Caspase Activation Events Research Articles

Decitabine promotes apoptosis in mesenchymal stromal cells isolated from patients with myelodysplastic syndromes by inducing reactive oxygen species generation

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells, resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless, the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here, we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P < 0.00085), cleaved caspase (−3, −8, and −9) activity, and mitochondrial membrane potential reduction. Additionally, DAC-induced apoptosis was inhibited by Kp7-6, a FasL/Fas antagonist, indicating a crucial role of FasL/Fas, a cell death receptor, in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore, N-acetyl-L-cysteine (NAC), a widely accepted ROS scavenger, efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P < 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore, ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs, which serves as a crucial factor for mitochondrial membrane potential reduction, and DAC induces cell death prior to FasL/Fas stimulation.

Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.

25(OH) Vitamin D3 Affects Acute Myeloid Leukemia Cell Proliferation

We recently demonstrated that acute myeloid leukemia (AML) patients with insufficient serum 25(OH) vitamin D3 levels had a worse outcome following intensive induction therapy. However, the mechanisms by which 25(OH) vitamin D3 and its metabolites exert these effects are poorly understood. We utilized in vitro systems with orthogonal genomic and computational approaches to delineate leukemia-specific vitamin D molecular targets that may contribute to its activities. To achieve this goal, we systematically investigated vitamin D anti-proliferative effects using a genomically-annotated panel of human leukemia cell lines, including lines with genetic alterations that are commonly observed in patients. Since the genetic diversity of these cell lines reflects many of the alterations found in AML patients, we hypothesized that they collectively represent a model system to replicate candidate anti-leukemia drug effects. Specifically, we cultured 40 leukemia cell lines with and without 25(OH) vitamin D3 in a dose- and time-dependent manner for 72 hours and used high-throughput (HT) cell-based assays to simultaneously measure multiple modalities/ parameters related to cell proliferation and cytotoxicity (i.e. combining several HT assay chemistries to simultaneously assess cell number, viability, cytotoxicity and caspase activation events). Six AML cell lines (THP-1, MV4;11, MOLM-6, MOLM-13, EOL-1, JK-1) that responded to 25(OH) vitamin D3 treatment were identified and selected for multi-parametric RNA-Seq studies (NB 200nM Vitamin D treatment or vehicle control for 24 hours). Total RNA was extracted and analyzed by the Illumina HiSeq 2500 sequencing platform and mRNA, microRNA and ncRNAs were unambiguously quantitated. Utilizing gene expression profiles and in silico computational modeling of canonical or putative vitamin D receptor elements (VDRE) transcription factor promoter binding sites, we (1) identified a conserved/core gene transcriptional signature and (2) subsequently developed a vitamin D – genetic inference model. Gene enrichment analysis of the inferred network model identified genes involved in abnormal hematopoiesis, proliferation and differentiation, interleukin secretion, leukocyte migration, and bone marrow cell morphology and/or development. Notably, our data identified several 25(OH) vitamin D3 transcriptional regulatory cascades that converge on ETS2, JUND, NCOA4, RXR4, WT1, and ZFP30 transcription factors. Furthermore, we also demonstrate temporal and spatial regulation of protein kinases CAMK1 and FGFR, cytokine and growth factors (cAMP, CCL2, IL-8 and TNF), and cell differentiation markers (CD14, CD97, FCGR2A, IL3RG, IL7R, ITGAM, THBD). In summary, these data suggest that 25(OH) vitamin D3 exerts both direct anti-proliferative effects on leukemia cells and direct/indirect leukemia inhibitory microenvironment reprograming activities and better clarify the complex molecular mechanisms underlying the therapeutic activity of 25(OH) vitamin D3 in AML. Disclosures:No relevant conflicts of interest to declare.

Phosphorylation of eIF2 alpha in Sf9 cells: a stress, survival and suicidal signal

An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2alpha) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2alpha phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2alpha phosphorylation. Infection of Sf9 cells by wt and a mutant Deltapk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2alpha phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Deltapk2 baculovirus that lacks pk2, an inhibitor of eIF2alpha kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2alpha phosphorylation, thereby suggesting that eIF2alpha phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2alpha phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2alpha phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2alpha, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2alpha phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.

