Free Radical Biology & Medicine, Vol. 36, No. 4, pp. 507 –516, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2003.11.028

Original Contribution

DEPLETION OF DEOXYRIBONUCLEOSIDE TRIPHOSPHATE

POOLS IN TUMOR CELLS BY NITRIC OXIDE

BEATRICE ROY, OLIVIER GUITTET, CLAIRE BEUNEU, GENEVIEVE LEMAIRE, and MICHEL LEPOIVRE

UMR CNRS 8619, IBBMC, Universite de Paris XI, F-91405 Orsay Cedex, France

(Received 28 July 2003; Revised 10 November 2003; Accepted 26 November 2003)

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Abstract—Nitric oxide displays pro- and anti-tumor activities, prompting further studies to better understand its precise

role. Nitric oxide inhibits ribonucleotide reductase (RnR), the limiting enzyme for de novo dNTP synthesis. We report here

the first detailed analysis of dNTP variations induced in tumor cells by NO. NO prodrugs induced a depletion in dNTP

pools and an activation of the pyrimidine salvage pathway, as did hydroxyurea, the prototypic RnR inhibitor. In the

presence of dipyridamole, which blocked salvaged dNTP synthesis, depletion of dNTP pools was also observed in tumor

cells cocultured with macrophages expressing the high-output iNOS activity. This effect was rapid, reversible, blocked by

NO scavengers, and cGMP independent. It was quantitatively correlated to iNOS activity. In the absence of dipyridamole,

NO still induced a decrease in dATP concentration in tumor cells cocultured with macrophages, whereas surprisingly,

concentrations of dCTP and dTTP expanded considerably, resulting in a strong imbalance in dNTP pools. NO prodrugs

did not cause such an increase in pyrimidine dNTP, suggesting that pyrimidine nucleosides were released by NO-injured

macrophages. Altered dNTP levels have been reported to promote mutagenesis and apoptosis. It is suggested that

abnormal changes in dNTP pools in tumors might contribute to NO-dependent toxicity. D 2004 Elsevier Inc. All rights

reserved.

Keywords—Nitric oxide, Deoxyribonucleotide, Macrophage, Ribonucleotide reductase, Leukemia, Free radicals

INTRODUCTION

Ribonucleotide reductase (RnR) is crucial for DNA

replication and repair, because it is the only enzyme that

catalyzes the reduction of ribonucleotides into deoxyri-

bonucleotides in the de novo pathway for dNTP synthe-

sis [1]. A salvage pathway that phosphorylates deoxy-

nucleosides imported from the extracellular milieu

provides an alternative route for dNTP production, but

cannot compensate for RnR deficiency [2]. RnR inacti-

vation by a variety of pharmaceutical inhibitors such as

hydroxyurea and gemcitabine leads to dNTP depletion

and has been successfully applied to anticancer chemo-

therapy for many years [3,4].

dress correspondence to: Michel Lepoivre, UMR CNRS 8619,

0, University of Paris XI, F-91405 Orsay Cedex, France. Fax:

)-69-85-37-15; E-mail: michel.lepoivre@bbmpc.u-psud.fr.

507

Eukaryotic RnR is an a2h2 complex. The small h2

homodimer, called R2, is a storage form for a stable

free radical essential for activity and is supposed to be

transferred to the catalytic site on a2 (R1) to initiate a

radical-driven reduction of the substrate. The free

radical is located on a tyrosyl residue buried in the

core of protein R2 and close to a diferric iron center.

Previous studies have shown that the reactive radical

NO inactivates RnR [5,6]. Different inhibitory mecha-

nisms have been proposed, but the most probable is a

loss of the R2-centered free radical consecutive to a

radical–radical reaction between NO and the tyrosyl

free radical [7–9]. This event has been detected by

EPR spectroscopy in vitro and in cell culture. We

proposed that cessation of tumor cell proliferation

induced by the high-output iNOS activity arises in

part from RnR inhibition [5]. This would cause dNTP

depletion and hence, inhibition of DNA synthesis.

However, only one report from our laboratory docu-

mented the effects of NO on dNTP pools [10].

