From Unité 571, Centre National de la Recherche
Scientifique, Bâtiment 430, Université Paris-Sud, France,
¶ Unité 350, Institut National de la Santé et de la
Recherche Médicale, Institut Curie, Orsay, France, and the
Department of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Ribonucleotide reductase is essential for DNA synthesis in cycling cells. It has been previously shown that the catalytically competent tyrosyl free radical of its small R2 subunit (R2-Y·) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate a loss of R2-Y· induced either by ·NO or peroxynitrite in vitro. The ·NO effect is reversible and followed by an increase in ferric iron release from mouse protein R2. A similar increased iron lability in radical-free, diferric metR2 protein suggests reciprocal stabilizing interactions between R2-Y· and the diiron center in the mouse protein. Scavenging of R2-Y· by peroxynitrite is irreversible and paralleled to an irreversible loss of R2 activity. Formation of nitrotyrosine and dihydroxyphenylalanine was also detected in peroxynitrite-modified protein R2. In R2-overexpressing tumor cells co-cultured with activated murine macrophages, scavenging of R2-Y· following NO synthase II induction was fully reversible, even when endogenous production of peroxynitrite was induced by triggering NADPH oxidase activity with a phorbol ester. Our results did not support the involvement of peroxynitrite in R2-Y· scavenging by macrophage ·NO synthase II activity. They confirmed the preponderant physiological role of ·NO in the process.
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INTRODUCTION |
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Nitric oxide is a cell-permeable small radical molecule synthesized in diverse organisms by ·NO synthase enzymes (NOS),1 which are P450 self-sufficient hemoproteins using L-arginine and dioxygen as substrates (reviewed in Ref. 1). In mammals, physiological functions have been attributed to NOS activities in cardiovascular, neural, gastrointestinal, genitourinary and immune systems (2, 3). So called "neuronal" NOS (NOS I) and "endothelial" NOS (NOS III), usually constitutively expressed, are transiently activated by elevation of intracellular Ca2+ concentration (4). A cytokine-inducible NOS (NOS II) is transcriptionally regulated, producing ·NO at basal Ca2+ levels for up to several days (4). Among cytotoxic and pathophysiological functions, NOS II was rapidly recognized to support a nonspecific antiproliferative activity capable of limiting the growth of invading agents, including viruses, bacteria, parasites, and tumor cells, consistent with the wide distribution of the isoform in different cell types (3, 4).
Cytostatic antitumor effector mechanisms of macrophages have been shown
to rely on induction of NOS II activity, which severely alters energy
production, iron metabolism, and DNA synthesis in tumor target cells
(5, 6). There is still a debate about the identity of the cytotoxic
nitrogen oxide(s) acting under physiological conditions. For instance,
both ·NO and peroxynitrite ONOO have been reported
to inhibit mitochondrial respiratory chain, and to regulate iron
regulatory protein 1 function, the keystone in cell iron homeostasis
(5, 7-10). Ribonucleotide reductase (RR) inhibition by a NOS II
product, probably ·NO, has been demonstrated in tumor cells
co-cultured with macrophages (11, 12), or in tumor cells stimulated
with cytokines for endogenous NOS II induction (13). Previous studies
also indicated that RR inhibition supported an early,
NOS-dependent inhibition of DNA synthesis in tumor cells
(11, 12).
RR enzymes have been classified in at least four different groups (14, 15). They catalyze the reduction of ribonucleotides, providing the cells with deoxyribonucleotides required for DNA synthesis. The small homodimeric R2 subunit of class I RR in mammals and some prokaryotes like Escherichia coli harbors a free radical localized on a tyrosyl residue (R2-Y·) (16-18). X-ray structures of mouse and E. coli protein R2 have been determined (19, 20). Production of R2-Y· by a one-electron oxidation of Y122 (E. coli) or Y177 (mouse), and stability of the existing radical are dependent on a proximal nonheme diiron center which interacts electromagnetically with R2-Y· (21). The radical/metal center of protein R2 is required for activity, since it triggers a radical-driven reduction of the substrate through a long-range electron transfer between protein R2 and protein R1, the other homodimeric subunit of RR containing binding sites for substrates and allosteric effectors (see Refs. 19 and 20, and references therein). In different models, R2-Y· has been identified as a target for nitrogen oxides derived from NOS II activity (12, 22), but the chemical species involved have not been clearly identified. There is good evidence that ·NO participates in the reaction since HbO2, which binds ·NO, inhibits R2-Y· quenching induced by NOS II activity or ·NO donors (22-24). Also, a reversible radical-radical coupling reaction of ·NO with R2-Y· has been proposed to account for the disappearance of the tyrosyl free radical of E. coli protein R2 induced by thionitrites (24). However, so far, this experiment has not been reproduced with authentic ·NO gas under anaerobic conditions, to demonstrate unambiguously the role of ·NO in R2-Y· scavenging. Moreover, little is known about the reactivity of other nitrogen oxides toward RR.
