Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.     

IDO induction in IFN-γ activated astroglia: A role in improving cell viability during oxidative stress

Abstract

In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-γ increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-γ in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-γ significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.     

IDO induction in IFN-gamma activated astroglia: a role in improving cell viability during oxidative stress

In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-gamma increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-gamma in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-gamma significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.     

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.     

Combined effect of 3-aminobenzamide and N-acetylcysteine on HIV replication in chronically infected U937 cells

Summary

The existence of a close relationship between apoptosis associated with oxidative stress and the increase of viral progeny in chronically HIV-infected cells has been previously reported. The possibility of modulating both phenomena by using an antioxidant such as N-acetylcysteine (NAC) has also been demonstrated. The present investigation was designed to study the role of the nuclear enzyme poly-(ADP-ribose)-polymerase (PARP) when HIV- infected cells are treated with tumour necrosis factor alpha (TNFα), a cytokine capable of inducing both apoptosis and intracellular oxygen free radical production. PARP overexpression may result in a rapid drop of intracellular NAD⁺ and ATP concentration, thus contributing to cellular redox imbalance. We have used the specific PARP inhibitor 3- aminobenzamide (3-ABA), alone or in a combination with NAC. 3-ABA was only partially capable of inhibiting viral replication and apoptosis induced by TNFα. In contrast, the combination of NAC and 3-ABA led to an inhibition of apoptosis as well as to a marked decrease in viral particle production, with a parallel replenishment of intracellular reduced glutathione content. The results reported here confirm the potential role of antioxidant drug treatment in specific phases of HIV infection.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Poly(ADP-ribose) Polymerase Activation and Cell Injury in the Course of Rat Heart Heterotopic Transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 &#117 min). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 &#117 min of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Poly(ADP-ribose) polymerase activation and cell injury in the course of rat heart heterotopic transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 λmin). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 λmin of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells.

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.     

Beneficial Effects of Poly (ADP-ribose) Polymerase Inhibition Against the Reperfusion Injury in Heart Transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Beneficial effects of poly (ADP-ribose) polymerase inhibition against the reperfusion injury in heart transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.     

Modulation of cell and DNA damage by poly(ADP)ribose polymerase in lung cells exposed to H(2)O(2) or asbestos fibres

Poly(ADP)ribose polymerase (PARP) may participate in cell survival, apoptosis and development of DNA damage. We investigated the role of PARP in transformed human pleural mesothelial (MeT-5A) and alveolar epithelial (A549) cells exposed from 0.05 to 5mM hydrogen peroxide (H(2)O(2)) or crocidolite asbestos fibres (1-10 microg/cm(2)) in the presence and absence of 3-aminobenzamide (ABA), a PARP inhibitor. The cells were investigated for the development of cell injury, DNA single strand breaks and depletion of the cellular high-energy nucleotides. Compared to H(2)O(2), fibres caused a minor decrease in cell viability and effect on the cellular high-energy nucleotide depletion, and a marginal effect on the development of DNA strand breaks when assessed by the single cell gel electrophoresis (the Comet assay). Inhibition of PARP transiently protected the cells against acute H(2)O(2) related irreversible cell injury when assessed by microculture tetrazolium dye (XTT) assay and potentiated oxidant related DNA damage when assessed by the Comet assay. However, PARP inhibition had no significant effect on fibre-induced cell or DNA toxicity with the exception of one fibre concentration (2 microg/cm(2)) in MeT-5A cells. Apoptosis is often associated with PARP cleavage and caspase activation. Fibres did not cause PARP cleavage or activation of caspase 3 further confirming previous results about relatively low apoptotic potential of asbestos fibres. In conclusion, maintenance of cellular high-energy nucleotide pool and high viability of asbestos exposed cells may contribute to the survival and malignant conversion of lung cells exposed to the fibres.     

