Journal of Cell Science 110, 2315-2322 (1997) 2315 Printed in Great Britain © The Company of Biologists Limited 1997 JCS4287

Differential induction of apoptosis in Swiss 3T3 cells by nitric and the nitrosonium cation

Shazia Khan1,2, Midori Kayahara1,2, Umesh Joashi2, Nicholas D. Mazarakis2, Catherine Sarraf3, A. David Edwards2, Martin N. Hughes1 and Huseyin Mehmet2,* 1Department of Chemistry, King’s College London, Strand, London WC2R 2LS, UK 2Weston Laboratory, Department of Paediatrics and Neonatal Medicine, and 3Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK *Author for correspondence (e-mail: [email protected])

SUMMARY

We have investigated the effect of (NO) on decomposition to NO. The apoptotic effect of NO was apoptosis in Swiss 3T3 fibroblasts and compared it to the reduced in the presence of the NO scavenger oxyhaemo- effect of the nitrosonium cation (NO+). Both species globin, or the antioxidants N-acetylcysteine and ascorbic induced apoptosis, confirmed by electron microscopy, acid, whereas in the case of NO+ these antioxidants poten- propidium iodide staining, DNA laddering and activation tiated apoptosis. Glutathione also had a potentiating effect of caspases. The kinetics of triggering apoptosis were on the cytotoxicity of NO+. This suggests that cellular different for the two redox species: NO+ required only a 2 antioxidants may play a role in protecting the cell from NO- hour exposure, whereas NO required 24 hours. Three induced apoptosis while NO+ may trigger apoptosis inde- sources of NO were used: aqueous solutions of NO and two pendently of oxidative stress mechanisms. NO donors, S-nitrosoglutathione and S--N- acetylpenicillamine. The time course of apoptosis induced by these two S-nitrosothiols correlated with their rate of Key words: Apoptosis, Nitric oxide, Mitochondrion