In the present study, we compared the effects of ·NO and
ONOO against murine protein R2 in vitro. We
then used these data in order to identify the nitrogen oxide
responsible for R2-Y· quenching in co-cultures of macrophages
and tumor cells. Our results indicate that ONOO
is not a
physiological effector of the loss of R2-Y·.
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EXPERIMENTAL PROCEDURES |
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Reagents and Plasmids--
·NO gas was purchased from Air
Liquide (Moissy-Cramayel, France).
Isopropyl--D-thiogalactopyranoside was from Eurogentec
(Angers, France). [5-3H]CDP with a specific activity of
655 GBq/mmol was purchased from Amersham-France (Les Ulis). All other
reagents were from Sigma (L'isle d'Abeau Chesnes, France). Plasmids
pETM2 (25) and pVNR2 (26) were kindly given by Prof. L. Thelander
(University of Umeå, Umeå, Sweden) and Prof. M. Fontecave
(Université Joseph Fourier, Grenoble, France), respectively.
Expression of Recombinant Mouse R2 and R1 Protein--
Bacterial
protein R2 was prepared from an E. coli K12 strain
expressing the pVNR2 plasmid, using a purification procedure already
published (27). Recombinant mouse R2 protein was prepared as described
previously (25), with slight modifications. The mouse protein R2
expression vector pETM2 was transfected by electroporation into the
E. coli BL21(DE3)pLysS (CamR) strain. The resulting strain was named M2pLysS. For each preparation, 3 liters of LB medium supplemented with carbenicillin (50 µg/ml) and chloramphenicol (27 µg/ml) were infected by an overnight culture (1/100 dilution), and
shaken at 200 rpm at 37 °C. When absorbance at 590 nm was between
0.4 and 0.6, expression of mouse protein R2 was induced by addition of
isopropyl-1-thio--D-galactosidase, 0.5 mM.
After 4 h, cultures were chilled on ice and centrifuged at
2500 × g for 15 min at 4 °C, then resuspended in 30 ml of a 50 mM Tris·HCl buffer, pH 7.6, containing 1 mM EDTA. The resulting suspension was frozen in liquid
nitrogen and stored at
80 °C. Recombinant mouse protein R1 was
overexpressed in a baculovirus system and purified by affinity
chromatography as described in Ref. 28.
Production of Active and Radical-free Protein R2--
Presence
of the pLysS plasmid, encoding the T7 lysozyme, allowed spontaneous
lysis of M2pLysS bacteria during thawing. Purification of apoR2 was
performed as described in Ref. 25. The purity of the material was
checked by electrophoresis on a 10% SDS-polyacrylamide gel.
Reconstitution of the diferric center and regeneration of the tyrosyl
radical was done using an anaerobic procedure already described (25).
The average radical content per monomer was measured by EPR
spectroscopy. Alternatively, the concentration of R2-Y· was
determined from the difference in absorbance at 417 nm between active
protein R2 and metR2 ( = 3256 M
1·cm
1). Iron content was
evaluated by prolonged incubation of protein R2 with hydroxyurea (4 mM), which reduces and releases iron from the protein.
Released iron was quantified by desferrioxamine (
= 2500 M
1·cm
1 at 430 nm, see below). Active R2 protein contained 0.65 ± 0.06 radical
and 2.0 ± 0.08 iron atoms per monomer (mean ± S.E.,
n = 4). Protein metR2 was prepared by treatment of
native protein R2 at ambient temperature with 4-propoxyphenol in
stoichiometric equivalent versus the radical
concentration.
Saturated Solution of ·NO Gas and Synthesis of
Peroxynitrite--
·NO gas was allowed to bubble gently through
1 M NaOH before passing into an argon-deoxygenated
Tris·HCl buffer, 50 mM, pH 7.6, for 1 h. Nitric
oxide concentration in the buffered solution was measured by the
oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, 25 mg/ml, using = 15,000 M
1
cm
1 at 750 nm (29). Alternatively, oxidation of
oxyhemoglobin into methemoglobin was also used, according to Ref. 30,
with similar results.
Effect of ·NO and ONOO- on the Free Radical Content of Protein R2-- A reactivated sample of murine protein R2 was deaerated with an argon flux for 1 h at 0 °C, in 50 mM Tris·HCl, pH 7.6. Aliquots were transferred anaerobically into 1.5-ml microtubes kept in ice. Increasing volumes of a ·NO-saturated solution were added to a microtube series. After vortexing, 200 µl of each sample was transferred immediately and anaerobically into EPR quartz tubes and frozen in liquid nitrogen. Twenty microliters were used for the RR activity assay. The 250-µl aliquot remaining in each microtube was degassed for 45 min at 0 °C to eliminate ·NO, then processed as described above for analysis of R2-Y·.
