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Carcinogenesis, Vol. 23, No. 6, 929-941, June 2002
© 2002 Oxford University Press

CANCER BIOLOGY

Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to nontransformed fibroblasts

Stefanie Heigold1, Christine Sers2, Wibke Bechtel1, Boris Ivanovas1, Reinhold Schäfer2 and Georg Bauer1,3

1 Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79104 Freiburg and
2 Institut für Pathologie, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, D-10117 Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) mediates apoptosis induction in fibroblasts with constitutive src or induced ras oncogene expression, whereas nontransformed parental cells and revertants are not affected. This direct link between the transformed phenotype and sensitivity to NO-mediated apoptosis induction seems to be based on the recently described extracellular superoxide anion generation by transformed cells, as NO-mediated apoptosis induction in transformed cells is inhibited by extracellular superoxide dismutase (SOD), by SOD mimetics and by apocynin, an inhibitor of NADPH oxidase. Furthermore, nonresponsive nontransformed cells can be rendered sensitive for NO-mediated apoptosis induction when they are supplemented with xanthine oxidase/xanthine as an extracellular source for superoxide anions. As superoxide anions and NO readily interact in a diffusion-controlled reaction to generate peroxynitrite, peroxynitrite seems to be the responsible apoptosis inducer in NO-mediated apoptosis induction. In line with this conclusion, NO-mediated apoptosis induction in superoxide anion-generating transformed cells is inhibited by the peroxynitrite scavengers ebselen and FeTPPS. Moreover, direct application of peroxynitrite induces apoptosis both in transformed and nontransformed cells, indicating that peroxynitrite is no selective apoptosis inducer per se, but that selective apoptosis induction in transformed cells by NO is achieved through selective peroxynitrite generation. The interaction of NO with target cell derived superoxide anions represents a novel concept for selective apoptosis induction in transformed cells. This mechanism may be the basis for selective apoptosis induction by natural antitumor systems (like macrophages, natural killer cells, granulocytes) that utilize NO for antitumor action. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression.

Abbreviations: NO, nitric oxide; NOS, NO synthases; S/N-1, 13-morpholino sydonimine; SNP, sodium nitroprusside; SOD, superoxide dismutase.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is a free radical with diverse biological functions of central importance (for review see ref. 1) NO arises from the guanodino group of L-arginine in a NADPH-dependent reaction catalyzed by constitutively expressed or inducible NO synthases (NOS). NO represents a relatively long-lived and far ranging radical, with the ability to pass cellular membranes (2); to decrease the intracellular glutathione pool (3), to regulate gene expression via interaction with the zinc finger transcription factor SP1 (4) and to upregulate p53 gene expression (5).

NO and superoxide anions form peroxynitrite in a diffusion controlled reaction (6–8). Peroxynitrite and its protonated form peroxynitrous acid are highly reactive species with the potential to oxidize sulfhydryl groups (9), to nitrate tyrosine and tryptophane (10), to perform hydroxylation reactions (11) and to nitrosylat either directly (12) or after interaction with NO, leading to the formation of the nitrosylating molecule dinitrogentrioxide (13). In addition, peroxynitrite shows the potential for lipid peroxidation (14). Peroxynitrite is scavenged by glutathione and is readily inactivated by glutathione peroxidase, the glutathione peroxidase mimetic ebselen (15,16) and the decomposition catalyst FeTPPS (17,18). In turn, peroxynitrite can inactivate enzymes that might inhibit its generation, like SOD (19), or that might block its activity, like glutathione peroxidase (20). Peroxynitrite seems to be actively involved in many physiological and pathophysiological conditions (21,22).

NO represents a central tool used by most natural antitumor defense mechanisms. Two different strategies for NO-based antitumor mechanisms are known. (i) Cytokines secreted by effector cells like macrophages may induce the expression of iNOS in tumor cells. Subsequent NO synthesis becomes deleterious for the tumor cell itself (23). (ii) Effector cells like natural killer cells, monocytes, macrophages, Kupffer cells, microglia, neutrophils, endothelial cells or TGF-beta-treated fibroblasts release NO that mediates apoptosis induction in tumor cells (for review see ref. 24).

NO and peroxynitrite, the reaction product between NO and superoxide anions, have both been reported to induce apoptosis. Due to the multiple biological activities induced by NO, direct apoptosis induction by NO may depend on the cell type and the cellular redox state, as well as on the concentration and flux of NO. Apoptosis induction by NO has been reported for thymocytes (25), human leukemia cells (26) and mesangial cells (5). In the study by Sandau and Bruene (5), the apoptosis-inducing effect of NO was efficiently antagonized by the simultaneous presence of superoxide anions. In many other cellular systems, peroxynitrite rather than NO seems to be the responsible apoptosis inducer (21,22,27). This conclusion is based on several independent arguments. (i) Apoptosis can be induced in HL-60 cells by either addition of NO (28) or peroxynitrite (29). A HL-60 subline, resistant to NO-mediated apoptosis, showed increased activity of SOD, pointing to the role of superoxide anions for NO-mediated apoptosis, i.e. the necessity for peroxynitrite formation (30). (ii) Upregulation of mitochondrial SOD (i.e. removal of superoxide anions and prevention of peroxynitrite formation) caused resistance to NO-mediated apoptosis (31), whereas downregulation of intracellular SOD augmented NO-mediated apoptosis (32). (iii) Peroxynitrite has the potential for lipid peroxidation, a central step in apoptosis induction (14,33), whereas NO rather inhibits lipid peroxidation (33,34). (iv) Peroxynitrite-induced apoptosis depends on activation of caspase-3 (35). NO inhibits caspases through nitrosylation (36). (v) Mitochondrial permeability transition represents a central step during execution of apoptosis. It can be efficiently triggered by peroxynitrite, but not by NO or superoxide anions alone (37).

Constitutive extracellular superoxide anion production seems to be a central feature of transformed fibroblasts (38,39). Membrane associated NAD(P)H oxidases channel intracellular electrons (from NAD(P)H) to extracellular molecular oxygen, resulting in the production of extracellular superoxide anions. The activity of NADPH oxidases is controlled by rac, a small GTP-binding protein (40). The necessity for rac activity for superoxide anion production is proven by the inhibitory effect of dominant negative rac on superoxide anion production (38,40). The activity of rac itself is controlled by the oncogene ras (38), and the ras–rac signalling pathway is connected to the activity of src (41). Thus extracellular superoxide anion production is linked to oncogene activation and represents a distinct biochemical marker of transformed cells. It seems to be involved in the control of proliferation and in the maintenance of the transformed state (38,39,42). Furthermore, the presence of membrane-associated NADH oxidase has been shown to represent a rather uniform biochemical feature of tumor cells derived from different lineages (43,44). Therefore, constitutive extracellular superoxide anion production seems to represent a characteristic feature of transformed cells. This finding allowed to study directly whether peroxynitrite formation through the interaction of transformed cell-derived extracellular superoxide anions with effector cell-released NO represents an apoptosis inducing signal that is specifically generated in transformed cells and thus warrants selective killing of transformed cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
NO donors: The NO donor sodium nitroprusside (SNP) was obtained from Sigma (Deisenhofen, Germany). It was kept as a 200 mM stock solution in medium at –20°C. SNP represents a classical NO donor, used in numerous experimental and clinical studies (a literature research in BIOSIS showed >1100 papers with SNP as NO donor, both in experimental and clinical studies). Release of NO from SNP has been directly determined (45).

