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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1216.)
© 2000 American Heart Association, Inc.

Vascular Biology

Doxazosin Inhibits Retinoblastoma Protein Phosphorylation and G₁S Transition in Human Coronary Smooth Muscle Cells

Ulrich Kintscher; Shu Wakino; Sarah Kim; Simon M. Jackson; Eckart Fleck; Willa A. Hsueh; Ronald E. Law

From the Department of Medicine (U.K., S.W., S.K., S.M.J., W.A.H., R.E.L.), Division of Endocrinology, Diabetes and Hypertension, School of Medicine, University of California, Los Angeles, and the Department of Medicine/Cardiology (U.K., E.F.), Virchowklinikum, Humboldt University Berlin, and German Heart Institute Berlin, Berlin, Germany.

Correspondence to Ronald E. Law, PhD, UCLA School of Medicine, Division of Endocrinology, Diabetes and Hypertension, Warren Hall, Second Floor, Suite 24-130, 900 Veteran Ave, Box 957073, Los Angeles, CA 90095. E-mail rlaw{at}med1.medsch.ucla.edu

	Abstract

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Abstract—Previous studies have demonstrated that the ₁-adrenergic receptor antagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity of Dox occurs through a novel mechanism unrelated to its blocking the ₁-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G₁S progression of human coronary artery smooth muscle cells (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88±10.5% in 20 ng/mL platelet-derived growth factor and 1 µmol/L insulin (P+I)–stimulated cells (P<0.01 for 10 µmol/L Dox versus P+I alone) and 52±18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 µmol/L Dox versus 10% FBS alone). Inhibition of G₁ exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G₁ arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G₁ and enter the S phase. E2F-mediated G₁ exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibitory protein p27 is important for maximal activation of G₁ cyclin/cyclin-dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb. In Western blot analysis, p27 levels decreased after mitogenic stimulation (after P+I, 43±1.8% of quiescent cells [P<0.01 versus quiescent cells]; after 10% FBS, 55±7.7% of quiescent cells [P<0.05 versus quiescent cells]), whereas the addition of Dox (10 µmol/L) markedly attenuated its downregulation (after P+I, 90±8.3% of quiescent cells [P<0.05 versus P+I alone]; after 10% FBS, 78±8.3% of quiescent cells [P<0.05 versus 10% FBS alone]). Furthermore, Dox inhibited cyclin A expression, an E2F regulated gene that is essential for cell cycle progression into the S phase. The present study demonstrates that Dox inhibits CASMC proliferation by blocking cell cycle progression from the G₀/G₁ phase to the S phase. This G₁S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation.

Key Words: vascular smooth muscle cells • proliferation • cell cycle • retinoblastoma • doxazosin

	Introduction

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Proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the progression of atherosclerosis and in the development of restenosis after coronary angioplasty. After endothelial injury, VSMCs migrate into the intimal layer of the arterial wall, where they leave their quiescent state and reenter the cell cycle in response to growth factors and cytokines.¹ Progression through the mammalian mitotic cycle is coordinated by the expression and/or activation of multiple holoenzymes composed of a catalytic cyclin-dependent kinase (CDK) and a cyclin-regulatory subunit.² Different cyclin/CDK complexes are temporally activated at specific phases of the cell cycle. Progression through the first gap phase (G₁) requires cyclin D–dependent (CDK4 and CDK6) and cyclin E/CDK2 activity.³ Functional cyclin A/CDK2 is assembled and activated late in G₁ and during DNA synthesis (S phase).⁴ The kinase activity of the cyclin/CDK complexes can be negatively regulated by CDK-inhibitory proteins (CDKI), including p15^INK4 (p15), p16^INK4 (p16), p21^waf1/cip1 (p21), and p27^kip1 (p27).⁵ Cyclin/CDK holoenzymes phosphorylate the retinoblastoma tumor suppressor protein (Rb), resulting in inactivation of Rb and release of sequestered E2F transcription factors.⁶ E2F induces the transcription of genes encoding proteins that are required for S-phase DNA synthesis.⁷ Recent studies have illustrated the feasibility of using existing drugs in clinical use, whose safety has already been established, to target cell cycle–regulatory molecules in vascular cells as a novel therapeutic approach.^{8 9}