B. Roy et al.508

Moreover, several studies have demonstrated the exis-

tence of NO-dependent cytostatic mechanisms indepen-

dent of RnR inhibition [11–13]. Another aspect of the

question concerns the amounts of NO necessary to

exert antiproliferative effects. In fact, the role of NO in

tumor growth is a matter of debate (reviewed in

[14,15]). In several studies, NO has been shown to

promote carcinogenesis and tumor progression, in

particular via deleterious reactions on DNA [16] and

stimulation of angiogenesis [14]. Other reports have

established that sustained overproduction of NO, typ-

ically by cells expressing the iNOS isoform, can cause

cytostasis and apoptosis of tumor cells. Very different

concentrations of NO in the tumor microenvironment

might explain this discrepancy as well as differential

susceptibilities of tumor cells to NO-induced damage.

Clearly, more quantitative data are necessary to better

understand the paradoxical actions of NO on tumor

growth.

In the present report, we show that dNTP pools are

drastically modified by NO prodrugs or macrophage

iNOS activity. The extent of dNTP depletion is propor-

tional to NO output. In the presence of macrophages,

there is a profound alteration in the dNTP concentration

equilibrium. Imbalance in dNTP levels may help ex-

plain NO-dependent mutagenic effects.

EXPERIMENTAL PROCEDURES

Biochemicals and cytokines

LPS from Salmonella enteritidis, human oxyhemo-

globin, human recombinant IL-1h, hydroxyurea, and

dipyridamole were obtained from Sigma. Carboxy-

PTIO, S-ethylisothiourea (SEITU), and 1H-[1,2,4]oxa-

diazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased

from Calbiochem. [5-3H]Deoxycytidine (sp act 18.4

Ci/mmol) was from Amersham Pharmacia Biotech.

(Z)-1-[N-(3-Aminopropyl)-N-(4-(3-aminopropylammo-

nio)butyl)amino]diazen-1-ium-1,2-diolate (SPER-NO)

and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)ami-

no]diazen-1-ium-1,2-diolate (DETA-NO) were obtained

from Cayman Chemicals. Human recombinant TNF-a

and IFN-g were generous gifts from Rhone-Poulenc

Rorer and Roussel-Uclaf (France), respectively. Mu-

rine recombinant IFN-g was provided by Dr. Adolf

(Ernst–Boehringer Institut fur Arzneimittel Forschung,

Austria).

Macrophages and cell lines

Murine peritoneal macrophages elicited by i.p.

injection of thioglycolate broth were obtained and

purified by adherence as previously described [17].

The L-1210 murine lymphoblastic leukemia and the

human T4-lymphoblastoid CEM-SS cell lines were

maintained in RPMI 1640 medium (Invitrogen) sup-

plemented with 25 mM Hepes, pH 7.4, L-glutamine,

antibiotics, and 5% heat-inactivated fetal calf serum

(FCS). The human colon carcinoma cell line DLD-1

was maintained in Dulbecco’s modified Eagle’s medi-

um with added antibiotics and 10% FCS [18].

Coculture of macrophages with tumor cells for dNTP

measurements

In experiments designed to measure dNTP concen-

tration in tumor cells, macrophages were seeded into

six-well culture plates (Nunc) at a nonsaturating density

(typically 2 � 106 cells/well) in culture medium. After

adherence and washings, they were activated overnight

with 20 U/ml murine IFN-g and 100 ng/ml LPS, in the

presence of 50 AM SEITU, to reduce NO production

and NO-dependent toxicity. Then, nonadherent cells

were eliminated by two vigorous washings and 2 �106 tumor cells were introduced into 5 ml of fresh

culture medium. When required, carboxy-PTIO (150

AM), HbO2 (175 AM), aminoguanidine (2 mM), or

dipyridamole (1.2 AM) was also added. At the end of

the coculture period, nonadherent cells were gently

removed and collected. An aliquot was counted after

trypan blue staining to evaluate cytotoxicity and the

remaining cell pellet was used for nucleotide analysis.

These cells were washed twice in ice-cold phosphate-

buffered saline. The cell pellet was rapidly resuspended

in cold 60% methanol (250 Al) and the resulting

suspension was allowed to stand for 2 min in boiling

water. Then, the cell extract was frozen at �80jC for at

least 1 day before analysis.

Coculture of macrophages with tumor cells for

ribonucleotide measurements

Experiments performed to measure ribonucleotide

pools were done under the conditions used for dNTP as-

say, but modified to handle a higher number of tumor cells.

