Caspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3-/- TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.
During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.
- MeSH
- Apoptosis physiology MeSH
- Cell Division physiology MeSH
- Epithelial Cells cytology MeSH
- Apoptotic Protease-Activating Factor 1 deficiency MeSH
- Caspase 9 deficiency MeSH
- Mesoderm physiology MeSH
- Molar embryology physiology MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Proliferating Cell Nuclear Antigen analysis MeSH
- Dental Enamel embryology physiology MeSH
- Tooth Germ anatomy & histology embryology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Apaf1 protein, mouse MeSH Browser
- Apoptotic Protease-Activating Factor 1 MeSH
- Caspase 9 MeSH
- Proliferating Cell Nuclear Antigen MeSH
In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (Deltapsi(m)) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.
- MeSH
- Enzyme Activation MeSH
- Apoptosis * MeSH
- Cell Line MeSH
- Time Factors MeSH
- Cytochromes c metabolism MeSH
- Cytosol metabolism MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Microscopy, Fluorescence MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases metabolism MeSH
- Microscopy, Confocal MeSH
- Culture Media pharmacology MeSH
- Membrane Potentials MeSH
- Mitochondria pathology MeSH
- Mice MeSH
- Proteins metabolism MeSH
- Flow Cytometry MeSH
- Reactive Oxygen Species MeSH
- Subcellular Fractions MeSH
- Protein Transport MeSH
- Blotting, Western MeSH
- Iron metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Apaf1 protein, mouse MeSH Browser
- Casp3 protein, mouse MeSH Browser
- Casp9 protein, mouse MeSH Browser
- Cytochromes c MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- Culture Media MeSH
- Proteins MeSH
- Reactive Oxygen Species MeSH
- Iron MeSH
The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in non-small cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and procaspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently up-regulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme B-generated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c+dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase-3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol.
- MeSH
- Actins biosynthesis MeSH
- Enzyme Activation drug effects MeSH
- Apoptosis physiology MeSH
- Cytochromes c metabolism MeSH
- Cytosol metabolism MeSH
- Deoxyadenine Nucleotides metabolism MeSH
- Adult MeSH
- Gene Expression MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Transcription, Genetic MeSH
- Granzymes MeSH
- Immunoblotting MeSH
- Caspase 3 MeSH
- Caspase 7 MeSH
- Caspases biosynthesis genetics metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA, Messenger biosynthesis genetics MeSH
- Cell Line, Tumor MeSH
- Lung Neoplasms enzymology genetics metabolism pathology MeSH
- Carcinoma, Non-Small-Cell Lung enzymology genetics metabolism pathology MeSH
- Enzyme Precursors biosynthesis MeSH
- Proteins genetics MeSH
- Protein Biosynthesis * MeSH
- Aged MeSH
- Serine Endopeptidases metabolism MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- 2'-deoxyadenosine triphosphate MeSH Browser
- Actins MeSH
- APAF1 protein, human MeSH Browser
- CASP3 protein, human MeSH Browser
- CASP7 protein, human MeSH Browser
- Cytochromes c MeSH
- Deoxyadenine Nucleotides MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Granzymes MeSH
- GZMB protein, human MeSH Browser
- Caspase 3 MeSH
- Caspase 7 MeSH
- Caspases MeSH
- RNA, Messenger MeSH
- Enzyme Precursors MeSH
- Proteins MeSH
- Serine Endopeptidases MeSH
Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.
- Keywords
- alternative splicing, inhalation, splice junction expression, zinc oxide nanoparticles,
- MeSH
- Alternative Splicing drug effects MeSH
- Administration, Inhalation MeSH
- Apoptosis drug effects MeSH
- Immunity, Cellular drug effects MeSH
- Cell Cycle drug effects MeSH
- Gene Expression drug effects MeSH
- Mice, Inbred ICR MeSH
- Mice MeSH
- Nanoparticles toxicity MeSH
- DNA Repair drug effects MeSH
- Zinc Oxide toxicity MeSH
- Oxidative Stress drug effects MeSH
- Lung metabolism pathology MeSH
- Inflammation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Zinc Oxide MeSH
INTRODUCTION: Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes. METHODOLOGY: We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates. RESULTS: The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2-associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and pro-apoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO. DISCUSSION: Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish.
- Keywords
- apoptosis, cell death, fertilization, membrane integrity, trypan blue, zebrafish,
- Publication type
- Journal Article MeSH
PURPOSE: Tumour cells killing by cytotoxic therapies largely depends on triggering the intrinsic apoptosome-mediated caspase activation pathway but it had never been evaluated whether the expression of transcripts encoding the core components of apoptosome pathway is altered in non-small cell lung carcinoma (NSCLC). METHODS: We investigated the expression status of several apoptosome pathway-related transcripts including Apaf-1, procaspase-9, -3, -6, -7 and Smac in tumour and lung tissue samples from 65 surgically treated NSCLC patients and in 10 NSCLC cell lines with using real time RT-PCR. RESULTS: NSCLC tissues and cell lines showed significantly increased expression of procaspase-9, -3, -6 and Smac mRNAs as compared to the lungs and expression of these transcripts was simultaneously upregulated in a subset of NSCLCs belonging to different histopathological type, grade and stage categories. The expression of procaspase-7 mRNA in NSCLC tissues and cell lines and lungs was not significantly different. By contrast, the expression of Apaf-1 mRNA was frequently downregulated in the tumours as compared to matched lungs. Nevertheless, the examined NSCLC cell lines showed significantly higher expression of Apaf-1 mRNA than the lungs. The expression of Apaf-1, procaspase-9 and -6 mRNAs was higher in lung adenocarcinomas as compared to squamous cell lung carcinomas but the expression levels of the studied apoptosome pathway-related transcripts in the tumours were independent of tumour's grade and stage. CONCLUSIONS: The results of the present study suggest that there is a subgroup of NSCLCs, which may be intrinsically primed for apoptosis through upregulated expression of transcripts encoding the apoptosome pathway components.
- MeSH
- Actins metabolism MeSH
- Apoptosis * genetics MeSH
- Adult MeSH
- Down-Regulation MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Transcription, Genetic * MeSH
- Intracellular Signaling Peptides and Proteins genetics metabolism MeSH
- Caspase 3 MeSH
- Caspase 6 MeSH
- Caspase 7 MeSH
- Caspase 9 MeSH
- Caspases genetics metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Mitochondrial Proteins genetics metabolism MeSH
- Biomarkers, Tumor genetics metabolism MeSH
- Lung Neoplasms genetics metabolism MeSH
- Carcinoma, Non-Small-Cell Lung genetics metabolism MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Enzyme Precursors metabolism MeSH
- Apoptosis Regulatory Proteins MeSH
- Proteins genetics metabolism MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Aged MeSH
- Up-Regulation MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Actins MeSH
- APAF1 protein, human MeSH Browser
- CASP3 protein, human MeSH Browser
- CASP7 protein, human MeSH Browser
- CASP9 protein, human MeSH Browser
- DIABLO protein, human MeSH Browser
- Apoptotic Protease-Activating Factor 1 MeSH
- Intracellular Signaling Peptides and Proteins MeSH
- Caspase 3 MeSH
- Caspase 6 MeSH
- Caspase 7 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- RNA, Messenger MeSH
- Mitochondrial Proteins MeSH
- Biomarkers, Tumor MeSH
- Enzyme Precursors MeSH
- Apoptosis Regulatory Proteins MeSH
- Proteins MeSH