Peroxynitrite was added to protein R2 in 50 mM Tris·HCl buffer, pH 7.6, under aerobic conditions. The concentration of R2-Y· was immediately measured by EPR spectroscopy and R2 activity was evaluated from a 20-µl aliquot, as described above. The pH of the buffer was not modified after addition of ONOOEPR Quantification of Tyrosyl Free Radical Concentration-- Solutions of protein R2 or L1210-R2 cell pellets were analyzed for R2-Y· content at 77 K using a Varian E109 spectrometer, calibrated with the radical 1,1-diphenyl-2-picrylhydrazyl exhibiting an EPR signal centered at g = 2.0036, which was also used after double integration to quantify the tyrosyl free radical concentration in untreated, ·NO- or peroxynitrite-treated R2 protein samples. The amplitude of the trough at g = 1.994 was used to quantitate R2-Y· in cell samples, due to the presence of a partially overlapping strong signal from nitrosyl iron complexes at g values of 2.041 and 2.015. Microwave power was 10 mW, field modulation frequency was 9.18 GHz, and modulation amplitude was 1 millitesla.
Measurement of RR Activity-- RR activity was assayed following the conversion of [3H]CDP into [3H]dCDP. Magnesium chloride (15 mM), 100 mM KCl, 10 mM dithiothreitol, 3 mM ATP, 200 µM CDP, 74 kBq of [5-3H]CDP, and a 5-fold molar excess of protein R1 in 50 mM Tris·HCl buffer, pH 7.6, were mixed with 20 µl of protein R2 (0.5-2 µM) in a final volume of 90 µl. Samples were incubated at 37 °C for 10 min. Control experiments have shown that the reaction was linear over the 10-min assay. The reaction was stopped by heating for 2 min. CDP and dCDP were then dephosphorylated for 2 h at 37 °C using 200 µg/ml rattlesnake venom from Crotalus adamanteus. After boiling and centrifugation, supernatants were analyzed by radio-high performance liquid chromatography as described elsewhere (13). Specific activity was expressed as nanomoles of dCDP formed per minute per mg of protein R2.
Iron Release from Mouse Protein R2--
A volume of 500 µl of
20 µM protein R2 was deoxygenated with argon at room
temperature in a quartz cuvette closed with a rubber septum. After
1 h, a UV visible absorption spectrum of the protein was recorded.
Concentration of the R2-Y· radical was determined from the
absorbance at 417 nm (see above). ·NO or 4-propoxyphenol (used
to obtain metR2) were introduced with an argon-degassed syringe, in a
ratio [·NO]:[R2-Y·] of 8 or
[4-propoxyphenol]:[R2-Y·] of 1. Then, 10 µl of a
deoxygenated solution of desferrioxamine were added to a final
concentration of 1 mM to monitor ferric iron release by the
Fe3+-desferrioxamine complex absorbance at 430 nm ( = 2500 M
1·cm
1),
as previously reported (32). In these experiments, the free radical
content of untreated control protein R2 decreased from 0.65 to
approximately 0.5 radical per monomer after the 1-h flush with
argon.
Co-culture of Macrophages and Tumor Cells--
Peritoneal
macrophages elicited by intraperitoneal injection of 50 µg of
trehalose dimycolate to (C57BL/6xDBA/2)F1 mice were obtained as described (33, 34). The R2-overexpressing L1210-R2 cell
line was cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum, as described previously (12). Macrophages (35 × 106) were allowed to adhere for
3 h in 14-cm culture dishes (Nunclon), then washed three times
with phosphate-buffered saline. Lipopolysaccharide was added at 100 ng/ml in 20 ml of fresh culture medium to induce expression of NOS II
activity. After 4 h, macrophages were washed again with
phosphate-buffered saline and L1210-R2 cells in exponential growth
phase were added in RPMI 1640 medium containing 5% fetal calf serum,
at a macrophage:tumor cell ratio of 0.55. Cells were co-cultured for
5 h, with lipopolysaccharide. In some experiments, TPA (2 nM) was added with tumor target cells to trigger
O2 and H2O2 production by macrophage
NADPH oxidase activity (33). Nonadherent L1210-R2 cells were then
gently harvested and packed by centrifugation into an EPR quartz tube
(4 mm outer diameter), as previously mentioned (12). A first
determination of R2-Y· concentration in the cell pellet was
obtained. Cells were then thawed and R2-Y· was reactivated with
a solution of 100 mM dithiothreitol and 100 µM ammonium iron(II) sulfate added in a small volume,
representing approximately 1/10 of the cell pellet, according to Ref.
35. Cells were mixed with a glass rod, air bubbles were introduced with
a syringe, and the mixture was incubated at 37 °C for 10 min. Cells
were then centrifuged at 500 × g for 10 min and the reactivation procedure was repeated once, except that iron and the
reductant were not added. Finally, cells were frozen for a second
determination of R2-Y· content. In preliminary experiments, this
procedure was shown to allow optimal regeneration of R2-Y·.