Macrophages were initially seeded in larger culture dishes

(80 mm; Nunc) at a similar density. After macrophage

activation and elimination of nonadherent cells, 13 � 106

CEM-SS cells were added in 40 ml of culture medium.

Coculture of DLD-1 and CEM-SS cells

Experiments were carried out as already reported for

dNTP assay, except that 2.4 � 106 DLD-1 cells were

initially seeded into six-well culture plates. After 1 day,

cells were activated overnight with IL-1h (2 ng/ml),

human TNF-a (102 U/ml), and IFN-g (103 U/ml). Then,

2 � 106 CEM-SS cells were added and processed as

described above for dNTP extraction.

Depletion of dNTP pools by NO 509

Analysis of ribonucleotides

A Waters 600 multisolvent delivery system, a Waters

486 absorbance detector set at 260 nm, and Winflow

radiochromatography software (Berthold) were used for

the separation and quantification of ribonucleotides. Cell

extract (100–150 Al) was injected onto a Whatman

Partisil SAX 5 AM (250 � 4.6 mm) equilibrated in

buffer A (10 mM ammonium phosphate, pH 5.4), at a

flow rate of 2 ml/min. Elution started in 100% buffer A

for 7 min, then proceeded into 98% buffer A, 2% buffer

B (700 mM ammonium phosphate, pH 3.5) over 3 min,

and then switched to 100% buffer B using a linear

gradient over 40 min. The column was then regenerated

in buffer C (350 mM ammonium phosphate, pH 5.4) for

15 min before the next analysis was begun. Nucleotides

were identified by their retention time, compared to those

of authentic standards.

Analysis of dNTP

Quantification of dNTP in cell extracts was carried

out according to an enzymatic assay already described

[19]. This assay is based on the elongation of 32P-5V-end-labeled oligonucleotide primers annealed to com-

plementary oligonucleotide templates. Incorporation

within the primer/template was catalyzed by the Klenow

fragment of Escherichia coli, under conditions under

which the concentration of the dNTP to be analyzed is

limiting. The elongated products were separated after

electrophoresis on a denaturing 10% polyacrylamide

gel.

Determination of the pyrimidine salvage pathway activity

CEM-SS cells (3 � 106) were incubated for up to 6

h in culture medium supplemented with 0.1 AM 4 ACi[3H]deoxycytidine (dC*) and 100 AM SPER-NO, alone

or in combination. At different time intervals, cells were

collected and processed as described above for dNTP

analysis. About one-fifth of the material was used for

dCTP determination. The rest was used for the quantifi-

cation of radioactive dC metabolites (dC* and the

corresponding mono-, di-, and triphosphate derivatives).

HPLC separations were performed on an ion-exchange

Partisil 10-SAX column. The eluting buffers were 0.01

M potassium phosphate, pH 3.5 (buffer A), and 0.5 M

potassium phosphate, pH 3.5, containing 0.8 M KCl

(buffer B). The elution program of dC* and metabolites

was 8 min of buffer A followed by a 20 min linear

gradient to 100% buffer B and a 10 min isocratic elution

with 100% buffer B. The flow rate of the elution buffer

was 1 ml/min. Radioactivity was monitored by running

Quickszint Flow 306 scintillator (Zinsser Analytic)

through a radiomatic detector at a ratio of 2:1.

Measurement of nitrite production

Nitrite concentration was determined in cell culture

supernatants with the Griess reagent [7].

RESULTS

Depletion of dNTP pools induced by NO prodrugs

In a first set of experiments, structurally different

NO prodrugs were compared to HU in their ability to

deplete dNTP pools in human leukemia CEM-SS cells

(Fig. 1). Because RnR activity controls de novo

deoxynucleotide synthesis, dNTP concentrations are

indicative of intracellular RnR activity. HU (100 AM)

induced a pronounced and persistent decrease in dCTP

and dATP levels. The dTTP pool increased during the

first 4 h and then returned progressively to control

level. The dGTP concentration was not modified

during the first 6 h. All these variations were consis-

tent with previous observations [2,20]. The diazenium-

diolate DETA-NO is a long-lived NO donor (t1/2 = 20

h). When used at 1 mM, it induced dNTP variations

that closely resembled the HU profile. A small differ-

ence was a delay in maximal dTTP increase or in

dCTP decrease, probably reflecting the time before

sufficient NO amounts were released by DETA-NO

to inhibit RnR. The S-nitrosothiol SPER-NO at 100

AM had a transient effect on dNTP pools, which was

in accordance with its short half-life (t1/2 = 115 min in

RPMI medium at 37jC). This was particularly evident

on dCTP and dATP concentrations, which contracted

during the first 4 to 6 h and then returned to nearly

normal levels at 9 h. From these experiments, we

concluded that the NO prodrugs DETA-NO and

SPER-NO inhibited RnR activity, with a time course

related to their respective half-lives.

