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Am J Physiol Heart Circ Physiol 277: H1799-H1807, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 5, H1799-H1807, November 1999

Differential regulation of DNA synthesis by nitric oxide and hydroxyurea in vascular smooth muscle cells

Ruth Bundy, Nándor Marczin, Adrian H. Chester, and Magdi Yacoub

Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the influence of nitrovasodilators on DNA synthesis in cultured human aortic smooth muscle cells and explored the hypothesis that nitric oxide (NO) is directly involved in mediating the inhibitory effects of hydroxyurea on DNA synthesis. Both NO and hydroxyurea inhibited ongoing DNA synthesis and S phase progression in our cells. Exogenous deoxynucleosides partially reversed this inhibition, suggesting that ribonucleotide reductase is a primary target for both NO and hydroxyurea. Nitrovasodilators inhibited DNA synthesis by releasing NO, as detected by chemiluminescence and as shown by the reversal of DNA synthesis inhibition by NO scavengers. This inhibition appears to occur via a cGMP-independent mechanism. In contrast, hydroxyurea did not produce a detectable NO signal, and NO scavengers had no influence on its inhibition of DNA synthesis, suggesting that NO does not mediate the inhibitory action of hydroxyurea in our system. Furthermore, the action of nitrovasodilators and hydroxyurea on DNA synthesis differed according to redox sensitivity. The redox agents N-acetyl-L-cysteine and ascorbate reversed NO inhibition of DNA synthesis and had no effect on DNA synthesis inhibition caused by hydroxyurea.

deoxyribonucleic acid synthesis; aortic smooth muscle cell; ribonucleotide reductase; redox


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXCESSIVE PROLIFERATION OF smooth muscle cells (SMC) plays an important role in a number of vascular proliferative diseases, such as formation of vascular lesions associated with atherosclerosis (30), accelerated atherosclerosis after transplantation, and restenosis after balloon angioplasty (23, 31). There is increasing evidence that the proliferation of SMC can be significantly influenced by nitric oxide (NO), an endogenously produced vasodilator. Increasing the availability of NO at the site of vascular injury, using pharmacological NO donors and gene transfer of NO synthase, results in a decrease in SMC proliferation in vivo (28, 35, 37). In addition, the augmentation of endogenously produced NO by supplementation with L-arginine, the precursor of NO, also inhibits formation of proliferative lesions after balloon angioplasty (26) and vein grafting (4). Furthermore, attenuation of NO activity by the administration of inhibitors of NO synthase or by cholesterol has been shown to increase arterial lesion size after balloon angioplasty (26) and hypercholesterolemia (2). These data suggest a pivotal role of endogenously produced NO in controlling SMC during lesion formation.

Studies using chemical donors of NO and NO gas itself in cultured animal SMC have identified a variety of mechanisms whereby NO inhibits SMC growth (8, 14). In rat aortic SMC, NO has been shown to affect at least two stages of the cell cycle, early G1 phase and DNA synthesis (33, 34). Mitogen-induced activation of early cell signaling, such as activation of mitogen-activated protein kinase and associated G1 phase progression, is inhibited by NO (19) and appears to be mediated via activation of soluble guanylate cyclase and increased accumulation of cGMP (33). This is supported by the finding that analogs of cGMP mimic NO in inhibiting G1 progression (33). There is however evidence for cGMP-independent mechanisms of NO inhibition of the cell cycle. NO has been demonstrated to affect G1 progression by altering the expression of cyclins (12) and especially by inducing the cyclin inhibitor p21 (13). Furthermore, in mammalian tumor cells, NO has been shown to inhibit ribonucleotide reductase (17, 21), the enzyme essential for DNA synthesis, bringing about inhibition of ongoing DNA synthesis and cell cycle arrest at the G1/S boundary and S phase (33, 34).

The action of NO on DNA synthesis resembles that of hydroxyurea (HU), a well-known inhibitor of ribonucleotide reductase. HU is currently used as a therapeutic agent to inhibit cell proliferation in patients with leukemia and some forms of head and neck malignancies (15, 36). Studies using electron paramagnetic resonance spectroscopy reveal that HU reversibly binds to and inactivates the tyrosyl radical of ribonucleotide reductase (24). In addition to both NO and HU targeting ribonucleotide reductase, a more intricate relationship between NO and HU has been suggested in that NO itself would directly mediate the actions of HU. Kwon et al. (17) demonstrated that, in the presence of catalysts such as copper or copper-containing proteins, HU undergoes hydrogen peroxide-dependent oxidation, resulting in the release of an NO-like species. It has been postulated that this reaction could mediate the pharmacological effects of HU in cells, which have the capacity to oxidize it.

