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Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.

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.

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.

An early transient burst of poly(ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines (Simbulan-Rosenthal, C., Rosenthal, D., Iyer, S., Boulares, H., and Smulson, M. (1998) J. Biol. Chem. 273, 13703-13712) followed by cleavage of poly(ADP-ribose) polymerase (PARP), catalyzed by caspase-3. This inactivation of PARP has been proposed to prevent depletion of NAD (a PARP substrate) and ATP, which are thought to be required for later events in apoptosis. The role of PARP cleavage in apoptosis has now been investigated in human osteosarcoma cells and PARP -/- fibroblasts stably transfected with a vector encoding a caspase-3-resistant PARP mutant. Expression of this mutant PARP increased the rate of staurosporine and tumor necrosis factor-alpha-induced apoptosis, at least in part by reducing the time interval required for the onset of caspase-3 activation and internucleosomal DNA fragmentation, as well as the generation of 50-kilobase pair DNA breaks, thought to be associated with early chromatin unfolding. Overexpression of wild-type PARP in osteosarcoma cells also accelerated the apoptotic process, although not to the same extent as that apparent in cells expressing the mutant PARP. These effects of the mutant and wild-type enzymes might be due to the early and transient poly(ADP-ribose) synthesis in response to DNA breaks, and the accompanying depletion of NAD apparent in the transfected cells. The accelerated NAD depletion did not seem to interfere with the later stages of apoptosis. These results indicate that PARP activation and subsequent cleavage have active and complex roles in apoptosis.

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.