Germinal center B cell apoptosis requires both caspase and cathepsin activity.

Follicular dendritic cells (FDCs) select B cells during germinal center (GC) reactions. The B cells that are able to bind to the FDCs receive a signal that leads to the termination of endonuclease activity in the nuclei of those B cells. This signal must be in addition to the signals transferred through the cross-linkage of the B cell receptors and signals resulting from the interactions of the adhesion molecules lymphocyte function-associated Ag-1 and very late Ag-4 with ICAM-1 and VCAM-1, respectively. In this report, we present evidence that the FDCs silence all apoptotic processes in GC B lymphocytes and additionally switch off pre-present endonuclease activity. We also show that GC B cell apoptosis requires cathepsin activity downstream of caspase-3. This cathepsin activity is directly connected to endonuclease activity and therefore may be an interesting target for the antiapoptotic factors produced by FDCs.     

Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione S-transferase regulation

Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me(2)SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes and in vivo thioacetamide-induced liver failure, we showed that Me(2)SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me(2)SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST M1/2 and GST Alpha1/2 dropped under apoptotic conditions; furthermore transfection of GST M1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me(2)SO-protected hepatocytes. Together, these results demonstrate that Me(2)SO-mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection.     

Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.     

LIGHT sensitizes IFNγ-mediated apoptosis of HT-29 human carcinoma cells through both death receptor and mitochondria pathways

LIGHT [homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM/TR2)] is a new member of TNF superfamily. The HT-29 colon cancer cell line is the most sensitive to LIGHT-induced, IFNγ-mediated apoptosis among the cell lines we have examined so far. Besides downregulation of Bcl-XL, upregulation of Bak, and activation of both PARP [poly (ADP-ribose) polymerase] and DFF45 (DNA fragmentation factor), LIGHT-induced, IFNy-mediated apoptosis of HT-29 cells involves extensive caspase activation. Caspase-8 and caspase-9 activation, as shown by their cleavages appeared as early as 24 h after treatment, whereas caspase-3 and caspase-7 activation, as shown by their cleavages occurred after 72h of LIGHT treatment. Caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone) and a broad range caspase inhibitor Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) were able to block LIGHT-induced, IFNγ-mediated apoptosis of HT-29 cells. The activity of caspase-3, which is one of the major executioner caspases, was found to be inhibited by both Z-DEVD-MFK and Z-VAD-FMK. These results suggest that LIGHT-induced, IFNy-mediated apoptosis of HT-29 cells is caspase-dependent, and LIGHT signaling is mediated through both death receptor and mitochondria pathways.     

Paclitaxel targets mitochondria upstream of caspase activation in intact human neuroblastoma cells

We previously reported that paclitaxel acted directly on mitochondria isolated from human neuroblastoma SK-N-SH cells. Here, we demonstrate that the direct mitochondrial effect of paclitaxel observed in vitro is relevant in intact SK-N-SH cells. After a 2 h incubation with 1 microM paclitaxel, the mitochondria were less condensed. Paclitaxel (1 microM, 1-4 h) also induced a 20% increase in respiration rate and a caspase-independent production of reactive oxygen species by mitochondria. The paclitaxel-induced release of cytochrome c was detected only after 24 h of incubation, was caspase-independent and permeability transition pore-dependent. Thus, paclitaxel targets mitochondria upstream of caspase activation, early during the apoptotic process in intact human neuroblastoma cells.     

Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity.

Gangliosides shed by tumors enhance tumor formation, possibly by suppressing host antitumor immune function, and gangliosides purified from animal tissues and cultured cells inhibit human cellular immune function in vitro. Determination of immunosuppressive activity of highly purified gangliosides, to uncover structure-activity relationships, is therefore important. Here we have studied a series of gangliosides obtained from human tissue and determined their effects on human natural killer (NK) activity. Total gangliosides from human brain tissue were moderately inhibitory; 100 nmol/ml reduced NK activity of human nonadherent PBMC by 43%. The influence of carbohydrate structure upon inhibitory activity was determined by study of eight highly (HPLC) purified individual gangliosides. Of these, we unexpectedly found that the two minor brain gangliosides with the simplest carbohydrate structures, GM2 and GM3, were very active inhibitors (75 and 47%, respectively, at 50 nmol/ml). In contrast, the structurally more complex major species, GM1, GD1a, GD1b, GT1b, and two other minor gangliosides, GD2 and GD3, were inactive. Reduced effector-target binding in a single-cell binding assay by GM2 but not GM3 suggests different mechanisms of inhibition by these two active gangliosides. Since GM2 and GM3 are present in high concentrations in, and are shed by, several common human tumors (e.g., neuroblastoma, melanoma, and glioma), their ability to inhibit NK cytotoxicity supports the hypothesis of a role of shed tumor gangliosides in the enhancement of tumor formation.     