The NO donors DEA NONOate, PAPA NONOate, Spermine NONOate and S-nitrosoglutathione (NO-S-glutathione) were obtained from Alexis (Lausen, Switzerland). They were dissolved in medium at a final concentration of 20 mM and stored at –20°C. Care was taken to minimize the time between preparation of the stock solution (on ice) and freezing the aliquots, and between thawing the aliquots and their addition to the assays. Whereas SNP represents a very slowly decaying NO donor (46), DEA NONOate, Spermine NONOate and PAPA NONOate represent rather rapidly decaying NO donors with half-life times at 37°C of 2 min, 39 min and 15 min, respectively (47).

Peroxynitrite (synthesized from isoamylnitrite and hydrogen peroxide) was obtained as a 200 mM stock solution from Calbiochem. It was kept at –70°C until use.

The peroxynitrite generator 13-morpholino sydnonimine (SIN-1) was obtained from Sigma (Deisenhofen, Germany) and kept as a 200 mM stock solution in medium at –20°C. SIN-1 decomposes in two steps, thereby releasing superoxide anions and NO. Therefore, incubated SIN-1 solutions contain peroxynitrite, but in addition also free NO.

SOD (from bovine erythrocytes, Sigma, Deisenhofen, Germany) stock solutions (30 000 Units/ml in PBS) were kept at –20°C and only used once per aliquot. SOD represents an efficient scavenger of superoxide anions, in a two-step reaction:

SOD(Cu++) + O2o- -> SOD (Cu+) + O2; SOD(Cu+) H+ + H+ + O2•- -> SOD (Cu++) + H2O2. SOD is not cell-permeable (48,49) and therefore allows to demonstrate the functional role of extracellular superoxide anions.

As shown in this paper, NO-mediated apoptosis induction is inhibited by SOD, pointing to the role of superoxide anions and peroxynitrite formation. Care has to be taken to optimize the inhibitory concentration of SOD. When SOD is present in high concentrations compared with superoxide anions, the SOD(Cu+)-form may prevail and reduce NO to the nitroxyl ion (50). The nitroxyl ion may interact with molecular oxygen and form peroxynitrite in this way. A detailed study of this problem will be presented elsewhere (Zerweck et al., in preparation).

4-Hydroxy-3-methoxyacetophenone (Acetovanillone, Apocynin) was obtained from Calbiochem and kept as a stock solution of 2.5 mg/ml (in medium) at –20°C. Apocynin represents a specific inhibitor of NADPH oxidase (51–53). It has been shown to prevent peroxynitrite formation in macrophages through inhibition of NADPH oxidase (53).

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS) was obtained from Calbiochem. Stock solutions (10 mM) were kept at –20°C. FeTPPs represents a specific decomposition catalyst for peroxynitrite (17,18). Specificity control experiments showed that the FeTPPS concentrations applied in our studies did not scavenge superoxide anions or hydrogen peroxide, but efficiently decomposed chemically pure peroxynitrite.

Mn(II)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) and Mn(II)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) were obtained from Calbiochem and stored as 10 mM stock solutions at –20°C. MnTBAP and MnTMPyP represent cell-permeable SOD mimetics and also decomposition catalysts for peroxynitrite. Both activities (stated in the product data sheet of Calbiochem) were confirmed in our specificity experiment (Figure 6CGo).



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  		Fig. 6. NO-mediated apoptosis depends on superoxide anions and peroxynitrite generation. Experiments A and B test for the role of apocynin (a NADPH oxidase inhibitor), MnTBAP and MnTMPyP (two SOD mimetics and peroxynitrite decomposition catalysts) and FeTPPS (a peroxynitrite decomposition catalyst without SOD activity) on Spermine NONOate-mediated apoptosis induction in transformed cells. Experiment C represents a specificity control of the inhibitors used. (A and B) 15 000 transformed 208 F src3 cells were seeded per well in Costar 24 well tissue culture clusters and treated as indicated on the horizontal axis. The following concentrations were applied: Spermine NONOate (0.5 mM); Apocynin (50 µg/ml); MnTBAP (200 µM); MnTMPyP (50 µM); FeTPPS (200 µM in experiment A and 50 µM in experiment B). All assays were performed in duplicate. The percentages of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) were determined by inverse phase contrast microscopy after 12 h. (C) Test for specificity of the inhibitors. 15 000 transformed 208 F src cells in Costar 24-well tissue culture clusters were either treated with 100 µM BSO in the absence or presence of the indicated inhibitors (apocynin 50 µg/ml; MnTBAP 200 µM; catalase 77 U/ml, FeTPPS 200 µM) for 22 h or with 100 µM chemically synthesized peroxynitrite in the absence or presence of 50 µg/ml apocynin, 100 µM MnTBAP, 100 µM MnTMPyP, or 100 µM FeTPPS for 4 h. Control assays received no addition, or 50 µg/ml apocynin, 200 mM MnTBAP, 77 U/ml catalase, 200 µM FeTTPS and were incubated for 22 h. The percentages of apoptotic cells were determined as described above. All assays had been performed in duplicate. BSO treatment causes glutathione depletion and leads to apoptosis induction in transformed cells through ferrous ion-catalyzed Fenton reaction of extracellular hydrogen peroxide, derived from extracellular superoxide anions (Schimmel and Bauer, in press). This process depends on superoxide anions for hydrogen peroxide generation and oxidation of ferric to ferrous ions. Experiment C confirms that FeTPPS effectively decomposes peroxynitrite, but does not affect superoxide anions or hydrogen peroxide. MnTBAP and MnTMPyP show SOD activity as well as decomposition of peroxynitrite. Apocynin selectively blocks the superoxide anion-dependent process in BSO-treated cells, but has no effect on peroxynitrite.

 
Ebselen was obtained from Sigma. It was kept as a 20 mM stock solution in DMSO at –20°C. It was applied at a concentration of 4 µM during inhibitor experiments. Control experiments ruled out that the extremely low concentration of DMSO (0.02%) had any toxic effect or inhibitory effect on NO or peroxynitrite-mediated apoptosis induction (data not shown). Ebselen represents a very efficient scavenger of peroxynitrite (15), but can also interact with hydrogen peroxide (54).

Xanthine oxidase was obtained from Sigma. 3 mU/ml of the enzyme in the presence of 0.4 mM xanthine produced sufficient superoxide anions to drive NO-mediated apoptosis induction in nontransformed cells but was not toxic in the absence of the NO donor. Xanthine was obtained from Sigma. It was directly applied to medium in order to reach a 0.4 mM solution.

Isopropyl beta-D-thiogalactoside (IPTG) was obtained from Sigma, It was stored as a 2 M stock solution in medium at –20°C. It was added at a final concentration of 20 mM for induction of ras in IR-1 cells.

Cell culture
Cells were kept in Eagle's Minimal Essential Medium, containing 5% fetal calf serum that had been heated for 30 min at 56°C prior to use. Medium was supplemented with penicillin (40 U/ml), streptomycin (50 µg/ml), neomycin (10 µg/ml), moronal (10 U/ml) and glutamine (280 µg/ml). Cell culture was performed in plastic tissue culture flasks. Cells were passaged once or twice weekly.