Doxazosin (Dox) is a selective ₁-adrenergic receptor antagonist that is used extensively in the treatment of systemic hypertension and benign prostatic hyperplasia. Recent studies have demonstrated that Dox markedly inhibits platelet-derived growth factor (PDGF)-stimulated, epidermal growth factor–stimulated, angiotensin II–stimulated, thrombin-stimulated, and serum-stimulated proliferation of human VSMCs.¹⁰ This antiproliferative activity is physiologically significant because Dox reduced intimal hyperplasia in a rabbit balloon-injury model.¹¹ Dox blocked mitogenesis even after pretreatment with phenoxybenzamine, an irreversible ₁-adrenergic receptor antagonist. Inhibitory effects of Dox on DNA synthesis were also observed in PDGF-stimulated NIH 3T3 fibroblasts, which do not express any ₁-adrenergic receptor.¹⁰ Together, these findings suggest that the antiproliferative activity of Dox is mediated through a novel mechanism unrelated to its blocking the ₁-adrenergic receptor. Furthermore, the inhibition of multiple mitogenic pathways by Dox implies that it blocks the function of a fundamental component of the cell cycle machinery. The purpose of the present study was to determine the mechanism by which Dox inhibits DNA synthesis by examining its effect on cell cycle regulators in human coronary artery smooth muscle cells (CASMCs).

	Methods

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Materials
PDGF-BB, terazosin, dimethyl sulfoxide (DMSO), phenoxybenzamine, propidium iodide, DMEM, glutamine, and antibiotics were purchased from Sigma Chemical Co. Insulin was obtained from Lilly. FBS was purchased from Irvine Scientific. Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane, horseradish peroxidase–linked anti-rabbit and anti-mouse antibody, and ECL Western blotting detection reagents were from Amersham Life Sciences. Dox was kindly provided by Pfizer Inc. CASMCs and a smooth muscle growth medium-2 (SmGM-2) kit were purchased from Clonetics. NIH 3T3 fibroblasts were purchased from American Type Culture Collection.

Cell Culture
CASMCs were cultured in SmGM-2 containing 5% FBS, 2 ng/mL human basic fibroblast growth factor, 0.5 ng/mL human epidermal growth factor, 50 µg/mL gentamicin, 50 ng/mL amphotericin B, and 5 µg/mL bovine insulin. CASMCs from passages 4 to 8 were used for experiments. NIH 3T3 fibroblasts were grown in DMEM with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 mmol/L glutamine. For all experiments, cells were grown to 60% to 70% confluence and made quiescent by serum starvation (0.4% FBS/SmGM-2 or DMEM) for 24 hours.

Flow Cytometry
Quiescent CASMCs were pretreated for 30 minutes with Dox or vehicle (DMSO), followed by the addition of growth factors (20 ng/mL PDGF-BB+1 µmol/L insulin or 10% FBS). After 24 hours, cells were trypsinized, centrifuged at 1500 rpm for 3 minutes, washed with PBS, and then treated with 20 µg/mL RNase A (Calbiochem). DNA was stained with 100 µg/mL propidium iodide for 30 minutes at 4°C and protected from light, and 1x10⁶ cells were then analyzed with a FACScan (Becton Dickinson). DNA histogram analysis was performed by using ModFitLT software (Becton Dickinson). Experiments were repeated at least 3 times.

Adenoviral Infection of CASMCs
Adenovirus encoding human E2F-1 (Ad-E2F-1), driven by the cytomegalovirus (CMV) promoter, was kindly provided by R.W. MacLellan (University of California, Los Angeles).¹² Adenovirus containing CMV-driven green fluorescent protein (Ad-GFP) was generously provided by C.C. Hedrick (University of California, Los Angeles). Quiescent CASMCs were infected with Ad-E2F-1 or Ad-GFP at a multiplicity of infection of 200. After 120 minutes, Dox (10 µmol/L) or vehicle (DMSO) was added to the cells. After 48 hours of incubation, CASMCs were prepared for FACScan experiments as described above. Infection efficiency of CASMCs was >90%, as determined by GFP expression and visualized by fluorescence microscopy.

