Biochemical zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pharmacology. Vol. 41. No. 6/l. pp. 1045-10.54. 1991. Printed in Great Britain. ODO&2952/91 $3.00 + 0.00 0 1991. Pergamon Press plc INFLUENCE OF 8-(N,N-DIETHYLAMINO)OCTYL-3,4,5- TRIME~OXYBENZOATE (TMB-8) ON CELL CYCLE PROGRESSION AND PROLI~~~ON OF CULTURED ARTERIAL SMOOTH MUSCLE CELLS zyxwvutsrqponmlkjihgfed CLAUDE DESGRANGES,*t MICHEL CAMPAN,+ ALAIN-PIERRE GADEAU,* NATHALIE GUERINEAU,$ PATRICE MOLLARD$. and GABRIEL RAZAKA* *Unite de Cardiologie INSERM US, avenue du Haut-Leveque, F-33600 Pessac, France; and *Laboratone de Neurophysiologie, CNRS URA 1200, Universitd de Bordeaux 2, F-33076 Bordeaux Cedex, France (Received 10 September 1990; accepted 7 November 1990) Abs~ct~(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-I), a putative inhibitor of intra- cellular calcium mobilization, causes a dose-dependent inhibition of serum-induced proliferation of arteriat smooth muscle cells in culture. Neither early rise in cytosolic calcium concentration nor induction of early induced cell cycle dependent genes (c-fos, omithine de~rboxylase) are inhibited after serum stimulation in presence of 1oOpM TMB-8. In contrast, expression of thymidine kinase, a gene normally induced in late-G, phase, is entirely inhibited by TMB-8. Taken together with flow cytometry studies, these results indicate that TMB-8 blocks cell cycle progression in mid- or late-G, phase by a mechanism not directly related to early responses to serum stimulation since TMB-8 is also effective when introduced several hours after serum stimulation. Proliferation of intimal smooth muscle cells (SMCs) is a key event in development of atherosclerotic lesions [l, 21. Many agonists have been shown to induce arterial SMC proliferation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in uirro [for reviews 1,3,43. In spite of their heterogeneity, these factors share several common mechanisms involved in the signal tr~sduction into the cell, such as ph~phatidylinositol hydrolysis, increase in intracellular cytosolic calcium and pH, activation of protein kinase C, calmodulin and other pathways that lead to DNA synthesis and cell proliferation by more or less defined mechanisms [for reviews 5,6]. In vitro, the use of specific inhibitors has demonstrated the part of each of these pathways during the proliferative process. Indeed, inhibitors of receptor tyrosine kinase, of GTP-binding proteins, of Na+-H+ antiport, of protein kinase C, of calcium channels and of calmodulin decrease in different ways the proliferation of various cell types [7-91 including that of arterial SMC [l&14$ In addition, ~(~,~-diethylamino)~yl - 3,4,5 - trimethoxy- benzoate (TMB-8), a putative inhibitor of calcium release from sarcoplasmic reticulum in arterial SMCs [U-18] inhibits SMC DNA-synthesis induced by platelet-derived growth factor (PDGF) [17]. Despite its complex composition, serum is the reference substance to induce a complete progression through the cell cycle from quiescence exit to mitosis. Serum stimulation of arterial SMCs is generally accompanied by a transient rise in cytosolic calcium, resulting both from the release of stored intracellular calcium and from an influx of external calcium (19,201. The present study reports that TMB-8 inhibits serum- t Author to whom ~o~es~ndence should be addressed at Unit6 8 de i’INSERh4, avenue du Haut-Lkveque, F-33600 Pessac, France. induced proliferation of cultured arterial SMCs essentially by blocking cell cycle progression in mid- Gr phase. Although the inhibitory mechanisms of this product are not clearly defined, it appears that TMB-8 acts by a mechanism not directly related to the transient intracellular calcium increase induced after serum stimulation, since TMB-8 (i) does not inhibit this early seem-induced rise in calcium and (ii) continues to be active when introduced several hours after stimulation or in continuously cycling SMCs. MATERIALS ANDMETHODS Cell culture. Arterial SMCs were isolated from thoracic and abdominal aortas of male Wistar rats by enzymatic dissociation. Aortas were removed aseptically and cleaned of adventitia, and after cutting into small fragments, were incubated in 10 mL Dulbecco modified Eagle’s Medium (DMEM) containing 1 mg/mL collagenase and 0.1 mg/mL elastase for successive 30min periods at 37” in a stirring Aask. Single cell suspensions were separated from undigested tissues and centrifuged (lOmin, 200g) after addition of 10% fetal calf serum (FCS). Cell pellets were resuspended in DMEM supplemented with 10% FCS and seeded into 25 cm* flasks. The medium was changed every 2 days and confluency was reached about 10 days after seeding. Secondary cultures were obtained from these primary cultures by serial passages after harvesting the cells with trypsin-EDTA. These cells were stained by zyxwvutsrq Ll , a monoclonal antibody that recognizes a surface antigen specifically expressed in cultured rat arterial SMCs [21]; furthermore, they were stained by lA4, a monoclonal antibody recognizing exclusively ry- smooth muscle actin [22], suggesting the smooth