Analysis of apoptosis in cell-free systems

Cell-free systems have been instrumental in the identification of several important components of the cell death machinery such as cytochrome c, APAF-1, ICAD/CAD (DFF45/DFF40) and Smac/Diablo. Such systems have also proved invaluable for the detailed analysis of caspase activation mechanisms, caspase activation cascades, proteolysis of caspase substrates, apoptosis-associated chromatin condensation and internucleosomal DNA fragmentation. Here, we describe a cell-free system that we have used routinely in our laboratory for the analysis of caspase activation and associated events. Caspase activation in this system can be triggered either through assembly of the APAF-1 apoptosome by addition of cytochrome c/dATP, or alternatively, by addition of the cytotoxic lymphocyte protease, granzyme B. In both cases, the order of caspase activation events has been established and the relative importance of individual caspases to apoptosis-associated nuclear events, as well as substrate proteolysis, is known. Cell-free systems are therefore very useful for screening potential caspase-inhibitory compounds or other agents that may positively or negatively affect caspase-dependent events in apoptosis.

Bcl-xL Deamidation a Trigger for Apoptosis

Using a transgenic mouse as a model for T cell lymphoma, Zhao et al . provide evidence that defective deamidation of Bcl-xL contributes to the apoptosis resistance and oncogenesis of thymocytes in these animals. These mice ( CD45 −/− lck F505 ) are deficient in the phosphatase CD45 and have a mutant form of the kinase Lck. DNA damage does not cause apoptosis in the thymocytes of these mice despite a normal p53 response. Bcl-xL sequesters the proapoptotic BH3 domain proteins, such as Bim and Puma, and deamidation, which converts Asn 52 and Asn 66 to isoaspartate (Iso-Asp), has been suggested to prevent this interaction, allowing apoptosis to proceed. Zhao et al . show that Bcl-xL deamidation (monitored by retardation on native gel electrophoresis) was not prevented by a pan-caspase inhibitor or by knockdown of Bax and Bak (two proteins required for mitochondrial-mediated apoptosis). Thus, Bcl-xL deamidation appears to occur before caspase activation and not as a consequence of caspase activation. Coimmunoprecipitation experiments confirmed that Bcl-xL did not bind Bim or Puma and that the interaction between Bcl-xL and these two proteins was not lost in response to DNA damage in the cells from the CD45 −/− lck F505 mice. Anion-exchange chromatography was used to separate native from deamidated Bcl-xL, and the lack of interaction between Bim and the deamidated forms was confirmed. The authors noted that deamidation occurs more readily at higher pH and found that DNA damage raises the intracellular pH of wild-type thymocytes. When the pH of wild-type cells was artificially elevated, Bcl-xL deamidation occurred to a greater extent than at neutral pH, and when DNA damage was induced under conditions that prevented the elevation in pH, Bcl-xL deamidation was prevented, and the number of apoptotic cells was reduced. The resistance to apoptosis in response to DNA damage was overcome in the CD45 −/− lck F505 thymocytes when the pH was increased to 7.55 or above. DNA damage triggers an increase in the abundance of the Na + /H + exchanger 1 (NHE-1) through increased gene expression in wild-type cells, and this is not observed in the CD45 −/− lck F505 cells. Overexpression of NHE-1 in the CD45 −/− lck F505 and wild-type cells caused Bcl-xL deamidation and triggered apoptosis; whereas pharmacological inhibition of NHE-1 activity blocked alkalinization, Bcl-xL deamidation, and apoptosis. Using cells from patients with chronic lymphocytic leukemia, the authors showed that enforced alkalinization caused an increase in Bcl-xL deamidation and apoptosis. R. Zhao, D. Oxley, T. S. Smith, G. A. Follows, A. R. Green, D. R. Alexander, DNA damage-induced Bcl-x L deamidation is mediated by NHE-1 antiport regulated intracellular pH. PLoS Biol. 5 , e1 (2007). [PubMed]