The aim of our study was to investigate the influence of NO on DNA synthesis in human aortic SMC and to explore the possibility that NO is directly involved in the effects of HU on DNA synthesis in these cells.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. DMEM, FBS, trypsin-EDTA, glutamine, penicillin-streptomycin, PBS, HU, N-acetyL-L-cysteine (NAC), N-acetyl-L-serine, L-ascorbic acid, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), IBMX, 2'-deoxyadenosine, 2'-deoxyguanosine, smooth muscle alpha -actin, HHF35 human actin, and myosin were purchased from Sigma Chemicals. Radiolabeled [3H]thymidine was from ICN, and 125I-labeled cGMP was obtained from Amersham Life Sciences. Rabbit anti-cGMP was purchased from Calbiochem. S-nitroso-L-glutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), (Z)-1-[2-aminoethyl]-N-(2-ammonioethyl)aminodiazen-1-ium 1,2-diolate (DETA-NO), 1H-[1,2,4]oxadiazole[4,3-alpha ]quinoxalin-1-one, and carboxy 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) were obtained from Alexis Biochemicals.

Cell culture. Human aortic SMC were cultured by the explant method from aortas of human donor heart allografts. First, the endothelial layer was removed with a scalpel blade, and strips of SMC from the tunica media were peeled off with forceps and cut into small pieces. The aorta pieces were then washed with DMEM supplemented with 10% FBS and were evenly distributed and secured in petri dishes by pins. DMEM, supplemented with 2 mM glutamine, penicillin-streptomycin (1 U/ml and 1 mg/ml, respectively), and 10% FBS, was added to the dishes, and they were incubated in a humidified atmosphere at 37°C in 5% CO2 in air. Cells started to grow out from the explants after 4-7 days in culture. Adherent cells were allowed to grow to confluence and were then subcultured every 5-7 days by releasing them with trypsin-EDTA and diluting them 1:4 in DMEM containing 10% FBS. Subcultured cells were given fresh DMEM supplemented with 20% FBS every 48 h. The human aortic SMC were identified by their typical hill-and-valley morphology in culture and their characteristic immunocytochemical staining for smooth muscle alpha -actin, HHF35 human actin, and myosin. Experiments were performed on cells derived from three different donors at passages lower than 10.

Cell synchronization, DNA synthesis, and S phase progression. Confluent SMC were treated with 0.6 mM HU for 12 h, and, after removal of HU, cells were incubated for a further 4-h period in the absence or presence of experimental agents. The cells were then pulse-labeled between 3 and 4 h with [3H]thymidine (1 µCi/ml). Incorporation of [3H]thymidine into acid-insoluble macromolecules was determined by extracting the cells with 10% TCA at 4°C for 30 min. Acid-insoluble macromolecules were solubilized in 0.1% SDS-0.3 mol/l NaOH for 1 h. Radioactivity was measured by scintillation counting (TRI-CARB 1600TR, liquid scintillation counter). Cell progression through the cell cycle was measured using flow cytometric analysis. After removal of HU from synchronized cells, the cells were incubated in the absence and presence of experimental agents for 4 h. They were then harvested by trypsinization and were washed with PBS. The pellet was fixed and permeabilized with absolute ethanol at 4°C for 10 min. After repeated washing with PBS, cells were treated with 400 µg/ml RNase A and labeled with 600 µg/ml propidium iodide for 30 min at 37°C. Propidium iodide binding and determination of DNA content were analyzed by a flow cytometer (Coulter Epics XL).

NO analysis by chemiluminescence. Ongoing release of NO from S-nitrosothiols was monitored by the chemiluminescence principle, using an NO analyzer (Sievers NO analyzer, model 270B). Conditions were set to detect NO gas itself as opposed to biologically inactive stable degradation products of NO, such as nitrite and nitrate. A continuous stream of N2 gas was bubbled through 5 ml of serum-free tissue culture medium in the purge vessel to deliver any released NO gas to the analyzer. Under these conditions, injection of NO donors (1-1,000 µM) or NO gas itself resulted in a positive NO signal. Furthermore, injection of sodium nitrite, up to 10 mM, into the purge vessel did not result in an NO signal, but a signal was readily detectable under acidic conditions.