Compensatory proliferation induced by cell death in the Drosophila wing disc requires activity of the apical cell death caspase Dronc in a nonapoptotic role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%-60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc-in the absence of cell death-results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well.     

Methyl jasmonate-induced cell death in grapevine requires both lipoxygenase activity and functional octadecanoid biosynthetic pathway

Grapevine (Vitis vinifera L., cv. Limberger) leaf tissues and suspension-cultured cells were induced to undergo programmed cell death (PCD) by exogenously added methyl jasmonate (MeJA). The elicitor signaling pathway involved in MeJA-induced PCD was further investigated using pharmacological, biochemical and histological approaches. Pharmacological dissection of the early events preceding the execution of MeJA-triggered PCD indicated that this process strongly depends on both, de novo protein and mRNA synthesis. Treatment of leaf discs and cell suspensions with lipase inhibitor Ebelactone B and specific lipoxygenase inhibitor Phenidone blocked MeJA-induced PCD. These results suggest that some chloroplast membrane-derived compound(s) is required for MeJA-induced PCD in grapevine. The progression of MeJAtriggered PCD may be further inhibited by the use of metabolic inhibitors of key enzymes of octadecanoid biosynthesis including AOS, AOC, and OPR indicating that the functional jasmonate biosynthetic pathway is an integral part of the MeJA-induced signal transduction cascade that results in the coordinate expression of events leading to PCD. Finally, the activation of the octadecanoid pathway, as a critical point in MeJA-induced PCD, was independently demonstrated with cellulysin, a macromolecular elicitor acting via octadecanoid signaling. The cellulysin was shown to be a very potent enhancer of MeJA-triggered PCD in grapevine cells.     

Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death

Apoptosis plays an important role in the pathogenesis of many viral infections. Despite this fact, the apoptotic pathways triggered during viral infections are incompletely understood. We now provide the first detailed characterization of the pattern of caspase activation following infection with a cytoplasmically replicating RNA virus. Reovirus infection of HEK293 cells results in the activation of caspase-8 followed by cleavage of the pro-apoptotic protein Bid. This initiates the activation of the mitochondrial apoptotic pathway leading to release of cytochrome c and activation of caspase-9. Combined activation of death receptor and mitochondrial pathways results in downstream activation of effector caspases including caspase-3 and caspase-7 and cleavage of cellular substrates including PARP. Apoptosis is initiated by death receptor pathways but requires mitochondrial amplification producing a biphasic pattern of caspase-8, Bid, and caspase-3 activation.     

The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions

Programmed cell death (PCD) or apoptosis process in a hybridoma cell line induced by the deprivation of one of the main nutrients, glutamine, has been studied. The use of caspase inhibitors has enabled maintenance of cell viability during a significant period of time, when glutamine depletion was maintained in the culture. Two caspase inhibitors partially suppressed the progress of PCD under glutamine deprivation: Ac-DEVD-cho and z-VAD-fmk. Indeed, as a consequence of this protection, the number of viable cells decreased by 10% (for z-VAD-fmk) and by 80% (for Ac-DEVD-cmk) after 36 h of culture, while it decreased by 90% for a control culture in the absence of protective compounds. However, when the culture was exposed to non-apoptotic conditions after this period of time under apoptosis protection conditions, a normal growth pattern was not recovered. Interestingly, the simultaneous use of both inhibitors made the recovery of the cell culture possible even after a period of 36 h under glutamine depletion, indicating that the inhibition of the effector caspases occurs upstream of the point in which hybridoma cells enter into the commitment step of the death programme.     

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.     

Targeting of human dendritic cells by autologous NK cells

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.     

Heparin antiproliferative activity on bovine pulmonary artery smooth muscle cells requires both N-acetylation and N-sulfonation

The antiproliferative activity of Heparin (HP) on bovine pulmonary artery smooth muscle cells (BPASMC) in vitro requires both N-acetylation and N-sulfonation. This was demonstrated by quantifying the relative N-acetylation of three commercial heparins of known antiproliferative activities, using their Fourier-transform infrared (FTIR) band areas at 1381-1378 and 1320-1317 cm(-1), which combined resulted in 1.0, 1.0 and 1.3 cm2 for Choay, Elkins-Sinn and Upjohn HP, respectively. These results show that Upjohn HP, which is at least 44% more antiproliferative than the other two, is 30% more N-acetylated. Upjohn HP was also N-desulfonated chemically, and its antiproliferative activity was determined. Its total sulfonate (--SO 3 -) content (O- and N-sulfonate) was quantified using the FTIR band area at 1260-1200 cm(-1) for the S=O stretching; a drop in sulfonate content from 21.87% (w/w) before N-desulfonation to 16.51% (w/w) after N-desulfonation, resulted in a 67% decrease in its inhibitory potency. In addition to the requirement that approximately 24% of the sulfonate content be bonded to N, the data show a direct correlation between the extent of Upjohn HP N-acetylation and its antiproliferative activity on BPASMC.     