INVITED COMMENTARY

The dogma with respect to apoptosis and the biological fate of cardiac myocytes implicates the eventual death of those cells. Graded, suspended, or reversible apoptosis is a relatively unexplored entity in the setting of heart failure or cardiomyopathy. If cells that are expressing apoptotic genes (eg, caspase-3) could be “rescued” or recruited back into the functional myocardium using selective gene therapy emphasizing expression of genes that promote survival, myocytic drop-out in damaged hearts may conceivably be ablated or attenuated. Narula and coworkers have previously described myofibrolytic cells (MFLs) as those with little sarcomeric structure but with intact nuclei [1Narula N, Arbustina E, Haider N, et al. Although apoptotic, myofibrillarlytic myocytes demonstrate survival instincts. United States and Canadian Academy of Pathology, Chicago; February 2002. Mod Pathol 2002:38A.Google Scholar, 2Communal C. Sumandea M. de Tombe P. et al.Functional consequences of caspase activation in cardiac myocytes.Proc Natl Acad Sci USA. 2002; 99: 6252-6256Crossref PubMed Scopus (317) Google Scholar]. Presumably these cells are former cardiac myocytes that have undergone the loss of sarcomeric proteins. As they claim that these cells may reside for extended periods within the damaged heart, it could be argued that reversibility of the programmed cell death presents an intriguing putative mechanism for myocardial salvage. Certainly the distinction between total cell apoptosis versus cells with little else other than viable nuclei per se is an interesting finding worth pursuing. In the current issue, Narula and coworkers have proposed that MFLs are characterized by upregulation of both caspase-3 and Bcl2 [3Narula N. Narula J. Zhang P.J. et al.Is the myofibrillarlytic myocyte a forme fruste apoptotic myocyte.Ann Thorac Surg. 2005; 79: 1333-1337Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Whereas the coexpression of pro-apoptotic and anti-apoptotic genes in the evolution of tissue development (eg, neural crest tissue) is not unusual; the existence of this mechanism in post-myocardial infarction heart is likely to generate controversy. Evidence that MLFs are metabolically active with intact nuclei (upregulation of proliferating cell nuclear antigen) provide the springboard for future work to fully characterize the MFL type. This may include sequential tracking studies to monitor the loss of myofibrillar proteins, and more intriguing, experiments to force their “restoration” to a myocytic phenotype. Finally, if MFLs appear frequently within the border zone of the infarct, then is the infarct and border zone region characterized by cells that exhibit graded anti-apoptotic and pro-apopotic gene expression? These questions may provide fertile ground for further research.

Molecular Ordering of the Caspase Activation Cascade Initiated by the Cytotoxic T Lymphocyte/Natural Killer (CTL/NK) Protease Granzyme B

Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.

PLANT PROGRAMMED CELL DEATH, ETHYLENE AND FLOWER SENESCENCE

Programmed cell death (PCD) applies to cell death that is part of the normal life of multicellular organisms. PCD is found throughout the animal and plant kingdoms; it is an active process in which a cell suicide pathway is activated resulting in controlled disassembly of the cell. Most cases of PCD described in animal systems take the form of apoptosis, a cell death process characterised by specific features such as cell shrinkage, blebbing of the plasma membrane, condensation and fragmentation of the nucleus and internucleosomal cleavage of DNA. The final stage of apoptosis is the fragmentation of the cell into cellular debris-containing vesicles called `apoptotic bodies` that are being phagocytosed by other cells. A specific class of cell death- associated cystein proteases (caspases) has been identified. Generally, apoptotic cell death involves a sequence of caspase activation events in which initiator caspases activate down-stream executioner caspases that process a variety of target proteins eventually leading to the apoptotic phenotype. The occurrence of hallmarks of animal apoptosis was studied in tomato cells treated with the anticancer drug and inducer of apoptosis, camptothecin (cpt). It was shown that cpt-induced cell death is accompanied by nuclear condensation, the appearance of TUNEL-positive nuclei, DNA laddering and formation of DNA-containing (apoptotic) bodies and was greatly inhibited by inhibitors of animal caspases. Together the results indicate that cpt induced a cell death pathway with similarities to caspase-mediated (apoptotic) cell death in animal systems. We used cpt-treated cells to study the possible involvement of ethylene in cell death. Treatment of the cells with relatively high concentrations of ethylene did not have any effect on viability of the cells. However, when ethylene was applied in combination with cpt, a significant increase in cell death was observed as compared to cpt treatment alone. Experiments with inhibitors of ethylene production or ethylene action showed that ethylene is an essential factor mediating cpt-induced cell death. Flower senescence is accompanied by rapid death of large numbers of cells. In situ DNA degradation was studied in gypsophila petals using TUNEL. We showed that TUNEL positive nuclei appear well before the onset of the increase in ethylene production and visible signs of senescence. The role of PCD in flower senescence is discussed.

