The Caspase-Activated DNase promotes cellular senescence
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ABSTRACT:
SUBMITTER: Dr. Aladin Haimovici
PROVIDER: S-SCDT-10_1038-S44318-024-00163-9 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.
| S-EPMC21302 | biostudies-literature
Project description:Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes.
| S-EPMC2840077 | biostudies-literature
Project description:The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.
| S-EPMC2377037 | biostudies-literature
Project description:Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.
| S-EPMC9177556 | biostudies-literature
Project description:Caspase-activated DNase (CAD) is an endonuclease that is activated by active caspase 3 during apoptosis and is responsible for degradation of chromatin into nucleosomal units. These nucleosomal units are then included in apoptotic bodies. The presence of apoptotic bodies is considered important for the generation of autoantigen in autoimmune diseases, such as systemic lupus erythematosus (SLE), that are characterized by the presence of antinuclear antibodies. The present study was carried out to determine the role of CAD in SLE and to investigate the ability of lupus autoantibodies to bind to CAD-deficient or CAD-sufficient apoptotic cells.The Sle1, Sle123, and 3H9 mouse models of SLE, in which autoimmunity is genetically predetermined, were used. To determine the role of chromatin fragmentation in SLE, CAD deficiency was introduced in these mouse models.Deficiency of CAD resulted in increased anti-double-stranded DNA antibody titers in lupus-prone mice. Surprisingly, the absence of CAD exacerbated only genetically predetermined autoimmune responses. To further determine whether nuclear modifications are needed in order to maintain tolerance to nuclear autoantigens, we used the 3H9 mouse, an anti-DNA heavy chain knockin; in this model, the autoreactive B cells are tolerized by anergy. In accordance with findings in the CAD-mutant Sle1 and Sle123 mice, CAD-deficient 3H9 mice spontaneously generated anti-DNA antibodies. Finally, we showed that autoantibodies with specificities toward histone-DNA complexes bind more to CAD-deficient apoptotic cells than to CAD-sufficient apoptotic cells.We propose that in mice that are genetically predisposed to lupus development, nuclear apoptotic modifications are needed to maintain tolerance. In the absence of these modifications, apoptotic chromatin is abnormally exposed, facilitating the autoimmune response.
| S-EPMC3292632 | biostudies-literature
Project description:BackgroundAtherosclerosis is a chronic disease that is closely related to inflammation and macrophage apoptosis, which leads to secondary necrosis and proinflammatory responses in advanced lesions. Caspase-activated DNase (CAD) is a double-strand specific endonuclease that leads to the subsequent degradation of chromosome DNA during apoptosis. However, whether CAD is involved in the progression of atherosclerosis remains elusive.Methods and resultsCAD-/-ApoE-/- and ApoE-/- littermates were fed a high-fat diet for 28 weeks to develop atherosclerosis. Human specimens were collected from coronary heart disease (CHD) patients who were not suitable for transplantation. CAD expression was increased in the atheromatous lesions of CHD patients and high-fat diet-treated ApoE-deficient mice. Further investigation demonstrated that CAD deficiency inhibited high-fat diet-induced atherosclerosis, as evidenced by decreased atherosclerotic plaques, inhibited inflammatory response, and macrophage apoptosis, as well as enhanced stability of plaques in CAD-/-ApoE-/- mice compared to the ApoE-/- controls. Bone marrow transplantation verified the effect of CAD on atherosclerosis from macrophages. Mechanically, the decrease in the phosphorylated levels of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-regulated kinase 1 and 2 (MEK-ERK1/2) that resulted from CAD knockout and the activation of nuclear factor kappa B signaling mediated by CAD stimulation that was suppressed by inhibiting ERK1/2 phosphorylation revealed the potential association between the role of CAD in atherosclerosis and the MAPK signaling pathway.ConclusionsIn conclusion, CAD deficiency protects against atherosclerosis through inhibiting inflammation and macrophage apoptosis, which is partially through inactivation of the MEK-ERK1/2 signaling pathway. This finding provides a promising therapeutic target for treating atherosclerosis.
| S-EPMC5210397 | biostudies-literature
Project description:BackgroundNuclear factor-κB is a multi-subunit transcription factor that plays a central role in cellular senescence. We previously reported that an increase in the p52 subunit is seen in senescent cells and aged tissue. In the current work, we examined the mechanism by which p52 is activated and whether the increase in p52 promotes senescence.ResultsUsing both primary mouse embryonic fibroblasts (MEFs) and WI-38 human lung fibroblasts, we examined cells after serial passage and following prolonged culture. An increase in p52 was found in the nucleus relative to pre-senescent cells. The increase in p52 protein was not reflected by an increase in NFKB2 mRNA or by an increase in the abundance of upstream activating kinases, IKKα and NIK. To examine whether p52 promotes senescence, we over-expressed mature p52 in primary MEFs. Significantly more senescence was seen compared to control, a finding not seen with p52 mutated at critical DNA binding residues. In addition, blocking p52 nuclear translocation with the peptide inhibitor, SN52, decreased β-galactosidase (β-gal) formation. Subsequent filtration studies demonstrated that proteins in conditioned media (CM) were necessary for the increase in p52 and mass spectrometry identified S100A4 and cyclophilin A (CYPA) as potential factors in CM necessary for induction of p52. The requirement of these proteins in CM for induction of p52 was confirmed using depletion and supplementation studies. In addition, we found that activation of STAT3 signaling was required for the increase in p52. Finally, genome wide ChIP-sequencing analysis confirmed that there is an increase in p52 chromatin enrichment with senescence and identified several downstream factors whose expression is regulated by increased p52 binding.ConclusionsThese results demonstrate that p52 nuclear translocation is increased in senescent cells by factors in conditioned media and that mature p52 induces cellular senescence. The data are consistent with the prior observation that p52 is elevated in aged tissue and support the hypothesis that p52 contributes to organismal aging.
| S-EPMC8981737 | biostudies-literature
Project description:Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1.
| S-EPMC1462972 | biostudies-literature
Project description:DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development.
| S-EPMC2423419 | biostudies-other
Project description:We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP-CAD from the nuclei in approximately 50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.
| S-EPMC135751 | biostudies-literature