Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence.

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

The apoptosome molecular timer synergises with XIAP to suppress apoptosis execution and contributes to prognosticating survival in colorectal cancer.

The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.

LncRNA POU3F3 promotes proliferation and inhibits apoptosis of cancer cells in triple-negative breast cancer by inactivating caspase 9.

It has been reported that lncRNA POU3F3 was upregulated in esophageal squamous-cell carcinomas, indicating its role as an oncogene in this disease. However, the mechanism of its function and its involvement in other malignancies is unknown. In the present study we found that expression levels of lncRNA POU3F3 were higher in tumor tissues than in adjacent healthy tissues of triple negative breast cancer (TNBC) patients and were significantly and inversely correlated with levels of cleaved caspase 9 only in tumor tissues. In addition, plasma levels of lncRNA POU3F3 were higher in TNBC patients than in healthy controls and were significantly and inversely correlated with levels of cleaved caspase 9 only in TNBC patients. In addition, treatment of exogenous Cleaved Caspase-9 significantly attenuated the effects of lncRNA POU3F3 overexpression on cancer cell proliferation and apoptosis. lncRNA POU3F3 may promote proliferation and inhibit apoptosis of cancer cells in triple-negative breast cancer.

Role of toll-like receptor 4 and caspase-3, -8, and -9 in lipopolysaccharide-induced delay of apoptosis in equine neutrophils.

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Roles of Bcl-2 and caspase-9 and -3 in CD30-induced human eosinophil apoptosis.

BACKGROUND/PURPOSE: Activation of cell surface CD30 by immobilized anti-CD30 monoclonal antibodies (mAb) induces strong apoptosis in human eosinophils. This anti-CD30 mAb-induced eosinophil apoptosis is inhibited by the addition of inhibitors of p38, ERK1/2 mitogen-activated protein kinases, and phosphatidylinositol 3-kinase. However, there is little data investigating the role of Bcl-2 and caspases in eosinophil apoptosis induced by anti-CD30 mAb. We sought to determine whether anti-CD30 mAb induces human eosinophil apoptosis via Bcl-2 and caspase pathways. METHODS: Peripheral blood was drawn from 37 healthy volunteers. The CD30 expression on eosinophils was measured at various time points. Eosinophils were then cultured in plates precoated with anti-CD30 mAb (clone Ber-H8), isotype control immunoglobulin G1, interleukin (IL)-5, or dexamethasone. Western blot analysis was performed to determine the expression of Bcl-2, procaspase-8, -9, and -3, and caspase-8, -9, and -3 after cross-linking of CD30. Human eosinophils were also cultured in plates precoated with anti-CD30 mAb (clone Ber-H8) in the presence or absence of caspase-9 or -3 inhibitors. Eosinophil apoptosis was assessed using flow cytometry. RESULTS: The addition of anti-CD30 mAb significantly increased eosinophil apoptosis compared with controls. In western blot analysis, the addition of anti-CD30 mAb significantly decreased the expression of Bcl-2 and procaspase-9 and -3 and increased the expression of caspase-9 and -3. The addition of caspase-9 or -3 inhibitors decreased anti-CD30 mAb-induced human eosinophil apoptosis. Procaspase-8 or caspase-8 expression was not changed in response to various stimuli. CONCLUSION: Anti-CD30 mAb-induced human eosinophil apoptosis is likely to be mediated through Bcl-2 and caspase-9 and -3.

Acute cytotoxicity of MIRA-1/NSC19630, a mutant p53-reactivating small molecule, against human normal and cancer cells via a caspase-9-dependent apoptosis.

