic cells under Tunnel assay, and the external display of phosphatidylserine. More importantly, Z-VAD survival effects on immortalized brown adipocytes concur with a downregulation of Bcl-xS mRNA/protein and an upregulation of Bcl-2 protein content. Ultimately, Z-VAD prevents the loss of mitochondrial membrane potential.     

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.     

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Effects of insulin-like growth factor-1 on okadaic acid-induced apoptosis in SH-SY5Y cells

The effects of insulin-like growth factor-1 (IGF-1) on the cytotoxicity and apoptosis induced by okadaic acid (OA) in SH-SY5Y cells were investigated. Cell viability was measured using the MTT (3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide) assay. Early and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide (PI) double-staining. Caspase-3 activation was detected by Western blot analysis. Preincubation with IGF-1 for 24 h prevented cytotoxicity induced by 40 nM OA given for 24 h, and the MTT value significantly increased. Incubation with 20 nM OA for 24 h caused a marked increase in the percentage of early apoptotic and late apoptotic/necrotic cells, which was not dependent on the activation of caspase-3. OA-induced apoptosis was significantly decreased by pretreatment with 10 ng/ml of IGF-1 for 24 h. The results supported the hypothesis that IGF-1 may be useful in the treatment of Alzheimer's disease.     

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.     

Control of late apoptotic events by the p38 stress kinase in l‐glutamine‐deprived mouse hybridoma cells

l ‐Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0‐Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress‐activated kinase p38 mitogen‐activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln‐starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal‐induced apoptosis was blocked by the overexpression of the anti‐apoptotic protein Bcl‐xL or by the caspase inhibitor Z‐VAD‐fmk. Interestingly, Bcl‐xL expression inhibited p38 activation, but Z‐VAD‐fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti‐oxidant N‐acetyl‐l ‐cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln‐starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Caspase-3/7 inhibition alters cell morphology in mitomycin-C treated chondrocytes

Apoptosis may play a role in osteoarthritis (OA). Apoptosis can proceed via two different pathways depending on the stimulus. However, both pathways converge upon the effector caspases, caspases-3 and -7. In some systems inhibition of caspases-3 and -7 can prevent apoptosis and may therefore have important therapeutic implications. To confirm this, apoptosis was induced in canine chondrocytes with mitomycin-c (MMC), either in the presence or absence of the general caspase inhibitor, Z-VAD FMK, or a specific caspase-3/7 inhibitor. Z-VAD FMK prevented MMC induced cell death. In contrast, inhibition of caspases-3 and -7 in the presence of MMC induced morphological changes that could be described as necrotic-like or paraptotic-like but did not prevent cell death. The addition of an inhibitor of caspase-8 or caspase-9 along with inhibitor of caspase-3/7 was required to reduce cell death. The morphological changes did not occur in the presence of the caspase-3/7 inhibitor alone and could be prevented by addition of Z-VAD FMK. These data lead to the conclusion that, if the apoptotic program cannot be completed, the cells are pushed into a necrotic or other nonapoptotic mode of death which may involve caspase-8 and/or caspase-9.     

Caspase-dependent apoptosis in THP-1 cells exposed to oxidized low-density lipoproteins

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. We investigated the apoptotic process in human monocytic THP-1 cell line, exposed to oxLDL generated by treatment of native LDL either with hypochlorous acid (HOCl), mainly affecting the protein moiety, or with copper sulfate (CuSO(4)), mainly affecting the lipid moiety. After incubation with both types of oxLDL, we observed: (i) microscopy signs of apoptosis in THP-1 cells, (ii) a significant increase of apoptotic cells proportional to LDL protein concentration, either by annexin V or by cell cycle phase analysis with propodium iodide flow cytometry, (iii) a reduction of THP-1 cell apoptosis in presence of the caspase inhibitor Z-VAD.fmk, (iv) the resistance of THP-1 cells apoptosis after PMA-elicited differentiation. In conclusion, HOCl-oxLDL are as potent as Cu-oxLDL to induce high rates of apoptosis in monocytes through a caspase-dependent pathway. Moreover, the resistance of differentiated THP-1 cells to oxLDL-induced apoptosis is compatible with the hypothesis that mature macrophages have prolonged survival and thereby enhance the atherogenic process.     

Study of caspase inhibitors for limiting death in mammalian cell culture

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.