Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclophosphamide (CYC) is a metabolically activated, DNA-alkylating, antitumor agent that causes pulmonary fibrosis. BALB/cN (B) mice are sensitive and C57Bl/6N (C) mice are resistant to CYC-induced fibrosis. Pulmonary bioactivation may contribute to strain sensitivity. Therefore, we tested the intrinsic susceptibility of murine lung slices to cell injury by direct exposure to CYC for 2-8 hr. Injury was measured by release of lactate dehydrogenase (LDH). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP, EC 2.4.2.30), causing depletion of its substrate, NAD. NAD can also be decreased by phosphorylation to NADP, as seen with oxidative stress. Depletion of NAD can lead to loss of ATP. Thus, we measured LDH release, PAP activation, NAD, NADP and ATP in slices incubated with or without the PAP-inhibitor, 3-aminobenzamide (3-AB). CYC (0.1 to 1.0 mg/mL for 4-8 hr) caused LDH release in slices from both murine strains, but LDH release was significantly greater in B lung slices than in C slices. After an 8-hr incubation 63.9 +/- 3.7% (mean +/- SEM) of total LDH was released from B lung slices with 1.0 mg CYC/mL, whereas only 45.8 +/- 2.6% was released from C lung slices (P < 0.05). 3-AB reduced LDH release to 44.7 +/- 2.4% in B slices and 28.1 +/- 2.0% in C slices (P < 0.05 vs CYC only). PAP activity in nuclei isolated from CYC-treated B lung slices was increased 2- to 4-fold after 2 hr of incubation with 0.5 and 1.0 mg CYC/mL. PAP activation was delayed and reduced with incubation in 3-AB. PAP was activated 2-fold in nuclei from C slices treated with 0.5 mg CYC/mL for 2 hr. NAD was decreased at 2 and 4 hr in B slices treated with 0.5 and 1.0 mg CYC/mL, and at 4 hr with 0.1 mg CYC/mL. NAD depletion occurred only at 4 hr in the resistant C slices treated with 1.0 mg CYC/mL. CYC increased NADP by a similar extent in B and C lung slices. In B slices, NAD losses were approximately 4 times the increases in NADP. CYC did not decrease ATP in B slices and ATP dropped 25% only after 4 hr in the resistant C slices. We conclude that CYC is directly toxic to lung tissue and observe that strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo. PAP activation and oxidative stress may contribute to this toxicity.
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PMID:Acute pneumocyte injury, poly(ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide. 798 Jun 45

We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.
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PMID:Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells. 916 25

Apoptotic changes occurred specifically in a macrophage-like cell line, J774.1, treated with lipopolysaccharide (LPS) and cycloheximide (CHX) prior to the release of lactate dehydrogenase (LDH). The addition of 100 ng/ml LPS and 10 microg/ml CHX induced both the formation of DNA nicks and elevation of caspase-3-like activity (DEVDase) after 75 min, and then the cleavage of poly(ADP-ribose) polymerase (PARP) into 28-kDa fragments, formation of apoptotic bodies, and DNA ladder formation. These apoptotic changes were reversible until 60 min, however, later than 75 min after LPS and CHX addition, the apoptosis proceeded normally even on extensive washing of the macrophages, which removed the LPS and CHX. These results suggest that there is a "point of no return" in the apoptotic processes in macrophages induced by LPS and CHX and that DNA nicks and activation of DEVDase are critical for these processes.
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PMID:Apoptotic changes preceding necrosis in lipopolysaccharide-treated macrophages in the presence of cycloheximide. 963 79