Detection of Dihydroxyphenylalanine Residues-- Protein R2 from E. coli treated with various concentrations of peroxynitrite was first desalted on a Bio-Gel polyacrylamide column (Bio-Rad). The protein was then transferred onto a nitrocellulose membrane by dot-blotting. Detection of catechol structures was performed according to Ref. 36. The membrane was immersed in a 2 M potassium glycinate solution, pH 10, containing 0.24 mM nitro blue tetrazolium, and incubated in the dark for 45 min. Membranes were irreversibly stained by immersion at 4 °C in 0.1 M sodium borate, pH 7.0.
Immunodetection of Nitrotyrosine-- Mouse or E. coli protein R2 treated with ·NO or ONOO was transferred onto a nitrocellulose membrane by dot blotting. The membrane was then blocked for 1 h with 3% skim milk in phosphate-buffered saline. A mouse monoclonal antinitrotyrosine (Upstate Biotechnologies, Lake Placid, NY) was added at a dilution of 1:2000, and the membrane was incubated overnight at 4 °C in the blocking buffer. After three washings, a secondary goat anti-mouse IgG-horseradish peroxidase-linked antibody was added at a 1:5000 dilution and incubated with the membrane for 90 min in the blocking buffer, at room temperature. Blots were revealed by enhanced chemiluminescence using a kit from Amersham.
Protein Determination-- Protein concentration was measured using the Bradford protein assay (Bio-Rad). Bovine serum albumin was taken as a reference.
Nitrite and Citrulline Assays-- Nitrite concentration in the co-culture medium of macrophages and L1210-R2 tumor cells was measured with the Griess reagent, as described previously (22). Citrulline production from L-[U-14C]arginine was measured over 5 h in macrophage cultures (1 × 106/ml/well), as reported elsewhere (13).
Determination of Hydrogen Peroxide and Peroxynitrite Production
by Macrophages--
Macrophages were cultivated in 24-well culture
plates (1 × 106/well), washed, activated with
lipopolysaccharide for 4 h, and stimulated with TPA, as described
above. Hydrogen peroxide production was measured in 500 µl of Hanks'
balanced salt solution by the peroxidase-catalyzed oxidation of phenol
red (34). Peroxynitrite synthesis was estimated from the superoxide
dismutase- and aminoguanidine-inhibitable oxidation of DHR (31), in 500 µl of serum-free RPMI 1640 culture medium without phenol red. Two
hours after TPA addition, production of rhodamine was measured at 500 nm ( = 74,500 M
1·
cm
1).
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RESULTS |
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Effects of ·NO on Mouse Protein R2-- Previous studies have shown that the free radical of the small R2 subunit of RR was scavenged by chemical ·NO donors in the presence of oxygen, allowing ·NO autoxidation to higher nitrogen oxides. The present experiments were conducted under anaerobic conditions to study the effect of authentic ·NO gas. Other experiments with peroxynitrite were performed in the presence of oxygen, since identical results on R2-Y· were obtained with or without oxygen in preliminary studies. The tyrosyl free radical of mouse protein R2 was quenched similarly by ·NO and by peroxynitrite (Fig. 1). The EPR spectrum of partially quenched R2-Y· recorded at 77 K in the presence of either nitrogen oxide did not reveal any modification of the radical profile, except its reduced amplitude. The disappearance of R2-Y· induced by ·NO was concentration-dependent for both ·NO (Fig. 2) and protein R2 (not shown). The apparent stoichiometry of the reaction was clearly higher than 1:1. For instance, complete loss of the R2-Y· EPR signal occurred at a ratio [·NO]:[R2-Y·] estimated to be 8.5 ± 1.0, from three independent experiments. The reaction was fast, going to completion within the time required to mixture R2 with ·NO and to transfer the solution into EPR tubes (about 30 s). The reaction was also reversible. If ·NO was purged from the solution with argon, R2-Y· reappeared in samples kept carefully at 0 °C to minimize iron loss (see below). With an 8-fold excess of ·NO, the recovery was nearly complete and reproducible (81.6 ± 6.0% of control, n = 3). The EPR profile of the recovered radical was identical to a native R2 control (not shown). Fig. 2 also shows that activity of protein R2 was not significantly reduced by ·NO. This result was unexpected, since the tyrosyl free radical quenched by ·NO is required for R2 activity. We therefore suspected that conditions used for assaying R2 activity (10 mM DTT, O2, and a 4.5-fold dilution of the sample) might have promoted the regeneration of R2-Y·. In fact, we observed in one control experiment that, after it had been totally quenched by ·NO, R2-Y· reappeared by 60% within 1 min under RR assay conditions.