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

Calbindin-D28K prevents drug-induced dopaminergic neuronal death by inhibiting caspase and calpain activity

Calbindin-D28K protects against apoptotic and necrotic cell death; these effects have been attributed to its ability to buffer calcium. In this study, we investigated the mechanisms underlying the neuroprotective effects of calbindin-D28K in staurosporine (STS)-induced apoptosis and 1-methyl-4-phenylpyridinium (MPP⁺)-induced necrosis. Treatment of the dopaminergic neuronal cell line MN9D with STS or MPP⁺ induced cell death that was associated with increased levels of free intracellular calcium. However, only MPP⁺-induced death was inhibited by co-treatment of the cells with a calcium chelator or a sodium/calcium antiporter inhibitor. Overexpression of calbindin-D28K prevented MPP⁺-induced MN9D cell death, which occurs in the absence of any detectable caspase activation. These pro-survival effects of calbindin-D28K were associated with the inhibition of calcium-mediated calpain activation, as determined by processing of Bax. Overexpression of calbindin-D28K also blocked STS-induced MN9D death. However, this effect was accompanied by the inhibition of capase-3 cleavage, poly(ADP-ribose)polymerase cleavage, and caspase activity. These findings suggest that calbindin-D28K protects against both types of cell death by inhibiting caspase- or calcium-mediated death signaling pathway.     

Immune rejection of a large sarcoma following cyclophosphamide and IL-12 treatment requires both NK and NK T cells and is associated with the induction of a novel NK T cell population

Combined immunotherapy with cyclophosphamide (Cy) and IL-12, but not IL-12 alone, stimulates eradication of a large established solid tumor (20 mm), MCA207, a methylcholanthrene-induced murine sarcoma. In these studies we demonstrate that NK1.1(+) cells and CD1d-dependent NK T cells each play important yet distinct roles in regression of a large tumor in response to Cy and IL-12, and we define a novel NK T cell subset, selectively increased by this treatment. Mice depleted of NK1.1(+) cells demonstrated more rapid initial tumor growth and prolonged tumor regression following treatment, but tumors were eventually eradicated. In contrast, initial tumor regression following therapy was unimpaired in CD1d(-/-) mice, which are deficient in most NK T cells, but tumors recurred. No tumor regression occurred following Cy and IL-12 therapy in CD1d(-/-) mice that were depleted of NK1.1(+) cells. We found that Cy and IL-12 induced the selective increase in liver and spleen lymphocytes of a unique NK T subpopulation (DX5(+)NK1.1(-)CD3(+)). These cells were not induced by treatment in CD1d(-/-) mice. Our studies demonstrate a contribution of both NK and NK T cells to the Cy- and IL-12-stimulated anti-tumor response. We describe the selective induction of a distinct NK T cell subset by Cy and IL-12 therapy, not seen following IL-12 therapy alone, which we suggest may contribute to the successful anti-tumor response induced by this immunotherapeutic regimen.     

TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death.

Tumor necrosis factor (TNF)alpha is considered to play a key pathogenetic role in inflammatory bowel diseases. In this study we analyzed the mechanisms by which TNFalpha induces intestinal epithelial cell apoptosis. TNFalpha alone, and more potently in combination with IFNgamma, induced a high degree of IEC-6 cell apoptosis. This effect was more than 100-fold stronger if both of the TNF-R were stimulated, compared to stimulation of the p55-TNF-R alone, indicating an important apoptosis enhancing effect of the p75-TNF-R. TNFalpha-induced apoptosis required activation of ICE caspases and was completely abolished by its inhibitor, zVAD-fmk. Specific inhibition of caspase-3 with zDEVD-fmk did not alter the effect of TNFalpha. Western blot analyses confirmed that caspase-3 was not activated in response to TNFalpha. In the presence of complete inhibition of the caspase cascade with zVAD-fmk (>/=50 microM), TNFalpha induced cell necrosis rather than apoptosis. Our data reveal that TNFalpha can trigger enterocyte cell death via apoptosis or necrosis, depending upon the activation or blockade of specific caspases.     

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity

Osteosarcoma and Ewing’s sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.