Activation of the pyrimidine salvage pathway by

SPER-NO

Inhibition of RnR activity by HU induces an

activation of the pyrimidine salvage pathway [2]. We

investigated whether such a regulatory mechanism was

induced by NO, by measuring incorporation of radio-

labeled dC into the dCTP pool. Addition of 0.1 AMradiolabeled dC to feed the salvage pathway attenuated

notably the decrease in dCTP concentration induced

by SPER-NO (Fig. 2A). An enhancement of the

amount of radiolabeled metabolites of dC was visible

in cells cultivated with tritiated dC and SPER-NO for

1 to 3 h, compared to controls with dC alone (Fig.

2B). The specific activity of dCTP (Fig. 2C, calculated

from the data in Figs. 2A and 2B) exhibited a

maximum between 1 and 3 h, coincident with the

Fig. 2. Activation of the pyrimidine salvage pathway by SPER-NO.CEM-SS cells were cultivated for up to 6 h in the presence of 100 AMSPER-NO to inhibit RnR activity. Activation of the salvage pathwaywas quantified by uptake and phosphorylation of radiolabeled dC* (0.1AM, 4 ACi). (A) Variations in dCTP concentration induced by SPER-NO or SPER-NO and dC*, compared to control levels in untreatedcells. (B) Radioactivity of all dC* metabolites (squares) or recoveredas dCTP (circles) or dCDP (diamonds) in cells incubated with dC*alone (open symbols) or with dC* and SPER-NO (closed symbols).(C) Specific radioactivity of the dCTP pool labeled with dC* alone orwith dC* and SPER-NO. Similar results were obtained in anotherexperiment.

Fig. 1. Variations in dNTP pools induced by NO prodrugs. CEM-SScells were incubated in the presence of HU (top), DETA-NO(middle), or SPER-NO (bottom). At the times indicated, they wereharvested for dNTP measurement: dTTP (closed circles), dCTP(open circles), dGTP (open squares), and dATP (closed squares).Results are expressed as the percentages of dNTP concentrations incontrol cells cultured alone. One experiment representative of threeis shown.

B. Roy et al.510

drop in dCTP caused by SPER-NO alone. When the

NO prodrug lost its activity (i.e., 5 h after SPER-NO

addition), the specific activity of dCTP decreased to

the control value. Overall, these results indicate that

activation of the pyrimidine salvage pathway was

induced by SPER-NO in response to dNTP depletion.

Identical results were obtained with DETA-NO, except

that phosphorylation of dC was more durable, consis-

tent with the much longer half-life of the pro-drug

(data not shown).

Depletion of dNTP pools induced by iNOS-expressing

macrophages

To examine the effects of physiological amounts of

NO on dNTP pools, mouse L-1210 or human CEM-SS

cells were cocultivated with macrophages previously

stimulated by IFN-g/LPS for induction of iNOS expres-

sion. Replenishment of the dCTP and dTTP pools via

Fig. 3. Depletion of dNTP pools by macrophage iNOS. L-1210 cellswere first cocultivated for up to 4 h with activated macrophages in thepresence of dipyridamole. At the time indicated by an arrow, target cellswere removed from the macrophage monolayer and cultivated in freshmedium for up to 90 min. In duplicate samples (open symbols),removed L-1210 cells were incubated in the presence of 10 mMhydroxyurea. To prevent dNTP synthesis by the salvage pathway,dipyridamole was present throughout the experiment. In addition,nucleoside-free dialyzed serum was used during the recovery phase. Atthe times indicated, leukemia cells were harvested for measurement ofintracellular concentrations of dNTPs. (A) dGTP (squares) and dATP(circles). (B) dTTP (diamonds) and dCTP (triangles). One experimentrepresentative of three is shown.