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Effects of ONOO on Mouse Protein
R2--
Peroxynitrite was added to protein R2 in the presence of
oxygen. A concentration-dependent destruction of
R2-Y· by ONOO
was observed between 100 and 400 µM (Fig. 5). On a molar
basis, peroxynitrite was approximately 40 times less efficient than
·NO under anaerobiosis (IC50 = 124.7 ± 10.8, n = 4). The phenomenon was independent of the
concentration of R2-Y· in the range 0.5 to 4.0 µM
(not shown), suggesting a reaction limited by peroxynitrite
instability. Results from reverse-order experiments (addition of
ONOO
allowed to decompose before adding protein R2)
confirmed that peroxynitrite was the effector (radical content after
400 µM ONOO
: 79.3 ± 2.3% of control,
n = 3). Attempts to regenerate R2-Y· in
ONOO
-treated R2 protein were unsuccessful, even after
addition of 10 mM DTT and 10 µM
Fe2+ to reconstitute the radical/metal center (Fig. 5,
inset). Furthermore, no regeneration of R2-Y· was
observed when a submaximal concentration of peroxynitrite (200 µM) was used. Thus, destruction of R2-Y· by
peroxynitrite was irreversible, contrasting with the reversible quenching of the radical by ·NO. Peroxynitrite also inactivated
R2 activity. There was a good parallelism between loss of R2-Y·
and loss of activity, suggesting a causal relationship. However, treatment of radical-free, iron-free apoR2 protein with 400 µM peroxynitrite prevented subsequent formation of the
metal/radical center (not shown). Thus, R2-Y· was not the only
target for ONOO
in the protein. Formation of active R2
protein was observed in control reverse-order experiments with apoR2
protein. Since peroxynitrite has been reported to induce nitration and
hydroxylation of aromatic amino acids, we searched for the presence of
nitrotyrosine and dihydroxyphenylalanine in peroxynitrite-modified R2
proteins, using immunostaining and colorimetric assays, respectively. A concentration-dependent formation of nitrotyrosine was
detected in mouse and E. coli R2 proteins incubated with
inactivating concentrations of peroxynitrite (Fig.
6). The mouse subunit was less nitrated than the protein from E. coli. Reverse-order treatment was
not effective, nor was incubation of protein R2 with a high
concentration of ·NO ([·NO]:[R2-Y·] = 25:1).
Dihydroxyphenylalanine structures were detected in bacterial R2 protein
incubated with 100-400 µM (Fig.
7). The mouse protein R2 was not
significantly colored using this technique.
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Quenching of R2-Y· in Tumor Cells by NOS II
Activity--
It was concluded from these experiments in
vitro that effects of ·NO and ONOO on mouse
protein R2 could be clearly distinguished from each other on the basis
of the reversibility of R2-Y· quenching. Scavenging of the
radical by ·NO is spontaneously reversible and apoR2 protein
that forms thereafter, upon prolonged incubation of protein R2 with
·NO, can be easily regenerated into active R2 by reintroduction of the metal/radical center. By contrast, when incubated with peroxynitrite as active protein or apoprotein, the R2 subunit undergoes
irreversible modifications that are not compatible with the existence
of R2-Y·. This criteria of reversibility was therefore used in
cell culture experiments as a probe to identify the nature of the
nitrogen oxide derived from NOS II activity that induced R2-Y·
scavenging in tumor cells. Effector cells were murine peritoneal macrophages elicited by trehalose dimycolate in vivo and
stimulated in vitro with LPS to induce NOS II expression
(Fig. 8). TPA triggered in these cells a
respiratory burst that lasts for 90 to 120 min. It was monitored by
H2O2 production in arginine-free medium, to avoid trapping of O
2 by NO. The activity was dependent on TPA concentration (Fig. 8, inset) so that we were able to adjust
the production of reactive oxygen species to match NO synthesis, since highest yields of peroxynitrite are obtained when the two reactants NO
and O
2 are produced at similar rates (37, 38). In the absence
of TPA, the tyrosyl free radical EPR signal of L1210-R2 cells
co-cultured with macrophages was almost completely quenched within a
few hours, in agreement with previous reports (Fig.