Although numerous studies have focused on the mechanisms of action of the candidate chemotherapeutic drug MIRA-1/NSC19630, initially described as a mutant p53-reactivating small molecule, the issue of its toxicological evaluation remains open. Here, we devised a strategy to examine the effects of MIRA-1 on a variety of human normal cells and cancer cell lines. First, we demonstrated a massive and rapid (within 2 hours) MIRA-1 apoptotic effect on human normal primary epithelial cells as shown using an intestinal mucosa explant assay. MIRA-1 was also cytotoxic to primary and subcultured human mesenchymal cells. Interestingly these effects were restricted to actively proliferating cells. Second, MIRA-1 acute toxicity was independent of p53, since it occurred in human normal cells with increased or silenced p53 expression level, in cancer cells derived from solid or liquid tumors, with either mutated or wt TP53, and in cancer cells devoid of p53. Third, combined pharmacological and genetic approaches showed that MIRA-1 acute cytotoxicity was mediated by a caspase-9-dependent apoptosis. In conclusion, our strategy unveils the limitations of the targeted action of a small molecule designed to reactivate mutant p53.

Caspase-9 is required for normal hematopoietic development and protection from alkylator-induced DNA damage in mice.

Apoptosis and the DNA damage responses have been implicated in hematopoietic development and differentiation, as well as in the pathogenesis of myelodysplastic syndromes (MDS) and leukemia. However, the importance of late-stage mediators of apoptosis in hematopoiesis and leukemogenesis has not been elucidated. Here, we examine the role of caspase-9 (Casp9), the initiator caspase of the intrinsic apoptotic cascade, in murine fetal and adult hematopoiesis. Casp9 deficiency resulted in decreased erythroid and B-cell progenitor abundance and impaired function of hematopoietic stem cells after transplantation. Mouse bone marrow chimeras lacking Casp9 or its cofactor Apaf1 developed low white blood cell counts, decreased B-cell numbers, anemia, and reduced survival. Defects in apoptosis have also been previously implicated in susceptibility to therapy-related leukemia, a disease caused by exposure to DNA-damaging chemotherapy. We found that the burden of DNA damage was increased in Casp9-deficient cells after exposure to the alkylator, N-ethyl-nitrosourea (ENU). Furthermore, exome sequencing revealed that oligoclonal hematopoiesis emerged in Casp9-deficient bone marrow chimeras after alkylator exposure. Taken together, these findings suggest that defects in apoptosis could be a key step in the pathogenesis of alkylator-associated secondary malignancies.

A systems biology analysis of apoptosome formation and apoptosis execution supports allosteric procaspase-9 activation.

The protease caspase-9 is activated on the apoptosome, a multiprotein signal transduction platform that assembles in response to mitochondria-dependent apoptosis initiation. Despite extensive molecular research, the assembly of the holo-apoptosome and the process of caspase-9 activation remain incompletely understood. Here, we therefore integrated quantitative data on the molecular interactions and proteolytic processes during apoptosome formation and apoptosis execution and conducted mathematical simulations to investigate the resulting biochemical signaling, quantitatively and kinetically. Interestingly, when implementing the homodimerization of procaspase-9 as a prerequisite for activation, the calculated kinetics of apoptosis execution and the efficacy of caspase-3 activation failed to replicate experimental data. In contrast, assuming a scenario in which procaspase-9 is activated allosterically upon binding to the apoptosome backbone, the mathematical simulations quantitatively and kinetically reproduced all experimental data. These data included a XIAP threshold concentration at which apoptosis execution is suppressed in HeLa cervical cancer cells, half-times of procaspase-9 processing, as well as the molecular timer function of the apoptosome. Our study therefore provides novel mechanistic insight into apoptosome-dependent apoptosis execution and suggests that caspase-9 is activated allosterically by binding to the apoptosome backbone. Our findings challenge the currently prevailing dogma that all initiator procaspases require homodimerization for activation.

ER stress responses in the absence of apoptosome: a comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts.

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9(+/+) and casp9(-/-) MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9(-/-) cells as compared with casp9(+/+) MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9(-/-) MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.

Genome-wide pathway analysis in attention-deficit/hyperactivity disorder.