Depletion of dNTP pools by NO 511

the pyrimidine salvage pathway might partly mask the

inhibition of de novo dNTP synthesis caused by RnR

inhibition. Therefore, unless otherwise stated, the fol-

lowing experiments were performed in the presence of

dipyridamole, an inhibitor of Na+-independent nucleo-

side carriers. Furthermore, a short coculture period was

selected to minimize extracellular accumulation of

deoxyribonucleosides released by NO-injured cells

(see below).

A progressive depletion of the four dNTP pools

was observed in the leukemia cells during the 4 h they

were cocultivated with macrophages, in the presence

of dipyridamole (Figs. 3A and 3B). As already ob-

served with HU and NO prodrugs, the dATP and

dCTP pools were more depleted than dGTP and dTTP.

There was no decrease in dNTP pools in tumor cells

cocultivated with unstimulated macrophages, which did

not express iNOS (data not shown). Analysis of ADP,

GDP, UDP, CDP, and ATP, which are respectively the

four diphosphate substrates and the major positive

effector of RnR, did not reveal significant variations

in CEM-SS cocultivated for 8 h with activated macro-

phages (data not shown). Therefore, we excluded the

possibility that dNTP depletion might result from a

decreased synthesis of some ribonucleotides important

for RnR activity. The reaction of NO with the RnR-

centered tyrosyl free radical is readily reversible [8,9].

To determine the reversibility of NO effects on dNTP

levels, the target cells were then withdrawn from the

macrophage monolayer and allowed to recover in fresh

medium, in the presence of cycloheximide to prevent

RnR neosynthesis (Figs. 3A and 3B). A very rapid

rise in the four dNTP concentrations was observed in

L-1210 cells recovering from iNOS injury. The deficit

in purine dNTPs was totally suppressed after 1 h. The

recovery of pyrimidine dNTPs was slower, but still

rapid. To verify that replenishment of dNTP pools

during this period was due to reactivation of RnR, HU

was added to duplicate L-1210 cell samples at the

beginning of the culture in fresh medium (indicated by

an arrow in Figs. 3A and 3B). Hydroxyurea not only

prevented the rise in dNTP concentrations, but even

prolonged the decrease in dNTP concentration caused

by iNOS. Similar results were obtained using CEM-SS

as target cells (data not shown). These results indicat-

ed that efficient depletion of dNTPs by NOS activity

required a long-lasting production of NO, because

RnR inhibition by NO is easily reversible.

NO-dependent but cGMP-independent inhibition of

dNTP synthesis

Physiological effects of NO are frequently mediated

by activation of soluble guanylate cyclase. Our pre-

vious studies using a purified enzyme have docu-

mented a direct inhibitory effect of NO on RnR.

Nevertheless, in a more complex cell coculture system,

we could not ignore the possibility of a cGMP-

dependent inhibition of dNTP synthesis. This hypoth-

esis was ruled out, based on the complete ineffective-

ness of ODQ, a potent inhibitor of guanylate cyclase,

on the decrease in dATP and dCTP pools induced by

activated macrophages (Table 1). The cell-permeable

cGMP analog 8-Br-cGMP was also without effect on

Table 1. Depletion of dNTP Pools by iNOS Is cGMP Independent

Treatmenta dNTP (% of control)b

dATP dCTP

MA 72.5 F 1.5 37.7 F 2.9MA + ODQ 72.9 F 1.7 41.6 F 0.3MA + C-PTIO 111 F 4.2c 93.4 F 3.1c

MA + HbO2 95.6 F 5.6c 84.6 F 6.2c

a Human CEM-SS cells were cocultured for 6 h with activated

macrophages (MA) in the presence of dipyridamole to induce dNTP

depletion. This effect was not affected by the guanylate synthase

inhibitor ODQ (25 AM), but was prevented by the NO scavengers C-

PTIO (150 AM) and HbO2 (175 AM).b Intracellular dNTP levels measured in CEM-SS cells at the end of

the coculture period were compared to those of tumor cells cultured

alone. Means F SD of three determinations.c Statistically different from tumor cells incubated with macrophages,

by Student’s t test ( p < .05).

Fig. 4. Quantitative relationship between depletion of dNTP pools andNO output. CEM-SS cells were cocultured with different densities ofactivated macrophages, ranging from 0.25 to 2 � 106/well, in thepresence of dipyridamole. (A) Nitrite production in culture medium wasplotted as a function of time. The slope of this straight line (in AM/h)was represented as a function of macrophage density. (B and C)Variations in the levels of dATP and dCTP in tumor cells incubated for2, 4, or 8 h with macrophages, as a function of the rate of nitriteproduction by the macrophage monolayer. Results are expressed aspercentages of dNTP concentration in control cells cultured alone. Dataare a compilation of four to five independent experiments.