9 and Ref. 12). The tumor cell pellet was
then thawed and supplemented with ferrous iron and DTT in a small
volume and finally incubated for 10 min at 37 °C to allow the
regeneration of the radical/metal center. Under these conditions,
R2-Y· was regenerated with a high efficiency (Figs. 9 and
10), supporting the previously proposed
hypothesis that ·NO was mainly, if not exclusively involved in
the reaction (12). In a second set of experiments, TPA was added at a
low concentration to trigger a production of
H2O2 equivalent to the production of nitrite
(Fig. 8A). Since nitrite flux is approximately one-third to
one-half of total ·NO synthesis in macrophage cultures and since
H2O2 results from the dismutation of two
molecules of O
2, it was considered that comparable rates of
nitrite and H2O2 production should reflect comparable rates of ·NO and superoxide anion synthesis,
favorable to peroxynitrite production during the first half of the
co-culture period. Measurement of citrulline production, as a mirror of
NO synthesis, was also performed in macrophage cultures incubated
without target cells in 24-well culture plates. Citrulline synthesis
was not modified by 2 to 8 nM TPA (not shown). However, the
nitrite:citrulline ratio in the absence of TPA, equal to 0.31 ± 0.04, increased to 0.49 ± 0.1 upon TPA addition (mean ± S.E., n = 4). The reason for this increase in nitrite
yield after TPA is obscure. It might be indicative of peroxynitrite
production, since at physiological pH, peroxynitrite decay produces
substantial amounts of nitrite (39). That peroxynitrite was effectively
produced during the first 2 h was confirmed by
TPA-dependent oxidation of DHR, inhibited by superoxide
dismutase and aminoguanidine (Fig. 8B). Increasing superoxide dismutase concentration up to 2000 units/ml did not improve
the inhibition of DHR oxidation, but combining superoxide dismutase
(2000 units/ml) and aminoguanidine was about 20% more inhibitory than
aminoguanidine alone (data not shown). The mean value for TPA-triggered
superoxide dismutase and aminoguanidine-inhibited rhodamine production
from 500 µM DHR was 0.41 ± 0.06 nmol/106 macrophages (n = 3). A significant
TPA-independent formation of rhodamine was also noticed, probably due
to metal-catalyzed oxidation of DHR, or NADPH oxydase-independent
production of O
2 (Fig. 8B). Under similar
experimental conditions, the mean yield of DHR (500 µM)
oxidation by synthesized peroxynitrite added as a bolus at 10, 20, or
40 µM was 8.3 ± 1.6%. If the yield of the reaction
in the presence of the continuous flux of peroxynitrite produced by the
macrophage culture is not very different from this estimated value, it
could indicate that macrophages might have produced approximately 5 nmol/106 cells of peroxynitrite during the first 2 h
of the co-culture. When L1210-R2 tumor cells were co-cultured with
macrophages stimulated with TPA, R2-Y· in tumor cells was
quenched efficiently (Fig. 9). Regeneration of the metal/radical center
with iron and DTT addition leads to a complete recovery of the free
radical (Fig. 10). Thus, no irreversible loss of R2-Y· was
observed in tumor target cells co-cultured with macrophages triggered
for peroxynitrite production, indicating that macrophage-derived peroxynitrite was not efficient against the small R2 subunit of RR
under our conditions.
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DISCUSSION |
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Previous reports demonstrated a radical-radical addition reaction between ·NO and phenoxyl free radicals of diverse origins, including small model compounds, oxidized free L-tyrosine, N-acetyl-L-tyrosine, or tyrosine-containing peptides, and tyrosyl free radicals in prostaglandin H synthase, in the R2 subunit of bacterial RR, and in photosystem II (24, 40-44). A unique and surprising property of this radical-radical combination reaction is its reversibility. It occurred independently of a particular radical environment, like metal centers frequently located in the vicinity of the radical in proteins, as it has been observed using simple free molecules (40). Results from the present work, demonstrating a very fast and reversible scavenging of mouse R2-Y· by ·NO, are in good agreement with the existence of a coupling reaction between the two radicals. Contrasting with previous studies (13, 24) these experiments were performed under anaerobiosis, precluding the formation of higher nitrogen oxides derived from ·NO autoxidation (NO2, N2O3). Reduction of the free radical by ·NO is excluded, since, among other factors, the reintroduction of the free radical in protein metR2 would have required a reductant like DTT and oxygen (18). In a few models, further oxidation of the EPR-silent Tyr-NO adduct gave rise to iminoxy radicals and, eventually, stable nitrotyrosine (42, 44). Neither nitrotyrosine nor iminoxy radicals were detected after incubation of mouse R2 with ·NO (data not shown), suggesting that, under our experimental conditions, the Tyr-NO adduct is not further oxidized.
A ratio ·NO:Tyr· close to 8:1 was necessary to observe
the complete scavenging of R2-Y·. This is significantly higher
than the 1:1 ratio expected from the coupling reaction. Although we
cannot exclude nonspecific binding of ·NO to poorly defined
sites, such as metal contaminants in the buffer or adsorbed onto the
protein, it is conceivable that this apparent high stoichiometry may
reflect a competition between the coupling reaction and the
dissociation of the Tyr-NO adduct. The rate constant for the coupling
reaction of ·NO with tyrosyl-centered radicals is very fast, of
the order of 1 × 109 M1
s
1 (41), but the rate of the reverse reaction has not
been determined. Yet, the weakness of the Tyr-NO complex is illustrated
by the very rapid reappearance of R2-Y· in experiments using a
short-lived chemical precursor of ·NO (24). A very rapid
dissociation of the coupled radicals is also supported by the absence
of a significant decrease in R2 activity, measured immediately after
addition of ·NO. Since R2-Y· is required for activity,
its scavenging by ·NO was expected to inactivate the enzyme. A
fast and spontaneous regeneration of R2-Y· within the first
seconds of the assay is therefore suspected. It may have been further
promoted by the 4.5-fold dilution of ·NO concentration before
assaying R2 activity and also by the reaction of ·NO with oxygen
during the test. Additional experiments are required to elucidate this
important question.
