Activation Of DNase Research Articles

Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks

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.

Interactions between Viral and Prokaryotic Pathogens in a Mixed Infection with Cardiovirus and Mycoplasma

In the natural environment, animal and plant viruses often share ecological niches with microorganisms, but the interactions between these pathogens, although potentially having important implications, are poorly investigated. The present report demonstrates, in a model system, profound mutual effects of mycoplasma and cardioviruses in animal cell cultures. In contrast to mycoplasma-free cells, cultures contaminated with Mycoplasma hyorhinis responded to infection with encephalomyocarditis virus (EMCV), a picornavirus, but not with poliovirus (also a picornavirus), with a strong activation of a DNase(s), as evidenced by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) immunofluorescence assay and electrophoretic analysis of host DNA. This degradation was reminiscent of that observed upon apoptosis but was caspase independent, judging by the failure of the specific pan-caspase inhibitor Q-VD-OPh to prevent it. The electrophoretic mobility of the enzyme responsible for DNA degradation and dependence of its activity on ionic conditions strongly suggested that it was represented by a DNase(s) of mycoplasma origin. In cells not infected with EMCV, the relevant DNase was dormant. The possibility is discussed that activation of the mycoplasma DNase might be linked to a relatively early increase in permeability of plasma membrane of the infected cells caused by EMCV. This type of unanticipated virus-mycoplasma "cooperation" may exemplify the complexity of pathogen-host interactions under conditions when viruses and microorganisms are infecting the same host. In the course of the present study, it was also demonstrated that pan-caspase inhibitor zVAD(OMe).fmk strongly suppressed cardiovirus polyprotein processing, illustrating an additional pitfall in investigations of viral effects on the apoptotic system of host cells.

Activation of DNA-degrading enzymes during apoptosis.

Cell death by apoptosis requires a precise plan of destruction of DNA and proteins. In this paper, we review the current knowledge on the different DNA-degrading enzymes which are activated in apoptotic cells. The activation of DNases by upstream proteases is also discussed.

Gelsolin Regulates Cardiac Remodeling After Myocardial Infarction Through DNase I–Mediated Apoptosis

Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN(-/-)) and wild-type littermates (GSN(+/+)) to left anterior descending coronary artery ligation or sham surgery. We found that GSN(-/-) mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN(+/+) mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN(-/-) mice. Absence of gelsolin markedly reduced DNase I-induced apoptosis. The association of hypoxia-inducible factor (HIF)-1alpha with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1alpha was similar to that of gelsolin, and HIF-1alpha was detected in the gelsolin complex by coprecipitation and HIF-1alpha bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN(-/-) mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1alpha and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.

Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells?

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 μM CdSO4. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.

Mechanism of DNA Fragmentation During Hypoxia in the Cerebral Cortex of Newborn Piglets

We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets.

Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani ( Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.

Merbarone Induces Activation of Caspase-Activated DNase and Excision of Chromosomal DNA Loops from the Nuclear Matrix

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.

Melatonin reduces the severity of anesthesia-induced apoptotic neurodegeneration in the developing rat brain

General anesthetics cause widespread apoptotic neurodegeneration in many regions of the developing rat brain. The activation of mitochondria-dependent apoptotic pathway is important in the early stages of anesthesia-induced developmental neuroapoptosis. To investigate potential means of protecting against this type of damage, we studied melatonin, a sleep-promoting agent and antioxidant known to inhibit apoptotic-type neuronal damage by improving mitochondrial homeostasis and stabilizing the inner mitochondrial membrane. When 7-day-old rats (the peak of synaptogenesis) were exposed to a commonly used and highly pro-apoptotic anesthesia cocktail (midazolam, isoflurane, nitrous oxide) in combination with the escalating doses of melatonin (from 1 to 20 mg/kg, s.c.), the severity of anesthesia-induced damage was reduced in a dose-dependent manner in two most vulnerable brain regions--the cerebral cortex and anterior thalamus. Melatonin-induced neuroprotection was mediated, at least in part, via the inhibition of mitochondria-dependent apoptotic pathway since melatonin caused an up-regulation of the anti-apoptotic protein, bcl-X(L), reduction in anesthesia-induced cytochrome c release into the cytoplasm and a decrease in anesthesia-induced activation of caspase-3, an important step in the activation of DNAses and the formation of the apoptotic bodies.

