Activation of interleukin-1beta-converting enzyme by nigericin is independent of apoptosis

Interleukin-1beta-converting enzyme (ICE) is believed to be one of the key proteases involved in apoptosis. Since the precursor form of interleukin-1beta (pre-IL-1beta) is one of the well known substrates for ICE, and a potassium/proton ionophore, nigericin, enhances IL-1beta processing, the authors hypothesized that nigericin induces apoptosis through the activation of ICE. In a lipopolysaccharide (LPS)-stimulated and nigericin-treated human monocytic cell line, THP-1, apoptosis was induced, as assessed as to a decrease in cell size, chromatin condensation, exposure of phosphatidylserine and DNA fragmentation. Under exactly the same conditions, nigericin also induced IL-1beta processing in these cells, which was significantly inhibited by an ICE inhibitor, acetyl-Tyr-Val-Ala-Asp-CHO. On the contrary, treatment with this inhibitor at the same concentration did not inhibit nigericin-induced apoptosis, assessed as to the decrease in cell size, chromatin condensation and DNA fragmentation. Although apoptosis induced by nigericin was also observed for LPS-stimulated human peripheral blood mononuclear cells and a mouse T lymphoma cell line, EL-4, the ICE inhibitor did not inhibit the apoptosis in the cells. These results suggest that activated ICE is not involved in the apoptosis induced by nigericin. Since apopain activity was not augmented under the same conditions, neither ICE nor apopain may play any role in the nigericin-induced apoptosis.     

Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2

Apoptosis requires the activation of caspases (formerly interleukin 1beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27(kip1), and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2.     

Increased mucosal B-lymphocyte apoptosis during polymicrobial sepsis is a Fas ligand but not an endotoxin-mediated process

Sepsis is reported to induce an increase in the rate of apoptosis (Ao), in immature lymphoid cells residing in hematopoietic tissues such as the thymus and bone marrow. Alternatively, secondary lymphoid tissue, such as the spleen exhibit little innate (unstimulated) Ao. However, it is unknown whether or not polymicrobial sepsis has any effects on the frequency of Ao in mucosal lymphoid tissue and what, if any, are the functional consequences of such a change. To assess this, Peyer's patch cells were harvested from C3H/HeN (endotoxin-sensitive) mice killed 12 or 24 hours after the onset of polymicrobial sepsis (cecal ligation and puncture [CLP]). The results indicate that the percentage of cells that were Ao+ as determined by flow cytometry were markedly increased at 24 hours, but not at 12 hours post-CLP. This correlates well with evidence of increased DNA fragmentation as well as histological changes observed both at a light and transmission electron microscopic level of the Peyer's patch Ao. Phenotypically, these changes were restricted to the B220+ (B-cell) population that also exhibited a marked increase of Fas/Apo-1 antigen expression. The functional consequence of this increased apoptosis appears to be associated with the endogenous stimulation (activation) of IgA production by mucosal B lymphocytes and increased nuclear c-Rel expression. Furthermore, we found that Peyer's patch lymphocytes isolated from C3H/HeJ-Faslgld (endotoxin-tolerant/Fas ligand- [FasL] deficient) as opposed to C3H/HeJ (endotoxin-tolerant) inbred mice did not exhibit increased Ao after CLP. These findings indicate that increased B-cell Ao appears to be a FasL-Fas antigen-mediated process, but is not due to endotoxin sensitivity. In conclusion, we speculate that the increased Fas-associated apoptosis detected in mucosal B cells (as opposed to splenic or bone marrow B cells) may be due to increased luminal antigens other than endotoxin, released due to gut barrier integrity breakdown during sepsis.     

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.     

Activation of the Xenopus oocyte mitogen-activated protein kinase pathway by Mos is independent of Raf

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.     

Maturation of Qa-1b class I molecules requires beta 2-microglobulin but is TAP independent

Two conformationally distinct and stable forms of Qa-1b, one strongly associated with beta 2-microglobulin (beta 2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable beta 2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without beta 2m, there was no maturation of either form in cells from beta 2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires beta 2m for an initial folding step. However, beta 2m is not essential for subsequent processing of Qa-1b molecules.     

Socially mediated reduction of isolation distress in rat pups is blocked by naltrexone but not by Ro 15-1788.

[Correction Notice: An erratum for this article was reported in Vol 104(4) of Behavioral Neuroscience (see record 2008-10515-001). On page 462, the last paragraph of the first column was incorrect. The corrected paragraph is provided in the erratum.] The presence of a single anesthetized littermate significantly reduced the rate of ultrasonic vocalization by 10-day-old pups isolated in a novel environment. Naltrexone (1.0 mg/kg) returned the vocalization rate to the level of pups tested alone and disrupted the maintenance of body contact between the test pup and a companion. This suggests that the companion exerts comforting effects through endogenous opioid mechanisms. Although chlordiazepoxide is as effective as morphine in the quieting of isolation distress, the benzodiazepine (BDZ) antagonist Ro 15-1788 (5, 10, or 20 mg/kg) was ineffective in blocking the comfort effect and facilitated quiet contact with the companion. In isolated pups, Ro 15-1788 caused a significant, but not a dose-related, decrease in vocalization, a possible indication of the displacement of an endogenous anxiogenic ligand at the BDZ receptor complex. [An erratum for this article will appear in Behavioral Neuroscience, 1990 (Aug), Vol 104:4. The erratum concerns an error in the last paragraph of the first column on page 462.] (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Activation of T cells by superantigen: cytokine production but not apoptosis depends on MEK-1 activity