Nontransformed rat fibroblasts 208 F, 208 F cells transformed through constitutive expression of v-src (termed `208F src3' cells) and 208 F cells with an inducible H-ras oncogene (termed `IR-1 cells') have been established in the laboratory of R. Schäfer. For the establishment of IR-1 cells, immortalized 208 F rat fibroblasts had been transfected with the HRAS oncogene under the control of the SV40 promoter and the lac operator sequence, the lac repressor gene and pSV2neo. Ras oncogene induction through addition of 20 mM IPTG (isopropyl beta-D-thiogalactoside) caused the expression of the transformed phenotype within 24–48 h (55). Ras protein was detectable in induced cells when the western blot technique was applied, but was undetectable in control cells (data not shown). 208 F src3 cells and IPTG-treated IR cells show criss-cross morphology, form colonies in soft agar and are sensitive for intercellular induction of apoptosis (55,56), whereas parental 208 F cells do not exhibit these features of transformed cells. Revertants from transformed 208 F src3 cells have been recently isolated and characterized (56), bcl-2 overexpressing 208 F src3 cells and 208 F src3 cells, containing a control plasmid without BCl-2 were obtained through transfection of 208 F src3 cells with either the pRc neo plasmid containing the bcl-2 gene at the polylinker cloning site or with the pRc neo plasmid, using the standard calcium phosphate technique. Plasmid-containing cells were selected through cultivation in the presence of 1 mg/ml G 418. For determination of Bcl-2 expression, cells were fixed with methanol/acetone (1:1) at –20°C for 20 min. A 1:5 dilution in PBS of a commercially available monoclonal antibody directed against Bcl-2 (Dianova, Hamburg, Germany) was added, and incubation at 4°C was performed overnight. After washing with PBS (three times, 5 min), biotinylated rabbit anti-mouse IgG was added and incubation at 37°C was performed for 1 h. After washing, FITC-labelled streptavidin was added and incubated for 20 min at 37°C. The dishes were washed three times with PBS before fluorescence microscopy. The cell population showed a homogenous expression of bcl-2.

Apoptosis induction
Cells were seeded at a density of 15 000 cells per assay (24 well Costar tissue culture cluster). (The density of cells is critical for the efficiency of NO-mediated apoptosis induction. High cell-density causes inhibition of NO mediated apoptosis. This effect and the underlying biochemical reactions will be described elsewhere (72).

NO donors or SIN-1 were prediluted and added at the indicated final concentration. Determination and quantitation of apoptosis was based on the classical morphological criteria membrane blebbing, nuclear condensation and nuclear fragmentation. These were determined using inverted phase contrast microscopy, as recently described (56,57). The percentage of apoptotic cells was determined from at least 200 cells categorized per assay. Apoptotic cells were either attached or rounded and showed (a) membrane blebbing; or (b) membrane blebbing and nuclear condensation/fragmentation; or (c) nuclear fragmentation/condensation without blebbing (these cells seem to represent later stages of apoptosis where the blebs have been already lost). Care was taken to differentiate apoptotic cells from nonapoptotic rounded cells with intact nuclei, reflecting mitotic stages.

All quantitative data in this paper were derived using this method. In parallel, control assays ensured that apoptotic cells characterized by morphological criteria as described above showed a positive TUNEL reaction, indicative of free 3' hydroxyl groups of the DNA, one of the hallmarks of apoptotic cells.

DNA strand breaks (free 3' hydroxyl groups) were detected by the TUNEL reaction (56–58), using a commercially available detection kit (Boehringer, Mannheim, Germany). Cells were fixed with 3% paraformaldehyde in PBS for 30 min at room temperature, washed with PBS for 30 min and then permeabilized in 0.1% Triton X-100/0.1% sodium citrate in PBS for 2 min on ice. After two washing steps with PBS, the TUNEL assay kit (containing terminal deoxynucleotidyl transferase, fluorescein-labelled dUTP and suitable buffer components) was added. Incubation at 37°C was for 60 min, followed by three washing steps with PBS. Cells were then stained with 1 µg/ml bisbenzimide in PBS for 30 min at room temperature, followed by two washing steps with PBS. The assays were then inspected, using an inverted fluorescence microscope with suitable filter sets.

In some assays, the exposure of phosphatidyl serine at the outside of apoptotic cells was demonstrated through staining with fluorescent annexin V-Cy3, using a commercially available kit (Sigma).

Determination of mitochondrial depolarization
Cells were stained with 5 µg/ml rhodamine 123 (Sigma, Deisenhofen, Germany) for 30 min in medium at 37°C. Medium was removed and the cells were washed two times with PBS before they were inspected by inverted fluorescence microscopy. In control cells, mitochondria were stained bright red, whereas cells with mitochondrial depolarization showed no staining.

For the detection of caspase-3 activity, a commercially available test kit (PhiPhiLux-G2D2; Alexis) was used. It is based on the cleavage of a specific caspase-3 substrate (GDEVDGI), containing two rhodamines. The cleaved product shows red fluorescence.

Nitrotyrosine (a marker for the interaction of peroxynitrite with proteins) was detected using a monoclonal antibody directed against nitrotyrosine (Alexis). In order to restrict detection of nitrotyrosine at the outside of the cell membrane, cells were neither fixed nor permeabilized, but only gently washed with PBS. Incubation with monoclonal antibody (1.5 µg/ml) in PBS was at 37°C for 1 h, followed by two wash steps with PBS. Bound antibody was detected by incubation with biotinylated anti-Mouse-IgG (45 min, 37°C), followed by a wash step and incubation with CY3-labelled streptavidin. After a final wash step, cells were inspected using an inverted fluorescence microscope. This procedure led to a very low background and a diffuse staining on the surface of positive cells. However, the loss of many detached apoptotic cells during the washing procedures was unavoidable.

Statistical analysis
In all experiments, assays were performed in duplicate. The mean values (from duplicate assays within the same experiment) and the empirical standard deviations were calculated and are shown in the figures. Absence of standard deviation bars for certain points indicates that the standard deviation was too small to be reported by the graphic program, i.e. that results obtained in parallel were nearly identical. Empirical standard deviations were calculated merely to demonstrate how close the results were obtained in parallel assays within the same experiment and not with the intention of statistical analysis of variance, which would require larger numbers of parallel assays.

The key experiments have been repeated more than ten times, involving different investigators. The Yates continuity corrected chi-square test was used for the statistical determination of significances.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Apoptosis induction mediated by the NO donor sodium nitroprusside is strictly correlated to the expression of the transformed phenotype and depends on the generation of superoxide anions
Src-oncogene transformed fibroblasts or the nontransformed parental cell line 208 F were incubated with the NO donor sodium nitroprusside (SNP). As can be seen in Figure 1Go, SNP induced apoptosis selectively in transformed 208 F src3 cells in a concentration- and time-dependent manner. In contrast, nontransformed 208 F cells only showed a marginal apoptotic response during the time of the experiment. Apoptosis induction by SNP in transformed cells was completely blocked by extracellular superoxide dismutase (SOD). This finding indicates that extracellular superoxide anions (which are known to be generated by transformed fibroblasts (Irani et al., 1997) are necessary for apoptosis induction by the NO donor SNP.