Western Immunoblot
Quiescent cells were exposed to Dox, terazosin, or vehicle (DMSO) 30 minutes before stimulation with the indicated growth factors. After 24 hours, protein isolation, electrophoresis, and blotting were performed as previously described.¹³ Blots were incubated with specific antibodies against total Rb (No. 14001A, Pharmingen), phospho Rb Ser807/811 (No. 9308S, New England BioLabs Inc), phospho Rb Ser795 (No. 9301S, New England BioLabs Inc), cyclin D1 (sc-481, Santa Cruz), cyclin E (sc-753, Santa Cruz), cyclin A (sc-751, Santa Cruz), CDK2 (sc-6248, Santa Cruz), CDK4 (sc-749, Santa Cruz), CDK6 (sc-7181, Santa Cruz), and CDKI p27 (sc-1641, Santa Cruz) at a 1:200 concentration. Immunoreactive bands were visualized with the use of horseradish peroxidase–conjugated secondary antibodies (1:1000 dilution). The peroxidase reaction was developed by use of an ECL detection system (Amersham Corp). Band intensity was analyzed by densitometry.

Statistics
ANOVA with paired or unpaired t tests was performed for statistical analysis, as appropriate. Values of P<0.05 were considered to be statistically significant. Data are expressed as mean±SEM.

	Results

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Dox Inhibits G₁S Progression of Human CASMCs
The effect of Dox on cell cycle progression was determined by flow cytometry. Subconfluent CASMCs were synchronized by serum starvation for 24 hours, with 92±2.9% of cells arrested in G₀/G₁ phase and 5.9±2.9% present in S phase. Mitogenic stimulation with the combination of PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS for 24 hours stimulated the CASMC progression into S phase by 29±1.6% and 24±3.9%, respectively (Figure 1). Treatment of cells with Dox inhibited mitogen-induced G₁S progression of human CASMCs in a dose-dependent manner, with a maximal reduction of S-phase transition of 88±10.5% in PDGF- and insulin-stimulated cells (P<0.01 for 10 µmol/L Dox [3.5% S-phase transition] versus PDGF+insulin alone [29% S-phase transition]) and 52±18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 µmol/L Dox [11.6% S-phase transition] versus 10% FBS alone [24% S-phase transition]), as seen in Figure 2. Dox had a lesser effect to inhibit G₁S progression in serum-stimulated versus PDGF+insulin–treated cells, which could be due to drug binding to the albumin present in serum.

View larger version (22K): [in this window] [in a new window]   Figure 1. Dox inhibits mitogen-induced G1S progression of human CASMCs. Cells were starved in SmGM-2 containing 0.4% FBS for 24 hours to be synchronized in G0/G1 phase. Quiescent CASMCs were stimulated by treatment with PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS. Cells were preincubated with Dox (10 µmol/L) or DMSO for 30 minutes before the addition of mitogens. After 24 hours, DNA was stained with propidium iodide (PI), and 1x106 cells were analyzed by flow cytometry. The x- and y-axes represent the intensity of PI fluorescence and cell number, respectively. The figure shows representative DNA histograms.

View larger version (23K): [in this window] [in a new window]   Figure 2. Dox inhibits mitogen-induced G1S progression of human CASMCs in a dose-dependent manner. Treatment of cells and measurement of DNA content were performed as described in Figure 1. Mean±SEM is expressed as percentage of cells in S phase (left y-axis) and percentage of S-phase transition (right y-axis, percentage of treated cells in S phase minus quiescent cells [Q] in S phase). *P<0.05 vs quiescent cells; #P<0.05 and ##P<0.01 vs mitogen-stimulated cells (n=3).