Measurement of cGMP. Accumulation of cGMP was studied in cultured human and rat aortic SMC or in fresh aorta pieces, which were denuded of endothelium and cleaned from adventitia. SMC were preincubated for 10 min with the phosphodiesterase inhibitor IBMX (1 mM) to prevent degradation of cGMP and were further treated with increasing concentrations of NO donors or HU in the presence of IBMX for 15 min or 4 h. The cells were then washed, and cGMP was extracted with 0.1 M HCl for 1 h. Cellular cGMP content was quantified by standard RIA performed according to Brooker et al. (1), with samples and standards acetylated by 2:1 mixture of triethylamine-acetic anhydride. We used a rabbit polyclonal antibody to cGMP and separated the antibody-bound and unbound radioactive cGMP by the charcoal method.

Experimental design. To investigate the influence of agents on DNA synthesis throughout these studies, cells were initially synchronized by HU to cause cell cycle arrest at the G1/S boundary. Synchronized cells were used as opposed to cycling cells to eliminate any influence that NO might exhibit on other stages of the cell cycle, such as early G1 signaling and G1 phase progression. Cells were then allowed to restore DNA synthesis activity by removal of HU and to progress through S phase in a synchronized manner 3-4 h after removal of HU. The influence of experimental agents such as NO donors and HU on DNA synthesis and S phase progression was evaluated by thymidine uptake and flow cytometric analysis, respectively, in this time frame and experimental setting.

The influence of NO on thymidine uptake and S phase progression was evaluated using different NO donors, such as S-nitrosothiols (GSNO and SNAP), DETA-NO, and NO gas itself. The ongoing release of NO from these donors was assessed by chemiluminescence. To assess if ribonucleotide reductase is a target of NO, exogenous deoxynucleosides, end products of ribonucleotide reductase, were administered to bypass the enzyme and to provide substrate for DNA synthesis. We used a combination and concentration of the deoxynucleosides deoxyadenosine and deoxyguanosine, both at 0.4 mM, which was found to be optimal in reversing NO inhibition of DNA synthesis in other systems (17). A similar strategy was used to investigate the influence of HU on DNA synthesis and to establish ribonucleotide reductase as its target in human aortic SMC.

The postulated role of NO in mediating the action of HU was tested by three approaches. First, to evaluate the potential oxidation of HU by cellular components to produce NO, chemiluminescence analysis was performed in the absence or presence of human aortic SMC. Second, the formation of biologically active NO-like species from HU was monitored by evaluation of soluble guanylate cyclase activation. The activation of this enzyme and the subsequent accumulation of cGMP is one of the most characteristic and sensitive intracellular mechanisms that respond to NO. This response in culture, however, depends on the ability of passaged cells to maintain soluble guanylate cyclase activity. This appears to be problematic in human aortic SMC, as they have been reported to lose soluble guanylate cyclase activity during culture (25). To overcome this difficulty, we used either fresh aortic pieces or cultured rat aortic SMC, a cell line that has been shown to express soluble guanylate cyclase activity in culture (29). In addition, to further clarify the role of cGMP in inhibiting DNA synthesis in our human cultured cells, the influence of 8-BrcGMP, a cell-permeable analog of cGMP, on DNA synthesis was tested. Third, we reasoned that, if NO species are involved in mediating the effect of HU on DNA synthesis, then NO scavengers should partially or fully reverse the effect of HU. Therefore, the experiments on HU-induced inhibition of DNA synthesis were performed in the presence of extracellular or intracellular scavengers of NO.

We recently observed redox sensitivity of the inhibitory action of NO on DNA synthesis. To study further similarities between NO and HU in inhibiting DNA synthesis in human aortic SMC, we therefore evaluated the influence of chemically distinct redox agents NAC and ascorbic acid on DNA synthesis inhibition by NO donors and HU.

Statistics. Data are presented as means ± SE of three to four separate experiments. One-way ANOVA was used to determine significance among groups, after which the modified t-test with the Bonferroni correction was used for comparison between individual groups. A value of P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of HU and NO on DNA synthesis. To synchronize human aortic SMC at the G1/S phase boundary, cells were treated with 0.6 mM HU for 12 h. The DNA synthetic rate measured by thymidine incorporation was reduced 12 h after HU treatment compared with the control cycling cells (419 ± 44 vs. 18,981 ± 3,672 counts/min). DNA content, measured by flow cytometry, showed synchronization of HU-treated cells at the G1/S boundary (Fig. 1B). After removal of HU and re-addition of 5% serum, the cells resumed their DNA synthesis, as thymidine incorporation increased and reached a maximum 3-4 h after washout of HU (4,030 ± 1,056 and 28,114 ± 1,911 for 1 and 4 h, respectively). Flow cytometry showed a synchronized progression of cells through S phase resulting in accumulation of cells in G2 phase after 4 h (Fig. 1C).