This study aimed to (1) to identify candidate single-nucleotide polymorphisms (SNPs) and mechanisms of attention-deficit/hyperactivity disorder (ADHD) and (2) to generate SNP-to-gene-to-pathway hypotheses. An ADHD genome-wide association study (GWAS) dataset that included 428,074 SNPs in 924 trios (2,758 individuals) of European descent was used in this study. The Identify candidate Causal SNPs and Pathways (ICSNPathway) analysis was applied to the GWAS dataset. ICSNPathway analysis identified 11 candidate SNPs, 6 genes, and 6 pathways, which provided 6 hypothetical biological mechanisms. The strongest hypothetical biological mechanism was that rs2532502 alters the role of CD27 in the context of the pathways of positive regulation of nucleocytoplasmic transport [nominal p < 0.001; false discovery rate (FDR) = 0.028]. The second strongest mechanism was the rs1820204, rs1052571, rs1052576 → CASP9 → mitochondrial pathway (nominal p < 0.001; FDR = 0.032). The third mechanism was the rs1801516 → ATM → CD25 pathway (nominal p < 0.001; FDR = 0.034). By applying the ICSNPathway analysis to the ADHD GWAS data, 11 candidate SNPs, 6 genes that included CD27, CASP9, ATM, CD12orf65, OXER1, and ACRY, and 6 pathways were identified that may contribute to ADHD susceptibility.

Oxymatrine triggers apoptosis by regulating Bcl-2 family proteins and activating caspase-3/caspase-9 pathway in human leukemia HL-60 cells.

With the objective of identifying promising antitumor agents for human leukemia, we carried out to determine the anticancer ability of oxymatrine on the human leukemia HL-60 cell line. In vitro experiments demonstrated that oxymatrine reduced the proliferation of HL-60 cells in a dose- and time-dependent manner via the induction of apoptosis and cell cycle arrest at G2/M and S phases. The proteins involved in oxymatrine-induced apoptosis in HL-60 cells were also examined using Western blot. The increase in apoptosis upon treatment with oxymatrine was correlated with downregulation of anti-apoptotic Bcl-2 expression and upregulation of pro-apoptotic Bax expression. Furthermore, oxymatrine induced the activation of caspase-3 and caspase-9 and the cleavage of poly(ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, pretreatment with a specific caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) inhibitor significantly neutralized the pro-apoptotic activity of oxymatrine in HL-60 cells, demonstrating the important role of caspase-3 and caspase-9 in this process. Taken together, these results indicated that oxymatrine-induced apoptosis may occur through the activation of the caspase-9/caspase-3-mediated intrinsic pathway. Therefore, oxymatrine may be a potential candidate for the treatment of human leukemia.

Bergapten prevents lipopolysaccharide mediated osteoclast formation, bone resorption and osteoclast survival.

PURPOSE: This study was designed to investigate the potential effect of bergapten on lipopolysaccharide (LPS)-mediated osteoclast formation, bone resorption and osteoclast survival in vitro. METHODS: After osteoclast precursor RAW264.7 cells were treated with bergapten (5, 20, 40 µmol/L) for 72 hours in the presence of LPS (100 ng/ml), osteoclastogenesis was identified by tartrate-resistant acid phosphatase (TRAP) staining, and the number of TRAP-positive multinucleated cells [TRAP(+)MNCs] per well were counted. To investigate the effect of bergapten on osteoclastic bone resorption, RAW264.7 cells were treated with bergapten for six days in the presence of LPS, and the area of bone resorption was analyzed with Image Pro-Plus. Next, we examined apoptosis of RAW264.7 cells after bergapten incubation for 48 hours by flow cytometer using annexin V/propidium iodide (PI) double labeling. Finally, osteoclast survival was observed by Hoechst 33342 labeling and Western blotting after bergapten treatment for 24 hours. RESULTS: Data showed that bergapten (5-40 µmol/L) dose-dependently inhibited LPS-induced osteoclast formation and bone resorption. Treatment with bergapten triggered apoptotic death of osteoclast precursor RAW264.7 cells in a dose-dependent manner. Furthermore, bergapten significantly reduced the survival of mature osteoclast, as demonstrated by emergence of apoptotic nuclei and activation of apoptotic protein caspase 3/9. CONCLUSIONS: These findings suggest that bergapten effectively prevents LPS-induced osteoclastogenesis, bone resorption and survival via apoptotic response of osteoclasts and their precursors. The study identifies bergapten as an inhibitor of osteoclast formation and bone resorption and provides evidence that bergapten might be beneficial as an alternative for prevention and treatment of inflammatory bone loss.

Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis.

BACKGROUND: Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochrome c release is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined. RESULTS: Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochrome c, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM. CONCLUSIONS: Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.

Role of the intrinsic apoptotic pathway in cataractogenesis in type 2 diabetic patients.

PURPOSE: To determine and compare the expression of caspase-3 and caspase-9 in human lens epithelial cells of cataract patients with and without diabetic retinopathy (DR). SETTING: St. Mary's Hospital, Catholic University of Korea, Seoul, South Korea. DESIGN: Randomized prospective study. METHODS: Patients were classified into 3 groups as follows: patients without diabetes mellitus (DM) (Group 1), patients with DM but without DR (Group 2), and patients with DM and DR (Group 3). An anterior capsulotomy specimen was obtained from each patient during cataract surgery, and immunohistochemical staining was performed for caspase-3 and caspase-9. The results were observed using confocal microscopy and were analyzed by reactivity scoring. The duration of DM and the hemoglobin A1c (HbA1c) levels were recorded. RESULTS: The study evaluated 60 patients. The level of caspase-3 and caspase-9 expression was highest in Group 3 followed by Group 2 and Group 1 (both P < .00). Expression of caspase-3 and caspase-9 was higher with a longer duration of DM and with higher HbA1c levels. CONCLUSION: The intrinsic apoptotic pathway may be associated with diabetic cataract in patients with type 2 DM. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Melatonin induces apoptosis through a caspase-dependent but reactive oxygen species-independent mechanism in human leukemia Molt-3 cells.

Melatonin is a naturally occurring indoleamine synthesized in the pineal gland that exhibits an extensive repertoire of biological activities. An increasing number of studies indicate that melatonin protects normal cells, while it reducing cancer cell proliferation. In this study, we investigated the effect of melatonin on the growth of the human leukemia cells and found that it efficiently reduced the number of cells in a concentration- and time-dependent manner. Thus, incubation with the indoleamine increased the percentage of cells with a hypodiploid DNA content, augmented the number of annexin V-positive cells, and also provoked ultrastructural changes that are features of apoptotic cell death. Evaluation of caspases revealed that caspase-3, caspase-6, caspase-7, and caspase-9, but not caspase-8 and caspase-2, were quickly activated (3-6 hr). The increase in the activity of these proteases was associated with up-regulation of the pro-apoptotic factor Bax and also with the release of cytochrome c from mitochondria. Pretreatment of the cells with the general caspase inhibitor z-VAD-fmk, reduced melatonin-induced apoptosis, but it did not block cell death suggesting that melatonin activates an alternative cell death modality in the absence of caspase activity. Thus, the activation of caspases was preceded by a fast (<30 min) increase in reactive oxygen species (ROS). Rotenone and antimycin A reduced the levels of ROS stimulated by melatonin, indicating that the complex I and the complex III of the mitochondrial electron transport chain are important sources of these chemical species. However, the role of ROS in melatonin-induced cell death remains elusive because anti-oxidants that were shown to decrease ROS levels (glutathione, N-acetyl-l-cysteine and Trolox) were unable to abrogate melatonin-induced cell death.

