Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes

Nuclear factor kappaB (NFkappaB) regulates the expression of various genes essential for cell survival. Here we demonstrate that suppression of NFkappaB nuclear import with SN50 peptide carrying the nuclear localization sequence (NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50 induces irreversible cell loss due to the reduction in the mitochondrial transmembrane potential (DeltaPsim) followed by disruption of the cell membrane, hallmarks of necrosis. These observations demonstrate that although inhibition of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent apoptosis in T-PBL, cell death may still proceed in the absence of functional caspase activity. The availability of downstream caspases appears to determine the mode of cell death in NFkappaB defective cells.     

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-alpha-induced apoptosis. STS activated I kappa B kinase and nuclear factor-kappa B (NF-kappa B) nuclear translocation and DNA binding but inhibited transactivation of I kappa B-alpha, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-kappa B reporter in transfected Huh-7 cells.     

Caspase-3-like protease influences but is not essential for DNA fragmentation in Blastocystis undergoing apoptosis

Blastocystis hominis undergo apoptosis after treatment with a cytotoxic monoclonal antibody (MAb), 1D5, by mechanisms that are not fully understood, although our previous study demonstrated that caspase-3-like protease activity is involved. To elucidate the mechanism of MAb 1D5-induced apoptosis, we inhibited Blastocystis caspase-3-like protease to investigate if there would be a concomitant decrease in in situ DNA fragmentation. However, MAb 1D5-induced apoptosis, evidenced by DNA fragmentation, was not completely blocked by pretreating with specific caspase-3 inhibitor, Ac-DEVD-CHO, indicating that caspase-independent apoptotic pathways might also be involved. Our results also revealed that the treatment with MAb 1D5 resulted in the loss of mitochondrial membrane potential (deltapsim), independent of Ac-DEVD-CHO pretreatment. In conclusion, this study demonstrates that MAb 1D5-induced apoptosis in B. hominis is not wholly dependent on caspase-3-like protease activity and is associated with mitochondrial dysregulation. This is the first report showing evidence for complex apoptotic pathways in a unicellular parasite.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Elevated calcium in preneoplastic cells activates NF-kappa B and confers resistance to apoptosis

Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup-). Sup+ cells, which undergo apoptosis when cultured in low serum, show little or no DNA binding activity to nuclear factor (NF)-kappa B either in 10% or 0.2% serum. In contrast sup- cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-kappa B. The constitutive activation of NF-kappa B observed in sup- cells is not due to loss of I kappa B alpha. We considered that the activation of NF-kappa B in sup- cells might be secondary to an increase in cytosolic Ca(2+), since sup- cells have a cytosolic Ca(2+) level that is double that in sup+ cells. In support of a role for Ca(2+), lowering cytosolic Ca(2+) in sup- cells by addition of the cell-permeable Ca(2+) chelator 1,2 bis(O-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) reduced cytosolic Ca(2+) by approximately 31% relative to untreated sup- cells, concomitant with a 65% reduction in NF-kappa B DNA binding activity and a reduction in I kappa B kinase (IKK) activity. In sup- cells in low serum, addition of BAPTA-AM also resulted in a significant ( approximately 50%) increase in caspase-3 activity. Raising extracellular Ca(2+) in sup+ cells resulted in a slight activation of I kappa B kinase and in enhanced NF-kappa B DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-kappa B in sup- cells is largely proteasome-independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca(2+) in sup- cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-kappa B in sup- cells; however, the predominant effect of Ca(2+) appears to be mediated by Ca(2+)-enhanced degradation by calpain.     

The NF-kappa B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes

We explored the role of the NF-kappa B pathway in the survival of primary human CD4+ T lymphocytes during CD28 costimulation. Transduction of proliferating CD4+ T cells with a tetracycline-regulated retrovirus encoding for a dominant-interfering, degradation-resistant I-kappaBalpha (inhibitor of kappa B alpha factor) mutant induced apoptosis. Using DNA arrays, we show that Bcl-xL features as a prominent anti-apoptotic member among a number of early CD28-inducible genes. A 1.2-kb segment of the proximal Bcl-xL promoter, linked to a luciferase reporter, responded to CD3/CD28 stimulation in Jurkat cells. Mutation of an NF-kappa B site around -840 decreased, while ectopic expression of I-kappa B kinase-beta (IKK beta) enhanced reporter gene activity. Na+-salicylate and cyclopentenone PGs, direct inhibitors of IKK beta, interfered in the activation of the Bcl-xL promoter and induced apoptosis in CD28-costimulated CD4+ T cells. Moreover, salicylate blocked nuclear localization of NF-kappa B factors that bind to the NF-kappa B binding site in the Bcl-xL promoter, as well as the expression of Bcl-xL protein. HuT-78, a lymphoblastoid T cell line with constitutive NF-kappa B activity, contained elevated levels of Bcl-xL protein and, similar to proliferating CD4+ T cells, was resistant to apoptotic stimuli such as anti-Fas and TNF-alpha. In contrast, the same stimuli readily induced apoptosis in a Jurkat T cell clone with no detectable Bcl-xL expression. Jurkat BMS2 cells also differed from HuT-78 in collapse of mitochondrial membrane potential and superoxide generation in the mitochondrium. Taken together, these data demonstrate that CD3/CD28-induced activation of IKK beta and expression of Bcl-xL promote the survival of primary human CD4+ T lymphocytes.     

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases(also termed caspases) have been discovered and implicated as the common effectors of apoptosis. Inthis report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectruminhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events werefollowed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetictetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, suchas morphological characteristics and DNA fragmentation. These results suggest that staurosporine-inducedapoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

NF-kappa B2 p100 is a pro-apoptotic protein with anti-oncogenic function

Nuclear factor-kappa B (NF-kappa B) promotes cell survival by upregulating expression of anti-apoptotic genes, a process that is antagonized by inhibitors of kappa B (I kappa B) factors. The only NF-kappa B family member known to be mutated in human cancer is NF-kappa B2 p100 (ref. 2), a factor with I kappa B activity. Here, we report the isolation from irradiated mouse tumour cells of a complex that induces caspase-8 activity in cell-free assays and identify p100 as an essential component of this complex. Expression of p100 profoundly sensitizes cells to death-receptor-mediated apoptosis through a pathway that is independent of I kappa B-like activity. The carboxyl terminus of p100 contains a death domain that is absent from all known tumour-derived mutants. This death domain mediates recruitment of p100 into death machinery complexes after ligand stimulation and is essential for p100's pro-apoptotic activity. p100 also sensitizes NIH3T3 cells to apoptosis triggered by oncogenic Ras, resulting in a marked inhibition of transformation that is rescued by suppression of endogenous caspase-8. These observations thus identify an I kappa B-independent apoptotic activity of NF-kappa B2 p100 and help explain its unique tumour suppressor role.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.     

Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.