Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.     

Involvement of both caspase-like proteases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin

We investigated the involvement of caspases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in U937 cells. We found that caspase-3- and caspase-6-like activities, but not caspase-1-like activity, increased during toxin-induced apoptosis. Z-D-CH2-DCB, a caspase-like inhibitor, completely inhibited the generation of caspase-3- and caspase-6-like activities and blocked all features of apoptosis induced by toxins: nuclear morphological changes, DNA fragmentation, and cytotoxicity. However, three caspase-specific inhibitors, Ac-YVAD-CHO, Ac-DEVD-CHO, and Ac-VEID-CHO, had no effect, even though Ac-DEVD-CHO and Ac-VEID-CHO inhibited the increased caspase-3- and caspase-6-like activity, respectively. These results suggest that the generation of caspase-3- and caspase-6-like activities is redundant, and other caspases distinct from caspase-3 and -6 may be important in toxin-induced apoptosis. Furthermore, serine protease inhibitor, 3,4-dichloroisocoumarine (DCI), abolished the apoptotic cell death and DNA fragmentation caused by toxins, without affecting the increased caspase-3- and caspase-6-like activities. Our results suggest that multiple proteases with different preferences for apoptotic substrates participate in toxin-induced apoptotic death of U937 cells.     

A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9 cells

We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.     

DNA fragmentation into 200-250 and/or 30-50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone

Internucleosomal cleavage of DNA has often been regarded as the biochemical hallmark of apoptosis. We now demonstrate in isolated rat liver nuclei that DNA is initially cleaved into > or = 700, 200-250 kbp and 30-50 kbp fragments via a multi-step process, which is activated by Mg2+ and Mg2+(+)Ca2+ but not by Ca2+ alone. The subsequent internucleosomal cleavage requires both cations. These findings demonstrate that a key event in the apoptotic process is the fragmentation of DNA into large kbp fragments by either a Mg(2+)-dependent process (which can be potentiated by Ca2+) and/or by a Ca2+/Mg2+ activated endonuclease(s).     

Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes

Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymocytes in response to dexamethasone and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca2+,Mg(2+)-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosomal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.     

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.     

Ca2+/Mg(2+)-dependent endonuclease in marrow CD34 positive and erythroid cells in myelodysplasia

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34+ and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg(2+)-dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca2+/Mg(2+)-dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34+ and erythroid cells. Our results suggested that Ca2+/Mg(2+)-dependent endonuclease is related to ineffective erythropoiesis in MDS.     

Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).     

Detection of Mg(2+)-dependent endonuclease activity in myeloid leukemia cell nuclei capable of producing internucleosomal DNA cleavage

We detected Mg(2+)-dependent, Ca(2+)-independent endonuclease activity in non-apoptotic myeloid leukemia cell nuclei using autodigestion method which cleaved the chromatin of the autologous leukemia cells to an oligonucleosomal length pattern. Similar endonuclease activity could be successfully recovered in the protein extracts of the human leukemia cell nuclei. The extracts consistently elicited characteristic DNA cleavage of another leukemia cell (KG-1) nuclei as the target, the enzyme activity of which had been inactivated. We propose that this method is a useful tool for the study of endonucleases involved in apoptosis.     

Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis

Although the commonly activated death protease caspase-3 appears not to be essential for apoptosis during development except in the brain, it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence. We have addressed this question with MCF-7 breast carcinoma cells that we recently showed lack caspase-3 owing to the functional deletion of the CASP-3 gene. Tumor necrosis factor- or staurosporine-induced apoptosis of caspase-3-deficient MCF-7 cells resulted in cleavage of the death substrates PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45, but not alpha-fodrin. In contrast, all these substrates including alpha-fodrin were cleaved in apoptotic HeLa cells expressing caspase-3. Introduction of CASP-3 cDNA, but not CASP-10 cDNA, into MCF-7 cells restored alpha-fodrin cleavage. In addition, tumor necrosis factor- or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in alpha-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VAD-fmk and Z-DEVD-fmk prevented apoptosis of MCF-7 cells, we were unable to detect activation of caspases 2 and 7, which are known to be inhibited by Z-DEVD-fmk. Together our results suggest that caspase-3 is essential for cleavage of alpha-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspases other than caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells.     

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.     