To study the effects of Dox on progression to G₂/M phase, we performed flow cytometry after 72 hours of mitogenic stimulation. Consistent with the 24-hour data, CASMCs treated with Dox for 72 hours had a higher percentage of cells in G₁ phase and a smaller percentage in S phase compared with the values for cells treated with mitogens alone. No increase in the number of cells in G₂/M phase was observed, which suggests that the antiproliferative activity of Dox results from targeting events in the G₁ phase (data not shown).

Dox Inhibits Phosphorylation of Rb
To elucidate the mechanism for the G₁ arrest by Dox, we examined its effect on Rb phosphorylation. Rb migrates in an SDS-polyacrylamide gel as multiple, closely spaced bands reflecting varying degrees of phosphorylation. After 24-hour mitogenic stimulation with PDGF+insulin or 10% FBS, a mobility shift of Rb was observed indicative of increased phosphorylation. Dox inhibited the mobility shift in a dose-dependent manner (Figure 3A). We also examined the effect of Dox on specific phosphorylation sites in Rb, Ser795, and Ser807/811, which mediate CDK-dependent regulation of Rb function.^{14 15} Quiescent CASMCs exhibited low levels of phosphorylated Ser807/811 and Ser795 (Figure 3B and 3C). Phosphorylation of Ser807/811 and Ser795 increased substantially after a 24-hour stimulation with growth factors (PDGF+insulin or 10% FBS). Dox inhibited phosphorylation of Ser807/811 (Figure 3B) and Ser795 (Figure 3C). Mitogenic stimulation of CASMCs induced a modest increase in the level of Rb, which was not affected by Dox. These data demonstrate that Dox blocks an early G₁ event required for Rb phosphorylation.

View larger version (36K): [in this window] [in a new window]   Figure 3. Dox inhibits phosphorylation of Rb. Cells were starved in SmGM-2 containing 0.4% FBS for 24 hours. Quiescent CASMCs were stimulated by treatment with PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS. Cells were preincubated with Dox at different concentrations or DMSO (-) for 30 minutes before the addition of mitogens. After 24 hours, whole-cell proteins (40 µg) were assayed by Western immunoblotting with use of anti-Rb antibody. A, Hyperphosphorylated Rb (pRb)and hypophosphorylated Rb (Rb). B, Anti–phospho Rb Ser807/811 antibody (top) and densitometric analysis of immunoblots with anti-phospho Rb Ser807/811 antibody shown as percentage of mitogen-stimulated cells (bottom). Results are presented as mean±SEM (n=3). *P<0.05 and **P<0.01 vs mitogen-stimulated cells. C, Representative Western immunoblot (n=3) with use of anti–phospho Rb Ser795.

Dox Has No Effect on Expression of CDKs and G₁ Cyclins D1 and E but Inhibits Cyclin A Induction During G₁ Phase
To understand the mechanism by which Dox inhibits Rb phosphorylation, we examined its effect on the expression of CDKs for which Rb is a physiological substrate. CDK2 levels were low in quiescent cells, increased after 24-hour mitogenic stimulation, and did not change with Dox (Figure 4A). Quiescent CASMCs expressed CDK4 and CDK6, which did not change after either mitogenic stimulation or treatment with Dox (Figure 4A). We next examined the effect of Dox on protein expression of G₁-phase cyclins D1 and E, because their association with CDKs is required for CDKs to phosphorylate Rb and other substrates. Both cyclins, D1 and E, were expressed at low levels in quiescent CASMCs, which increased after a 24-hour stimulation with PDGF+insulin or 10% FBS. Treatment with Dox had no effect on the induction of cyclins D1 and E by mitogens (Figure 4A). To confirm the effect of Dox to inhibit early G₁ cyclin/CDK phosphorylation of Rb, we also examined its effect on a late G₁/S-phase cyclin. Cyclin A is essential for cell cycle progression in S phase, and its expression is regulated through the E2F transcription factor, whose activity is controlled by the phosphorylation status of Rb and early G₁ cell cycle events.^{4 16} In quiescent CASMCs, low levels of cyclin A protein were detected, which increased after a 24-hour mitogenic stimulation. Dox effectively inhibited cyclin A expression in a dose-dependent manner, consistent with its acting in early G₁ phase to suppress cell cycle progression (Figure 4B).