Oxidant-induced cell injury has been implicated in the pathogenesis of several forms of acute renal failure. The present studies examined whether activation of poly(ADP-ribose)polymerase (PARP) by oxidant-induced DNA damage contributes to oxidant injury of renal epithelial cells. H2O2 exposure resulted in an increase in PARP activity and decreases in cell ATP and NAD content. These changes were significantly inhibited by 10 mM 3-aminobenzamide (3-ABA), a PARP inhibitor. In contrast, H2O2-induced DNA damage was not prevented by 3-ABA. Exposure of LLC-PK(1) cells to 1 mM H2O2 for 2 h induced necrotic cell death as measured by increased lactate dehydrogenase (LDH) release. 3-ABA completely prevented the H2O2-induced LDH release. Live/dead fluorescent staining confirmed the protection by 3-ABA. These results are consistent with the view that oxidant-induced DNA damage activates PARP and that the subsequent ATP and NAD depletion contribute to necrotic cell death. Of note, although protected from necrosis, cells treated with H2O2 and 3-ABA underwent apoptosis as evidenced by DNA fragmentation and bis-benzimide staining. In conclusion, activation of PARP contributes to oxidant-induced ATP depletion and necrosis in LLC-PK1 cells. However, PARP inhibition may target cells toward an apoptotic form of cell death.
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PMID:Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK1 cells. 1048 26

Amyloid beta protein (Abeta) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Abeta-treatment of cultured neurons. Treatment of rat primary cortical culture with Abeta 25-35, an active fragment of Abeta, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Abeta 25-35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Abeta-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and alpha-fodrin, were produced by Abeta-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Abeta-induced DNA fragmentation and cleavage of alpha-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH(2)-DCB, additionally prevented Abeta-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Abeta-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Abeta-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Abeta-induced neuronal death still occurred with different morphological features.
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PMID:Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons. 1052 27

The exact causes of the extensive cell death in nigral transplants are still unknown. Since poly-(ADP-ribose) polymerase (PARP) overactivation has been implicated in neuronal death, we examined the effects of PARP on the survival of nigral grafts by using donor tissue from PARP knock-out or wild-type mice. Eight hours after preparation of the nigral cell suspension, cell damage was quantified by measurement of lactate dehydrogenase release, DNA fragmentation and caspase activation. At this stage, PARP deletion had no protective effect. Moreover, neither the survival of transplanted dopaminergic neurons, nor the functional recovery of hemiparkinsonian graft recipients were improved by the absence of PARP. We conclude that cell death in embryonic nigral grafts is not affected by the absence of PARP activation.
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PMID:Neuronal death in nigral grafts in the absence of poly (ADP-ribose) polymerase activation. 1059 43

The protective effect of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) against ischemia-reperfusion-induced injury was studied in the Langendorff heart perfusion system. To understand the molecular mechanism of the cardioprotection, the effect of BGP-15 on ischemic-reperfusion-induced reactive oxygen species (ROS) formation, lipid peroxidation single-strand DNA break formation, NAD(+) catabolism, and endogenous ADP-ribosylation reactions were investigated. These studies showed that BGP-15 significantly decreased leakage of lactate dehydrogenase, creatine kinase, and aspartate aminotransferase in reperfused hearts, and reduced the rate of NAD(+) catabolism. In addition, BGP-15 dramatically decreased the ischemia-reperfusion-induced self-ADP-ribosylation of nuclear poly(ADP-ribose) polymerase(PARP) and the mono-ADP-ribosylation of an endoplasmic reticulum chaperone GRP78. These data raise the possibility that BGP-15 may have a direct inhibitory effect on PARP. This hypothesis was tested on isolated enzyme, and kinetic analysis showed a mixed-type (noncompetitive) inhibition with a K(i) = 57 +/- 6 microM. Furthermore, BGP-15 decreased levels of ROS, lipid peroxidation, and single-strand DNA breaks in reperfused hearts. These data suggest that PARP may be an important molecular target of BGP-15 and that BGP-15 decreases ROS levels and cell injury during ischemia-reperfusion in the heart by inhibiting PARP activity.
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PMID:BGP-15, a nicotinic amidoxime derivate protecting heart from ischemia reperfusion injury through modulation of poly(ADP-ribose) polymerase. 1069 58