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  		Fig. 1.   Cell synchronization by hydroxyurea (HU) and influence of S-nitro-L-glutathione (GSNO) on S phase progression after washout of HU in human aortic smooth muscle cells. DNA content of human aortic smooth muscle cells was monitored by flow cytometric analysis. A: control; typical DNA frequency profile of cycling cells labeled with propidium iodide. The majority of cells are in G1 phase, few cells are in S phase undergoing DNA synthesis, and a small population of cells are in G2, having doubled their DNA content. B: 0 h after washout of hydroxyurea; synchronization of cells at G1/S phase after exposure to 0.6 mM HU for 12 h. C: 4 h after washout of HU, a significant portion of cells passed through S phase to G2, exhibiting double their DNA content. D: 4 h after washout of hydroxyurea + GSNO; attenuation in progression through S phase in GSNO-treated cells. Data are representative of 4 different experiments.

To investigate the influence of NO donors and HU on ongoing DNA synthesis and S phase progression, cells were treated with these compounds immediately after HU washout for 4 h. Incubation of cells with GSNO for 4 h after washout of HU produced a concentration-dependent inhibition of DNA synthesis, as shown by thymidine incorporation (82.8 ± 4.9, 15.1 ± 2.9, and 2.5 ± 0.2% of control for 100, 250, and 1,000 µM, respectively; Fig. 2A). Higher concentrations (1 mM) of SNAP were required to produce a significant inhibition of DNA synthesis (40.8 ± 14.4% of control for 1 mM). Similarly to the nitrosothiols, DETA-NO and NO gas produced concentration-dependent inhibition of thymidine incorporation (84.2 ± 3.2, 71.7 ± 2.6, and 27.2 ± 0.6% of control for 250, 400, and 600 µM, respectively, for DETA-NO and 68.0 ± 0.6, 41.9 ± 4.6, and 20.1 ± 1.0% of control for 1:4, 1:2, and 1:1 dilutions of saturated solution of NO, respectively). Flow cytometry also revealed that GSNO attenuated S phase progression, as GSNO-treated cells exhibited less propidium iodide binding (Fig. 1D). HU also showed a concentration-dependent inhibition of thymidine incorporation (72.3 ± 1.8, 29.1 ± 0.9, 15.9 ± 0.3% of control for 200, 400, and 600 µM, respectively; Fig. 2B) and inhibition of S phase progression, as characterized by the majority of HU-treated cells exhibiting less propidium iodide binding, measured by flow cytometry (data not shown).


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  		Fig. 2.   Influence of GSNO and HU on ongoing DNA synthesis of human aortic smooth muscle cells. Ongoing DNA synthesis was monitored by [3H]thymidine incorporation into acid-insoluble macromolecules in cells progressing through S phase after synchronization at G1/S boundary as described in METHODS. A: concentration-dependent inhibition of DNA synthesis by GSNO when present during S phase progression (0-4 h). B: concentration-dependent inhibition of DNA synthesis by HU when present during S phase progression (0-4 h). Thymidine incorporation data are means ± SE from a representative experiment of 3 using 8 replicates. * P < 0.05 from control cells in the absence of GSNO or HU.

Effect of deoxynucleosides on S-nitrosothiol and HU-induced inhibition of DNA synthesis. To assess the role of ribonucleotide reductase in HU-induced inhibition of DNA synthesis, cells were treated with HU in the presence of 2'-deoxyadenosine and 2'-deoxyguanosine (0.4 mM each) after synchronization of cells at the G1/S boundary. In the absence of deoxynucleosides, HU concentration dependently inhibited DNA synthesis as before (Fig. 3B). The combination of 2'-deoxyadenosine and 2'-deoxyguanosine restored DNA synthesis to 43-60% compared with 7-32% in the absence of deoxynucleosides for 50-600 µM HU (Fig. 3A). This occurred despite the fact that the deoxynucleosides themselves inhibited DNA synthesis (31.8 ± 0.8%). Similarly to HU, deoxynucleosides also restored DNA synthesis inhibition caused by increasing concentrations of GSNO (50-600 µM, Fig. 3A).