INTRODUCTION NO to be readily oxidised or reduced to species with chemi- cally distinct properties and potentially different biological Nitric oxide ( monoxide, NO)1 is a key molecule in effects. For example, in the nervous system, NO can be neu- intracellular signal transduction. Its ability to activate rotoxic, in part through its reaction with the superoxide ion − − guanylate cyclase is well recognised but NO may also regulate (O2 ) to form peroxynitrite (ONO2 ). This is a highly reactive other signalling pathways; for example, NO can modify molecule that can induce lipid peroxidation or inactivate iron- function by nitrosating thiol groups in cellular sulphur clusters (Radi et al., 1991; Hausladen and Fridovich, (Ignarro, 1992; Stamler, 1994) and can itself act as a 1994). In contrast, the nitrosonium cation (NO+) can act as a neurotransmitter in both the central and peripheral nervous protective signal (Lipton et al., 1993), suggesting that the redox systems (Snyder, 1992). Since NO can also act as a cytotoxic state of NO influences NO-mediated effects. agent in the immune and nervous systems (Hibbs et al., 1987; NO+ is produced by the one-electron oxidation of NO. Dawson et al., 1991) this property, together with its role in Although it is an effective nitrosating agent at low pH, at phys- intracellular signalling, is consistent with NO inducing cell iological pH NO+ is a transient species with an estimated half- death by apoptosis. life of 10−10 seconds, being immediately hydrolysed to . Apoptotic cell death is an active physiological process However, carriers of the NO+ group may exist within the cell; distinct from necrosis and is characterised by defined morpho- these include nitrosylhaems and S-nitrosothiols (Wade and logical and biochemical features including membrane Castro, 1990; Barnett et al., 1995). blebbing, cytoplasmic shrinkage, nuclear pyknosis and DNA The aim of this study was to investigate the toxicity of NO fragmentation (Kerr et al., 1972; Martin et al., 1994). However, and NO+ on Swiss 3T3 fibroblasts. These cells are well char- the signalling pathways that lead to apoptosis remain poorly acterised at the intracellular signalling level and can be understood. NO might induce apoptosis through its interactions reversibly arrested in the G0 phase of the cell cycle, thus with membrane or cytosolic targets, or act as an intracellular removing the heterogeneity of response that may exist in an messenger in the apoptotic cascade. However, recent reports asynchronous population (Rozengurt, 1986). Aqueous have shown that, while NO can induce apoptosis in activated solutions of NO, or the NO-donors S-nitrosoglutathione macrophages, it prevents apoptosis in other cell lines (Cui et (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were al., 1993; Mannick et al., 1994; Farinelli et al., 1996). used. The effect of NO+ was investigated using the nitroprus- 2− These conflicting data could be explained by the ability of side anion ([Fe(CN)5(NO)] ). This molecule contains a co- 2316 S. Khan and others ordinated NO+ ligand and acts as a donor of NO+ at physio- All experiments with SNP were performed in the dark to prevent logical pH (Bates et al., 1991). NO or NO+-induced apoptosis photolysis of the nitroprusside anion to produce NO. Cycloheximide was also investigated in the presence of the NO-scavenger oxy- (CHX, 5 µg/ml), a protein synthesis inhibitor that induces apoptosis haemoglobin, or the antioxidants N-acetylcysteine, glutathione (Searle et al., 1975), was used as a positive control. and ascorbic acid. Time-course experiments with nitric oxide donors A freshly prepared solution of GSNO in 10% CM (1 mM) was added to the cell monolayers for defined incubation times from 0 to 24 hours. MATERIALS AND METHODS For SNAP experiments, a freshly prepared solution (1 mM) in 10% CM was added to quiescent cell monolayers at 24, 48 and 72 hour Materials intervals. Unless stated, all chemicals were purchased from Sigma (Poole, Dorset, UK) and used without further purification. Nitric oxide and trapping experiments Preparation of aqueous solutions of nitric oxide The NO scavenger oxyhaemoglobin and the antioxidants ascorbic NO was generated by reacting concentrated H2SO4 with a saturated acid or N-acetylcysteine were each diluted in 10% CM (to the con- solution of and purified by passing the resulting gas centrations specified in the figure legends) and preincubated with through 10 M NaOH and distilled H2O. Saturated solutions of NO either GSNO for 24 hours, or SNP for 2 hours. In studies using sodium were prepared by bubbling purified NO through deoxygenated nitroprusside (SNP), the antioxidant glutathione was also tested. Dulbecco’s