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  		Fig. 1. The NO donor SNP mediates apoptosis induction selectively in transformed fibroblasts, in a reaction that is dependent on the concentration of SNP and on the generation of superoxide anions. 15 000 transformed 208 F src3 or nontransformed parental 208 F cells were seeded in Costar 24-well tissue culture clusters. They were treated with the indicated concentrations of SNP, in the absence or presence of 150 U/ml SOD. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically, using inverted phase contrast microscopy. All assays were performed in duplicate. The left panel shows the kinetics of apoptosis induction in controls and in the presence of 0.06, 0.25 and 1 mM SNP. The right panel shows the correlation between apoptosis induction measured at 70 h after SNP addition and the concentration of SNP, in the absence or presence of SOD.

 
If sensitivity for NO-mediated apoptosis was causally correlated to the transformed phenotype, revertants from src-transformed cells should have lost their sensitivity for NO-mediated apoptosis in parallel with their loss of the transformed state. In addition, cells with an inducible ras oncogene should only be sensitive, when the oncogene was expressed. Both predictions were verified by our experiments and allowed to correlate the transformed phenotype to sensitivity for NO-mediated apoptosis induction (Figure 2Go): Three revertants (R2, R4, R5) derived from src-transformed fibroblasts that had lost the transformed phenotype (56), showed background apoptosis induction when challenged with the NO donor SNP, whereas the src-transformed cell line 208 F src3 showed a strong apoptotic response. When oncogene expression was induced in IR1 cells (208 F cells carrying a plasmid with an inducible ras oncogene), the cells acquired sensitivity for NO-mediated apoptosis induction in parallel to the expression of the transformed state, whereas the noninduced cell population only showed a marginal response (Figure 2Go). NO-mediated apoptosis induction in the ras-expressing cells was completely blocked by SOD and therefore seemed to be strictly dependent on extracellular superoxide anion production.



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  		Fig. 2. Apoptosis induction mediated by the NO donor SNP is strictly correlated to the expression of the transformed phenotype. 15 000 transformed 208 F src3 cells or revertants R2, R4, R5 derived from 208 F src3 cells (69) were seeded in Costar 24 well tissue culture clusters and treated with the indicated concentrations of the NO donor SNP. After 49 h, the percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined, using inverted phase contrast microscopy. All assays were performed in duplicate. In a parallel experiment, IR1 cells (208 F cells harbouring an IPTG-inducible ras oncogene) were pretreated with 20 mM IPTG for 2 days (to induce ras expression) (`IR1 + IPTG') or remained untreated (`IR1'). Then the cultures were trypsinized and 15 000 cells/assay were seeded in Costar 24-well tissue culture clusters. IPTG treatment was continued in the case of ras-expressing cells. The assays received the indicated concentrations of SNP (open circles), as well as additional SOD (150 U/ml) in some of the assays, as indicated (closed circles). The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined after 64 h, using inverted phase contrast microscopy. All assays were performed in duplicate.

 
Different NO donors induce apoptosis selectively in transformed fibroblasts: indication that NO represents the active principle involved in apoptosis induction mediated by NO donors
The experiments shown so far had been performed with the NO donor SNP. If mediation of apoptosis induction by SNP was due to the NO moiety released from the molecule (and not by the side products), chemically different NO donors should all mediate apoptosis induction and this induction should be selective with respect to the transformed state. As can be seen in Figure 3Go, the NO donors PAPA NONOate, Spermine NONOate, S-nitrosoglutathione, and DEA NONOate induced apoptosis selectively in src transformed fibroblasts, but not in parental nontranformed 208 F cells. This finding verifies the selectivity of NO mediated apoptosis induction for transformed cells. As these NO donors release NO much faster than SNP (46,47), the kinetics of apoptosis in transformed cells mediated by these compounds reached its maximum already at 10–12 h after addition of the respective compound.



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  		Fig. 3. Selective apoptosis induction in transformed fibroblasts by different NO donors indicates the role of NO to mediate selective apoptosis induction. 15 000 transformed 208 F src3 cells or nontransformed 208 F cells were seeded in Costar 24 well tissue culture clusters and treated with 0.5 mM of the following NO donors: PAPA NONOate, Spermine NONOate, N-nitrosoglutathione, DEA NONOate and SNP. DEA NONOate was added stepwise (0.1 mM, five times, intervals of 1 h). Controls remained without additions. The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically, using inverted phase contrast microscopy. All assays were performed in duplicate. The upper part of the graph shows the kinetics of apoptosis induction in transformed fibroblasts by Spermine NONOate. Similar kinetics were obtained for PAPA NONOate, S-nitrosoglutathione and DEA NONOate (data not shown). The lower part of the graph demonstrates the percentages of apoptotic transformed and nontransformed fibroblasts determined at 22 h after addition of the NO donors.

 
To test for apoptosis induction more stringently, src-transformed fibroblasts were treated with the NO doner Spermine NONOate or not and then the cells were inspected by inverted phase contrast microscopy (Figure 4A,BGo). In addition, their nuclear morphology was evaluated after bisbenzimide staining (Figure 4C,DGo) and DNA strand breaks were detected through the TUNEL reaction (Figure 4E,FGo). As shown in Figure 4Go, the cells showed the characteristic spindle-shaped morphology of transformed cells, with substantial criss-cross growth (A). Some rounded (mitotic) cells with intact nuclei were seen. In the assays treated with the NO donor, many cells were rounded and showed condensed or fragmented nuclei. In addition, many cells showed membrane blebbing. Bisbenzimide staining confirmed that control cells had intact nuclei (Figure 4CGo) and the TUNEL assay demonstrated the absence of DNA strand breaks (Figure 4EGo). In NO-treated cultures, a substantial number of cells with condensed or fragmented nuclei (Figure 4DGo) were seen and these were positive in the TUNEL reaction (Figure 4FGo). These data show that NO mediates membrane blebbing, nuclear condensation, nuclear fragmentation and DNA strand breaks, which are hallmarks of apoptosis.



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  		Fig. 4. Cell death mediated by the NO donor Spermine NONOate is characterized as apoptosis. Transformed 208 F src3 fibroblasts were seeded in Costar 24 well tissue culture clusters (15 000 cells per well) and treated with 0.5 mM Spermine NONOate for 12 h. Control assays did not receive Spermine NONOate. Control assays (A) and Spermine NONOate-treated cultures (B) were then inspected by inverted phase contrast microscopy. In parallel, the TUNEL reaction (E: control cells; F: Spermine NONOate-treated cells) was performed and in addition, the assays were stained with bisbenzimide (C: control cells; D: Spermine NONOate-treated cells) as described under Materials and methods.