Hyporesponsiveness of CD4 T Cells Induced in Oral Tolerance Is Maintained by Selective Impairment in the TCR‐Induced Calcium/NFAT Signaling Pathway Resulting from Caspase Activation

We examined intracellular signaling of hyporesponsive CD4 T cells induced by continuous feeding with high-dose antigen in a TCR transgenic mouse system. The results demonstrated a selective impairment in their TCR-induced calcium/NFAT-signaling pathway. Proteomic analysis revealed caspase activation in these cells, which resulted in cleavage of GADS. Further analysis of the TCR-signaling complex showed that GADS-LAT-SLP-76-associated PLC-gamma1 was decreased in both phosphorylation and association. Thus, as a consequence of caspase activation, orally tolerant CD4 T cells could not form normal TCR signaling complexes associated with GADS and showed downregulated PLC-gamma1 activation, which resulted in impairment of TCR-induced calcium signaling.

Enhancement of death-receptor induced caspase-8-activation in the death-inducing signalling complex by uncoupling of oxidative phosphorylation

Signalling through the death receptor CD95 induces apoptosis by formation of a signalling complex at the cell membrane and subsequent caspase-8 and caspase-3-activation. Treatment of Jurkat T cells with protonophores across the mitochondrial membrane such as 2,4-dinitrophenol (DNP) enhances the death-inducing capacity of CD95. In this study, we show that this enhancement is due to the specific acceleration of caspase-8-processing and activation at the CD95-receptor. DNP-treatment did not affect NF-κB-induction by CD95. Immunoprecipitation experiments showed that the amounts of the adapter FADD/MORT1 and pro-caspase-8 at the CD95-receptor were not altered by DNP. Subcellular fractionation studies revealed that the amount of mature caspase-8 but not pro-caspase at the membrane was increased following CD95-stimulation in the presence of DNP. As a consequence of caspase-activation, c-FLIP-levels in the cytosol decreased. In Jurkat cells overexpressing c-FLIP S, DNP was still able to enhance caspase-activation. The enhancing capacity of DNP was seen in some cell lines (Jurkat, CEM and HeLa) but not in SKW6 cells and was also found in mitogen-stimulated human T cells. Furthermore, the enhancement extended to TRAIL-induced caspase-activation. Thus, a mechanism exists by which caspase-8-activation can be accelerated at death receptors and this mechanism can be triggered by targeting mitochondrial oxidative phosphorylation.

708 MSD-EACR research award. Celecoxib activates a novel mitochondrial apoptosis signaling pathway

Cell-free systems have been instrumental in the identification of several important components of the cell death machinery such as cytochrome c, APAF-1, ICAD/CAD (DFF45/DFF40) and Smac/Diablo. Such systems have also proved invaluable for the detailed analysis of caspase activation mechanisms, caspase activation cascades, proteolysis of caspase substrates, apoptosis-associated chromatin condensation and internucleosomal DNA fragmentation. Here, we describe a cell-free system that we have used routinely in our laboratory for the analysis of caspase activation and associated events. Caspase activation in this system can be triggered either through assembly of the APAF-1 apoptosome by addition of cytochrome c/dATP, or alternatively, by addition of the cytotoxic lymphocyte protease, granzyme B. In both cases, the order of caspase activation events has been established and the relative importance of individual caspases to apoptosis-associated nuclear events, as well as substrate proteolysis, is known. Cell-free systems are therefore very useful for screening potential caspase-inhibitory compounds or other agents that may positively or negatively affect caspase-dependent events in apoptosis.

Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation.

Fluorochrome-labeled inhibitors of caspases (FLICA, e.g., FAM-VAD-FMK, FITC-VAD-FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes. Apoptosis of HL-60, Jurkat, MCF-7 and T-24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H(2)O(2)). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase-3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z-VAD-FMK or z-DEVD-FMK. FLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase-3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM-VAD-FMK, FITC-VAD-FMK or FAM-DEVD-FMK binding to apoptotic cells could not be inhibited by z-VAD-FMK or z-DEVD-FMK, respectively, when the unlabeled inhibitors were added post-induction of apoptosis. FLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases.

Dying to Make Platelets

Two groups, de Botton et al. and Clarke et al. , showed that platelet formation occurs through a variation of apoptosis involving compartmentalized caspase activation. Megakaryocytes, the platelet precursor cells, undergo a morphological reorganization in which they extend processes called proplatelets, which develop nodes that are eventually released as mature platelets. De Botton et al. found that caspase inhibitors blocked thrombopoietin-stimulated proplatelet formation in cultured human megakaryocytes, as did overexpression of Bcl-2 (an antiapoptotic protein). The authors used Western analysis to demonstrate proteolytic maturation of caspase-3 and caspase-9 and cleavage of caspase substrates during megakaryocyte maturation. Confocal microscopy analysis of immunofluorescently labeled cells indicated that punctate, localized caspase-3 activation occurred before proplatelet formation. Furthermore, early localized caspase-3 activation was associated with mitochondrial release of cytochrome c but not DNA fragmentation or cell death. Clarke et al. found that caspase inhibitors blocked platelet production and proplatelet formation in a human megakaryoblastic cell line that spontaneously generated functional platelets. Fluorescence analysis indicated that the cell bodies of proplatelet-bearing cells contained activated caspases but the proplatelets did not. Cells developing proplatelets showed heterochromatin condensation consistent with early apoptosis. Fas ligation stimulated proplatelet formation, platelet production, and nuclear condensation, and augmented platelet production by primary cultures of mouse megakaryocytes and human bone core explants. The authors used a fluorescent mitochondrial dye to show that proplatelet mitochondria maintained their membrane potentials in cells showing nuclear condensation. Dying platelets cultured in the absence of survival factors, however, showed collapsed mitochondrial membrane potentials and released cytochrome c. Western analysis together with caspase-activating treatments suggested that platelets lacked caspase-9. S. de Botton, S. Sabri, E. Daugas, Y. Zermati, J. E. Guidotti, O. Hermine, G. Kroemer, W. Vainchenker, N. Debili, Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 100 , 1310-1317 (2002). [Online Journal] M. C. H. Clarke, J. Savill, D. B. Jones, B. S. Noble, S. B. Brown, Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J. Cell Biol . 160 , 577-587 (2003). [Abstract] [Full Text]

