Competence effect of PDGF on Ki-67 antigen and DNA contents, and its inhibition by trichostatin-A and a butylydene phthalide BP-421 in primary smooth muscle cells of rat aorta by flow cytometry.
We investigated with flow cytometry the platelet-derived growth factor (PDGF)-induced competence in primary cultured smooth muscle cells (SMC) of rat thoracic aorta. A cytogram was obtained by a double staining technique with fluorescein isothiocyanate-conjugated mouse monoclonal antibody against the proliferation-associated nucleus antigen Ki-67, and propidium iodide for total DNA content. (1) The nearly confluent SMC after 6 d-culture with 5% fetal bovine serum (FBS) were further cultured under serum starvation for 2d. (2) SMC was cultured with 5% FBS for 4 d, and then with 5% FBS+trichostatin-A (TS-A) (1 microgram/ml) for 2d. The cytogram showed the broadening of Ki-67 antigen signal for G0/G1 phase by TS-A compared with that of SMC under serum starvation, suggesting the existence of early and late phases of G1. (3) After serum starvation, the preculture with PDGF (100 ng/ml) for 3 h which was followed by a further 15 h-culture with 3% FBS caused significant more entry into S phase than control culture. The extent was greater than that with 15 h-culture with 10% FBS. (4) 15 h-culture with 1% FBS after PDGF (30 ng/ml) pretreatment stimulated entry into S phase cells, which was inhibited by TS-A (1 microgram/ml) and by a butylydene phthalide derivative BP-421 (3 micrograms/ml). Flow cytometric analysis demonstrate that PDGF pretreatment stimulates the entry into S phase by 20% of total SMC having early and late phases of G1, and that the PDGF-competence is inhibited by TS-A and BP-421.
Okabe
M
M
Department of Chemical Pharmacology, Toyama Medical and Pharmaceutical Sciences, Japan.
Kimura
I
I
Kimura
M
M
D000818:Animals; D001011:Aorta / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis*; D005434:Flow Cytometry; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007399:Interphase / Q000187:drug effects; D019394:Ki-67 Antigen; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D009687:Nuclear Proteins / Q000096:biosynthesis*; D010796:Phthalic Anhydrides / Q000494:pharmacology*; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors*; D051381:Rats; D017208:Rats, Wistar
C098816:3-heptylidene-5,6-dihydroxyphthalide; D016895:Culture Media, Serum-Free; D006877:Hydroxamic Acids; D019394:Ki-67 Antigen; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010796:Phthalic Anhydrides; D010982:Platelet-Derived Growth Factor; C012589:trichostatin A; D004247:DNA