Hypoxia induces apoptosis in human neuroblastoma SK-N-MC cells by caspase activation accompanying cytochrome c release from mitochondria

We have attempted to elucidate the mechanism of apoptotic cell death induced by hypoxia (very low oxygen conditions) in neuronal cells. Human neuroblastoma SK-N-MC cells under hypoxic conditions resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent chromatin dye. Pretreatment with Z-Asp-CH2-DCB, a caspase inhibitor, suppressed the DNA ladder in response to hypoxia in a concentration-dependent manner. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm the involvement of caspase-3 during apoptosis, Western blot analysis was performed using anti-caspase-3 antibody. The 20- and 17-kDa proteins, corresponding to the active products of caspase-3, were generated in hypoxia-challenged lysates in which processing of the full length form of caspase-3 was evident. With a time course similar to this caspase-3 activation, hypoxic stress caused the cleavage of PARP, yielding an 85-kDa fragment typical of caspase activity. In addition, caspase-2 was also activated by hypoxia, and the stress elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that caspase activation and cytochrome c release play roles in hypoxia-induced neuronal apoptosis.     

Anatomical basis of a congenital hearing impairment: basilar papilla dysplasia in the Belgian Waterslager canary

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.     

Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD+ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD+. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purified caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.     

Long-term trends in tuberculosis. Comparison of age-cohort data between Hong Kong and England and Wales

Apoptosis is cellular suicide functionally opposite of mitosis. It plays an important role in tissue growth control and removal of damaged and premalignant cells. The decrease in death suppressor Bcl-2 protein level was implicated in the many types of apoptotic cell death. Because Bcl-2 protein was recently found to be cleaved during apoptosis induced by Fas ligation, IL-3 withdrawal, and alphavirus infection, we assessed whether Bcl-2 protein was also cleaved during the anticancer drug (VP-16)-induced apoptotic cell death in U937 cells. We found that Bcl-2 protein was cleaved in vivo and in vitro after the treatment of VP-16. We also found that caspase-3/CPP32, which was activated after VP-16 treatment, was responsible for the direct cleavage of Bcl-2 protein. The overexpression of the cleaved Bcl-2 fragment increased the sensitivity to VP-16 and promoted apoptotic cell death. Therefore, caspase-3/CPP32 accelerates VP-16-induced U937 cell apoptosis by cleaving death suppressor Bcl-2 protein to produce a death promoter Bcl-2 fragment.     

Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.     

Protective effects of the antiparkinsonian drugs talipexole and pramipexole against 1-methyl-4-phenylpyridinium-induced apoptotic death in human neuroblastoma SH-SY5Y cells

Treatment of human neuroblastoma SH-SY5Y cells with 1 mM 1-methyl-4-phenylpyridinium (MPP+) for 3 days induced production of reactive oxygen species (ROS), followed by caspase-3 activation, cleavage of poly(ADP-ribose) polymerase (PARP), and apoptotic cell death with DNA fragmentation and characteristic morphological changes (condensed chromatin and fragmented nuclei). Simultaneous treatment with 1 mM talipexole slightly inhibited the MPP+-induced ROS production and apoptotic cell death. In contrast, pretreatment with 1 mM talipexole for 4 days markedly protected the cells against MPP+-induced apoptosis. However, this protective effect might not be mediated by dopamine receptors. The talipexole pretreatment induced an increase in antiapoptotic Bcl-2 protein level but had no effect on levels of proapoptotic Bax, Bak, and Bad. It also inhibited MPP+-induced ROS production, p53 expression, and cleavages of caspase-3 and PARP. Similarly, pramipexole pretreatment increased Bcl-2 and inhibited MPP+-induced apoptosis. Although pretreatment with bromocriptine also had a protective effect against MPP+-induced apoptosis, it had no effect on the protein levels of Bcl-2 family members. On the other hand, N6,2'-O-dibutyryl cAMP or calphostin C induced a decreased Bcl-2 level and enhanced MPP+-induced cell death. These results suggest that talipexole has dual actions: (1) it directly scavenges ROS, affording slight protection against MPP+-induced apoptosis, and (2) it induces Bcl-2 expression, thereby affording more potent protection, if it is administrated before MPP+. Pramipexole has similar effects, whereas bromocriptine seems to exhibit the former but not the latter effect.     

Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provided cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 was found to abolish UV-induced SAPK and p38 MAPK activity, and inhibit UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 was also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibited UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, did not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity.     

Virus- and interferon-induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.     

Coronary endothelial dysfunction after Kawasaki disease: evaluation by intracoronary injection of acetylcholine

1. Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli et al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS). 2. Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 microM), or S-nitrosoglutathione (GSNO, 300 microM), however, potentiated the DNA strand scission induced by 200 microM tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators. 3. SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or ATP. 4. The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells. 5. It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III.