Dying to Make Platelets

Two groups, de Botton et al. and Clarke et al., showed that platelet formation occurs through a variation of apoptosis involving compartmentalized caspase activation. Megakaryocytes, the platelet precursor cells, undergo a morphological reorganization in which they extend processes called proplatelets, which develop nodes that are eventually released as mature platelets. De Botton et al. found that caspase inhibitors blocked thrombopoietin-stimulated proplatelet formation in cultured human megakaryocytes, as did overexpression of Bcl-2 (an antiapoptotic protein). The authors used Western analysis to demonstrate proteolytic maturation of caspase-3 and caspase-9 and cleavage of caspase substrates during megakaryocyte maturation. Confocal microscopy analysis of immunofluorescently labeled cells indicated that punctate, localized caspase-3 activation occurred before proplatelet formation. Furthermore, early localized caspase-3 activation was associated with mitochondrial release of cytochrome c but not DNA fragmentation or cell death. Clarke et al. found that caspase inhibitors blocked platelet production and proplatelet formation in a human megakaryoblastic cell line that spontaneously generated functional platelets. Fluorescence analysis indicated that the cell bodies of proplatelet-bearing cells contained activated caspases but the proplatelets did not. Cells developing proplatelets showed heterochromatin condensation consistent with early apoptosis. Fas ligation stimulated proplatelet formation, platelet production, and nuclear condensation, and augmented platelet production by primary cultures of mouse megakaryocytes and human bone core explants. The authors used a fluorescent mitochondrial dye to show that proplatelet mitochondria maintained their membrane potentials in cells showing nuclear condensation. Dying platelets cultured in the absence of survival factors, however, showed collapsed mitochondrial membrane potentials and released cytochrome c. Western analysis together with caspase-activating treatments suggested that platelets lacked caspase-9.S. de Botton, S. Sabri, E. Daugas, Y. Zermati, J. E. Guidotti, O. Hermine, G. Kroemer, W. Vainchenker, N. Debili, Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 100, 1310-1317 (2002). [Online Journal]M. C. H. Clarke, J. Savill, D. B. Jones, B. S. Noble, S. B. Brown, Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J. Cell Biol. 160, 577-587 (2003). [Abstract] [Full Text]

Cleaved out of Action

The pathway by which the transcriptional regulator p53 activates apoptosis appears to diverge between invertebrates and vertebrates. In Drosophila , in response to such cellular stresses as DNA damage, p53 stimulates the expression of Reaper , thereby stimulating ubiquitin-mediated protein degradation of inhibitors of apoptosis (IAPs) and alleviating IAP inhibition of caspase activation. IAP homologs have been identified in mammals, but Reaper homologs have not. Instead, IAP inhibition in mammals is alleviated by Smac, which is released from mitochondria during the apoptotic cascade, or by HTRA2, a serine protease. Jin et al. investigated how a specific IAP, CIAP1, was inactivated in p53-mediated apoptosis and found that it was cleaved in response to DNA damage caused by etoposide treatment of HeLa cells. To determine whether CIAP1 cleavage was a consequence of caspase activation or occurred through a different mechanism, p53-independent apoptosis was triggered by activation of Fas. CIAP1 was cleaved in this apoptotic scenario but produced fragments of different size than those observed during p53-mediated apoptosis. Furthermore, caspase inhibitors did not block the p53-stimulated cleavage of CIAP1. Cleavage of CIAP1 produced by p53 processes required protein synthesis and could be inhibited by treatment of the cells with a serine protease inhibitor. In addition, serine protease inhibitors blocked etoposide-stimulated apoptosis of HeLa and primary mouse thymocytes, which indicates the importance of CIAP1 cleavage. Northern analysis showed that HTRA2 transcripts were increased after etoposide treatment or in response to overexpression of p53. These data suggest that HTRA2 may be a candidate for the serine protease responsible for CIAP1 cleavage during p53-mediated apoptosis. S. Jin, M. Kalkum, M. Overholtzer, A. Stoffel, B. T. Chiat, A. J. Levine, CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals. Genes Dev. 17 , 359-367 (2003). [Abstract] [Full Text]

Specific involvement of caspases in the differentiation of monocytes into macrophages

Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester-induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-(DL)-Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester-differentiated U937 cells and M-CSF-treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death.

Nitric Oxide Induces Caspase-dependent Apoptosis and Necrosis in Neonatal Rat Cardiomyocytes