modified Eagle’s medium (DMEM, ICN Biomedicals, These solutions were added to quiescent 3T3 cultures and incubated Thame, Oxon, UK) for 30 minutes at room temperature (20¡C). The (37¡C) for 2 or 24 hours. The 2 hour cultures were washed with pH of the medium was maintained between pH 7.2 and 7.4 by the serum-free DMEM and incubated for a further 22 hours with 10% CM addition of hydrochloric acid. The concentration of the saturated NO (0.5 ml/well). In a separate series of experiments, defined antioxidants solution at 20¡C is known to be 1.7 mM and NO solutions were were added after the NO species. For NO, cultures were exposed to diluted in conditioned medium (CM, spent medium removed from GSNO for 8 hours and then washed and the medium replaced with quiescent monolayers) and DMEM to give a final solution of NO in 10% CM alone or containing 0.3 mM ascorbic acid. For NO+, ascorbic 10% CM at the concentrations specified in the figure legends. acid (0.3 mM) was added to Swiss 3T3 cells at defined time points before or after SNP addition (1 mM); exposure to SNP was for a total Synthesis of S-nitrosoglutathione and S-nitroso-N- of 2 hours. For each NO species, apoptosis was measured 24 hours acetylpenicillamine after the start of the experiment as described in the text. GSNO was prepared by the reaction of acidified sodium nitrite solution with glutathione. The resulting solution was treated with Fluorescence microscopy acetone to precipitate the GSNO, which was washed then dried in At the end of each experiment, cell cultures were fixed by the addition vacuo (Hart, 1985). SNAP was synthesised by the reaction of acidified of one volume of methanol:glacial acetic acid (3:1 v/v) for 3 minutes sodium nitrite solution with N-acetylpenicillamine. The resulting at room temperature. This was aspirated and replaced with the same green solid was washed and then dried in vacuo (Moynihan and fixative (0.5 ml/well) for a further 3 minutes. Cultures were then Roberts, 1994). washed twice with phosphate-buffered saline (PBS) and incubated with propidium iodide (PI, 60 µg/ml) in the presence of DNAse-free Preparation of oxyhaemoglobin RNAse (300 µg/ml) for 30 minutes at 37¡C. PI-labelled nuclei were Oxyhaemoglobin was prepared according to the method of Ignarro et visualised with the aid of a Fluovert FU fluorescence microscope al. (1987). A solution of bovine crystalline haemoglobin in Krebs buffer (Leitz, Wetzlar, Germany) using a ×25 objective. Apoptosis was was reduced with sodium dithionite, with excess sodium dithionite scored by counting the proportion of small, pyknotic, highly fluor- being removed from the reaction by passage through a Sephadex G25 escent nuclei to normal, diffusely stained ones. A minimum of 200 column. The resulting solution was then exposed to air for 30 minutes. nuclei per field were counted, with at least six fields for each experi- The concentration of oxyhaemoglobin was determined by spectrome- mental point. try as described previously (Feelisch and Noack, 1987) and diluted in 10% CM to the concentrations specified in the figure legends. Electron microscopy Following treatment with NO or NO+ donors for 2 (NO+) or 16 (NO) Cell culture hours Swiss 3T3 cells (grown on coverslips on 24-well plates) were Swiss 3T3 fibroblasts were seeded at a density of 5×104 cells/dish fixed in 2% glutaraldehyde for 2 hours at 4¡C, washed, osmicated and onto 90 mm tissue culture dishes (Nunc, Roskilde, Denmark) in 10 dehydrated before embedding in Taab¨ resin. Coverslips were ml DMEM supplemented with the following (from Life Technologies, snapped off with liquid nitrogen and 1 µm sections were cut and Paisley, UK): 10% foetal calf serum (FCS), penicillin (100 units/ml) stained with toluidine blue for block selection at the light microscope and streptomycin (100 µg/ml). Cells were cultured in a humidified level. Sections of 100 nm thickness were then cut and collected on 10% CO2 atmosphere at 37¡C. After 3 to 4 days, subconfluent cells nickel grids, stained with uranyl acetate and lead citrate and examined were subcultured onto 24-well plates (Greiner, Dursley, Glos, UK) at by electron microscopy (CM-10, Phillips, Netherlands). a density of 1.75×103 cells/well in 0.5 ml DMEM/10% FCS. Cultures were grown to confluence and used when quiescent, after 7 to 8 days. DNA laddering Following treatment with NO or NO+ donors for 16 hours, Swiss 3T3 Experimental protocol cells (grown on 90 mm dishes) were lysed in situ and the DNA pre- Quiescent cultures of Swiss 3T3 cells were washed twice with serum- cipitated as described by Laird et al. (1991). Briefly, cells were washed free DMEM (37¡C). Defined concentrations of each compound were in 