Nitric oxide (NO) is a diffusible messenger involved in several patho-physiological processes including immune-mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO-synthase or by NO donors stimulates the expression of the tumor suppressor gene, ~53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO-synthase inhibitors such as No-monomethyl+arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since ~53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO-induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.Key words: Nitric oxide; ~53; Apoptosis; DNA fragmentation IntraductiollNitric oxide (NO) is a messenger molecule involved in several processes including relaxation of smooth muscle, neurotransmission, and tumor cell as well as bacteria killing [l-3]. However, induction of a high output system for NO in response to cytokines or a massive production of NO following accumulation of the excitatory neurotransmitter glutamate [4,5] can result in cell killing. Neurons [6], pancreatic B-cells [7] or macrophages [8,9] seem to be particularly sensitive to NO toxicity. While in some systems NO can react with other radicals and effectively cause cell death by necrosis, in others the progressive intra-or extracellular generation of NO has been suggested to cause apoptosis [8,10,11]. Mechanisms proposed for NO toxicity include its interaction with protein thiol groups [3,12] or iron-sulfur proteins [13], or by direct DNA damage [14]. The latter, regardless of whether it is induced by radiation or by drugs such as etoposide, can result in apoptosis [ 15,161. Expression of wild-type ~53, a tumor suppressor gene, seems to be closely linked to apoptosis caused by most of the DNA-damaging agents [15,16]. The wild-type nuclear phosphoprotein ~53, originally characterized as a tumor suppressor protein [17], acts as a checkpoint control in the cell cycle, permitting the repair of damaged DNA. The block in GJS transition which results from ~53 activation has been suggested to cause apoptosis in the case of severe DNA damage [18,19]. More recently, it has become apparent that the ~53 gene product can take part directly in the apoptotic process [20]. Materials and methods MaterialsThe mouse macrophage-like cell line RAW 264.7 was provided by Prof. A. Wendel, Faculty of Biology, University of Konstanz, Germany. LPS (Escherichia coli serotype 0127:B8), NMMA, protein A-Sepharose, and SNP were purchased from Sigma, Deisenhofen, *Corresponding author. Fa...

Nitric oxide (NO) generation initiates apoptotic cell death in different experimental systems. In RAW 264.7 macrophages the appearance of typical apoptotic markers is linked to inducible NO synthase induction. Mechanistically, accumulation of tumour suppressor p53 precedes apoptotic DNA fragmentation. With the use of S-nitroglutathione (GSNO) we correlated a dose-dependent p53 up-regulation to DNA fragmentation measured after 4 h and 8 h, respectively. Our studies revealed a linear correlation between the potency of five different NO donors with respect to apoptosis induction and p53 accumulation. Furthermore, we probed for NO-induced apoptosis after stable transfection of RAW 264.7 macrophages with plasmids encoding p53 antisense RNA. Clones with down-regulated p53 levels in response to GSNO exhibited a marked reduction in DNA fragmentation. Expression of the inducible NO synthase in response to lipopolysaccharide and interferon-gamma caused apoptosis in RAW 264.7 macrophages and neomycin-vector controls within 24 h. In contrast, p53 antisense RNA-expressing clones appeared highly resistant towards endogenous NO, although inducible NO synthase induction with concomitant nitrite production remained unchanged. For RAW 264.7 macrophages our results established a functional role of the tumour suppressor p53 during NO-induced apoptotic cell death. However, p53 antisense experiments and the use of the p53-negative cell line U937 substantiated p53-independent signalling pathways operative during NO-mediated apoptosis.

Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis.