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  		Fig. 3.   Effects of exogenous deoxynucleosides on GSNO- and HU-induced inhibition of DNA synthesis in human aortic smooth muscle cells. A: concentration-dependent inhibition of [3H]thymidine incorporation by GSNO 4 h after cell synchronization at G1/S boundary in control cells, which is partially reversed by a combination of 2'-deoxyadenosine and 2'-deoxyguanosine (0.4 mM each). B: concentration-dependent inhibition of [3H]thymidine incorporation by HU 4 h after cell synchronization at G1/S boundary and partial reversal by deoxynucleosides. Data are means ± SE from a representative experiment using 8 replicates. * P < 0.05 from control cells in the absence of deoxynucleosides.

Monitoring NO release from NO donors and HU by chemiluminescence. Injection of 250 µM GSNO to the purge vessel of the NO analyzer resulted in an immediate increase in NO signal followed by a steady state, signifying a constant rate of release of NO gas from the nitrosothiol (Fig. 4A). Interestingly, additions of human aortic SMC to the purge vessel further increased NO release from GSNO, suggesting increased metabolism of GSNO in the presence of human aortic SMC. In contrast, injection of HU (1.8 mM) caused no NO signal (Fig. 4B). Furthermore, addition of human aortic SMC to the chamber resulted in no detectable NO signal, providing no evidence for oxidation of HU to an NO-like species by these cells.


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  		Fig. 4.   Nitric oxide (NO) release from GSNO and HU measured by chemiluminescence. A: Injection of cell culture medium into the purge vessel of the chemiluminescence analyzer produced no NO signal, whereas GSNO (250 µM) resulted in an immediate increase in NO signal, followed by a steady state. Addition of human aortic smooth muscle cells (SMC) resulted in further increase in NO signal. B: Injection of HU (1.8 mM) into the purge vessel caused no detectable NO signal, and with addition of human aortic SMC the NO signal remained unaltered. Data are representative of 4 different experiments.

Effect of NO donors and HU on cGMP level. Treatment of cultured human aortic SMC with increasing concentrations of GSNO resulted in no increase in cGMP content (22.2 ± 3.9, 25.6 ± 4.4, and 28.25 ± 4.5 fmol/well for control and 250 and 500 µM GSNO, respectively). However, freshly obtained human aorta pieces produced high cGMP levels in response to NO donors (7.6 ± 0.9, 268.4 ± 87.6, 893.6 ± 176.1 fmol/mg wet wt for control and 10 and 100 µM GSNO, respectively; Fig. 5A), suggesting that culturing human aortic SMC results in loss of responsiveness to NO to produce cGMP. We therefore tested DNA synthesis and cGMP responses in cultured rat aortic SMC, a cell line that maintains its ability to respond to NO with production of cGMP (25). Similarly to human aortic SMC, both NO donors and HU inhibited [3H]thymidine uptake in rat aortic SMC. These cells also accumulated cGMP in response to GSNO (61.3 ± 26.4, 197.7 ± 10.2, and 330.0 ± 55.1 fmol/well for control and 10 and 100 µM GSNO, respectively; Fig. 5B); however, there was no change in response to increasing concentrations of HU (83.8 ± 14.9, 50.6 ± 20.2, and 49.6 ± 26.3 fmol/well for control and 0.6 and 1.8 mM HU, respectively; Fig. 5D). Furthermore, HU remained ineffective in increasing cGMP in freshly isolated human aorta pieces (7.6 ± 0.9, 6.8 ± 0.2, and 3.0 ± 0.6 fmol/mg for control and 0.6 and 1.8 mM HU, respectively; Fig. 5C).


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  		Fig. 5.   Effect of GSNO and HU on cGMP accumulation in native human aortic pieces and in cultured rat aortic SMC. Tissue and cultured cells were preincubated with IBMX for 10 min and were exposed to varying concentrations of GSNO or HU for 15 min. cGMP was then extracted by 0.1 mM HCl and assayed by RIA. A: concentration-dependent increase in cGMP levels by GSNO in human aorta pieces, but there is no increase in cGMP in response to HU (C). Rat aortic SMC also show large increases in cGMP in response to GSNO (B) but no change with HU (D). Data are means ± SE from a representative experiment using 3 replicates. * P < 0.05 from cells in the absence of GSNO or HU.