 
Peroxynitrite represents the responsible inducer during NO-mediated apoptosis induction
As superoxide anions and NO are known to form peroxynitrite in a diffusion driven reaction (6–8), peroxynitrite was the most likely candidate for apoptosis induction in the SOD-inhibitable, NO-mediated reaction shown for src- or ras-transformed fibroblasts in this paper. To clarify this point directly, several experimental approaches were applied: (i) the effect of the peroxynitrite scavenger ebselen on NO-mediated apoptosis induction was tested; (ii) the extracellular localization of peroxynitrite generation was tested through the inhibitory effect of glutathione, which is not cell-permeable; (iii) inhibitors that block superoxide anion generation (apocynin), decompose peroxynitrite (FeTPPS) or scavenge superoxide anions and decompose peroxynitrite (MnTBAP, MnTMPyP) were tested for their effects on NO-mediated apoptosis induction in transformed cells; (iv) chemically pure peroxynitrite and the peroxynitrite generator SIN-1 were tested for their apoptosis inducing effects on transformed as well as nontransformed cells; (v) nitrotyrosine formation, an indicator for peroxynitrite generation and interaction with proteins was tested on transformed cells treated with SpermineNONOate.

When the peroxynitrite scavenger ebselen (15) was added to assays containing src transformed fibroblasts and SNP, a marked inhibitory effect on apoptosis induction mediated by SNP or Spermine NONOate was seen (Table IGo). This finding points to a potential role of peroxynitrite in this process. Inhibition by ebselen was stronger for SNP-mediated apoptosis induction compared to Spermine NONOate-mediated apoptosis induction.


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  		Table I. NO-mediated apoptosis induction is inhibited by ebselen and extracellular glutathione
 
If apoptosis induction was in fact due to extracellular peroxynitrite formation, apoptosis induction mediated by NO should be inhibited by extracellular glutathione. Glutathione cannot penetrate cells. It interacts efficiently with peroxynitrite, but not with NO (for review see ref. 24). As can be seen in Table IGo (experiment B), addition of extracellular glutathione efficiently counteracted NO-mediated apoptosis induction, indicating that the responsible inducer was generated in the extracellular space and pointing to peroxynitrite as the potential responsible molecule.

However, ebselen as well as glutathione might have interacted with hydrogen peroxide (54), rather than with peroxynitrite directly. Hydrogen peroxide might have derived from one electron oxidant interaction of peroxynitrite with suitable substrates, followed by superoxide anion generation and dismutation (59). To test for an involvement of hydrogen peroxide in our studies, transformed cells were treated with SNP in the presence or absence of catalase. As can be seen in Figure 5Go, catalase had no inhibitory effect on SNP-mediated apoptosis induction, but rather enhanced it. Therefore hydrogen peroxide is not the apoptosis inducer effective in our system and the inhibitory effects by ebselen and glutathione seem to be indicative for the role of peroxynitrite rather than that of hydrogen peroxide during apoptosis induction.



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  		Fig. 5. SNP-mediated apoptosis induction in transformed fibroblasts is not inhibited by catalase. 15 000 transformed 208 F src3 fibroblasts were seeded per well in Costar 24-well tissue culture clusters. The assays received either no addition (`control'), 0.25 mM SNP (`SNP'), 0.25 mM SNP plus 77 U/ml catalase (`SNP + CAT') or 0.25 mM SNP plus 150 U/ml SOD (`SNP + SOD'). The percentage of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) was determined kinetically. All assays were performed in duplicate. Control assays with catalase or SOD alone showed no apoptosis inducing effect (data not shown).

 
For a further evaluation of the potential role of peroxynitrite formation during NO-mediated apoptosis induction in transformed cells, apocynin, MnTBAP, MnTMPyP and FeTTPs were tested for their effect on apoptosis induction mediated by the NO donor Spermine NONOate. As can be seen in Figure 6A and BGo, Spermine NONOate-mediated apoptosis induction was markedly inhibited by each of these compounds. In a specificity control experiment, the same inhibitors were tested for their effect on autocrine, ROS-mediated apoptosis induction in glutathione-depleted (BSO-treated) transformed fibroblasts (60) (this process depends on superoxide anions that reduce ferric ions to ferrous ions, which then catalyze the Fenton reaction) and on chemically pure peroxynitrite. As shown in Figure 6CGo, the NADPH oxidase inhibitor blocked the superoxide-anion-dependent process in BSO-treated cells, but had no effect on apoptosis induction by peroxynitrite directly. In contrast, the peroxynitrite decomposition catalyst FeTPPS blocked peroxynitrite-induced apoptosis, but had no effect on the superoxide anion-dependent process. In line with their product data sheet (Calbiochem Homepage), MnTBAP and MnTMPyP interfered with the superoxide anion-dependent process and also scavenged peroxynitrite directly. Taken together, the data shown in Figure 6Go demonstrate that NO-mediated apoptosis induction requires superoxide anion generation and is based on the formation of peroxynitrite.

Our data so far indicate that selective apoptosis induction in transformed fibroblasts by NO donors was based on their extracellular superoxide anion production and subsequent generation of peroxynitrite. Therefore, peroxynitrite seemed to be selectively generated by NO-treated transformed cells, but should not be a selective apoptosis inducer per se. Direct addition of peroxynitrite should then induce apoptosis in transformed as well as nontransformed cells. As can be seen in Figure 7Go, chemically pure peroxynitrite or the peroxynitrite generator SIN-1 caused apoptosis in parental (nontransformed) cells, in cells transformed by constitutive src oncogene expression, in cells harbouring an inducible ras oncogene, independent of ras expression and in revertants that have lost the transformed phenotype. In each case, apoptosis induction was blocked by the peroxynitrite decomposition catalyst FeTPPs. In contrast to peroxynitrite, the NO donor Spermine NONOate mediated apoptosis induction selectively in cells that expressed the transformed phenotype, in a reaction that was also blocked by FeTPPS. These data demonstrate that peroxynitrite represents an efficient apoptosis inducer that cannot differentiate between transformed and nontransformed cells. They confirm that NO-mediated apoptosis induction depends on selective peroxynitrite formation in transformed cells.



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  		Fig. 7. Peroxynitrite induces apoptosis nonselectively in transformed and nontransformed cells. Cells were seeded in Costar 48-well tissue culture clusters (7500 cells per assay) and were treated with 50 µM chemically synthesized peroxynitrite (PON) for 4 h (A), 0.5 mM of the peroxynitrite generator SIN-1 for 9 h (B), or 0.5 mM of the NO donor Spermine NONOate for 17 h (C). Parallel assays under all conditions contained 100 µM of the selective peroxynitrite decomposition catalyst FeTPPS. Control assays received no addition or only FeTPPS and were cultured for 17 h (D). At the indicated times, the percentages of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) were determined in duplicate experiments. Cell lines used: IR-1: nontransformed 208 F cells harbouring (but not expressing) an inducible ras oncogene; IR-1 + IPTG: ras oncogene expression had been induced 48 h before the addition of peroxynitrite or Spermine NONOate in IR-1 cells through IPTG. The inducer was present throughout the experiment, to maintain the transformed state; 208 F: nontransformed rat fibroblasts; 208 F src3 cells: 208 F cells transformed by constitutive expression of the src oncogene; R2, R4, R5: revertants from 208 F src3 cells that had spontaneously lost their transformed phenotype (56).

