Role of isoprenylation in the inhibitory action of lovastatin on proliferation of SV40 immortalized human saphenous vein smooth muscle cells.

Abstract

Lovastatin is a competitive inhibitor of HMG CoA reductase [I]. Inhibition of this enzyme blocks both cellular synthesis of cholesterol and other intermediates derived from mevalonate. These include a number of important intermediates such as the isoprenoids, famesol pyrophosphate and geranylgeraniol pyrophosphate [2]. The latter are usually involved in the lipid modification (isoprenylation) of a number of proteins including key signalling molecules such as members of the Ras superfamily [3]. Isoprenoid modification seems to be required for proper localization and functional activity of these proteins. Previous studies have shown that inhibition of HMG CoA reductase by statins decreases vascular smooth muscle cell proliferation despite the free availability of cholesterol and without gduction in cellular levels of cholesterol [4]. These effects were reversed by mevalonate suggesting that they may be due to inhibition of protein isoprenylation. In this study we examined the effect of lovastatin on proliferation and DNA synthesis in a cultured immortalized smooth muscle cell line. SV-40 large T antigen transformed SM 1 cells, an immortalized clonal smooth muscle cell line derived from cultured human saphenous vein cells isolated from a single individual were used for all experiments. SMI cells were routinely cultured in DMEM supplemented with 1OOIU/ml penicillin, IOOpg/ml streptomycin sulphate, geneticin ((3418; 0.2m ml), and 10% (v/v) foetal calf serum (FCS). SMI cells (-14x10 cells/cm*' were seeded onto 24 well plates and 9cm petri dishes for proliferation assays and flow cytometric (FACS) analysis respectively. All treatments were carried out either in serum-free medium or medium containing 10% (vlv) dialysed FCS. Cell cycle analysis and quantification of % hypodiploid (A& apoptotic cells was performed following harvesting [5]. Cells were processed using Cell Testm Plus DNA reagent kit (Becton Dickinson, CA USA). SMl proliferation was measured using a Coulter Counter. DNA synthesis was also assessed by [3Hmethyl] thymidine incorporation. For these studies cells (seeded at -6000 cells/well) were exposed to ['H-methyl] thymidine (5pCVml) for 4 hours after a 22-hour stimulation with 10% dialysed FCS containing lovastatin (1Op.M) or vehicle. Mevalonate (0. ImM), famesyl pyrophosphate (FPP; 1Op.M) and geranylgeranyl pyrophosphates (GPP; 1Op.M) were also coincubated with lovastatin in some studies. Cell proliferation was inhibited by lovastatin (Fig.]). This effect was prevented by co-incubation with mevalonate or GPP, but not with FP (0.OlmM) (Fig.l).FACS analysis indicated that in the presence of 10% FCS S-phase increased from 13i1% to 26d% over 48h. This increase was inhibited by lovastatin at 48h, but not at 24h. Similarly lovastatin inhibited [3H-methyl] thymidine incorporation at 48h and 72h in response to 10% FCS but had no significant effect at 24h (Fig. 3). The inhibitory effect of lovastatin on ['H-methyl] thymidine incorporation was prevented by coincubation with mevalonate, GPP, but not by FPP. Y Cell number (x 104