Engagement of the TCR may result in proliferation and cytokine release or programmed cell death. These two outcomes may be the consequence of distinct T cell receptor-coupled signal transduction pathways or may reflect quantitative differences in signaling strength via a single pathway. Here we show that genetic inhibition of MAP kinase kinase (MEK) by a dominant negative mutant or through chemical inhibition by PD98059 inhibits IL-2 secretion but not programmed cell death after TCR ligation by superantigen. This supports the hypothesis that T cell cytokine release and apoptosis result from signaling through distinct pathways and implies that the molecular signaling mechanisms regulating apoptosis of mature T cells and negative selection of thymocytes may be similar.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyl phosphorylation of the beta-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44mapk and p42mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44mapk and p42mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.     

Dissecting Fas signaling with an altered-specificity death-domain mutant: requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation

Fas is a cell surface death receptor that regulates peripheral tolerance and lymphoid homeostasis. In many pathologic conditions, ectopic Fas activation mediates tissue destruction. Several proteins that can bind to the cytoplasmic death domain of Fas have been implicated in Fas signal transduction. Here we show that FADD, which couples Fas to pro-caspase-8, and, Daxx, which couples Fas to the Jun N-terminal kinase pathway, bind independently to the Fas death domain. We have isolated a death domain mutant, termed FasDelta, that selectively binds Daxx but not FADD. In tranfected tissue culture cells, FasDelta activated Jun N-terminal kinase normally but was impaired in cell death induction. These results suggest that FADD and Daxx activate two independent pathways downstream of Fas and confirm the essential role of FADD binding in apoptosis induction.     

Fas and Fas ligand interaction is necessary for human osteoblast apoptosis

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoylphorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.     

Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, LLC-PK1 cells infected with CPV delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV delta SPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPV delta crmA, wt RPV, or RPV delta SPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.     

A mouse Fas-associated protein with homology to the human Mort1/FADD protein is essential for Fas-induced apoptosis

The Fas cell surface receptor belongs to the tumor necrosis factor receptor family and can initiate apoptosis in a variety of cell types. Using the Fas cytoplasmic domain as bait in a yeast two-hybrid screening, we isolated a mouse cDNA encoding a 205-amino-acid protein. Its predicted protein sequence shows 68% identity and 80% similarity with the sequence of recently described human Mort/FADD. This protein, most likely the mouse homolog of human FADD, associates with Fas in vivo only upon the induction of cell death. A fraction of this protein is highly phosphorylated at serine/threonine residues, with both phosphorylated and unphosphorylated forms being capable of binding to FAS. Stable expression of a truncated form of the Mort/FADD protein protects cells from Fas-mediated apoptosis by interfering with the wild-type protein-Fas interaction. Thus, mouse Mort/FADD is an essential downstream component that mediates Fas-induced apoptosis.     

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.     

Induction of Fas (Apo-1, CD95)-mediated apoptosis of activated lymphocytes by polyclonal antithymocyte globulins

Polyclonal horse antilymphocyte and rabbit antithymocyte globulins (ATGs) are currently used in severe aplastic anemia and for the treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs activate the human classic complement pathway and induce lysis of both resting and phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells. At low, submitogenic, concentration ATGs induce antibody-dependent cell cytotoxicity of PHA-activated cells, but not resting cells. They also trigger surface Fas (Apo-1, CD95) expression in naive T cells and Fas-ligand gene and protein expression in both naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and rapamycin. The data suggest that low doses of ATGs could be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.     

Activation of the K-ras oncogene in colorectal neoplasms is associated with decreased apoptosis

BACKGROUND: Recent in vitro data indicate that the oncogenic effects of activated ras genes may be mediated, at least in part, through inhibition of apoptotic cell death. To examine this proposition in vivo, the relationship between mutations of the K-ras gene and the frequency of apoptosis was studied in a series of 69 sporadic colorectal neoplasms (11 adenomas and 58 carcinomas). METHODS: Mutations in codon 12 of K-ras were determined by a single tube, enriched polymerase chain reaction. Apoptotic cells in tumor sections were identified by in situ end-labeling of fragmented DNA, whereas levels of bcl-2 and p53 proteins were determined by immunohistochemistry. RESULTS: Tumors with mutant K-ras had a significantly lower apoptotic index than those with the wild-type allele (P < 0.05). They were also more likely to exhibit positive bcl-2 staining (P < 0.05). Adenomas showed significantly greater bcl-2 positivity than carcinomas (89% and 51%, respectively; P < 0.05). The frequency of apoptosis in these tumors was not related to either bcl-2 positivity or p53 status. CONCLUSIONS: These findings suggest that activation of K-ras in colorectal carcinoma may inhibit apoptosis and thus favor tumor progression. Alternatively, this association may reflect an accumulation of K-ras mutations in cells in which normal apoptotic pathways have been impaired.