 
To check for peroxynitrite formation through a second assay, 208 F src3 cells treated with 0.5 mM Spermine NONOate for 7 h were tested for nitrotyrosine, using a monoclonal antibody (see Materials and methods for the procedure). To restrict the method to the detection of nitration at the outside of the cell membrane, cells were not fixed or permeabilized. Thus, the washing steps caused a marked loss of apoptotic cells, but still 39 out of 450 inspected cells showed nuclear condensation in the Spermine NONOate-treated assay, compared with five in the control. Thirty cells from 450 were positive for staining with the monoclonal antibody against nitrotyrosine in the Spermine NONOate-treated population (most of them were also apoptotic), whereas no nitrotyrosine-positive cells were seen in the control. This finding is in line with extracellular peroxynitrite formation by Spermine NONOate-treated transformed cells. The correlation between nitrotyrosine staining and apoptosis seems to indicate that cells that allow a marked peroxynitrite generation have a higher chance to die through apoptosis.

Supplementation of nonreactive nontransformed fibroblasts with an extracellular superoxide anion-generating system renders them sensitive for NO-mediated apoptosis induction
The data presented so far seem to indicate that selective apoptosis induction mediated by NO donors depends on extracellular peroxynitrite formation through the interaction of transformed cell-derived superoxide anions with NO, whereas nontransformed are unable to sustain peroxynitrite generation due to insufficient superoxide anion generation. Supplementation of nontransformed cells with an extracellular source of superoxide anions should therefore render them sensitive for NO-mediated apoptosis induction. As can be seen in Figure 8Go, nontransformed 208 F fibroblasts responded to apoptosis induction by all NO donors applied, when the superoxide anion generating system xanthine oxidase/xanthine was present in parallel. As shown for the reaction with SNP, the effect of xanthine oxidase/xanthine was due to superoxide anion generation, as it was blocked by SOD. These data confirm that NO has no direct apoptosis inducing potential for fibroblasts, but that its interaction with superoxide anions allows for apoptosis induction.



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  		Fig. 8. Supplementation of nontransformed cells with a superoxide anion-generating system allows NO-mediated apoptosis induction. 15 000 nontransformed 208 F fibroblasts per well were seeded in Costar 24-well tissue culture clusters. Medium contained 0.4 mM xanthine in all assays. Control assays received no NO donors. The NO donors PAPA NONOate, Spermine NONOate, S-nitrosoglutathione, DEA NONOate and SNP were added at a concentration of 0.5 mM. Control assays and assays containing the indicated NO donors were run either in the absence (`–') or the presence (`+') of 4 mU/ml xanthine oxidase (`XO') as superoxide anion-generating system. All assays were performed in duplicate. In parallel, two assays with SNP and xanthine oxidase received 150 U/ml SOD, to test whether the effect of xanthine oxidase addition was in fact due to superoxide anion generation. The percentages of apoptotic cells (characterized by membrane blebbing and/or nuclear condensation/fragmentation) were determined by inverse phase contrast microscopy after 22 h, except for the assays containing SNP, where apoptosis was determined after 71 h.

 
Signalling pathways during NO-mediated apoptosis induction
In order to clarify whether NO-mediated apoptosis requires the induction of signalling molecules or whether a pre-existing apoptosis machinery is used, the peroxynitrite generator SIN-1 was added to transformed cells in the absence and presence of the protein synthesis inhibitor cycloheximide. As peroxynitrite-induced apoptosis was not hampered by the presence of high concentrations of cycloheximide (Figure 9Go), all the direct and indirect targets for the peroxynitrite-based signalling and execution pathway for apoptosis induction seemed to be expressed in the cells already before addition of SIN-1.



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  		Fig. 9. Apoptosis induction by peroxynitrite does not depend on ongoing protein synthesis. 15 000 transformed 208 F src3 cells were seeded in Costar 24 well tissue culture clusters and were treated with 1 mM SIN-1, 1 mM SIN-1 plus 50 µg/ml of the protein synthesis inhibitor cycloheximide, 50 µg/ml cycloheximide alone or left untreated. After 24 h the percentage of apoptotic cells was determined. All assays were performed in duplicate.

 
The many signalling pathways utilized during apoptosis induction can be roughly categorized in two major categories: (a) signalling pathways from membrane associated death receptors to execution caspases and (b) pathways involving activation of the mitochondrial permeability transition pore, mitochondrial depolarization, cytochrome c and ROS release from mitochondria, leading to the activation of execution caspases through several steps (61,62). To test for a potential involvement of mitochondria during NO-mediated apoptosis, the effect of the NO donor SNP was tested in src-transformed fibroblasts and in src-transformed fibroblasts that overexpressed Bcl-2. (Bcl-2 has been shown to prevent activation of the mitochondrial permeability transition pore (63).) As can be seen in Figure 10Go, addition of SNP caused mitochondrial depolarization and apoptosis in parallel in transformed cells. Both effects were blocked in bcl-2-overexpressing cells. These data show that mitochondrial depolarization plays a functional role in NO-mediated apoptosis induction.



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  		Fig. 10. NO-mediated apoptosis induction depends on mitochondrial depolarization. 15 000 transformed 208 F src3 fibroblasts transfected with either the pRc neo control plasmid (`neo') or the pRc neo plasmid containing the bcl-2 gene at the polylinker cloning site (`bcl-2') were seeded in Costar 24-well tissue culture clusters. Assays received either 0.5 mM SNP (`SNP') or no addition (`Control'). After 56 hours the percentages of apoptotic cells were either determined by morphological criteria (membrane blebbing and/or nuclear condensation/fragmentation) using inverted phase contrast microscopy (`a') or by staining with annexin (`b'), using the commercially available APO–AC staining kit (Sigma, Deisenhofen, Germany). In parallel assays, rhodamine staining (see Material and methods) was used to determine the percentage of cells with depolarized mitochondria (Delta-psi-negative mitochondria). Note that SNP induced mitochondrial depolarization in control cells and that bcl-2 prevented both mitochondrial depolarization and apoptosis induction. The difference of 10% positive cells between morphological criteria and annexin-positivity in bcl-2 overexpressing cells reflects the fact that annexin stains early membrane changes during apoptosis that precede mitochondrial depolarization, membrane blebbing and chromatin condensation.

 
When Spermine NONOate-treated (0.5 mM) and untreated 208 F src 3 cells were tested for capase-3 activity, 32% of Spermine NONOate-treated cells compared to 4% of control cells showed caspase-3 activity 7 h after addition of the NO donor. At the same time point, 37% of Spermine NONOate-treated cells showed morphological signs of apoptosis compared to 4.4% in the control. These data indicate that the NO-mediated apoptosis induction through peroxynitrite formation is executed by caspase-3.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our data show that NO donors like SNP, S-nitrosoglutathione and the diazeniumdiolates DEA NONOate, SpermineNONOate and Papa NONOate mediate apoptosis induction selectively in transformed fibroblasts compared with nontransformed fibroblasts. As all of these compounds release NO, but generate different residual moieties, the role of NO for apoptosis induction in transformed cells is established. In line with the conclusion that NO represents the critical molecule to mediate apoptosis induction, the kinetics of apoptosis mediated by the rapidly decaying substances DEA NONOate, Spermine NONOate, PAPA NONOate and S-nitrosoglutathione showed an early onset and were rather fast, compared to the kinetics seen for the slowly decaying SNP (46) with a long delay seen in the apoptosis kinetics.