·NO also induces a 3-4-fold increase in the rate of ferric iron release from mouse protein R2, at room temperature. On a time scale basis, scavenging of R2-Y· by ·NO occurred first, followed by a slower release of iron from the protein. A direct effect of ·NO on the iron center of mouse R2 is unlikely, as it has been previously shown that the µ-oxo-bridged diferric center of hemerythrin and bacterial protein R2 do not react with NO (24, 45, 46). Reduction of Fe3+ has been previously shown to accelerate iron loss from mouse protein R2, since Fe2+ loosely binds to the protein (32). In experiments using bathophenanthroline to detect ferrous iron loss, we first thought that ·NO promoted R2 iron reduction, but careful analysis of this phenomenon later suggested that this was caused by a bathophenanthroline-driven artifactual reduction of free ferric iron by ·NO (data not shown). Consistent with the absence of R2 iron reduction by ·NO, a previous report indicated conversely a slow oxidation of reduced, diferrous protein R2 by ·NO (45). In fact, we observed that iron was released mostly as Fe3+ after ·NO treatment. It seemed therefore that the increased ferric iron lability of mouse protein R2 incubated with ·NO could be best rationalized as a direct consequence of ·NO coupling to R2-Y·. The absorption spectrum of protein R2 was modified upon addition of ·NO. Especially, the absorption bands of the metal center at 330 and 370 nm were significantly reduced, indicating that scavenging of R2-Y· by ·NO has modified the environment of the metal cofactor. A similar, featureless absorption spectrum is exhibited by a radical-free, diferric murine protein R2 (metR2), obtained by stoichiometric reduction of the tyrosyl free radical by propoxyphenol, in agreement with previously published data (47). Interestingly, the kinetics of ferric iron release by metR2 was much faster than iron loss from the native protein. The increased lability of the mouse metR2 iron site has been already described (48). Considering these results, we would like to propose the existence of unusual reciprocal interactions between R2-Y· and the metal cofactor in mouse protein R2, each component stabilizing the other one. As a consequence, loss of R2-Y·, either by reduction (metR2) or by coupling with ·NO, produces a radical-free protein with a very unstable metal center. Several arguments are in favor of this hypothesis. (i) Iron release is preceded by loss of R2-Y·. (ii) Kinetics of ferric iron release by metR2 and by R2 treated with ·NO are very similar. (iii) Recent reports have shown that, if the dinuclear iron center stabilizes the radical in protein R2, conversely, the presence of the radical strongly influences the reactivity and the stability of the metal core (47-49).(iv) The diferric center of E. coli protein metR2 is stable and ·NO did not labilize iron in this protein (24). In mouse, and probably in other mammals, formation of apoR2 protein is therefore promoted by a slow release of iron from active R2 (35) and by a faster one from met- or pseudomet-R2 proteins like nitrosylated R2. These results are consistent with earlier observations showing that mouse RR activity required a continuous supply of iron and oxygen (50), in order to regenerate the metal/radical center in apoR2 protein. They may also help to better understand the link observed between protein R2 levels and expression of ferritin, the cell iron-storage protein (51).
Peroxynitrite has been proposed to exert a key role in several
physiological and pathophysiological processes (reviewed in Ref. 52).
Peroxynitrite is formed from the reaction of superoxide anion with
·NO. The rate of the reaction (k = 6.7 × 109 M1 s
1) is fast
enough to compete with the coupling of R2-Y· with
·NO (41, 53). It was therefore of interest to evaluate the effects of peroxynitrite on RR, and especially on the small R2 subunit.
Peroxynitrite induced the irreversible loss of R2-Y· and
inactivated protein R2 irreversibly. A similar dose-response curve for
the two processes suggested that destruction of R2-Y· might
account for the loss of R2 activity. Nitration of tyrosyl residues and
hydroxylation of phenolic structures within E. coli and
mouse protein R2 were detected, consistent with the known reactivity of
ONOO
(52). These covalent modifications might involve the
amino acid carrying the free radical, thereby inhibiting R2 activity. This hypothesis is, however, probably too simplistic, considering the
multifaceted reactivity of peroxynitrite. Other reactive residues might
include the conserved Trp-48 and Tyr-356 (E. coli
numbering), which are close to the surface and crucial for the long
range electron transfer from R1 to R2-Y· (19). Covalent
modification of Tyr-307, located in the R2 surface area proposed to
interact with the R1 subunit (19), might also inactivate the protein.