View larger version (39K): [in this window] [in a new window]   Figure 4. A, Dox has no effect on expression of CDKs and G1 cyclins D1 and E. Cells were starved in SmGM-2 containing 0.4% FBS for 24 hours. Quiescent CASMCs were stimulated by treatment with PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS. Cells were preincubated with Dox (10 µmol/L) or DMSO (-) for 30 minutes before the addition of mitogens. After 24 hours, whole-cell proteins (40 µg) were assayed by Western immunoblotting by use of antibodies against CDK2, CDK4, CDK6, cyclin D1, and cyclin E. Blots are representative of 3 separate experiments. B, Dox inhibits cyclin A induction. Treatment of cells was performed as described in panel A. After 24 hours, whole-cell proteins (40 µg) were assayed by Western immunoblotting with use of anti–cyclin A antibody. Above the graph is a blot representative of 3 experiments. Densitometric analysis of cyclin A protein levels in Western blots is shown as percentage of mitogen-stimulated cells. Results are presented as mean±SEM (n=3). *P<0.05 and **P<0.01 vs mitogen-stimulated cells.

Dox Prevents Mitogen-Induced Downregulation of CDKI p27
CDKI p27 inhibits the activities of cyclin E/CDK2 and cyclin D1/CDK4 complexes.^{17 18} Downregulation of p27 during G₁ phase in response to mitogens is important for maximal activation of G₁ cyclin/CDK holoenzymes.¹⁹ Therefore, we investigated the effect of Dox on p27 expression after mitogenic stimulation. Western analysis of quiescent CASMCs revealed substantial p27 protein. Expression of p27 decreased markedly after a 24-hour stimulation with PDGF+insulin (43±1.8% of quiescent cells, P<0.01 versus quiescent cells) or 10% FBS (55±7.7% of quiescent cells, P<0.05 versus quiescent cells). Addition of Dox to mitogen-stimulated CASMCs attenuated downregulation of p27 levels (after PDGF+insulin+10 µmol/L Dox, 90±8.3% of quiescent cells [P<0.05 versus PDGF+insulin alone]; after 10% FBS+10 µmol/L Dox, 78±8.3% of quiescent cells [P<0.05 versus 10% FBS alone]), as seen in Figure 5. Dox had no effect on CDKIs p15, p16, or p21 (data not shown). These results suggest that Dox blocks G₁S progression of human CASMCs through its effects on CDKI p27.

View larger version (30K): [in this window] [in a new window]   Figure 5. Dox prevents mitogen-induced downregulation of CDKI p27. Cells were starved in SmGM-2 containing 0.4% FBS for 24 hours. Quiescent CASMCs were stimulated by treatment with PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS. Cells were preincubated with Dox at different concentrations or DMSO (-) for 30 minutes before the addition of mitogens. After 24 hours, whole-cell proteins (40 µg) were assayed by Western immunoblotting with use of anti-p27 antibody. Above the graph is a blot representative of 3 experiments. Graph shows results of densitometric analysis of p27 protein levels in Western blots presented as percentage of quiescent cells. Results are mean±SEM (n=3). *P<0.05 and **P<0.01 vs quiescent cells; #P<0.05 vs mitogen-stimulated cells.