Cell death induced in transformed fibroblasts after the addition of NO donors was characterized as apoptosis, as it showed the classical morphological signs of apoptosis like membrane blebbing, chromatin condensation and nuclear fragmentation. These morphological features have been shown to correlate with DNA strand breaks detectable by the TUNEL reaction. In addition, apoptotic cells were characterized by annexin staining, reflecting the exposure of phosphatidylserine to the outside of the cell membrane. NO-mediated apoptosis was characterized by mitochondrial depolarization and caspase-3 activation, indicating an involvement of mitochondria in the apoptotic process and characterizing the underlying signalling pathway as type II, according to a recent suggestion for classification of type I (death receptor-mediated apoptosis) and type II (mitochondria-controlled apoptosis) (61,62). The functional relevance of mitochondrial depolarization was overt as bcl-2 overexpression prevented mitochondrial depolarization as well as apoptosis. Bcl-2 has been shown to act directly at the mitochondrial permeability transition pore (63). After extended exposure to the NO donor, apoptotic cells changed their appearance towards the features of secondary necrosis. This may be due to a default in the energy supply or through direct damage of cellular membranes.

Sensitivity to NO-mediated apoptosis is strictly correlated to the transformed state of fibroblasts, as cells with a constitutively activated src oncogene or an induced ras oncogene are sensitive to apoptosis induction by the NO donor SNP, whereas nontransformed parental cells, parental cells harbouring, but not expressing an inducible ras oncogene, or revertants that have lost the transformed phenotype are not sensitive. These findings demonstrate a direct link between oncogene activity, transformed phenotype and sensitivity to NO-mediated apoptosis induction. This causal relationship may be the clue for selective destruction of transformed cells by natural antitumor systems that generate NO. As discussed below, the crucial biochemical parameter that defines the selective sensitivity of transformed fibroblasts for NO-mediated apoptosis seems to be their extracellular superoxide anion generation. Extracellular superoxide anion generation by transformed fibroblasts is in accordance with recent reports that demonstrated that ras activation causes rac-dependent activation of a membrane associated NADPH oxidase, whose flavocytochrome component channels electrons through the membrane and causes their interaction with extracellular molecular oxygen, resulting in superoxide anion generation (38). As the activity of src is connected to that of ras (41), it was to be expected that src-transformed fibroblasts show the same extracellular superoxide anion generation as ras-transformed cells. Extracellular superoxide anion generation by src and ras-transformed 208 F fibroblasts as used in this study has been recently directly confirmed in a functional assay which is based on the conversion of hydrogen peroxide (the dismutation product of superoxide anions) to hypochlorous acid by myeloperoxidase (MPO), followed by hydroxyl radical generation through HOCl/superoxide anion interaction according to the formula (64):

Hydroxyl radicals function as ultimate apoptosis inducers in this system.

The functional role of extracellular superoxide anion generation by transformed fibroblasts for their sensitivity to NO-mediated apoptosis induction was proven, as NO-mediated apoptosis was inhibited when either superoxide anion generation was blocked by apocynin (an inhibitor of NADPH oxidase) (51–53) or when superoxide anions were scavenged by extracellular SOD or by SOD mimetics like MnTBAP or MnTMPy. As SOD cannot enter cells (in contrast to certain low molecular weight SOD mimetics) (48,49), inhibition of NO-mediated apoptosis by SOD addition defines the extracellular location of superoxide anion generation by transformed cells. Inhibition of NO-mediated apoptosis induction by apocynin indicates that NADPH oxidase is the source for superoxide anions. This finding together with the extracellular location of superoxide anion generation point to the activity of a membrane associated NADPH oxidase, in line with the findings by Irani et al. (38). As seen in our specificity control experiments, the inhibitory effect of the two SOD mimetics MnTBAP and MnTMPy may not only be due to their superoxide anion scavenging potential, but also due to their ability to decompose peroxynitrite, the reaction product between superoxide anions and NO. However, both effects of these compounds allow the same conclusion, i.e. that superoxide anions are responsible for NO-mediated apoptosis induction through their interaction with NO and subsequent peroxynitrite formation.

The functional role of extracellular superoxide anions for NO-mediated apoptosis is not only proven by inhibition of NO-mediated apoptosis induction through inhibition of superoxide anion formation or scavenging of superoxide anions, but also by the positive effect of the superoxide anion-generating xanthine oxidase/xanthine on NO-mediated apoptosis induction in nontransformed cells in the presence of a variety of different NO donors. Whereas nontransformed cells only showed a minor apoptotic response to NO donors under control conditions (as they lack substantial superoxide anion generation, in contrast to oncogene-transformed cells (38,64)), supplementation with xanthine oxidase/xanthine as a source of superoxide anions rendered them sensitive for NO mediated apoptosis. As this reaction was blocked by SOD, the functional role of superoxide anions for NO-mediated apoptosis induction in this system is also demonstrated.

Our data show that superoxide anion-generating transformed cells are sensitive to NO-mediated apoptosis which can be blocked through inhibition of superoxide anion generation or scavenging superoxide anions. In contrast, nontransformed cells that lack sufficient extracellular superoxide anion generation are not responsive to NO-mediated apoptosis, but can be rendered sensitive when an exogenous source for superoxide anions is supplied. Therefore, NO does not seem to exhibit direct apoptosis inducing potential for fibroblasts; rather the reaction product between NO and superoxide anions seems to be the responsible apoptosis inducer. This conclusion is in line with several other findings like (a) inhibition of lipid peroxidation by NO (33,34); (b) inactivation of caspases by NO (36); and (c) the inability of NO to trigger the mitochondrial permeability transition pore (37). As superoxide anions and NO form peroxynitrite in a diffusion controlled reaction (6–8):

Hydroxyl radicals function as ultimate apoptosis inducers in this system.

The functional role of extracellular superoxide anion generation by transformed fibroblasts for their sensitivity to NO-mediated apoptosis induction was proven, as NO-mediated apoptosis was inhibited when either superoxide anion generation was blocked by apocynin (an inhibitor of NADPH oxidase) (51–53) or when superoxide anions were scavenged by extracellular SOD or by SOD mimetics like MnTBAP or MnTMPy. As SOD cannot enter cells (in contrast to certain low molecular weight SOD mimetics) (48,49), inhibition of NO-mediated apoptosis by SOD addition defines the extracellular location of superoxide anion generation by transformed cells. Inhibition of NO-mediated apoptosis induction by apocynin indicates that NADPH oxidase is the source for superoxide anions. This finding together with the extracellular location of superoxide anion generation point to the activity of a membrane associated NADPH oxidase, in line with the findings by Irani et al. (38). As seen in our specificity control experiments, the inhibitory effect of the two SOD mimetics MnTBAP and MnTMPy may not only be due to their superoxide anion scavenging potential, but also due to their ability to decompose peroxynitrite, the reaction product between superoxide anions and NO. However, both effects of these compounds allow the same conclusion, i.e. that superoxide anions are responsible for NO-mediated apoptosis induction through their interaction with NO and subsequent peroxynitrite formation.