100 mM Tris/HCl pH 8.5 and lysed in 0.5 ml of lysis buffer (100 prepared in 10% CM (except aqueous NO). The compounds dissolved mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS and 200 mM NaCl) in 10% CM were then added to the cell monolayers and incubated for containing proteinase K (100 µg/ml) and incubated for 16 hours at 2 or 24 hours at 37¡C. The 2 hour cultures were washed with serum- 55¡C. DNA was precipitated by adding one volume of isopropanol free DMEM (37¡C) and incubated for a further 22 hours with 10% followed by mixing for 15 minutes at room temperature. After cen- CM (0.5 ml/well). trifugation (5 minutes at 13000 g) the DNA pellet was air dried, resus- Induction of apoptosis by NO and NO+ 2317 pended in TE buffer and its concentration determined spectrophoto- studded cytosol, while mitochondria were distinctly elongated metrically. DNA (5 µg per lane) was loaded onto a 1% agarose gel in and sinuous. Apoptotic cells were largely absent from cultures 100 mM Tris-borate and the fragments resolved by electrophoresis maintained in 10% CM. Following treatment with either NO (70 V for 5 hours) and visualised by ethidium bromide staining. or NO+, cells dying by apoptosis were clearly visible. The sequence of apoptotic events was notable, cytoplasmic changes Poly (ADP-ribose) polymerase (PARP) cleavage clearly preceding nuclear transitions. In early apoptotic cells To investigate PARP cleavage in Swiss 3T3 cells, cultures treated with the cytoplasm fragmented into numerous, small apoptotic NO or NO+ donors for 1 hour at 37¡C were washed twice with PBS and lysed in situ with 1% SDS. The samples were then heated at 90¡C bodies (Fig. 1A), while nuclei remained electron lucent. for 5 minutes. Protein content was determined by the BCA method Following a 24 hour treatment with NO, cells lost their using a commercial kit (Pierce, Cheshire, UK) and lysates stored at extended phenotype in keeping with cytoplasmic contraction. −80¡C prior to use. In contrast to NO treatment, mitochondria were clearly Total cellular proteins (50 µg/lane) were separated on a 7.5% poly- modified following a 2 hour exposure to NO+ (Fig. 1B). These acrylamide gel and electrotransferred onto nitrocellulose membrane mitochondria appeared metabolically active and, furthermore, (Hybond ECL, Amersham, UK). Primary incubations were with measurements of succinate dehydrogenase activity indicated mouse monoclonal anti-PARP antibody C-2-10 (Transduction Lab- that NO+ treatment resulted in mitochondrial hypermeta- oratories, Kentucky, USA) diluted 1:3,000 for 1 hour at room tem- bolism at 2 hours, but caused death by 8 hours (data not perature. Secondary incubations were with HRP-linked anti-mouse shown), suggesting that these organelles were a primary target antibody (Amersham) at 1:1,000 dilution under the same conditions. + Peroxidase activity was visualised with an enhanced chemilumin- for NO . At later time points, typical apoptotic nuclei with escence substrate reagent (Supersignal HRP, Pierce) and serial condensed chromatin and nuclear blebbing were only observed exposures made to autoradiographic film (Hyperfilm ECL, in apoptotic bodies whose cytoplasm was in an advanced stage Amersham). of degradation, with disruption of the plasma membrane in- dicating the onset of necrosis secondary to primary apoptotic Statistical analysis events (Fig. 1C). Data distributions were inspected for normality and homoscedacity, and appropriate parametric or non-parametric tests applied. Intergroup Fluorescence microscopy comparisons were made by ANOVA or Kruskall-Wallis ANOVA on Swiss 3T3 fibroblasts incubated with 10% CM for 24 hours ranks, with multiple comparison testing by Student-Newman-Keul’s were viable and the nuclei appeared normal, showing charac- or Dunn’s Tests. teristic diffuse granular staining with PI (Fig. 2A). Cells treated with cycloheximide appeared apoptotic after a 24 hour exposure. Under these conditions, PI-stained nuclei were RESULTS highly fluorescent and appeared pyknotic (Fig. 2B). These features are characteristic of cells undergoing apoptosis and Electron microscopy this nuclear morphology was confirmed by electron Viewed with the electron microscope, healthy quiescent 3T3 microscopy (data not shown). Cells treated with either cells were spindle shaped with a high nucleus-to-cytoplasmic aqueous NO (Fig. 2C) or NO+ (Fig. 2D) for 24 hours ratio; nuclei were elongated and euchromatic. Cytoplasmic exhibited similar nuclear changes and apoptotic cells were organelles, such as the endoplasmic reticulum and Golgi morphologically indistinguishable from cultures treated with apparatus, were widely separated from each other by ribosome- cycloheximide.