Dox Has No Effect on S-Phase Transition Induced by E2F-1 Overexpression
Inhibition of Rb hyperphosphorylation leads to G₁ arrest by inactivating the E2F transcription factor through its binding to Rb.⁶ Overexpression of E2F-1 has been shown to induce quiescent cells to enter the S phase.²⁰ To test directly that the inhibitory effects of Dox on CASMC cell cycle progression are mainly mediated through effects on Rb and inactivation of E2F, we infected quiescent CASMCs with an Ad-E2F-1 and performed flow cytometry. Infection of CASMCs with Ad-E2F-1 resulted in 20.2±1.8% S-phase transition compared with 1.7±0.8% in Ad-GFP–infected cells (P<0.05, Figure 6). Moreover, the overexpression of exogenous E2F-1 could overcome the Dox-mediated G₁ arrest in CASMCs (after Ad-E2F-1+10 µmol/L Dox, 26±4.3% S-phase transition; after Ad-E2F-1 alone, 20.2±1.8% S-phase transition; P=NS), as seen in Figure 6; these findings support our conclusion that the effects of Dox on p27 and Rb phosphorylation are causally related to its inhibition of G₁S progression in CASMCs.

View larger version (17K): [in this window] [in a new window]   Figure 6. Dox has no effect on S-phase transition induced by E2F-1 overexpression. Quiescent CASMCs were infected with Ad-E2F-1 or Ad-GFP at a multiplicity of infection of 200. After 120 minutes, Dox (10 µmol/L) or vehicle (DMSO) was added to the cells. After 48 hours of incubation, CASMCs were prepared for flow cytometry as described in Figure 1. A, Representative DNA histograms are shown. B, Results are expressed as mean±SEM of percentage of S-phase transition. *P<0.05 vs Ad-GFP–infected cells.

Cell Cycle Effects of Dox Are Independent of ₁-Adrenergic Receptor Blockade
To determine whether the effects of Dox on cell cycle regulators were through its blockade of the ₁-adrenergic receptor, we examined the effects of terazosin, another ₁-adrenergic receptor antagonist, on the phosphorylation of Rb at Ser807/811 in CASMCs. Although Dox potently inhibited Rb phosphorylation, terazosin had no significant effect (Figure 7A). Additionally, we performed flow cytometry with CASMCs, which were preincubated with phenoxybenzamine (1 µmol/L), an irreversible ₁-receptor antagonist that inactivates ₁-receptors in VSMCs. Dox also significantly blocked the mitogen-induced G₁/S progression in these cells (data not shown). Because it is difficult to demonstrate that phenoxybenzamine blocked all ₁-receptors, we studied the effects of Dox in NIH 3T3 fibroblasts, which do not express any type of -receptor.¹⁰ Dox markedly inhibited PDGF+insulin– or 10% FBS–induced Rb phosphorylation in NIH 3T3 cells, whereas terazosin showed no effect (Figure 7B). Together, these findings strongly support an ₁-adrenergic receptor–independent mechanism of action for the inhibition by Dox of CASMC G₁S progression.

View larger version (25K): [in this window] [in a new window]   Figure 7. Cell cycle effects of Dox are independent of 1-adrenergic receptor blockade. Cells were starved in SmGM-2 (CASMCs) or DMEM (NIH 3T3 fibroblasts) containing 0.4% FBS. Quiescent CASMCs or NIH 3T3 fibroblasts were stimulated by treatment with PDGF (20 ng/mL) and insulin (1 µmol/L) or 10% FBS. Cells were preincubated with Dox (10 µmol/L), terazosin (10 µmol/L), or DMSO (-) for 30 minutes before the addition of mitogens. After 24 hours, whole-cell proteins (40 µg) were assayed by Western immunoblotting with use of anti–phospho Rb Ser807/811 antibody. A, Representative Western immunoblot of 3 experiments with CASMCs. B, Representative Western immunoblot of 3 experiments with NIH 3T3 fibroblasts.

	Discussion

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The present study demonstrates that Dox inhibits CASMC proliferation by blocking cell cycle progression from the G₀/G₁ phase to the S phase. This G₁S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation via an ₁-adrenergic receptor–independent mechanism.

