DNA fragmentation factor 45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we measured the expression of DFF45 during mouse development and compared DNA fragmentation and viability of DFF45-deficient cells with wild-type control cells after activation of apoptosis. We found that DFF45 is ubiquitously expressed throughout mouse development. Moreover, DFF45-deficient thymocytes are resistant to DNA fragmentation with in vivo dexamethasone treatment. Furthermore, primary thymocytes from DFF45 mutant mice are also more resistant to apoptosis than wild-type control cells on exposure to several apoptotic stimuli. Dying DFF45-deficient thymocytes exhibit different morphology than wild-type control cells in that they show reduced degree of chromatin condensation, absent nuclear fragmentation, intranuclear cytoplasmic invagination, and striking nuclear chromatin conglutination after release from disintegrating cells. These results indicate that DFF45 is essential during normal apoptosis.     

Dna fragmentation factor 45 mutant mice exhibit resistance to kainic acid-induced neuronal cell death

Excitotoxicity is a process where glutamate or other excitatory amino acids induce neuronal cell death. Emerging evidence suggests that apoptosis plays a key part in excitotoxic neurodegeneration. The DNA fragmentation factor 45 (DFF45 or ICAD) is a subunit of a heterodimeric DNase complex crucial for DNA fragmentation during apoptosis. Using a DFF45 mutant mouse model, we previously found that DFF45 deficient cells are more resistant to apoptosis than normal control cells. To investigate whether the lack of DFF45 may attenuate neuronal cell death induced by excitotoxicity, we compared kainic acid-induced seizure behavior and neuronal cell death in DFF45 mutant and wild-type control mice. We found that the mutant mice exhibit similar kainic acid-induced seizure severity compared to control mice. However, DFF45 mutant mice are more resistant than control mice to kainic acid-induced CA3 neuronal cell death. Interestingly, residual DNA degradation can be detected in the hippocampus of DFF45 mutant mice that exhibit KA-induced lesions. Our results suggest that a lack of DFF45 can lead to neuronal resistance to excessive activity-induced toxicity.     

Regional and Temporal Alterations in DNA Fragmentation Factor (DFF)-Like Proteins Following Experimental Brain Trauma in the Rat

DNA fragmentation, an early event in neuronal death following traumatic brain injury, may be triggered by the 40-kDa subunit of DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45-kDa subunit of DFF (DFF45), and activation of DFF40 may occur as a result of caspase-3-mediated cleavage of DFF45 into 30- and 11-kDa fragments. In this study, the intracellular distribution of DFF45 and DFF40 was examined following lateral fluid percussion brain injury of moderate severity (2.4-2.7 atm) in male Sprague-Dawley rats. In the cytosolic fraction (S1) of the injured cortex at 2 and 24 h postinjury, significant decreases in the intensities of DFF45-like proteins at 45- and 32-kDa bands and a concomitant increase in the 11-kDa bands were observed (p < 0.05 vs. uninjured controls). A significant decrease in the intensities of the 32-kDa band in the nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction (p < 0.05) and recovered by 24 h postinjury, whereas DFF40 was significantly decreased in the S1 and increased in the P1 fraction at both 2 and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase-3-DFF45/DFF40-DNA cleavage observed following traumatic brain injury.     

Lack of obvious 50 kilobase pair DNA fragments in DNA fragmentation factor 45-deficient thymocytes upon activation of apoptosis

The DNA fragmentation factor 45 (DFF45/ICAD) is a key subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation during apoptosis in vivo. To further assess the importance of DFF45 in chromosomal DNA degradation, we induced apoptosis in wild-type control and DFF45 deficient thymocytes and compared the cleavage of chromosomal DNA to 50 kilobase pair size fragments. We found that there is a lack of obvious large chromosomal DNA fragments upon treatments by various apoptotic agents in DFF45 deficient thymocytes. The major organ systems in the DFF45 mutant mice either two months or fifteen months of age appear normal. These results suggest that functional DFF45 is required for cleavage of DNA into both large size and oligonucleosomal size fragments in thymocytes during apoptosis. However, deficiency in DFF45 apparently does not significantly affect normal mouse development and tissue homeostasis.     

Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis

Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.     

Interaction of DNA fragmentation factor (DFF) with DNA reveals an unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.     

Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis.

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.     

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.     

The DFF40/CAD endonuclease and its role in apoptosis

The sequential generation of large-scale DNA fragments followed by internucleosomal chromatin fragmentation is a biochemical hallmark of apoptosis. One of the nucleases primarily responsible for genomic DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD). DFF40/CAD is a magnesium-dependent endonuclease specific for double stranded DNA that generates double strand breaks with 3'-hydroxyl ends. DFF40/CAD is activated by caspase-3 that cuts the nuclease's inhibitor DFF45/ICAD. The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. However, the nuclease hypersensitive sites can be detected and DFF40/CAD is potentially involved in large-scale DNA fragmentation as well. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis.     

Involvement of CD45 in DNA fragmentation in apoptosis induced by mitochondrial perturbing agents

CD45 is a type I transmembrane molecule with phosphatase activity which comprises up to 10% of the cell surface area in nucleated haematopoietic cells. We have previously demonstrated the absence of nuclear apoptosis in CD45-negative T cells after chemical-induced apoptosis. The aim of this study was to characterize the role of CD45 in nuclear apoptosis. In contrast to wild type CD45-positive T cells, the CD45-deficient T cell lines are resistant to the induction of DNA fragmentation and chromatin condensation following tributyltin (TBT) or H₂O₂ exposure, but not to cycloheximide-induced apoptosis. CD45 transfection in deficient cell lines led to the restoration of chromatin condensation and DNA fragmentation following TBT exposure. In both CD45-positive and negative T cell lines, TBT exposure mediates intracellular calcium mobilization, caspase-3 activation and DFF45 cleavage. Moreover, DNA fragmentation was also induced by TBT in cells deficient in expression of p56lck, ZAP-70 and SHP-1. Subcellular partitioning showed a decrease in nuclear localisation of caspase-3 and DFF40. Together, these results demonstrate for the first time, that CD45 expression plays a key role in internucleosomal DNA fragmentation and chromatin condensation processes during apoptosis. CD45 activity or its substrates’ activity, appears to be located downstream of caspase-3 activation and plays a role in retention of DFF40 in the nucleus. Philippe Desharnais and Geneviève Dupéré-Minier have contributed equally to this work.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

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.     

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.     

Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.     

BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells

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 Ca²⁺ 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.     

Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis

Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase-activated DFF40/ CAD nuclease, which is chaperoned and inhibited by DFF45/ICAD. CIDE proteins share a homologous regulatory CIDE-N domain with DFF40/CAD and DFF45/ ICAD. Here we report the solution structure of CIDE-N of human CIDE-B. We show that the CIDE-N of CIDE-B interacts with CIDE-N domains of both DFF40 and DFF45. The binding epitopes are similar and map to a highly charged bipolar surface region of CIDE-B. Furthermore, we demonstrate that the CIDE-N of CIDE-B regulates enzymatic activity of the DFF40/ DFF45 complex in vitro. Based on these results and mutagenesis data, we propose a model for the CIDE-N/ CIDE-N complex and discuss the role of this novel bipolar interaction in mediating downstream events of apoptosis.     

Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis.

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.     

Activation of caspase-3, proteolytic cleavage of DFF and no oligonucleosomal DNA fragmentation in apoptotic Molt-4 cells

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.