Effect of 8-BrcGMP on DNA synthesis. To investigate the influence of 8-BrcGMP, an analog of cGMP, on DNA synthesis, cells were initially synchronized at the G1/S boundary by treatment with 0.6 mM HU for 12 h, followed by incubation with increasing concentrations of 8-BrcGMP immediately after HU washout for 4 h. There was no noticeable inhibition of DNA synthesis by 8-BrcGMP, as shown by thymidine incorporation (114.5 ± 2.6, 113.2 ± 5.4, and 111.5 ± 5.9% of control for 400, 600, and 1,000 µM, respectively).

Effect of NO scavengers on HU- and GSNO-induced inhibition of DNA synthesis. To further clarify the role of NO in mediating inhibition of DNA synthesis induced by GSNO and HU, hemoglobin and the intracellular NO scavenger cPTIO were introduced. Cells synchronized at the G1/S boundary were incubated for 30 min with hemoglobin (10 µM) or cPTIO (100 µM), and inhibition of DNA synthesis by HU and GSNO was assessed as before in the presence or absence of the NO scavengers. The concentration-dependent inhibition of thymidine incorporation by GSNO (98.4 ± 3.8, 54.3 ± 1.4, and 31.4 ± 1.7% of control, for 200, 400, and 600 µM, respectively) was reversed by hemoglobin (104.7 ± 3.1, 97.3 ± 2.6, and 65.7 ± 1.4% of control, for 200, 400, and 600 µM, respectively; Fig. 6A). Similarly, in the presence of cPTIO, GSNO remained ineffective in inhibiting DNA synthesis (105 ± 3.7, 117 ± 6.9, and 101 ± 5.2% of control for 200, 400, and 600 µM, respectively; Fig. 6B). The inhibitory effect of HU on DNA synthesis in human aortic SMC was not affected, however, by either hemoglobin or cPTIO (Fig. 6, C and D).


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  		Fig. 6.   Influence of NO scavengers on GSNO- and HU-induced inhibition of DNA synthesis. Human aortic SMC synchronized at the G1/S boundary were preincubated with hemoglobin (Hb; 10 µM) or carboxy 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO; 100 µM) for 30 min. The influence of HU and GSNO on ongoing DNA synthesis was evaluated in cells progressing through S phase (0-4 h), either in the presence or absence of Hb and cPTIO. A and B: concentration-dependent inhibition of [3H]thymidine incorporation by GSNO was reversed by both Hb and cPTIO. C and D: concentration-dependent inhibition of DNA synthesis by HU was unaffected by Hb or cPTIO in human aortic SMC. Data are means ± SE from a representative experiment using 4 replicates. * P < 0.05 from cells in the absence of Hb and cPTIO.

Comparative effects of redox agents on NO and HU inhibition of DNA synthesis. The concentration-dependent inhibition of thymidine incorporation by GSNO (71.8 ± 12.3, 41.1 ± 7.3, and 27.6 ± 5.8% of control for 200, 400, and 600 µM, respectively) was reversed by 10 mM NAC (83.8 ± 5.5, 73.0 ± 5, and 70.5 ± 5.8% of control for 200, 400, and 600 µM, respectively; Fig. 7A). Ascorbic acid (10 mM) also prevented the inhibition of DNA synthesis induced by 600 µM GSNO (67.4 ± 5.0 vs. 19.0 ± 1.3% of control), despite the fact that ascorbic acid itself had an inhibitory effect on DNA synthesis (50.1 ± 7.5% of control). Contrary to NO donors, the concentration-dependent inhibition by HU (84.1 ± 2.5, 49.7 ± 0.7, and 22.4 ± 0.8% of control for 200, 400, and 600 µM, respectively) was not reversed by 10 mM NAC (87.1 ± 4.9, 56.1 ± 0.1, and 24.0 ± 0.4% of control for 200, 400, and 600 µM, respectively; Fig. 7B). Ascorbic acid also showed no reversal of HU-induced inhibition of DNA synthesis (5.7 ± 0.4 vs. 19.0 ± 1.3% of control for 600 µM HU).


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  		Fig. 7.   Effect of N-acetyl-L-cysteine (NAC) on inhibition of DNA synthesis induced by GSNO and HU. Human aortic smooth muscle cells were synchronized at the G1/S boundary. The influence of HU and GSNO on ongoing DNA synthesis was evaluated in cells progressing through S phase (0-4 h), either in the presence or absence of NAC (10 mM). DNA synthetic rate was monitored by [3H]thymidine incorporation into acid-insoluble macromolecules. A: concentration-dependent inhibition of [3H]thymidine incorporation by GSNO is prevented by NAC (10 mM). B: concentration-dependent inhibition of [3H]thymidine incorporation by HU is unaffected by NAC (10 mM). Data are means ± SE from a representative experiment of 4 using 4 replicates. * P < 0.05 from cells in the absence of NAC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major aim of our study was to explore the mechanism whereby NO regulates DNA synthesis in human aortic SMC and contrast it to that of HU. We undertook this by studying cells synchronized at the G1/S boundary and limited the experimental time frame to duration of S phase progression to eliminate NO influence on other stages of the cell cycle.