A B C

a

Fig. 1. Effect of NO and NO+ on subcellular morphology of Swiss 3T3 cells analysed by electron microscopy. Electron micrographs of Swiss 3T3 fibroblasts undergoing apoptosis following treatment with NO or NO+. (A) A Swiss 3T3 fibroblast (a) at an early stage of apoptosis following a 24 hour treatment with 1 mM GSNO. There is extensive cytoplasmic fragmentation (arrows) while the nucleus is healthy, without any chromatin condensation. ×4,500. (B) Mitochondrial changes in Swiss 3T3 fibroblasts following a 2 hour treatment with 1 mM SNP. The cytoplasm is organelle rich, but the most notable features are the mitochondria, which are uncommonly long, sinuous and electron dense. ×10,500. (C) Apoptotic Swiss 3T3 cell fragments following a 24 hour treatment with 1 mM GSNO. Here, chromatin condensation (arrow) and nuclear blebbing are visible, but by this stage, cytoplasm (C) is severely degraded, plasma membranes are disrupted and the cytoplasm is undergoing secondary necrosis. ×7,000. 2318 S. Khan and others

Fig. 2. Effect of NO and NO+ treatment on nuclear morphology of Swiss 3T3 fibroblasts examined by propidium iodide staining. Cultures were incubated with 10% CM alone (A), or in the presence of 5 µg/ml cycloheximide (B), or 1 mM NO in solution (C) or 1 mM SNP (D) for 24 hours. Nuclei were visualised following PI staining. Healthy (arrows) and apoptotic (arrowheads) nuclei could be easily distinguished in mixed populations (C). ×175.

