Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca(2+) chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation.
 ...
PMID:BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells. 1144 71

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.
 ...
PMID:Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis. 1146

The molecules participating in apoptosis induced by T-2 toxin in human leukemia HL-60 cells were investigated. The rank order of the potency of trichothecene mycotoxins to induce internucleosomal DNA fragmentation was found to be T-2, satratoxin G, roridin A >> diacetoxyscirpenol > baccharin B-5 >> nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, baccharin B-4=vehicle control. Western blot analysis of caspase-3 in T-2-treated cells clearly indicated the appearance of its catalytically active fragment of 17-kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, cells exposed to T-2 led to cleavage of PARP from its native 116-kDa form to the 85-kDa product. Moreover, DFF-45/ICAD were cleaved to give a 12.5-kDa fragment via T-2 treatment. T-2 caused the release of cytochrome c from mitochondria into the cytosol. Increased enzymic activity of caspase-9 on LEHD-AMC was shown. These data indicate that T-2-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through cytosolic accumulation of cytochrome c along with caspase-9 activation.
 ...
PMID:Apoptosis induction by T-2 toxin: activation of caspase-9, caspase-3, and DFF-40/CAD through cytosolic release of cytochrome c in HL-60 cells. 1157 12

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.
 ...
PMID:Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation. 1169 7

DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.
 ...
PMID:Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis. 1178 71

A variety of endonucleases has been implicated in apoptotic DNA fragmentation. DNA fragmentation factor (DFF) is one of the endonucleases responsible for DNA fragmentation. Since an oligonucleosomal DNA ladder is not induced in apoptotic Molt-4 cells, we investigated whether or not the absence of ladder formation is related to an inability of DFF endonuclease in the cells. Semiquantitative RT-PCR analysis showed that the mRNA level of DFF-40 and DFF-45 in Molt-4 cells was approximately the same, compared with in other cells, which exhibit different levels of the fragmentation in apoptosis. When Molt-4 cells were induced to undergo apoptosis by neocarzinostatin (NCS) treatment, both caspase-3 activation and DFF-45 cleavage were observed. Furthermore, DFF immunoprecipitated from Molt-4 cells exhibited DNA degradation activity. These results suggest that functional expression of DFF is not sufficient for the induction of DNA fragmentation in Molt-4 cells.
 ...
PMID:Activation of caspase-3, proteolytic cleavage of DFF and no oligonucleosomal DNA fragmentation in apoptotic Molt-4 cells. 1187 77

The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD and 45 kD subunits. The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the genomic DNA degradation into nucleosomal fragments during apoptosis. As an inhibitor for DFF40, the 45 kD subunit (DFF45) complexes with DFF40, inhibiting DNase activity until certain apoptosis signals are received. In cells undergoing apoptosis, the cleavage of DFF45 by activated caspase-3 frees DFF40from the complex and initiates the apoptosis-specific DNA fragmentation. In this report, the coding region of human DFF45 gene was amplified from the total RNA of HeLa cells by RT-PCR. The resulting 1 kb DNA fragment was cloned into the bacterial expression vector pET-28a(+) with a 6xhistidine tag fused to the N-terminus of DFF45, generating plasmid pET28a-DFF45, which was then used to transform E.coli BL21(DE3). Induced by IPTG, the recombinant DFF45 was expressed efficiently with a yield of 56.6% of total bacterial proteins. The product was purified to homogeneity through a nickel affinity column, followed by heat treatment, and approximately 4--6 mg of DFF was purified from 100 ml culture. Purified recombinant human DFF45, added into the apoptotic cell-free system of Xenopus egg extracts, could effectively inhibit both the digestion of lambdaDNA and the degradation of chromosomal DNA into nucleosomal fragments in the nuclei of chicken red blood cells. Our results demonstrated the existence of an apoptosis-specific endonuclease in this cell-free system, the activity of which could be inhibited by recombinant human DFF45.
 ...
PMID:Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts. 1205 94

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.
 ...
PMID:Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3. 1216 Dec 80

DNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)-induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3-activated CAD in a cell-free system. Peptide-binding domain-deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)-binding domain-deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.
 ...
PMID:Heat shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells. 1273 67

DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.
 ...
PMID:Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis. 1274 78


<< Previous 1 2 3 4 Next >>