Our data show that NO donors inhibit ongoing DNA synthesis and the progression of human aortic SMC through S phase by releasing NO. This appears to occur via a cGMP-independent mechanism that involves regulation of ribonucleotide reductase by a redox-sensitive process. After the original discovery of NO inhibition of DNA synthesis in cultured rat mesangial cells (7), this has been observed in a variety of models (8, 33). Our data extend these findings to human vascular tissue.

The role of NO in inhibiting DNA synthesis has been demonstrated using chemically distinct NO donors, which include nitrosothiols, DETA-NO, and NO gas itself. NO released from these NO donors was monitored by chemiluminescence, and the influence of NO donors on DNA synthesis was prevented by extracellular and intracellular scavengers of NO. Although relatively high concentrations of NO donors in the 100 µM range were required to inhibit DNA synthesis, the actual rate of NO release from the donors could be several magnitudes lower, as suggested by the relatively low steady-state NO levels detected by chemiluminescence and the relatively slow rate of decomposition of S-nitrosothiols monitored by spectrophotometry at 340 nm (data not shown). Although the inhibition of DNA synthesis by NO was reproducible, there was some variability in the magnitude of the response between experiments. This could be due to a variety of different factors, such as performing experiments using several distinct isolations from different donors, passage number, seeding density, and confluence of the cultures at the time of the experiment.

There has been an ongoing debate regarding the mediation of NO inhibition of DNA synthesis by cGMP (14, 33, 34). To clarify this issue, we measured cGMP accumulation in our human aortic SMC. In the cultured, multipassaged cells, we observed no increase in cGMP accumulation in response to millimolar levels of GSNO. However, when freshly obtained pieces of human aorta were used, we obtained large increases in cGMP levels in response to micromolar concentrations of NO donors. This suggests that the responsiveness of the cells to NO to produce cGMP was lost in culture, a phenomenon that was also previously observed by others (6). Nevertheless, the lack of cGMP accumulation together with data showing the inability of 8- BrcGMP to modulate DNA synthesis in these cells suggest that the observed inhibition of DNA synthesis by NO donors proceeds via a cGMP-independent mechanism. This suggestion is in agreement with published findings in other systems, where inhibition of DNA synthesis by NO has been concluded to be independent of activation of the enzyme soluble guanylate cyclase and cellular accumulation of cGMP (9, 33).

A proposed mechanism for the inhibition of ongoing DNA synthesis and S phase progression by NO is a direct action of NO on ribonucleotide reductase, the key enzyme that provides deoxynucleotides for DNA synthesis (17, 32, 33). We reasoned that exogenous deoxynucleosides should overcome the inhibition of DNA synthesis by bypassing the inhibited ribonucleotide reductase enzyme. Such an approach has been successful in identifying ribonucleotide reductase as the target of macrophage-derived NO in tumor cells (17). The optimal combination of deoxynucleosides, which reversed the inhibition of ribonucleotide reductase by NO in that study, also significantly reversed the DNA synthesis inhibition exhibited by both NO and HU in our system, despite the fact that they themselves inhibited DNA synthesis. These data are consistent with the hypothesis that ribonucleotide reductase is a primary and common target for both NO and HU in inhibiting DNA synthesis and S phase progression in cultured human aortic SMC. However, we realize some limitations in both our data and those reported by Kwon et al. (17) in that the reversal of inhibition by deoxynucleosides is not complete, and these substances inhibit DNA synthesis themselves. This could be due to deoxynucleosides competing with each other for intracellular transport and phosphorylation by nucleoside kinase, resulting in suboptimal restoration of intracellular nucleotide pools (17, 18).