The N‐terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin‐actin complex

Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin.

Structure and Function of Poly(ADP-ribose) Polymerase-1: Role in Oxidative Stress-Related Pathologies

Poly(ADP-ribosyl) ation is a reversible post-translational protein modification implicated in the regulation of a number of biological functions. Whereas an 18 member superfamily of poly(ADP-ribose) polymerase (PARP) enzymes synthesize poly(ADP-ribose) (PAR), a single protein, PAR glycohydrolase (PARG) is responsible for the catabolism of the polymer. PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells. PARP-1 activated by DNA breaks cleaves NAD(+) into nicotinamide and ADP-ribose and uses the latter to synthesize long branching PAR polymers covalently attached to acceptor proteins including histones, DNA repair enzymes, transcription factors and PARP-1. Whereas activation of PARP-1 by mild genotoxic stimuli may facilitate DNA repair and cell survival, irreparable DNA damage triggers apoptotic or necrotic cell death. In apoptosis, early PARP activation may assist the apoptotic cascade [e.g. by stabilizing p53, by mediating the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus or by inhibiting early activation of DNases]. In most severe oxidative stress situations, excessive DNA damage causes over activation of PARP-1, which incapacitates the apoptotic machinery and switches the mode of cell death from apoptosis to necrosis. Besides serving as a cytotoxic mediator, PARP-1 is also involved in transcriptional regulation, most notably in the NF kappaB and AP-1 driven expression of inflammatory mediators. Pharmacological inhibition or genetic ablation of PARP-1 provided remarkable protection from tissue injury in various oxidative stress-related disease models ranging from stroke, diabetes, diabetic endothelial dysfunction, myocardial ischemia-reperfusion, shock, Parkinson's disease, arthritis, colitis to dermatitis and uveitis. These beneficial effects are attributed to inhibition of the PARP-1 mediated suicidal pathway and to reduced expression of inflammatory cytokines and other mediators (e.g. inducible nitric oxide synthase).

Mitochondrial ceramide and the induction of apoptosis.

In most cell types, a key event in apoptosis is the release of proapoptotic intermembrane space proteins from mitochondria to the cytoplasm. In general, it is the release of these intermembrane space proteins that is responsible for the activation of caspases and DNases that are responsible for the execution of apoptosis. The mechanism for the increased permeability of the mitochondrial outer membrane during the induction phase of apoptosis is currently unknown and highly debated. This review will focus on one such proposed mechanism, namely, the formation of ceramide channels in the mitochondrial outer membrane. Ceramides are known to play a major regulatory role in apoptosis by inducing the release of proapoptotic proteins from the mitochondria. As mitochondria are known to contain the enzymes responsible for the synthesis and hydrolysis of ceramide, there exists a mechanism for regulating the level of ceramide in mitochondria. In addition, mitochondrial ceramide levels have been shown to be elevated prior to the induction phase of apoptosis. Ceramide has been shown to form large protein permeable channels in planar phospholipid and mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway with which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.

Lnterferon‐β gene therapy for cancer: Basic research to clinical application

Interferon-beta gene therapy for cancer is the first such protocol developed in Japan. Here we describe the development process of our interferon-beta gene therapy from basic research to clinical application. Interestingly, the biological and biochemical characteristics of interferon-beta gene therapy through transfer of the interferon-beta gene into tumor cells by means of cationic liposomes differed from those of conventional interferon-beta protein therapy. Interferon-beta gene transfer could induce apoptosis in interferon-beta protein-resistant tumor cells, such as glioma, melanoma, and renal cell carcinoma. Induction of apoptosis was related to the level of intracellular mRNA of interferon-beta, prolongation of the phosphorylation time of molecules in the interferon-beta signal transduction pathway, such as JAK1, Trk2, and STAT1, and activation of DNase gamma. In our preclinical study we developed lyophilized cationic liposomes containing interferon-beta gene (gene drug) for clinical use and confirmed their safety. Thereafter, we performed a pilot clinical trial in patients with malignant glioma and confirmed the safety and effectiveness of this interferon-beta gene therapy. In this review we also comment on the status of gene therapy regulation in Japan. Interferon-beta gene therapy is expected to become widely available for clinical use in cancer patients, and this new strategy might be extended to molecular targeting therapy, or used in combination with cell therapy or other therapies.

