title: Doxazosin inhibits retinoblastoma protein phosphorylation and G(1)-->S transition in human coronary smooth muscle cells.
abstract: Previous studies have demonstrated that the alpha(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 alpha(1)-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G(1)-->S 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 micromol/L insulin (P+I)-stimulated cells (P<0.01 for 10 micromol/L Dox versus P+I alone) and 52+/-18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 micromol/L Dox versus 10% FBS alone). Inhibition of G(1) exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G(1) arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G(1) and enter the S phase. E2F-mediated G(1) 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 G(1) 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 micromol/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 G(0)/G(1) phase to the S phase. This G(1)-->S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation.
authors.affiliation: Department of Medicine, Division of Endocrinology, Diabetes and Hypertension, School of Medicine, University of California, Los Angeles, USA.
authors.forename: U
authors.initials: U
authors.lastname: Kintscher
authors.forename: S
authors.initials: S
authors.lastname: Wakino
authors.forename: S
authors.initials: S
authors.lastname: Kim
authors.forename: S M
authors.initials: SM
authors.lastname: Jackson
authors.forename: E
authors.initials: E
authors.lastname: Fleck
authors.forename: W A
authors.initials: WA
authors.lastname: Hsueh
authors.forename: R E
authors.initials: RE
authors.lastname: Law
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D003331:Coronary Vessels / Q000166:cytology* / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D017292:Doxazosin / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects
chemical_list: D000317:Adrenergic alpha-Antagonists; D019925:Cyclin A; D019927:Cyclin E; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D018844:Cyclin-Dependent Kinases; D017292:Doxazosin