Nitration of the conserved Phe-208 and Phe-212, close to the free
radical and important for its stability (54), might also have led to
the observed loss of R2-Y· EPR signal. However, these two
hydrophobic amino acids, as well as R2-Y·, are deeply buried
within protein R2. They should be thus less accessible to
ONOO
/ONOOH than surface residues. It has been previously
reported that the superoxide anion radical O
2 irreversibly
converted E. coli protein R2 into an inactive form lacking
the tyrosyl free radical. But x-ray crystallographic studies
established that R2-Y· was not directly modified by O
2,
suggesting that oxidation at other site(s) might have caused both R2
inactivation and destruction of R2-Y· (55). Similarly, loss of
R2-Y· induced by peroxynitrite might be more an index of a
multisite reactivity of protein R2 rather than a marker of
peroxynitrite action directed specifically against the free radical.
Successful inhibition by peroxynitrite of apoR2 indicates that reactive
sites other than R2-Y· do exist in E. coli and mouse
R2 proteins. The rank order of nitration staining (E. coli
R2 > mouse R2), reflecting the higher tyrosyl amino acid content
of the bacterial protein (16 in E. coli and only 9 in mouse
R2), also supported a nonspecific inactivation of protein R2 by
ONOO
/ONOOH.
In our previous studies, R2-Y· was shown to be quenched in
R2-overexpressing tumor cells exposed to the products of NOS II activity (12, 22). This event has been linked to NO-induced inhibition
of DNA synthesis and provided a molecular basis for the
antiproliferative action of macrophages activated for tumor cytotoxicity (12). The cytostatic action of macrophages has been first
proposed to rely mainly on ·NO production (5, 6). However,
accumulating evidence now suggests that peroxynitrite could also play a
significant role (7, 8). Considering that both ·NO and
ONOO can induce the disappearance of R2-Y·
in vitro, it was of importance to determine which of the two nitrogen oxides was the relevant radical scavenger in vivo.
High, nonphysiological concentrations of peroxynitrite were required to
quench R2-Y·, but, owing to the very short half-life of the
molecule, it was not possible to determine a priori whether
lower but persistent levels of peroxynitrite generated from a long
lasting biological source might not be as efficient. Inasmuch as
peroxynitrite irreversibly inhibited R2 activity, the full
reversibility of R2-Y· scavenging induced in L1210-R2 cells by
murine peritoneal-activated macrophages, either stimulated
or not with TPA, provided a clear evidence that
ONOO
was not, or marginally, involved. De novo
synthesis of active R2 protein during the 3-4-h time interval
between cessation of O
2 production (and thus of peroxynitrite
synthesis) and cell collection might have masked partially an
inactivation of protein R2 by peroxynitrite. Since the half-life of
mouse protein R2 is 3 h (56), about one-half of
peroxynitrite-injured protein R2 still remained at the end of the
experiment. Assuming a 10% error in EPR measurements, full recovery of
R2-Y· in L1210-R2 cells co-cultured with TPA-stimulated
macrophages indicated that less than 20% of protein R2 is inactivated
by peroxynitrite. This ratio is probably even much less in the absence
of NADPH oxidase activation. In previous reports, indirect evidence
indicated substantial formation of peroxynitrite by rodent macrophages, either activated with TPA or cultured in
L-arginine-depleted medium (57, 58). In the present work,
DHR oxidation testified to peroxynitrite production by TPA-stimulated
macrophages. Autocrine nitration of intracellular proteins, or
nitration of extracellular compounds by macrophage-derived
peroxynitrite has been already described (57, 58). Under our
conditions, irreversible loss of R2-Y· in tumor cells was not
observed. Western blots carried out with lysates of L1210-R2 cells or
macrophages stimulated with 2 to 8 nM TPA were also
negative for nitrotyrosine detection (not shown). These results show
that, under our experimental conditions, peroxynitrite did not nitrate
intracellular proteins, including protein R2, suggesting a protective
effect of cellular antioxidant components.
We conclude from the present data that ·NO, and not peroxynitrite, is the main effector of the scavenging of R2-Y· by cytotoxic macrophages. Since the modifications introduced by ·NO in protein R2 (i.e. coupling with the free radical and subsequent acceleration of iron release) are easily reversible in eukaryotic cells, persistent loss of the catalytically competent R2-Y· is dependent on a sustained production of ·NO. The limitation of RR-dependent cytostatic activity to cells expressing NOS II, but not short-lasting NOS I and III, is in agreement with this conclusion.
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ACKNOWLEDGEMENTS |
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We thank Profs. L. Thelander and M. Fontecave for providing pETM2 and pVNR2 plasmids, and B. Wolfersberger for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Association pour la Recherche contre le Cancer Grant 6555 and Ministère del' Enseignement Superieur et de la Recherche Grant ACC-SV 5.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: ERS 571, Bât. 430, UPS Orsay, F-91405 Orsay Cedex, France. Tel.: 33-01-6915-7972; Fax: 33-01-6985-3715; E-mail: michel.lepoivre{at}bbmpc.u-psud.fr.
The abbreviations used are: NOS, nitric oxide synthase; DHR, dihydrorhodamine; RR, ribonucleotide reductase; DTT, dithiothreitol; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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REFERENCES |
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