Dox, a selective ₁-adrenergic receptor antagonist, has been recently shown to inhibit proliferation of human VSMCs by blocking multiple mitogenic signaling pathways.¹⁰ In addition, Dox has also been shown to inhibit the PDGF-induced proliferation of rat mesangial cells.²¹ Proliferating mammalian cells pass through several cell cycle checkpoints, mainly G₁-to-S and G₂-to-M transitions. The broad inhibitory effect of Dox exerted against multiple mitogenic growth factors strongly implied that it blocked cell cycle progression at one of these checkpoints. Using flow cytometric analysis, we demonstrated that Dox prevents cell cycle progression at the G₁-to-S transition.

Hyperphosphorylation of Rb by G₁ cyclin/CDKs is required for entry into S phase.⁶ Rb has multiple CDK phosphorylation sites that regulate its conformation and ability to bind to other cell cycle–regulatory proteins, such as E2F and c-Abl.²² Among 16 potential CDK phosphorylation sites in Rb, Ser807/811 and Ser795 have been reported to have critical effects on the function of Rb. Mutation of Ser807/811 prevents the efficient phosphorylation of Rb in a human cervical carcinoma cell line, and mutation of Ser795 to Ala renders Rb resistant to inactivation by CDK4 in a microinjection assay.^{14 22} Dox blocked mitogen-induced phosphorylation of Ser807/811 and Ser795. Thus, the antiproliferative effects of Dox in CASMCs are mediated by preventing phosphorylation of at least 2 major CDK sites in Rb.

Hypophosphorylation of Rb inactivates the E2F transcription factor, leading to G₁ arrest.⁶ Inactivation of the transcriptional activity of E2F by use of an oligonucleotide decoy of its binding site has been reported to inhibit smooth muscle cell proliferation in vivo, whereas overexpression of E2F induces quiescent cells to enter S phase.^{16 23} Together, these studies demonstrate the pivotal role of E2F in the regulation of cell proliferation. In the present study, adenoviral delivery of E2F-1 in CASMCs led to S-phase transition of cells, which was not inhibited by Dox. These data confirm that Dox is inhibiting a process in the G₁ phase of the cell cycle required for E2F release and progression into S phase.

To identify a G₁-phase target of Dox, we examined its effect on the expression of G₁ cyclins and CDKs, which (as holoenzymes) phosphorylate Rb. Protein expression of cyclin D, cyclin E, CDK2, CDK4, and CDK6 after mitogenic stimulation was not affected by Dox. Because functional inactivation of Rb necessitates sequential phosphorylations during G₁ phase, a logical target of Dox would be a cell cycle–regulatory protein that affects several G₁ cyclin/CDK complexes.²⁴ CDKIs negatively regulate the activities of multiple cyclin/CDK complexes.⁵ CDKI p27 plays a pivotal role in the control of cell proliferation by inhibiting CDK2 and CDK4 activity. Overexpression of p27 in nonvascular cells causes cell cycle arrest in G₁ phase.^{17 18} Moreover, it has been recently reported that overexpression of p27 cDNA in VSMCs inhibits mitogen-stimulated [³H]thymidine incorporation by 58%, indicating its important role also in the regulation of G₁/S progression in these cells.²⁵

Downregulation of p27 during the late G₁ phase is required for cell cycle progression from G₁ to S phase.^{26 27} Recent studies have demonstrated that mitogenic stimulation of quiescent VSMCs, which have high levels of p27, and the subsequent transit through the G₁/S restriction point are associated with reduced levels of p27.^{19 28} We observed a significant decrease of p27 protein expression after stimulation of quiescent CASMCs with PDGF+insulin or 10% FBS. Treatment with Dox resulted in a dose-dependent restoration of high p27 levels. These findings identify the prevention of p27 downregulation in response to mitogens as the likely mechanism by which Dox blocks Rb hyperphosphorylation and inhibits CASMC proliferation. After phosphorylation, p27 breakdown seems to be regulated by 2 mechanisms: the ubiquitin-proteasome–mediated degradation and a ubiquitin-independent pathway that abrogates p27 function by eliminating its cyclin-binding domain.^{29 30} Recently, the hydroxymethylglutaryl–coenzyme A reductase inhibitor lovastatin has been shown to prevent p27 downregulation by inhibiting the proteasome pathway, independent of its blockade of hydroxymethylglutaryl–coenzyme A reductase enzymatic activity.³¹ It remains to be determined whether the inhibitory activity of Dox is also mediated by interacting with these pathways.