B. Roy et al.512

CEM-SS cell DNA synthesis (data not shown). How-

ever, when NO was trapped by two structurally un-

related molecules, namely carboxy-PTIO and HbO2,

the drop in dCTP and dATP concentrations in CEM-

SS cells was prevented significantly. These results

demonstrate that inhibition of de novo dNTP synthesis

by activated macrophages is NO dependent, but cGMP

independent.

Quantitative relation between dNTP depletion and NOS

activity

The effects of iNOS activity on dNTP synthesis

were further explored on a more quantitative basis.

First, NO fluxes were quantified as a function of

macrophage density. Nitrite production (and therefore

NO release) by macrophages was linear with time for

at least 8 h (not shown). Thus, iNOS activity was

expressed as the mean rate of nitrite production over

8 h (in AM/h). This rate increased steadily with

macrophage density (Fig. 4A). Then, dNTP levels

measured at different times were plotted as a function

of the rate of nitrite synthesis. Because the dCTP and

dATP pools exhibited the highest sensitivity to iNOS,

we focused only on these two nucleotides. The small-

est iNOS activity tested was 0.32 AM nitrite per hour.

It was ineffective after 2 h and caused only a marginal

reduction of dCTP and dATP concentrations at 4 h.

Above this threshold, dATP concentration measured at

2 and 4 h diminished progressively as a function of

iNOS activity (Fig. 4B). The decrease in dATP was

much more important at 4 than at 2 h. At 8 h, the

lowest iNOS activities had the same effect as after 4

h, but the highest ones seemed to be slightly less

efficient. Variations of the dCTP concentrations were

quite different. There was an almost linear decrease up

to a nitrite flux of 2–2.5 AM/h (Fig. 4C). In this

linear region, the slopes at 2 and 4 h were similar. At

higher iNOS activities, the dCTP level plateaued or

exhibited a tendency to re-increase. This trend was

more pronounced at 8 h. Thus, at 8 h, higher iNOS

Depletion of dNTP pools by NO 513

activities were less effective than lower ones on dCTP

and dATP pools. This could be due to a residual

activity of the pyrimidine salvage pathway despite the

presence of dipyridamole, in conjunction with an

increased release of nucleosides by macrophages at

the most toxic iNOS activities (see below).

In one series of experiments, murine macrophages

were replaced by human DLD-1 cells, which, after

activation by cytokines, produced nitrite at a 10-fold

lower rate that murine macrophages on a per-cell basis.

There was no effect of DLD-1 cells on dNTP levels in

CEM-SS cells, for up to 8 h (data not shown).

Contribution of the salvage pathway to dNTP pool

imbalance

Pyrimidine deoxynucleosides are recycled into

dNTPs by the salvage pathway much more efficiently

than purine nucleosides. Therefore, inhibition of de novo

dNTP synthesis on one hand, and activation of the

salvage pathway on the other hand, may induce an

imbalance in intracellular dNTP pools if large amounts

of pyrimidine nucleosides are available in the medium.

Such an imbalance has been linked to apoptosis [21]. To

investigate whether RnR inhibition by NO caused a

dNTP imbalance, we analyzed dNTP variations in tumor

cells with an active salvage pathway (that is, in the

absence of dipyridamole). Results are presented in Table

2. As expected, we observed a decrease in dATP

concentration (by 60%) during the 16 h coculture period

with macrophages, which was consistent with our pre-

vious results. However, there was a huge increase in

pyrimidine nucleotide pools in the tumor cells and no

variation of dGTP level. That dGTP concentration was

Table 2. NO-Dependent RnR Inhibition and Activation of the PyrimidineSalvage Pathway

Additivea dNTP (% of control)b Nitrite

dCTP dTTP dATP dGTP

(AM)c

None 638 F 37d 681 F 53d 34 F 4d 103 F 15 31 F 0.3AG 148 F 10d 143 F 11d 104 F 6 136 F 5d NDe