Molecular evidence for apoptosis: DNA laddering GSNO at 1 mM or, as a positive control, with cycloheximide and PARP cleavage at 5 µg/ml (Fig. 3A). One of the hallmarks of apoptosis is the endonuclease- Activation of apoptotic proteases (caspases) is fundamental mediated degradation of chromatin, giving rise to characteris- in execution of the apoptotic programme (Lazebnik et al., tic DNA laddering (reviewed by Wyllie et al., 1992). To inves- 1994). Caspase activation was investigated by measuring tigate the effect of the NO or NO+ donors on DNA cleavage of PARP, a known substrate for caspase-3. Quiescent fragmentation, Swiss 3T3 fibroblasts were incubated with 10% Swiss 3T3 cells contained the intact 116 K PARP protein, CM alone, or in the presence of GSNO or SNP, and the DNA detected by western blotting. Following treatment with GSNO extracted and separated by agarose gel electrophoresis. Cells (NO) or SNP (NO+) or the protein kinase inhibitor stau- treated with 10% CM alone contained only high molecular rosporine for 1 hour, PARP was cleaved to yield two fragments weight DNA, while cells treated with 1 mM NO+ contained of 85 K and 50 K. This effect was dose-dependent for both NO low molecular weight DNA species that migrated as a ladder, and NO+ (Fig. 3B). Although GSNO was more effective at with fragments differing by approximately 200 bp (Fig. 3A). lower concentrations, SNP resulted in the highest level of Similar results were obtained when cells were treated with cleaved PARP: densitometric analysis revealed that treatment

A B

kDa

Fig. 3. Effect of NO and NO+ treatment on DNA laddering and PARP cleavage in Swiss 3T3 fibroblasts. (A) DNA laddering. Quiescent Swiss 3T3 fibroblasts were treated with 10% CM either alone (lane 1), or in the presence of 1 mM SNP (lane 2), 1 mM GSNO (lane 3) or 5 µg/ml cycloheximide (lane 4). Total cellular DNA was resolved by agarose gel electrophoresis and visualised with ethidium bromide. M denotes the lane containing defined molecular mass markers as indicated in kb. (B) PARP cleavage following treatment with NO or NO+. Quiescent Swiss 3T3 fibroblasts were treated with 10% CM either alone (C), or with increasing concentrations of NO species as indicated, or in the presence of 100 nM staurosporine (STS). PARP cleavage was determined by immunoblotting with a monoclonal antibody (C-2-10), which recognises an epitope present in both intact PARP (116 kDa) and the caspase-cleaved 85 kDa and 50 kDa fragments. Induction of apoptosis by NO and NO+ 2319

A B

100 100

80 80 NO Fig. 4. Effect of NO, GSNO, SNAP and SNP on GSNO 60 viability of Swiss 3T3 fibroblasts. (A) Cells were 60 SNP 2 h treated with increasing concentrations of aqueous SNAP 24 h 40 solutions of NO, GSNO, SNAP and SNP for 24 40 % apoptosis hours. (B) Cells were treated with aqueous solutions % apoptosis 20 of NO (1 mM), GSNO (1 mM) and SNP (1 mM) for 20 2 or 24 hours. The proportions of apoptotic nuclei were determined by PI staining after a total 0 0 incubation time of 24 hours. The data are presented 0.01 0.1 1 10 10% CM NO GSNO SNP as the mean and s.e.m. (n=6). concentration (mM) 10 % CM NO (1 mM) SNOG (1 mM) SNP (1 mM) with SNP or GSNO, resulted in a maximum of 64% and 45% (Fig. 4A). 50% apoptosis was observed at an SNP concentra- of PARP cleavage, respectively. This compared to 37% PARP tion of 0.02 mM, with the maximal effect (100% cell death) cleavage in staurosporine-treated cultures, although this observed at 1 mM. These results indicated that NO+ was a more treatment also caused an overall increase in intact PARP levels. potent inducer of apoptosis than NO. Similar results were obtained when cells were treated with 5 µg/ml cycloheximide as a positive control for apoptosis (not Time-dependent effect of NO+, aqueous NO, GSNO shown). and SNAP The kinetics of cell death induced by NO and NO+ were inves- Dose-dependent effects of aqueous NO, GSNO and tigated. NO+ induced apoptosis in Swiss 3T3 fibroblasts after SNAP after 24 hours only a 2 hour exposure (Fig. 4B, P<0.05). In contrast, both NO induced apoptosis in a dose-dependent manner after 24 aqueous NO and GSNO failed to induce apoptosis after 2 hours hours (Fig. 4A). The concentration of aqueous NO required but did so after a 24 hour exposure (Fig. 4B, P<0.05). for 50% apoptosis was 0.8 mM, although it could not be There is uncertainty over the stability of NO in solution over tested at concentrations higher than 1 mM (a saturated long time periods and so, since it was impractical to prepare solution of NO has a concentration of 1.7 mM). Conse- fresh solutions of NO at each time point, GSNO and SNAP quently, the maximum level of apoptosis observed with were used to investigate the time course of NO-induced aqueous NO was 60%. Similarly, GSNO induced apoptosis apoptosis in more detail. When quiescent Swiss 3T3 cells were in Swiss 3T3 cells in a dose-dependent manner after 24 hours incubated with GSNO (1 mM) for defined time periods, (Fig. 4A). The concentration of GSNO giving 50% apoptotic apoptotic cells could be observed by 12 hours, reaching a cell death was 0.7 mM and the maximum effect (80% maximum (80% apoptosis) by 24 hours (Fig. 5A). In contrast, apoptotic cell death) was observed at 1 mM. In contrast, SNAP had only a moderate effect up to 48 hours (27% of cells SNAP, the other S-nitrosothiol employed in this study as a were apoptotic) but induced apoptosis in 76% of the cells NO-donor, did not induce apoptosis in Swiss 3T3 cells after following a 72 hour exposure (Fig. 5B). a 24 hour exposure period (Fig. 4A). Effect of NO scavengers and antioxidants on NO Dose-dependent effect of NO+ after 24 hours and NO+-induced apoptosis Like NO, NO+ induced apoptosis in Swiss 3T3 fibroblasts in To investigate the mechanism of NO- and NO+-induced a dose-dependent manner following a 24 hour treatment period apoptosis, the effects of known NO scavengers and antioxi-