The addition of lipopolysaccharide (LPS) together with cycloheximide (CHX) induced apoptosis in a subline of a J774.1 macrophage-like cell line, JA-4, as judged by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-staining and poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP)-cleavage. Caspase activities were examined in these macrophages in vitro using fluorogenic substrates such as acetyl-DEVD-aminomethyl coumarine (Ac-DEVD-AMC, caspase-3-like), acetyl-YVAD-aminomethyl coumarine (Ac-YVAD-AMC, caspase-1-like), acetyl-VEID-aminomethyl coumarine (Ac-VEID-AMC, caspase-6-like), and carbobenzoxy-IETD-aminofluoro coumarine (Z-IETD-AFC; caspase-8-like). Kinetic studies revealed these caspase activities with different Km and Vmax values in extracts of apoptotic macrophages. In the course of apoptosis, caspase-3-like activity increased first at 75 min, simultaneously with the appearance of TUNEL staining and prior to PARP cleavage, and then caspase-6 and 8-like activities increased at 90 and 105 min, respectively. However, caspase-1-like activity did not change throughout the experiment. Furthermore, removal of LPS and CHX by extensive washing of the cells for 60 min completely abolished the apoptosis and the subsequent release of lactate dehydrogenase (LDH) during additional incubation until 4 h after LPS addition. However, washing of the cells after 75 min or later resulted in the progress of apoptosis and LDH release, which was coordinated with the elevation of caspase-3-like activity at 60 min and that of caspase-6 or 8-like activity at 90 min, but not with that of caspase-1-like activity. These results suggest that caspase-3-like activity represents the most apical caspase among these caspases in terms of the intiation of apoptosis in macrophages treated with LPS and CHX. In the present study, we also provide evidence on the relatively low specificities of a series of caspase inhibitors other than acetyl-DEVD-aldehyde (Ac-DEVD-CHO) which specifically inhibited the caspase-3-like activity.
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PMID:Changes of caspase activities involved in apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysaccharide (LPS) and cycloheximide. 1070 74

Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated protein important in DNA repair, maintenance of chromosomal stability and programmed cell death. Here we report that an increase in caspase 3-activity and cleavage of PARP serves as an early execution phase signal in human neuroblastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinase inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 microM) for 30 min showed an increase in caspase 3-activity and by 1 h an increase in PARP 116-kDa band and an 85-kDa cleavage product, which further increased in density with time after treatment. Quantitative analysis for condensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosporine treatment. This was further confirmed with a Klenow fragment of DNA polymerase I assay which primarily detects single-stranded DNA breaks. A significant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (LDH) release revealed a significant increase in membrane permeability 8 h after staurosporine (0.1 microM) or etoposide (10 microM) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrity was observed 12 h after staurosporine treatment. Our results show that activation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis or cytotoxicity.
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PMID:Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma. 1076 13

The effects of an oxidative insult on cell survival and tau metabolism were investigated in human neuroblastoma SH-SY5Y cells. In this treatment paradigm cells were exposed to the membrane permeant oxidant tert-butylhydroperoxide (tBHP) for 40 min, returned to fresh media and cell survival/death was monitored during the post-treatment period. Cell viability decreased significantly by 6 hr after tBHP exposure, and by 24 hr lactate dehydrogenase (LDH) release was 40.1 +/- 8.8% in tBHP treated cells compared to 8.1 +/- 4.7% in control cells. This oxidative stress paradigm also resulted in significant activation of caspase-3 by 2 hr post-treatment and nuclear apoptotic morphology. Furthermore, tBHP treatment also resulted in delayed tau proteolysis that was first evident 2 hr post-treatment. Treatment of the cells with the general caspase inhibitor Boc-Asp(OMe)-Fluoromethylketone (BAF) completely inhibited caspase-3 activation in response to tBHP, and delayed, but did not prevent cell death. BAF treatment also decreased tau proteolysis. In vitro, recombinant tau was readily proteolyzed by active recombinant caspase-3 into a stable breakdown product. Further tau in the cell lysates was cleaved by active recombinant caspase-3 at a rate, and to an extent similar to that observed for the well-established caspase-3 substrate poly(ADP-ribose)polymerase (PARP). These results suggest that oxidative stress-induced cell death occurs through both caspase-dependent and-independent pathways, and that tau is likely an in situ substrate of caspase-3.
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PMID:Transient oxidative stress in SH-SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis. 1095 21


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