Activation of a CPP32-like protease during L1210 cell apoptosis induced by thiol deprivation

Reduced thiols (e.g., cysteine) are important in the maintenance of lymphocyte cell viability and growth. L1210 monocytic leukaemia cells were known to have a limited ability to uptake cystine, and they require cysteine for cell growth. L1210 cells underwent apoptosis when cultured without thiol-bearing and dithiol-cleaving compounds, adding thiols suppressed the apoptosis and promoted cell growth. A specific inhibitor of interleukin-1 -converting enzyme (ICE)-like and CPP32-like proteases could suppress L1210 cell apoptosis induced by thiol deprivation. The cell lysates of apoptotic L1210 cells exhibited protease activity that could cleave DEVD-AMC, but not YVAD-AMC, and so CPP32-like proteases, but not ICE-like proteases, were activated and participated in apoptosis. The addition of thiols could suppress CPP32-like protease activation. Although the cell death-suppressor bcl-2-family proteins (bcl-2 and bcl-XL) were recently found to suppress the activation of CPP32-like proteases, the expression levels of death-suppressor bcl-2-family proteins did not change when thiols were added. These results suggest that reduced thiols maintain L1210 cell survival by inhibiting the activation of CPP32-like proteases without changing the anti-apoptotic bcl-2-family protein expression.     

Modulation of Fas-mediated apoptosis by lipid rafts in T lymphocytes

In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.     

Differential induction of Escherichia coli autolysis by penicillin and the bacteriophage phi X174 gene E product.

The behavior of the temperature-sensitive, penicillin-tolerant Escherichia coli mutant VC44 to endogenously induced autolysis by the bacteriophage phi X174 gene E product (gpE) was investigated. Expression of the cloned phi X174 lysis gene showed that cultures of strain VC44 grown at the restricted temperature were fully sensitive to endogenously induced autolysis. The results revealed that the modes of E. coli lysis induction by gpE and by penicillin differ and that the trigger mechanisms for autolysis depend greatly on the specific inducer used.     

Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR-/-) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC(50) of 127±21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.     

Inhibition of transforming growth factor beta1-induced hepatoma cell apoptosis by liver tumor promoters: characterization of primary signaling events and effects on CPP32-like caspase activity

The effects of the liver tumor promoters phenobarbital, clofibrate, dieldrin, and DDT on transforming growth factor-beta1 (TGFbeta)-induced apoptosis were studied in FTO-2B hepatoma cells. Inhibition of apoptosis by these compounds was strongly correlated with a decrease in CPP32-like caspase activity. Similar effects were obtained with insulin and dexamethasone. CPP32-like activity may thus provide a useful tool for quantiation of apoptosis under various treatment conditions. Diverse effects on apoptosis-associated cellular signaling proteins were observed: insulin led to an activation of the MAP kinases ERK1/2, of PKB/Akt and of NF-kappaB, phenobarbital and clofibrate enhanced NF-kappaB activity solely, while dexamethasone slightly enhanced NF-kappaB activity and increased the expression of Bcl-xL. Since inhibition of apoptosis was still detectable if the anti-apoptotic compounds were administered more than 10 h after TGFbeta, the diverse primary signals appear to converge at a presumably late stage of apoptosis, but upstream of activation of CPP32 or related caspases.     

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide,amiloride and omeprazole

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.     

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.     

Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage phi X174 determined by Raman spectroscopy: a model for phi X174 DNA packaging.

The single-stranded packaged genome (ssDNA) of bacteriophage phi X174 is shown by Raman spectroscopy to lack both the ordered phosphodiester backbone and base stacking, which are demonstrated for unpackaged, protein-free ssDNA. In solutions of moderate ionic strength, unpackaged ssDNA contains 36 +/- 7% of deoxyribosyl phosphate groups with conventional B-type backbone geometry [i.e., gauche- and trans orientations, respectively, for the 5'O-P (alpha) and 3'O-P (zeta) torsions], indicative of hairpin formation and intramolecular base pairing. Additionally, the bases of unpackaged ssDNA are extensively stacked. Estimates from Raman band hypochromic effects indicate that unpackaged ssDNA contains approximately 70% of the maximal base stacking exhibited in the linear, double-stranded, replicative form III of phi X174 DNA. Conversely, for the packaged phi X174 genome, ordered (B-type) phosphodiester groups are not present, and only 40% of the base stacking in RFIII DNA is observed. These results are interpreted as evidence that the substantial hairpin-forming potential of ssDNA is eliminated by specific and extensive ssDNA-protein interactions within the phi X174 virion. Comparison of the present results with studies of other packaged single-stranded nucleic acids suggests that proteins of the capsid shell (gpF + gpG + gpH) do not fully account for the conformational constraints imposed on ssDNA of phi X174. Accordingly, we propose a model for ssDNA packaging in which the small basic gpJ protein, which is packaged along with the genome, is involved stoichiometrically in binding to the ssDNA (approximately 90 nucleotides per subunit). The proposed gpJ-DNA interactions could prevent helical hairpin formation, restrict base stacking, and disfavor fortuitous base pairing within the capsid. The present analysis is based upon use of model nucleic acids of known conformation for calibration of the Raman intensity in the region 810-860 cm-1 in terms of specific secondary structures. The calibration curve allows quantitative determination of the percentage of ssDNA nucleotides for which the 5'O-P-O3' group is configured (g-,t) as in the B-form of DNA. The method proposed here is analogous to that employed by Thomas and Hartman (1973) for ssRNA and should be applicable to single-stranded DNA and to partially denatured forms of double- and multiple-stranded DNAs.     

Adduct formation is involved in radiosensitization, mediated by cytochrome c, of phi X174 DNA by misonidazole

Radiosensitization by misonidazole of biologically active phi X174 DNA, mediated by cytochrome c, is most probably at least partly due to formation of an adduct between sensitizer and DNA, which can be removed from the DNA by a mild alkaline treatment thereby restoring the activity of the DNA.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.