A B 80 80 10% CM 10% CM

GSNO SNAP 60 60

40 40 Fig. 5. Kinetics of apoptosis induced by GSNO and SNAP. Quiescent cells were treated with 10% CM % apoptosis either alone or in the presence of 1 mM GSNO (A), % apoptosis 20 20 or 1 mM SNAP (B) for the times indicated and the proportions of apoptotic nuclei determined by PI staining at each time point. The data are presented as 0 0 the mean and s.e.m. at each time point (n=3 (GSNO) 0 8 16 24 0 24 48 72 or n=6 (SNAP)). time (h) time (h) 2320 S. Khan and others

A B C 100 alone 60 alone 100 alone GSH OxyHb GSH 50 NAC NAC 80 NAC 80 Asc Asc Asc 40 60 60 30 40 40 20 20 20 10

0 0 0 10 % CM GSNO 10 % CM SNP 0.01 0.1 1 SNP concentration (mM)

Fig. 6. Effect of scavengers and antioxidants on apoptosis induced by NO or NO+. (A) Quiescent Swiss 3T3 fibroblasts were treated with GSNO (0.6 mM) alone or in the presence of oxyhaemoglobin (0.1 mM), N-acetylcysteine (0.3 mM) or ascorbic acid (0.3 mM) for 24 hours. The proportions of apoptotic nuclei were determined by PI staining. The data are presented as the mean and s.e.m. (n=6). (B) Quiescent Swiss 3T3 fibroblasts were treated with SNP (1 mM) alone or in the presence of N-acetylcysteine (1 mM), glutathione (1 mM) or ascorbic acid (1 mM) for 2 hours. Cultures were then washed and incubated in 10% CM alone for a further 22 hours and the proportion of apoptotic nuclei determined by PI staining. The data are presented as the mean and s.e.m. (n=6). (C) Cells were treated with 10% CM containing increasing concentrations of SNP, either alone or in the presence of glutathione (1 mM), N-acetylcysteine (1 mM) or ascorbic acid (1 mM) for 2 hours. Cultures were then washed and incubated in 10% CM alone for a further 22 hours and the proportion of apoptotic nuclei determined by PI staining. The data are presented as the mean and s.e.m. (n=5). dants were tested. Cells were treated in the absence or presence apoptosis (Fig. 6B, P<0.05 and Fig. 6C). However, at concen- of oxyhaemoglobin, N-acetylcysteine or ascorbic acid, together trations higher than 1 mM, ascorbic acid also induced with concentrations of NO or NO+ that resulted in approxi- apoptosis when added alone (data not shown). This may be due mately 50% of the cells undergoing apoptosis when added in part to the pro-oxidant properties of this molecule (Peterkof- alone. sky and Prather, 1977). The results indicated that all three compounds were effective To investigate this additive effect further, a subtoxic con- inhibitors of NO-induced apoptosis. Oxyhaemoglobin concen- centration of ascorbic acid (0.3 mM) was added to Swiss 3T3 trations as low as 0.1 mM significantly reduced the level of cells at defined time points before or after SNP (1 mM). The apoptosis induced by GSNO (Fig. 6A). In contrast, at 10-fold results showed that ascorbic acid potentiated the toxic effect of higher concentrations oxyhaemoglobin did not inhibit NO+- NO+ when added in the presence of SNP; however, after a 2 induced apoptosis (data not shown). NO-induced apoptosis hour exposure of cells to SNP, ascorbic acid (added after was also completely inhibited by the two antioxidants. In the washing the cell monolayers) had no potentiating effect (data presence of either N-acetylcysteine or ascorbic acid (each at not shown). These results indicate that ascorbic acid and NO+ 0.3 mM), apoptosis induced by aqueous NO or GSNO was react to form a more toxic species in solution. reduced to levels comparable to those in cultures treated with 10% CM alone (Fig. 6A, P<0.05). To investigate the mechanism of protection further, cultures were exposed to DISCUSSION GSNO for 8 hours and then washed and the medium replaced with 10% CM alone or in the presence of ascorbic acid. In The present investigation showed that both NO and NO+ can cultures exposed to GSNO alone 54% of cells were apoptotic trigger apoptosis in quiescent Swiss 3T3 fibroblasts. This was by 24 hours while, in the presence of ascorbic acid, this was confirmed by electron microscopy, nuclear pyknosis, DNA reduced to 22%, indicating that the protection did not depend laddering and PARP cleavage, a marker of caspase activation. on an extracellular scavenging effect of the antioxidant on NO Although both species induce apoptosis in these cells in a dose- (data not shown). dependent manner, there are clear differences between the In contrast, the induction of apoptosis by NO+ was markedly cytotoxic effects of NO and NO+. potentiated in the presence of antioxidants: the apoptotic effect First, at the subcellular level, treatment with NO+ results in of 1 mM SNP was increased significantly from 63% to 80% a rapid modification of mitochondrial structure, while exposure and 98% in the presence of N-acetylcysteine or glutathione to NO fails to produce morphological changes in these respectively (Fig. 6B, P<0.05). The addition of 1 mM glu- organelles. The mitochondrial alterations induced by NO+ are tathione shifted the dose-response curve of SNP to the left by consistent with these organelles being primary sites of free almost 2 orders of magnitude (Fig. 6C). Similarly, 1 mM N- radical attack mediated by NO species (Poderoso et al., 1996). acetylcysteine decreased the concentration of SNP giving 50% The hypermetabolism of mitochondria that we observed apoptosis from 0.7 mM to 0.1 mM (Fig. 6C). Ascorbic acid following NO+ treatment could result in the overproduction and also had a significant potentiating effect on NO+-induced subsequent release of cytochrome c into the cytoplasm, a Induction of apoptosis by NO and NO+ 2321 known trigger of apoptosis (Liu et al., 1996). We are currently al., 1991) this is unlikely to account for the induction of investigating this possibility in detail. apoptosis by NO+ since we found that a 2 hour exposure time Second, NO+ induces apoptosis after only 2 hours exposure, was sufficient for inducing apoptosis, whereas NO required at while NO cytotoxicity requires at least a 12 hour exposure. least 6 times longer. Our data are consistent with the transfer This kinetic difference suggests that NO and NO+ induce of the NO+ moiety from SNP to key thiol-containing proteins apoptosis by distinct mechanisms or at different points in the either on the cell surface or within the cytoplasm; for example, same signalling pathway. Finally, while NO+ is toxic at micro- our electron microscopy findings suggest that mitochondria molar concentrations, NO only triggers apoptosis in the mil- may be a primary target. In