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This work presents a sandwich-type electrochemical impedance immunosensor for detecting Clostridium difficile toxin A (TcdA) and toxin B (TcdB). Single domain antibody conjugated gold nanoparticles were applied to amplify the detection signal. Gold nanoparticles (Au NPs) were characterized by transmission electron microscopy and UV–vis spectra. The electron transfer resistance (Ret) of the working electrode surface was used as a parameter in the measurement of the biosensor. With the increase of the concentration of toxins from 1 pg/mL to 100 pg/mL, a linear relationship was observed between the relative electron transfer resistance and toxin concentration. In addition, the detection signal was enhanced due to the amplification effect. The limit of detection for TcdA and TcdB was found to be 0.61 pg/mL and 0.60 pg/mL respectively at a signal-to-noise ratio of 3 (S/N = 3). This method is simple, fast and ultrasensitive, thus possesses a great potential for clinical applications in the future.

Here, we investigated toxin domain functions that regulate the proinflammatory activity ofBlocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells.In this study, we identified a novel mechanism of regulating the biological activities of

Clostridium difficile virulence requires secretion of two exotoxins: TcdA and TcdB. The precise mechanism of toxin uptake and delivery is undefined, but current models predict that the cysteine protease domain (CPD)-mediated autocleavage and release of glucosyltransferase domain (GTD) are crucial for intoxication. To determine the importance of CPD-mediated cleavage to TcdB cytotoxicity, we generated two mutant toxins – TcdB-C698S and TcdB-H653A – and assayed their abilities to intoxicate cells. The CPD mutants include an intact GTD but lack the cysteine protease activity. The mutants had reduced potency in that their effect on cells was delayed and required higher concentrations than wild-type TcdB. They did eventually cause cell rounding, glucosylation of Rho GTPases, and apoptosis that was indistinguishable from that caused by TcdB. Although the mutant toxins caused a complete cell rounding, they failed to release their GTD into cytosol, whereas wild-type TcdB displayed significant autocleavage and release of GTD. We conclude that the cysteine protease-mediated autocleavage and release of GTD is not a prerequisite for the cytotoxic activity of TcdB, but rather limits the potency and speed of Rho GTPase glucosylation. Our findings revise and refine the current model for the mode of the action and cellular trafficking of TcdB.

Clostridium difficile infection (CDI) is the most common cause of antibiotic associated diarrhea and colitis in developed countries. The disease is mainly mediated via two major exotoxins TcdA and TcdB secreted by the bacterium. We have previously developed a novel, potently-neutralizing, tetravalent, and bi-specific heavy-chain-only single domain (VHH) antibody to both TcdA and TcdB (designated as ABA) that reverses fulminant CDI in mice (Yang et al, JID, 2014).Since ABA has a short serum half-life, in this study a replication-deficient recombinant adenovirus expressing ABA was generated and the long-lasting expression of functional ABA was demonstrated in vitro and in vivo . Mice transduced with one dose of the adenovirus displayed high levels of serum ABA for more than one month and were fully protected against systemic toxin challenges. More importantly, the ABA delivered by the adenovirus protected mice from both primary and recurrent CDI. Thus, replication-deficient adenoviral vector may be used to deliver neutralizing antibodies against the toxins in order to prevent C. difficile infection and recurrence.

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins1–4. Virulence is dependent on the autoactivation of a toxin cysteine protease5–9, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6)10–17. Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins18,19. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis mainly through two exotoxins TcdA and TcdB that target intestinal epithelial cells. Dendritic cells (DCs) play an important role in regulating intestinal inflammatory responses. In the current study, we explored the interaction of TcdB-intoxicated epithelial cells with mouse bone marrow-derived DCs. TcdB induced cell death and heat shock protein translocation in mouse intestinal epithelial CT26 cells. The intoxicated epithelial cells promoted the phagocytosis and the TNF-α secretion by DCs. Incubation with TcdB-intoxicated CT26 cells stimulated DC maturation. Moreover, TcdB-treated CT26 cells induced DC immigration when they were injected into mice subcutaneously. Taken together, these data demonstrate that TcdB-intoxicated intestinal epithelial cells are able to stimulate DC activation in vitro and attract DCs in vivo, indicating that epithelial cells may be able to regulate DC activation under the exposure of TcdB during C. difficile infection.

Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

The incidence of Clostridium difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. In this study, we generated a novel, potently neutralizing, tetravalent, and bispecific antibody composed of 2 heavy-chain-only VH (VHH) binding domains against both TcdA and TcdB (designated "ABA") that reverses fulminant CDI in mice infected with an epidemic 027 strain after a single injection of the antibody. We demonstrated that ABA bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo. Additionally, ABA was able to broadly neutralize toxins from clinical C. difficile isolates that express both TcdA and TcdB but failed to neutralize the toxin from TcdA(-)TcdB(+) C. difficile strains. This study thus provides a rationale for the development of multivalent VHHs that target both toxins and are broadly neutralizing for treating severe CDI.

Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Δψ). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Δψ and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death.Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Δψ suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Δψ suppression, and apoptosis and cell death induced by GraB, GraK, or perforin.In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-α, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4°C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

Dendritic cells (DCs) are the antigen-presenting cells capable of activating naive T cells. Although CD4+ T cells are crucial for Cryptosporidium parvum clearance, little is known about the role of DCs in the immune response to this parasite. In this study, the interaction between mouse DCs and C. parvum was investigated both in vitro and in vivo. For in vitro experiments, mouse bone marrow-derived dendritic cells (BMDCs) derived from wild-type C57B1/6 or MyD88-/- or C3H/HeJ mice and DC cell line DC2.4 were pulsed with C. parvum. Active invasion of parasites was demonstrated by parasite colocalization with host cell membranes and actin-plaque formation at the site of attachment. DC activation induced by the parasite invasion was demonstrated by upregulation of costimulatory molecules CD40, CD80, and CD86, as well as inflammatory cytokines IL-12, TNF-α, and IL-6. BMDCs derived from MyD88-/- and C3H/HeJ mice failed to produce IL-12 in response to C. parvum, suggesting the importance of TLR-dependent signaling pathway specially presence of a functional TLR4 pathway, for C. parvum-induced cytokine production. In vivo experiments showed that both parasite antigens and live parasites were transported to mice mesenteric lymph nodes. All together, these data suggest that DCs play a key role in host immune responses to C. parvum and pathogenesis of the disease.

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.

We report here the isolation and characterization of a new member of the ice/ced-3 family of cell death genes, named ich-3. The predicted amino acid sequence of Ich-3 protein shares 54% identity with murine interleukin-1beta converting enzyme (ICE). Overexpression of ich-3 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited by CrmA and Bcl-2. The mRNA and proteins of ich-3 are dramatically induced in vivo upon stimulation with lipopolysaccharide, an inducer of septic shock. The ich-3 gene product can be cleaved by cytotoxic T cells granule serine protease granzyme B, suggesting that Ich-3 may mediate apoptosis induced by granzyme B. Ich-3 does not process proIL-1beta directly but does promote proIL-1beta processing by ICE. These results suggest that Ich-3 may play a very important role in apoptosis and inflammatory responses and may be an upstream regulator of ICE.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

Granzymes are a family of granule-associated serine esterases that mediate apoptosis by cytotoxic T lymphocytes and natural killer cells. We have previously shown that cdc2, the mitosis-regulating cyclin-dependent kinase, is required for granzyme B-induced apoptosis in target cells. In addition, granzyme B induces premature activation and tyrosine dephosphorylation of cdc2 during apoptosis. Throughout most of the cell cycle and until the cell is prepared to enter mitosis, cdc2 kinase activity is negatively regulated by phosphorylation of a residue within its adenosine triphosphate-binding domain by Wee1, a nuclear kinase that maintains mitotic timing in eukaryotic cells. We have transiently expressed c-myc epitope-tagged Wee1 cDNA in BHK cells. Cells that expressed Wee1 in the nucleus became resistant to apoptosis induced by granzyme B and perforin. Wee1-transfected cells also exhibited markedly increased cdc2 tyrosine phosphorylation. Thus, Wee1 can rescue cells from granzyme-induced apoptosis by preventing cdc2 dephosphorylation.