DP 71 F 3d 122 F 11 24 F 1d 38 F 7d 31 F 0.9AG + DP 109 F 8 88 F 5 88 F 9 86 F 5 ND

a Human CEM-SS cells were cocultured for 16 h with activated

macrophages at a 1:1 ratio, without (None) and with the NOS inhibitor

aminoguanidine (AG), the nucleoside transporter inhibitor dipyridamole

(DP), or both (AG + DP).b Intracellular dNTP levels measured in CEM-SS cells at the end of

the coculture period were compared to those of tumor cells cultured

alone. Means F SD of triplicates.c Nitrite concentration in the culture medium.d Statistically different from tumor cells cultured alone, by Student’s

t test ( p < .05).e ND = not detectable.

not diminished might be explained by the 6-fold in-

crease in dTTP, because dTTP is a positive effector of

RnR for dGTP synthesis. All dNTP concentrations were

normalized by aminoguanidine, which completely

inhibited macrophage iNOS activity, as judged by un-

detectable nitrite production (Table 2). In particular, the

considerable expansion of dCTP and dTTP pools was

almost suppressed, suggesting that NO-injured cells

could be an important source of salvaged nucleosides.

Because such a dramatic increase in dTTP and dCTP

pools was not induced by NO prodrugs (Fig. 1), macro-

phages were probably the major source of nucleosides.

Indeed, significant amounts of dC (0.04–0.05 AM) and

dT (0.08–0.11 AM) were detected after 8 h in culture

supernatants from activated macrophages. Dipyridamole

efficiently reduced the rise in dTTP concentration and

allowed a marked decrease in the dCTP pool (Table 2),

confirming the important activity of the salvage pathway

in cells exposed to NO. Dipyridamole also induced a

decrease in the dGTP level and further reduced dATP

concentration.

We can now summarize the series of events affecting

deoxynucleotide metabolism in NO-injured cells. First,

sustained NO production inhibited de novo dNTP syn-

thesis at the level of ribonucleotide reduction. This in

turn activated the pyrimidine salvage pathway, to com-

pensate for RnR inhibition [2]. Activation of pyrimidine

recycling caused an increase in dCTP and dTTP levels.

The rise in pyrimidine dNTPs caused by macrophage

iNOS (Table 2) is much higher than the small increase

induced by NO prodrugs (Fig. 1), probably because

activated macrophages release significant amounts of

pyrimidine dNs. Because purine pools are not efficiently

replenished by the salvage pathway, the ratios

dCTP:dGTP and dTTP:dATP, close to 1 in CEM-SS

cells, were augmented respectively by a 5.6- and a 18-

fold factor in cells cocultured with macrophages. It

would be interesting to know whether this marked

asymmetry in dNTP concentration could contribute to

NO-dependent genotoxicity.

DISCUSSION

This study is the first detailed analysis of dNTP

variations in tumor cells exposed to NO generated either

from chemical NO prodrugs or from macrophage iNOS

activity. In both cases, there was a rapid depletion of

dNTP pools, especially dATP, that in turn activated the

pyrimidine salvage pathway. Because we excluded the

possibility that dNTP depletion might result from alter-

ations of ribonucleotide metabolism by NO, this work

provides direct evidence for NO-mediated RnR inactiva-

tion in intact cells. Similar variations in dNTP levels are

caused by pharmacological inhibitors of RnR [2,4,22].

B. Roy et al.514

However, the considerable expansion of dCTP and dTTP

concentrations induced by iNOS (but not by NO pro-

drugs) is unusual. Dipyridamole and NOS inhibitors

prevented this augmentation, suggesting that NO-injured

macrophages could release into the culture medium large

amounts of nucleosides that are salvaged by tumor cells.

Nitric oxide is known to induce apoptosis in macro-

phages [23,24]. Hence, hydrolysis of apoptotic DNA

fragments might produce pyrimidine deoxynucleosides.

Also, macrophages have been already reported to syn-

thesize important amounts of thymidine [25]. Thus,

macrophages could significantly contribute to pyrimidine

release in the medium and, therefore, to dNTP imbalance

in the target cells. Considering the promutagenic effects

of an abnormal expansion of dNTP pools [26] and the

presence of infiltrating macrophages in many tumors, it

would be valuable to know whether enhanced pyrimidine

availability and marked dNTP asymmetry could also

occur in a tumor tissue.