To corroborate that the inhibitory effects of Dox on G₁ CDKI/cyclin/CDK complexes and the concomitant disruption of Rb hyperphosphorylation impacted subsequent events in the cell cycle, we examined protein expression of cyclin A, which is transcriptionally regulated by E2F.¹⁶ Consistent with the inhibition by Dox of early G₁ events required for the release of E2F from Rb, we observed that cyclin A expression was effectively inhibited by Dox. Moreover, cyclin A associated with CDK2 has been shown to contribute to hyperphosphorylation of Rb in late G₁ phase.^{32 33} Therefore, downregulation of cyclin A protein levels might be an additional mechanism for the functional inactivation of Rb by Dox.

Our data are also consistent with the previous studies of Hu et al,¹⁰ who proposed that the antiproliferative activity of Dox is mediated through a novel mechanism unrelated to its activity as an ₁-adrenergic receptor antagonist. We demonstrated in 3 different experiments that the G₁/S progression or phosphorylation of a major functional Rb site is blocked by Dox via an ₁-adrenergic–independent pathway. Dox has been reported not to inhibit growth factor–induced receptor tyrosine kinase or cytoplasmic Src tyrosine kinase activity.¹⁰ Because it has been shown that the second messenger cAMP mediates growth arrest because of high levels of p27 protein, an increase of cAMP levels might be a logical inhibitory mechanism of Dox.³⁴ In contrast, cAMP-elevating agents have been reported to inhibit the activation of Raf and extracellular signal–regulated kinase in VSMCs and fibroblasts.^{35 36} This pathway is known to regulate mitogen-dependent induction of cyclin D1.³⁷ However, we did not observe any effects of Dox on cyclin D1 expression, and Hu et al showed no effect of Dox on the Ras-mitogen–activated kinase pathway.¹⁰ Thus, it is unlikely that Dox acts by increasing cAMP levels. Recent reports have identified members of the Rho family of GTPases as negative regulators of p27 expression.²⁸ Future studies are necessary to determine whether Dox interferes with this pathway. The direct molecular target of Dox and the postreceptor signal transduction pathways affected remain to be identified.

Consistent with our in vitro data, Dox has been shown in vivo to inhibit angiotensin II–induced DNA synthesis and intimal hyperplasia in animal balloon-injury models.^{11 38} Recent studies have illustrated the feasibility of targeting specific cell cycle regulators in cardiovascular cells as an alternative antiproliferative therapy.² One major approach is the use of modified viruses designed to carry a cell cycle–regulatory gene directly into the arterial wall. Localized arterial infection with an adenovirus encoding a nonphosphorylatable constitutively active form of Rb inhibited neointimal formation and VSMC proliferation in animal balloon-injury models.³⁹ Interventions based on targeting cell cycle regulators are warranted because their expression is altered after angioplasty. In a porcine balloon-injury model, p27 expression was markedly reduced in the intima and media early after angioplasty, whereas positive regulators of the cell cycle, such as cyclin A, were induced after angioplasty in rats, consistent with an injury-induced proliferative response.^{40 41} In combination, these studies suggest that Rb, p27, and cyclin A are promising targets for an antiproliferative therapy. Because Dox has been shown to inhibit VSMC proliferation by modulating these cell cycle regulators at concentrations comparable to the plasma levels achieved in hypertensive patients,⁴² it may provide a new therapeutic approach for proliferative vascular diseases.

	Acknowledgments

 
This study was supported by a national Institutes of Health grant HL-58328 to W.A.H and a grant from the Doxazosin Investigator and Consultants Educational Exchange to W.A.H. and R.E.L. S.W. was supported by a fellowship by the Mary K. Iacocca Foundation.

Received December 1, 1999; accepted February 2, 2000.

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