The functional role of extracellular superoxide anions for NO-mediated apoptosis is not only proven by inhibition of NO-mediated apoptosis induction through inhibition of superoxide anion formation or scavenging of superoxide anions, but also by the positive effect of the superoxide anion-generating xanthine oxidase/xanthine on NO-mediated apoptosis induction in nontransformed cells in the presence of a variety of different NO donors. Whereas nontransformed cells only showed a minor apoptotic response to NO donors under control conditions (as they lack substantial superoxide anion generation, in contrast to oncogene-transformed cells (38,64)), supplementation with xanthine oxidase/xanthine as a source of superoxide anions rendered them sensitive for NO mediated apoptosis. As this reaction was blocked by SOD, the functional role of superoxide anions for NO-mediated apoptosis induction in this system is also demonstrated.

Our data show that superoxide anion-generating transformed cells are sensitive to NO-mediated apoptosis which can be blocked through inhibition of superoxide anion generation or scavenging superoxide anions. In contrast, nontransformed cells that lack sufficient extracellular superoxide anion generation are not responsive to NO-mediated apoptosis, but can be rendered sensitive when an exogenous source for superoxide anions is supplied. Therefore, NO does not seem to exhibit direct apoptosis inducing potential for fibroblasts; rather the reaction product between NO and superoxide anions seems to be the responsible apoptosis inducer. This conclusion is in line with several other findings like (a) inhibition of lipid peroxidation by NO (33,34); (b) inactivation of caspases by NO (36); and (c) the inability of NO to trigger the mitochondrial permeability transition pore (37). As superoxide anions and NO form peroxynitrite in a diffusion controlled reaction (6–8):

peroxynitrite seems to represent the functional apoptosis inducer during NO-mediated apoptosis induction. This idea is verified as the peroxynitrite scavengers ebselen (15) and FeTPPS (17,18) inhibited NO-mediated apoptosis in transformed fibroblasts. The specificity of the peroxynitrite decomposition catalyst FeTPPS was directly confirmed in our study: FeTPPS inhibited peroxynitrite-dependent apoptosis but did not scavenge hydrogen peroxide or superoxide anions. The inhibitory effect of ebselen was due to its interaction with peroxynitrite and not due to a theoretically conceivable interaction with hydrogen peroxide, as catalase had no inhibitory effect but rather stimulated SNP-mediated apoptosis. The stimulatory effect of catalase is explained as removal of hydrogen peroxide (derived from spontaneous dismutation of transformed cell-derived superoxide anions) prevents consumption of NO through the interaction of NO with hydrogen peroxide as described by Nappi and Vass (65). A detailed study of this interaction will be presented elsewhere (72).

Generation of peroxynitrite during NO-mediated apoptosis induction in transformed cells was also demonstrated through the detection of nitrotyrosine on transformed cells treated with the NO donor SpermineNONOate. As peroxynitrite readily nitrates tyrosine residues, nitrotyrosine is indicative for the generation of peroxynitrite. Detection of nitrotyrosine on cells that had not been fixed and permealized is indicative for extracellular peroxynitrite formation, a finding in line with extracellular superoxide anion generation.

In line with the conclusion that NO-mediated apoptosis depended on peroxynitrite generation, chemically pure peroxynitrite and the peroxynitrite generator SIN-1 induced apoptosis independent of the transformed state of the target cells. Again, these reactions were completely blocked by FeTPPS. Therefore, peroxynitrite seems to represent a nonselective, ultimate apoptosis inducer. The selectivity of NO-mediated apoptosis induction in transformed cells therefore seems to depend on selective generation of peroxynitrite (through extracellular superoxide anion generation by transformed cells) rather than on a selective action of peroxynitrite. The apoptosis inducing potential of peroxynitrite has been demonstrated in many other cellular systems (21,22,27). It correlates with the ability of peroxynitrite to cause lipid peroxidation (14,33) and to activate the mitochondrial permeability transition pore (37).

The biochemical events between extracellular peroxynitrite formation and mitochondrial depolarization are enigmatic, so far. Lipid peroxidation, followed by intracellular local glutathione depletion, sphingomyelinase activation, ceramide formation and interaction of ceramides with the mitochondrial permeability transition pore might be one of the conceivable signalling pathways (24). However, this hypothesis still awaits experimental verification or falsification, as only the role of mitochondria for peroxynitrite-induced apoptosis is established so far. Peroxynitrite may exert its biological effects through oxidation, nitration, hydroxylation or nitrosylation (for review see ref. 24). Depending on the pH and the concentration of carbon dioxide, peroxynitrite may undergo a variety of chemical reactions, which have a strong impact on its reactivity (for review of the complex network of peroxynitrite-related reactions see ref. 24).

When treatment of nontransformed cells with NO was continued for >2 days and at high concentrations of SNP, the marginal apoptosis induction was eventually increasing, but appeared >20 h later than observed in transformed cells (data not shown). This effect was not seen at high cell densities and it was inhibitable by SOD. This indicates that the difference between transformed and nontransformed cells with respect to superoxide anion generation is not absolute, but that nontransformed cells produce much less superoxide anions than their transformed descendants. The low concentration of superoxide anions generated by nontransformed cells may be related to the regulation of proliferation (66). Superoxide anion production during stimulation of proliferation therefore seems to occur at a much lower level than during constitutive generation by transformed cells.

Apoptosis induction by peroxynitrite that has been primarily generated extracellularly through the interaction of NO with superoxide anions has been demonstrated here for glutathione competent fibroblasts. As NO readily passes cellular membranes (2), and as mitochondria are known as a source of intracellular superoxide anion generation, intracellular peroxynitrite generation may be assumed to occur in parallel, irrespective of the transformed state of the cells. As glutathione-competent nontransformed cells are not substantially affected by NO donors, peroxynitrite that is most likely generated intracellularly, seems to be effectively counterbalanced. In accordance with the work of Arteel et al. and Sies et al. (15,16), intracellular glutathione in cooperation with glutathione peroxidase may be responsible for protection of cells from the effects of intracellular peroxynitrite formation. Ongoing work shows that glutathione-depleted nontransformed cells can be rendered sensitive to NO-mediated apoptosis induction (Steinmann et al., in preparation).

Extracellular superoxide anion generation was characterized in our study as the responsible oncogene-dependent biochemical feature of transformed cells that caused their sensitivity for NO-mediated apoptosis induction. Extracellular superoxide anions thereby seemed to cause the conversion of the nontoxic radical NO into the ultimate apoptosis inducer peroxynitrite. This generation of a toxic signal, based on the interaction of a nontoxic, far ranging signalling molecule (i.e. NO) with a nontoxic, short-ranging target cell derived radical (i.e. the superoxide anion) (67,68), represents an efficient principle to direct the generation of apoptosis-inducing molecules directly to the desired site, i.e. the membrane of the transformed target cell. The novel part of this scenario is that not the attacking NO radical but superoxide anions from the target cell determine specificity, efficiency and site of action of the apoptotic signal. Application of this principle by NO-generating effector cells like macrophages (69), granulocytes (70), TGF-beta-treated fibroblasts (71) and others seems be one of their signalling pathways for selective destruction of transformed cells during the control of oncogenesis.


   Notes
 
3 To whom correspondence should be addressed Email: tgfb{at}ukl.uni-freiburg.de Back


   Acknowledgments
 
This work was supported by the `Wissenschaftliche Gesellschaft Freiburg' and the `Dr. Mildred Scheel Stiftung für Krebsforschung' (Grant 10-1177-Ba3).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 24, 2001; revised February 21, 2002; accepted February 22, 2002.


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