this context, the S- of limolar range, a concentration much higher than that measured mitochondrial proteins could then act as a direct and rapid within healthy cells (10 to 100 nM). Since at lower concentra- trigger of pro-apoptotic signalling pathways. We are currently tions NO behaves as an important physiological messenger, investigating this possibility in more detail. these findings suggest that apoptosis may be a mechanism for The results obtained with the antioxidants reinforce the con- eliminating cells that overproduce NO. clusion that NO and NO+ induce apoptosis by distinct mecha- What is the physiological relevance of NO+ toxicity? NO nisms. Glutathione and ascorbic acid are naturally occurring can occur in all three redox states in vivo (reviewed by Butler antioxidants; both can scavenge hydroxyl radicals while and Williams, 1993) and the NO+ ion has been shown to have ascorbic acid is also able to reduce thiyl radicals. This property direct effects, mediated by nitrosation of thiol groups, in of ascorbic acid may account for its toxic effect at higher con- reducing blood pressure (Bates et al., 1991) and regulating the centrations. Both glutathione and ascorbic acid are important activity of transcription factors (Hausladen et al., 1996; regulators of cellular redox potential. N-acetylcysteine is a Richardson et al., 1995). Moreover, several haem-containing membrane-permeable precursor of glutathione and has been mitochondrial proteins are known targets of S-nitrosation shown to protect neuronal cells from apoptosis (Ferrari et al., (reviewed by Cifone et al., 1995) and the electron microscopy 1995) and also from necrosis (Boobis et al., 1986). In the data presented here are consistent with these observations. present study, NO-induced apoptosis was inhibited irrespective The two S-nitrosothiols employed as NO-donors in this of the antioxidant used. This suggests that the interaction of study both induced apoptosis, but with different kinetics. NO with the superoxide ion, the peroxide ion or the hydroxyl Nitrosothiols release NO by homolytic cleavage of the RS-NO radical (OH) represents a major cytotoxic route. bond but can also act as nitrosating agents under certain con- Ascorbic acid, glutathione and N-acetylcysteine have a high ditions (Butler and Williams, 1993; Park et al., 1993; Arnelle affinity for nitrosating agents and it was anticipated that these and Stamler, 1995). The time course of apoptosis induced by would reduce SNP-induced apoptosis by reacting with its NO+ GSNO and SNAP suggests that their cytotoxicity correlates moiety. However, this was not the case; indeed, apoptosis with their decomposition to NO. GSNO, which has a half-life induced by NO+ was potentiated by the antioxidants used in of 2 to 3 hours in 10% CM (results not shown), induces this study. Our time-course data with the addition of ascorbic apoptosis after approximately 12 hours. SNAP, which is more acid at defined times of SNP treatment suggest that the reaction + stable than GSNO in 10% CM (t1/2 = 28 hours), only induces between NO and thiol groups leads to the formation of a more a significant degree of apoptosis in Swiss 3T3 fibroblasts after cytotoxic species. a 48 hour exposure. In summary, NO+ is more potent than NO in inducing The slow kinetics of induction of apoptosis by NO may be apoptosis in quiescent Swiss 3T3 fibroblasts. At higher con- a consequence of autoxidation of NO. At the high concentra- centrations, NO can induce apoptosis, possibly by interacting tions used, the oxidation of NO to N2O3 via nitrogen dioxide with cellular oxidants to form more reactive species (Volk et (NO2) and then to nitrite would proceed more readily than at al., 1995). This implies that NO is more likely to be toxic if physiological concentrations. It has been shown that the the cell is undergoing oxidative stress, whereas NO+ appears products of NO autoxidation are more cytotoxic than NO itself, to act independently of such conditions. However, we cannot which may reflect an increased reactivity towards cellular exclude the possibility that NO+ and NO act as early and late targets such as glutathione (Wink et al., 1994). The rapid signals respectively in a common apoptotic pathway in Swiss formation of GSNO by this route could effectively extend the 3T3 fibroblasts. life time of activity of NO and the relatively slow decompo- It has been proposed that, in the nervous system, NO+ may sition of this nitrosothiol to NO could then induce apoptosis be neuroprotective by nitrosating thiol groups on the NMDA over a longer period. This may explain why aqueous NO receptor, thereby inactivating it (Lipton et al., 1993). Our requires a long exposure time to induce apoptosis in Swiss 3T3 results indicate that NO+ is a potent inducer of apoptotic cell cells despite having a very short cellular half-life. Nitro- death in Swiss 3T3 fibroblasts (which do not express NMDA sothiols can, under certain conditions, also behave as NO+ receptors). Although our data suggest that mitochondria are donors in transnitrosation reactions, depending partly on the primary targets for NO+, it is also possible that nitrosation of nucleophilicity of the acceptor group. However, the kinetics of cell surface thiol groups by NO+ leads to the inactivation of GSNO cytotoxicity in the present study argue against this, cytokine receptors that transmit survival signals in Swiss 3T3 since transnitrosation of thiols by GSNO is a very fast reaction fibroblasts. A large number of tyrosine kinase receptors contain (Barnett et al., 1995). cysteine-rich regions and since apoptosis has been proposed as The more rapid and potent induction of apoptosis by NO+ a default pathway for all cells (Raff, 1992), NO and its redox- taken together with the distinct mitochondrial effects, suggests related species may trigger apoptotic cell death by directly or a different mechanism for NO+-induced apoptosis, or that this indirectly disrupting the basal signals from such receptors. molecule induces apoptosis downstream of NO in the same pathway. Although SNP can behave as a NO donor (Bates et We thank Mary Kozma for technical assistance with tissue culture 2322 S. Khan and others and Vivien Emons for technical assistance with the electron phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer microscopy. We also thank Jonathan Stamler for helpful comments on 26, 239-257. the manuscript. 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