T. Uchiyama, H. Otani, T. Okada, H. Ninomiya, M. Kido, H. Imamura, S. Nogi and Y. Kobayashi. Nitric Induces Oxide Caspase-dependent Apoptosis and Necrosis in Neonatal Rat Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2002) 34, 1049–1061. Excessive nitric oxide (NO) production has been implicated in the pathophysiology of cardiomyocyte (CMC) apoptosis and necrosis induced by ischemia/reperfusion, inflammation and NO-donating chemicals. Although caspases are known to be involved in apoptosis, the present study examined whether caspases also play a role in NO-induced CMC necrosis. Neonatal rat CMCs were labeled with Annexin-V and propidium iodide, and apoptosis and necrosis were analyzed by confocal images and fluorescence activated cell sorter analysis. CMC apoptosis and necrosis were also evaluated by determining DNA fragmentation in the cell and the supernatant fractions. Treatment of CMCs with the NO donor, diethylenetriamine NO (DETA/NO) or S-nitroso-N-acetyl-penicillamine (SNAP) at concentrations of 10 and 100μM for 24h induced predominantly apoptosis over necrosis, but a higher concentration (1mM) of DETA/NO or SNAP provoked both apoptosis and necrosis. The lower doses of DETA/NO-induced apoptosis was associated with a gradual increase in caspase-3 activity over 24h without appreciable activation of poly ADP-ribose polymerase (PARP), while the higher dose of DETA/NO induced a marked increase in caspase-3 activity and CMC apoptosis until 2h after the treatment, and increased necrotic CMCs thereafter associated with robust activation of PARP. The caspase inhibitor Z-DEVD-FMK but not the poly ADP-ribose polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) abolished caspase-3 activation and CMC apoptosis induced by 100μM DETA/NO. However, both Z-DEVD-FMK and 3-AB abolished PARP activation and CMC necrosis induced by 1mM DETA/NO. The amount of nicotinamide adenine dinucleotide (NAD) and adenine nucleotides in CMCs was not significantly affected by treatment with 10 and 100μM DETA/NO, but was significantly reduced by treatment with 1mM DETA/NO without a decline of adenylate energy charge. The depletion of NAD and adenine nucleotides was abrogated by Z-DEVD-FMK and 3-AB. These results suggest that caspase activation play a crucial role in CMC apoptosis induced by lower concentrations of NO as well as in CMC necrosis induced by a higher concentration of and a longer exposure to NO. NO-induced CMC necrosis is likely mediated by PARP activation which occurs as a consequence of caspase activation.

Platelet formation is the consequence of caspase activation within megakaryocytes

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of long and thin cytoplasmic extensions called proplatelets. After platelet release, the senescent MKs (nucleus surrounded by some cytoplasm) undergo cell death by apoptosis. To explore the precise role of apoptosis in proplatelet formation, we grew human MKs from CD34(+) cells and assessed the possible role of caspases. Proteolytic maturation of procaspase-3 and procaspase-9 was detected by immunoblots in maturing MKs as well as in proplatelet-bearing MKs and senescent MKs. Cleavage of caspase substrates such as gelsolin or poly adenosine diphosphate (ADP)-ribose polymerase (PARP) was also detected. Interestingly, activated forms of caspase-3 were detected in maturing MKs, before proplatelet formation, with a punctuate cytoplasmic distribution, whereas a diffuse staining pattern was seen in senescent and apoptotic MKs. This localized activation of caspase-3 was associated with a mitochondrial membrane permeabilization as assessed by the release of cytochrome c, suggesting an activation of the intrinsic pathway. Moreover, these MKs with localized activated caspase-3 had no detectable DNA fragmentation. In contrast, when apoptosis was induced by staurosporine, diffuse caspase activation was seen; these MKs had signs of DNA fragmentation, and no proplatelet formation occurred. The pan-caspase inhibitor z-VAD.fmk as well as more specific inhibitors of caspase-3 and caspase-9 blocked proplatelet formation, whereas an inhibitor of calpeptin had no effect. Overexpression of Bcl-2 also inhibited proplatelet formation in maturing MKs. Thus, localized caspase activation is causal to proplatelet formation. We conclude that proplatelet formation is regulated by a caspase activation limited to only some cellular compartments.

CARD games in apoptosis and immunity.

A bewildering array of proteins containing the caspase recruitment domain (CARD) have now been identified. Previously, CARD-CARD interactions have been shown to be involved in the assembly of protein complexes that promote caspase processing and activation in the context of apoptosis. However, as the family of CARD-containing proteins has grown, it has become apparent that the majority of these proteins do not recruit caspases or promote caspase activation. Instead, many participate in NF-kappaB signalling pathways associated with innate or adaptive immune responses. Here, we suggest a simplified classification of the CARD proteins based upon their domain structures and discuss the divergent roles of these proteins in the context of host defence.