The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

We recently reported the purification of a lymphocyte granule protein called "fragmentin," which was identified as a serine protease with the ability to induce oligonucleosomal DNA fragmentation and apoptosis (Shi, L., R. P. Kraut, R. Aebersold, and A. H. Greenberg. 1992. J. Exp. Med. 175:553). We have now purified two additional proteases with fragmentin activity from lymphocyte granules. The three proteases are of two types; one has the unusual ability to cleave a tripeptide thiobenzyl ester substrate after aspartic acid, similar to murine cytotoxic cell protease I/granzyme B, while two are tryptase-like, preferentially hydrolyzing after arginine, and bear some homology to human T cell granule tryptases, granzyme 3, and Hanukah factor/granzyme A. Using tripeptide chloromethyl ketones, the pattern of inhibition of DNA fragmentation corresponded to the inhibition of peptide hydrolysis. The Asp-ase fragmentin was blocked by aspartic acid-containing tripeptide chloromethyl ketones, while the tryptase fragmentins were inhibited by arginine-containing chloromethyl ketones. The two tryptase fragmentins were slow acting and were partly suppressed by blocking proteins synthesis with cycloheximide in the YAC-1 target cell. In contrast, the Asp-ase fragmentin was fast acting and produced DNA damage in the absence of protein synthesis. Using a panel of unrelated target cells of lymphoma, thymoma, and melanoma origin, distinct patterns of sensitivity to the three fragmentins were observed. Thus, these three granule proteases make up a family of fragmentins that activate DNA fragmentation and apoptosis by acting on unique substrates in different target cells.

We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin," was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that fragmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca2+ dependent. A role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD N alpha-CBZ-L-lysine thiobenzyl esterase, which copurified with fragmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis.

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.

Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, in the presence of the granule pore-forming protein perforin, it initiates the processing of caspases and apoptosis. GrB apoptosis is also activated by adenovirus, which can effectively replace perforin. Methods for the purification and quantitation of GrB and perforin, and the preparation and titration of adenovirus, are described. In addition, methods for application of these reagents to the initiation of apoptosis in tumor target cells, with several assays for detecting GrB apoptotic activity, are detailed.

We have identified Nix, a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Nip3. Human and murine Nix have a 56 and 53% amino acid identity to human and murine Nip3, respectively. The carboxyl terminus of Nix, including a transmembrane domain, is highly homologous to Nip3 but it bears a longer and distinct asparagine/proline-rich N terminus. Human Nip3 maps to chromosome 14q11.2-q12, whereas Nix/BNip3L was found on 8q21. Nix encodes a 23. 8-kDa protein but it is expressed as a 48-kDa protein, suggesting that it homodimerizes similarly to Nip3. Following transfection, Nix protein undergoes progressive proteolysis to an 11-kDa C-terminal fragment, which is blocked by the proteasome inhibitor lactacystin. Nix colocalizes with the mitochondrial matrix protein HSP60, and removal of the putative transmembrane domain (TM) results in general cytoplasmic and nuclear expression. When transiently expressed, Nix and Nip3 but not TM deletion mutants rapidly activate apoptosis. Nix can overcome the suppressers Bcl-2 and Bcl-XL, although high levels of Bcl-XL expression will inhibit apoptosis. We propose that Nix and Nip3 form a new subfamily of pro-apoptotic mitochondrial proteins.

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Activation of the serine-threonine kinase p34cdc2 at an inappropriate time during the cell cycle leads to cell death that resembles apoptosis. Premature activation of p34cdc2 was shown to be required for apoptosis induced by a lymphocyte granule protease. The kinase was rapidly activated and tyrosine dephosphorylated at the initiation of apoptosis. DNA fragmentation and nuclear collapse could be prevented by blocking p34cdc2 activity with excess peptide substrate, or by inactivating p34cdc2 in a temperature-sensitive mutant. Premature p34cdc2 activation may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus.

targets of miR-21 and miR-155 are PTEN (inhibited by miR-21) and SHIP1 and SOCS1 (inhibited by miR-155) and PTEN and SHIP1 are upstream of Akt signaling, we investigated the effect of NT on Akt activation by Western blot. NT activated Akt in HCT-116 cells and this effect was partially reversed by miR-21 and/or miR-155 antisense inhibition (p

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.

SummaryPerforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca2+ and small dyes into the cell. The Ca2+ flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.