The structural similarities between HU and N-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted Kwon et al. (17) to explore release of NO from HU. They provided chemical evidence that, even in the absence of NO synthase, NO release from HU could be achieved by oxidation in the presence of hydrogen peroxide and copper as a catalyst. Although not studied in intact cells, this mechanism has been postulated to account for the pharmacological effects of HU in cells that can oxidize it (17). We therefore explored this in intact human aortic SMC by a variety of means. We monitored the release of NO from HU by chemiluminescence and assessed soluble guanylate cyclase activation and associated cGMP accumulation as a characteristic response to NO-like species and finally evaluated NO scavengers in modulating the action of HU. As opposed to detectable NO signals from various NO donors, addition of high concentrations of HU produced no NO signal by chemiluminescence. Furthermore, the addition of human aortic SMC to the purge vessel of the analyzer, which caused a significant release of NO from the nitrosothiols, was ineffective in releasing NO from HU. These observations suggest that NO release from HU, if the release occurred in human aortic SMC at all, remained below the detectable limit of our chemiluminescence analysis (<100 pmol). One of the most sensitive intracellular responses induced by NO-like species is the activation of soluble guanylate cyclase and accumulation of cGMP. Whereas low micromolar concentrations of NO donors produced great increases in cGMP, there was no increase in cGMP, even to millimolar levels of HU in fresh aortic pieces. To further test whether HU acts by releasing NO, we studied the influence of extracellular and intracellular scavengers of NO on the inhibition of DNA synthesis by HU. Inasmuch as both scavengers were effective in preventing NO-dependent inhibition of DNA synthesis, they were incapable of modulating the inhibition of DNA synthesis by HU in our human aortic SMC. Therefore, we found no evidence for release and biochemical action of an NO-like species from HU, leading us to conclude that NO release is not involved in mediating cellular actions of HU in the cells used in the current study. Alternatively, the species released from HU could be a peculiar NO species not sharing biochemical reactions of classical NO donors.

Not only do we show that NO is unlikely to mediate the effects of HU, but our data with redox agents suggest that the action of these two agents in inhibiting DNA synthesis and ribonucleotide reductase can be distinguished by pharmacological means. Activity of ribonucleotide reductase is highly dependent on the catalytically competent tyrosyl radical, stabilized by a redox-regulated dinuclear iron center on its R2 subunit and an interaction with critical thiols on the R1 subunit. In Escherichia coli ribonucleotide reductase, HU has been shown to scavenge the tyrosyl radical in a one-electron transfer reaction, in the absence of an effect on the iron center (10). The action of HU on ribonucleotide reductase is complicated, however, by species differences. For example, HU has been shown to affect the iron radical center in mammalian ribonucleotide reductase, which can be regenerated by dithiothreitol conferring some redox sensitivity (10). Similarly, NO has been demonstrated to destroy the tyrosyl radical in the absence of an effect on the iron center in isolated E. coli R2 protein (21). However, in contrast, there is evidence for NO having an additional effect on the iron center in mouse R2 protein (11). One of the prominent physiological effects of NO is interaction with heme iron and nonheme iron proteins (20, 27). An action of NO on the redox-regulated iron center of ribonucleotide reductase in our human aortic SMC could explain the redox sensitivity of NO-induced DNA synthesis inhibition in our studies.

An additional redox-sensitive target of NO could involve the critical thiols on the R1 subunits. Although direct nitrosylation of these thiols on the R1 subunit remains to be clarified, S-nitrosylation is increasingly recognized as a biological mechanism to explain interactions between NO and cysteine-containing proteins (3). The reaction appears to play a critical role in mediating the influence of NO on important cellular responses, such as S-nitrosylation of respiratory chain proteins, to confer inhibition of cellular respiration by NO. S-nitrosylation of caspases has recently been documented to explain the influence of NO on apoptosis (16, 22) and S-nitrosylation of receptors (5), channels (38), and signal transduction proteins (13) to illustrate means whereby NO affects cell signaling. A potential effect of NO in S-nitrosylating the R1 subunits of ribonucleotide reductase could explain the differential redox sensitivity of NO and HU on DNA synthesis in our human aortic SMC. Further studies are warranted to test these hypotheses and to clarify the exact molecular mechanism whereby NO and HU modulate DNA synthesis in human vascular tissue. This effort could have important clinical and therapeutic implications in understanding control of human vascular SMC growth by NO under normal conditions and in targeting accentuated proliferation of SMC pharmacologically.


    ACKNOWLEDGEMENTS

These studies were partially supported by British Heart Foundation Project Grant PG/97200 (R. Bundy and N. Marczin) and Országos Tudományos Kutatási Alap from Hungary (N. Marczin). M. Yacoub is a British Heart Foundation Professor of Cardiothoracic Surgery.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Yacoub, Dept. of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK.

Received 26 February 1999; accepted in final form 9 June 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 277(5):H1799-H1807
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