Sodium nitroprusside and peroxynitrite effect on hepatic DNases: an in vitro and in vivo study

BackgroundIt has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg2+- and caspase-dependent) and acid-DNase. The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite.ResultsThe effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after in vivo treatment with either SNP or peroxynitrite. A difference observed between the in vivo and in vitro effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased.ConclusionAlkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis.

Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis.

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.

DNA damage in the early primordial anther is closely correlated with stamen arrest in the female flower of cucumber (Cucumis sativus L.).

To investigate the regulatory mechanisms of sex expression in cucumber, morphological observations and biochemical analyses were carried out on inappropriate stamen development of female flowers of cucumber. It was found that developmental arrest of the inappropriate stamen mainly occurs at the anther primordium. This arrest is closely correlated with DNA damage, as detected by TUNEL assay, and might result from anther-specific DNase activation. It was also found that the DNA damage does not lead to cell degeneration, although chromatin condensation is observed in the anther primordia.

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles

Magnetic bead cellulose particles and magnetic poly(HEMA–co–EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA–co–EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg 2+, Ca 2+, Mn 2+ and Co 2+) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co 2+ ions.

Characterization of Two DNase γ-Specific Monoclonal Antibodies and the in Situ Detection of DNase γ in the Nuclei of Apoptotic Rat Thymocytes

Two novel monoclonal antibodies (mAbs), hg302 and hg303, raised against a synthetic peptide corresponding to the basic domain of human DNase γ, are characterized in detail. In Western blot analysis, hg303 recognizes both wild type and C-terminal Myc-His-tagged human DNase γ, but does not cross-react with human DNase I family members, DNase I, DNase X, or DNAS1L2. On the other hand, dot blot analysis reveals the fine specificity of hg302; it recognizes human, mouse, and rat DNase γ, but not other DNase I family DNases under non-denaturing conditions. Furthermore, hg302 efficiently immunoprecipitates wild type, but not C-terminal Myc-His-tagged, human DNase γ from cell lysates. In immunohistochemical analysis, hg302 strongly recognizes DNase γ in the nuclei of X-ray irradiation-induced apoptotic, but not normal rat thymocytes. The specific detection of DNase γ in apoptotic nuclei is confirmed by indirect-immunofluorescence analysis using TNF-α-induced apoptotic HeLa S3 cells transfected with DNase γ. These results, together with the observations that DNase γ is present in normal thymocytes and its activity is unchanged during the apoptosis, suggest that some molecular change(s), which triggers the activation of DNase γ, occurs in response to apoptotic stimuli in the basic domain, and hg302 specifically recognizes the activated DNase γ in immunohistochemical analysis.

Determinants of the nuclear localization of the heterodimeric DNA fragmentation factor (ICAD/CAD).

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.

Molecular Mechanisms of Apoptosis Induced by an Immunomodulating Peptide on Human Monocytes

A short immunomodulating peptide (Pa) containing a defined structural motif present in a number of extracellular matrix proteins and autoantigens was found to stimulate human monocytes. Pa-induced apoptosis of isolated monocytes, as indicated by internucleosomal DNA cleavage, increased annexin V binding capacity and cleavage of caspase substrates, such as poly(ADP)ribosylpolymerase. In addition, Bcl-2 protein levels were downregulated during Pa-induced cell death. Nuclear extracts of monocytes incubated with Pa showed higher neutral, Ca2+-dependent DNase activity than those obtained from nontreated monocytes. Caspase inhibitors prevented Pa-induced apoptosis, Bcl-2 depletion, and DNase activation. Treatment of monocytes with Pa activated c-Jun N-terminal kinases and p38 kinase, in an acidic sphingomyelinase- and caspase-dependent fashion. Pa-induced apoptosis was blocked by selective inhibitors of p38 kinase (SB203580) and acidic sphingomyelinase (SR33557). These results indicate that JNK and p38 kinase stimulation as well as monocyte apoptosis induced by Pa could depend, at least in part, on early activation of acidic sphingomyelinase.