Several reports have established the importance of

RnR in tumor proliferation, invasiveness, and aggressiv-

ity. For instance, elevation of RnR activity has been

observed in malignant cells and is positively correlated

with tumor growth rate in rodents [27,28]. Increased

levels of R2 protein and message have been found in

malignant tumor tissues [29]. Deregulated expression of

protein R2 in cooperation with oncogenes has been

shown to enhance tumor transformation and malignant

potential [30]. In this context, it could be anticipated that

even partial inhibition of RnR could be detrimental to

tumors. It was therefore important to define the condi-

tions leading to RnR inhibition in intact cells. The

reversibility of RnR inactivation by NO suggested that

only sustained iNOS activities would be inhibitory, as

opposed to constitutive NOSs activated transiently. Our

results indicated that the low iNOS activity of human

DLD-1 cells is ineffective against RnR. Therefore, RnR

inhibition by NO could be induced by those cells able to

produce a high flux of NO. Many mouse and rat cells

have this capacity, upon stimulation with cytokines or

bacterial components. Several, but fewer, human cell

types have been also found to produce NO at a compa-

rable rate, including hepatocytes [31], airway epithelial

cells [32], and alveolar macrophages [33]. Endogenous

iNOS activities found in human tumors are much weaker

[34–37]. They are probably too low to influence RnR

activity and, hence, to affect tumor proliferation in an

RnR-dependent manner. Nevertheless, iNOS overexpres-

sion induced by immunomodulation or gene transfection

in some experimental cancers is accompanied frequently

by NO-mediated anti-tumor cytostatic effects [15,38,39].

The strong iNOS activities measured in these models are

in the range of those that would impair RnR activity.

Considering our results, we envisage that a reduction in

RnR activity might partly support the antiproliferative

effects observed in these models.

NO-mediated depletion of dNTP pools could lead to

cellular damage beyond cessation of cell growth. Several

studies have suggested a link between RnR inhibition and

apoptosis. Structurally unrelated RnR inhibitors were

shown to induce or enhance apoptosis in different exper-

imental models [22,40,41], strongly suggesting that RnR

inhibition could be a proapoptotic event. But the most

convincing data linking dNTP deficiency to apoptotic cell

death came from studies on the recently discovered

homolog of R2, named p53R2 because its expression is

p53 dependent [42–44]. This protein is implicated spe-

cifically in DNA repair. Inhibition of p53R2 in vitro or

genetic inactivation of p53R2 in mice caused enhanced

apoptosis [42,43,45]. There is a remarkable sequence

homology between R2 and p53R2. In particular the

tyrosyl residue carrying the free radical and the amino

acid ligands for the diiron center are conserved [44].

Because the radical is the primary target of NO in R2, it

is tempting to imagine a similar inactivation of p53R2 by

NO at the level of its free radical. This hypothesis is

currently under investigation in our laboratory. The pres-

ent work demonstrates that NO is a potent inhibitor of

RnR in tumor cells. Its effects on dNTP pools are nearly

identical to those of chemotherapeutic RnR inhibitors.

Notwithstanding its pleiotropic roles in the immune

system, NO might be thus considered as a natural equiv-

alent of, for instance, hydroxyurea. Other endogenous

inhibitors of RnR have already been identified. In yeast,

the protein Sml1 binds to the R1 subunit, thereby inacti-

vating the enzyme when dNTP synthesis is not required

[46]. In many other organisms, protein R1 binds dATP, a

potent feedback inhibitor of all RnR activities [1]. The

inhibitors Sml1 and dATP exert a regulatory role. They

are intrinsically different from iNOS activity, which

perturbs RnR function. In this regard, NO belongs to a

new category of natural RnR inhibitors, whose mode of

action resembles those of pharmacological inhibitors.

Acknowledgments—We thank B. Wolfersberger for excellent technicalassistance and J.-C. Drapier (ICSN, CNRS, Gif-sur-Yvette, France) forgiving us the DLD-1 cells. We also gratefully acknowledge M. Hours(IFR 46 CNRS) and M.-P. Courageot (UMR 8619 CNRS) for assistancewith cytometric analysis. This work was supported by Grant 5329 froml’Association pour la Recherche contre le Cancer (to G.L.) and twogrants from La Ligue Nationale contre le Cancer (to M.L. and O.G.).

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ABBREVIATIONS

dNTP—deoxyribonucleoside triphosphate

NOS—NO synthase

iNOS—inducible NOS

RnR—ribonucleotide reductase

SEITU—S-ethylisothiourea

HU—hydroxyurea