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 g/ml) and interferon-␥ (100 units/ml), respond with endogenous NO ⅐ formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO ⅐ -derived apoptosis was blocked by the NO ⅐ -synthase inhibitor N Gmonomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-␥, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO ⅐ , released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO ⅐ -mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.Nitric oxide (NO) 1 is recognized for its participation in diverse biological processes in nearly all aspects of life (1-3). The formation of NO ⅐ occurs under both physiological and pathophysiological settings. The molecule is synthesized by a family of enzymes termed NO ⅐ synthases (NOS), which utilize arginine as their substrate in the generation of NO ⅐ and stoichiometric amounts of citrulline (4). For convenience, two types of NOS are recognized; constitutive isoforms, which are active for a relatively short time in response to intracellular Ca 2ϩ fluctuations, and a cytokine-inducible isoform. For the latter to be active, mRNA translation and protein synthesis are required. The inducible NOS generates large amounts of NO ⅐ for an extended period. However, once NO ⅐ is produced by the action of NOS, it is extremely susceptible to both oxidation and reduction. This results in the concomitant formation of species with NO ϩ -like activity (nitrosonium ion) or NO Ϫ (nitroxyl anion), respectively (5). In addition to reacting with oxygen, superoxide, and transition metals, NO ⅐ causes biological signaling via interactions with sulfhydryl groups. Because multiple NO surrogates are formed, transduction pathways are classified as either cyclic GMP-dependent or -independent. Cyclic GMP formation is initiated by NO ⅐ binding to the heme group of soluble guanylyl cyclase, thus causing enzyme activati...

1 Exposure of human mammary carcinoma cell line MCF-7 to TNF-a leads to apoptotic cell death within 24 h. In search for apoptosis-preventing signals, we identi®ed glucocorticoids as potent deathpreventing compounds. Ten nM dexamethasone provided a signi®cant protective e ect whereas 100 nM dexamethasone roughly blocked 80 ± 90% of TNF-a-induced apoptosis.2 Surprisingly, dexamethasone exerted a protective e ect even when supplied several hours after TNF-a. This points to a powerful inhibition of even advanced apoptotic processes by dexamethasone. 3 To further pinpoint the anti-apoptotic glucocorticoid action, we investigated the expression levels of several members of the inhibitors of apoptosis (IAPs) family of proteins in response to TNF-a and dexamethasone. IAP proteins directly block caspase protease activities including caspase-3, caspase-7, and caspase-9. Exposure of MCF-7 cells to TNF caused an extensive downregulation of cIAP1, cIAP2, and XIAP protein levels. The decline of the IAP protein levels temporally paralleled the appearance of apoptotic DNA fragments which started 12 ± 14 h following TNF-a addition and maximal e ects were seen within 24 h. 4 Coincubation of cells with TNF-a and dexamethasone potently blocked cIAP1, cIAP2, and XIAP downregulation. 5 TNF-a-mediated IAP protein downregulation was not a ected by proteasome inhibitors like lactacystin, ALLN or ALLM, whereas it was blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk which also prevented TNF-a-induced apoptotic cell death. These data suggest that inhibition of IAP downregulation mediated by a caspase proteolytic activity constitutes the antiapoptotic action of glucocorticoids in MCF-7 carcinoma cells.

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Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor‐α (TNF‐α), TNF‐α/basic fibroblast growth factor (bFGF), TNF‐α/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations. In search for apoptosis‐preventing signals, we identified glucocorticoids as potent death preventing factors. Co‐treatment of cells with 10 nM dexamethasone and TNF‐α, TNF‐α/bFGF, TNF‐α/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. Similarly to dexamethasone (TNF‐α‐ and LPS‐induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF‐α and 0.7, 8, 100 and 500 nM for LPS, respectively. Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF‐α/LPS‐induced apoptosis (IC50 ∼500 nM for TNF‐α and ∼500 nM for LPS), whereas sex hormones, i.e. β‐estradiol and testosterone remained without effect. The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU‐486. Concerning TNF‐α and LPS signal transduction, we found that dexamethasone efficiently prevented TNF‐α‐ and LPS‐induced activation of caspase‐3‐like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways. British Journal of Pharmacology (1999) 127, 1633–1640; doi:

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