The NAD+ precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate

The bile salt, sodium deoxycholate (NaDOC), is a natural detergent that promotes digestion of fats. At high physiologic levels, NaDOC activates many stress-response pathways and induces apoptosis in various cell types. NaDOC induces DNA damage and activates poly(ADP-ribose) polymerase (PARP), an enzyme that utilizes NAD+ as a substrate to repair DNA. NaDOC also induces oxidative stress, endoplasmic reticulum (ER) stress and contributes to protein malfolding. The NAD+ precursors, nicotinic acid (NA) and nicotinamide (NAM) were found to protect cells against NaDOC-induced apoptosis. NA and NAM also decreased constitutive levels of both activated NF-kappaB and GRP78, two proteins that respond to oxidative stress. However, the mechanism by which NA and NAM protects cells against apoptosis does not involve a reduction in constitutive levels of oxidative stress. NA or NAM treatment increased the protein levels of glyceraldehyde-3-phosphate dehydrogense (GAPDH), a multi-functional enzyme, in the nucleus and cytoplasm, respectively. NAM did not activate the promoter/response elements of 13 stress response genes nor reduce intracellular non-protein thiols, suggesting that it is non-toxic to cells. NAM thus has promise as a dietary supplement to help prevent disorders involving excessive apoptosis.     

Proteasome activation by poly-ADP-ribose-polymerase in human myelomonocytic cells after oxidative stress

Cytotoxic action of a variety of antitumor drugs generate oxidatively modified proteins that are predominantly metabolized via the proteasome. In the present study, a differentiation-retrodifferentiation cell system was exposed to oxidative stress by hydrogen peroxide treatment. Thus, the activity of the nuclear proteasome in proliferating human U937 leukemic cells increased by 2.5-fold after hydrogen peroxide treatment. In contrast, growth-arrested differentiated U937 cells demonstrated 40% less constitutive proteasomal activity, which was not inducible after hydrogen peroxide exposure. After a retrodifferentiation process, however, in which differentiated U937 cells resume autonomous growth again, the proteasomal activity was indistinguishable from that in U937 control cells, both constitutively and after induction of oxidative stress. Moreover, cells of TUR, a differentiation-resistant U937 subclone, expressed an elevated constitutive proteasomal activity that increased by 2.5-fold after oxidative stress. Immunoblot analysis revealed that these differences in proteasomal activities did not correlate with proteasome protein expression but with protein levels of the nuclear enzyme poly-ADP-ribose-polymerase (PARP). Further studies using specific PARP inhibitors revealed that the noninducible proteasome activity in differentiated U937 cells was PARP independent, whereas the increased activity level in oxidatively stressed TUR cells was downregulated upon PARP inhibition. Immunoprecipitation experiments demonstrated a protein-protein interaction of the functional active PARP with the proteasome in correlation with the proteasome activity. Similar results were obtained by analyzing protein carbonyls after oxidative stress. Taken together, these data suggest that proliferating, rather than growth-arrested, cells metabolize oxidatively damaged nuclear proteins via the proteasome by expressing high levels of PARP.     

Hydrogen peroxide-induced DNA repair and death of resting human blood lymphocytes

DNA single-strand breaks induced by cell treatment with hydrogen peroxide are repaired and simultaneously trigger programmed cell death in resting human blood lymphocytes. Apoptosis is accompanied by special morphological changes in lymphocytes (15% of total cell number), internucleosomal DNA degradation, and p53 level elevation. According to morphological criteria, a major part (up to 40% of total cell number) displayed necrotic death features. Nicotinamide inhibited repair in cells with 2.5-fold elevation of the apoptotic cell proportion, whereas the fraction of cells with necrotic nuclear morphology decreased 4.5-fold. Both the inhibition of repair and the protective effect of nicotinamide against necrotic death indicate that the repair process and related poly(ADP-ribose)polymerase (PARP) activation induce a decrease in intracellular NAD+ and ATP contents below the threshold at which necrosis becomes the preferential mechanism of cell death. The mixed pattern of cell death induced by hydrogen peroxide observed in resting lymphocytes can be explained in the context of a concept of cell de-energization as a consequence of effective single-stand break repair during the first hours after removing the genotoxic agent.     

The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells

Treatment with 0.2 mM hydrogen peroxide (H(2)O(2)) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H(2)O(2), or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H(2)O(2) caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosis-inducing treatments and treatment with 2 mM H(2)O(2) caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H(2)O(2) did not. Bcl-2 overexpression reduced the generation of necrosis by H(2)O(2), but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.