OSU-03012, a non-Cox inhibiting celecoxib derivative, induces apoptosis of human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

OSU-03012 is a celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity. It was previously reported to inhibit the growth of some tumor cells through the AKT-signaling pathway. In the current study, we assessed the ability of OSU-03012 to induce apoptosis in human esophageal carcinoma cells and the mechanism by which this occurs. A cell proliferation assay indicated that OSU-03012 inhibited the growth of human esophageal carcinoma cell lines with an IC50 below 2 µmol/l and had the most effective cytotoxicity against Eca-109 cells. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and flow cytometry analysis showed that OSU-03012 could induce the apoptosis in Eca-109 cells. After treatment of Eca-109 cells with 2 µmol/l OSU-03012 for 24 h, the apoptosis index increased from 14.07 to 53.72%. OSU-03012 treatment resulted in a 30-40% decrease in the mitochondrial membrane potential and caused cytochrome c release into the cytosol. Further studies with caspase-9-specific and caspase-8-specific inhibitors (z-LEHDfmk and z-IETDfmk, respectively) pointed toward the involvement of the caspase-9 pathway, but not the caspase-8 pathway, in the execution of OSU-03012-induced apoptosis. Immunoblot analysis demonstrated that OSU-03012-induced cellular apoptosis was associated with upregulation of Bax, cleaved caspase-3, and cleaved caspase-9. Ser-15 of p53 was phosphorylated after 24 h of treatment of the cancer cells with OSU-03012. This increase in p53 was associated with the decrease in Bcl-2 and increase in Bax. An inhibitor of p53, pifithrin-α, attenuated the anticancer effects of OSU-03012 and downregulated the expression of Bax and cleaved caspase-9. Altogether, our results show that OSU-03012 could induce apoptosis in human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

p53 activation by Ni(II) is a HIF-1α independent response causing caspases 9/3-mediated apoptosis in human lung cells.

Hypoxia mimic nickel(II) is a human respiratory carcinogen with a suspected epigenetic mode of action. We examined whether Ni(II) elicits a toxicologically significant activation of the tumor suppressor p53, which is typically associated with genotoxic responses. We found that treatments of H460 human lung epithelial cells with NiCl2 caused activating phosphorylation at p53-Ser15, accumulation of p53 protein and depletion of its inhibitor MDM4 (HDMX). Confirming the activation of p53, its knockdown suppressed the ability of Ni(II) to upregulate MDM2 and p21 (CDKN1A). Unlike DNA damage, induction of GADD45A by Ni(II) was p53-independent. Ni(II) also increased p53-Ser15 phosphorylation and p21 expression in normal human lung fibroblasts. Although Ni(II)-induced stabilization of HIF-1α occurred earlier, it had no effect on p53 accumulation and Ser15 phosphorylation. Ni(II)-treated H460 cells showed no evidence of necrosis and their apoptosis and clonogenic death were suppressed by p53 knockdown. The apoptotic role of p53 involved a transcription-dependent program triggering the initiator caspase 9 and its downstream executioner caspase 3. Two most prominently upregulated proapoptotic genes by Ni(II) were PUMA and NOXA but only PUMA induction required p53. Knockdown of p53 also led to derepression of antiapoptotic MCL1 in Ni(II)-treated cells. Overall, our results indicate that p53 plays a major role in apoptotic death of human lung cells by Ni(II). Chronic exposure to Ni(II) may promote selection of resistant cells with inactivated p53, providing an explanation for the origin of p53 mutations by this epigenetic carcinogen.

Roles of 14-3-3η in mitotic progression and its potential use as a therapeutic target for cancers.

14-3-3 proteins are involved in several cellular processes, including the G1/S and G2/M cell cycle transitions. However, their roles during mitosis are not well understood. Here, we showed that depletion of 14-3-3η, a 14-3-3 protein isoform, enhanced mitotic cell death, resulting in sensitization to microtubule inhibitors and inhibition of aneuploidy formation. The enhanced mitotic cell death by depletion of 14-3-3η appeared to be both caspase-dependent and independent. Furthermore, enhanced mitotic cell death and a reduction in aneuploidy following 14-3-3η depletion were independent of the mitotic checkpoint, which is thought to be the primary signaling event in the regulation of the cell death induced by microtubule inhibitors. When 14-3-3η depletion was combined with microtubule inhibitors in HCT116 and U87MG cells, it sensitized both cancer cell lines to microtubule inhibitors. These results collectively suggest that 14-3-3η may be required for mitotic progression and may be considered as a novel anti-cancer strategy in combination with microtubule inhibitors.

FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells.

Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.