Inhibitors of sterol synthesis. Submicromolar 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol causes a major modification of the sterol composition of CHO-K1 cells and a marked change in cell morphology.

F.D Pinkerton,A Izumi,S.O Nelson,J.S Pyrek,G.J Schroepfer,P.J Neill,J H Smith

doi:10.1016/s0022-2275(20)39968-5

Abstract

Incubation of Chinese hamster ovary cells (CHO-K1) with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol (0.1 microM) in lipid-deficient medium led to a major change in cellular sterol composition, which was characterized by a very marked accumulation of C30 sterols (lanosterol and 24,25-dihydrolanosterol). The accumulation of C30 sterols was associated with a striking change in cell morphology. The change in cell shape (elongation) was similar to that described previously (A. W. Hsie and T. T. Puck, 1971. Proc. Natl. Acad. Sci. USA. 68: 358-361; and confirmed herein) for CHO-K1 cells incubated in the presence of dibutyryl cAMP (1 mM). This change in morphology, induced by dibutyryl cAMP, was not accompanied by a change in cellular sterol composition. The cell elongation and accumulation of C30 sterols, induced by the 14 alpha-ethyl diol, were prevented by the addition of cholesterol (10 microM or 100 microM) and were reversed by removal of the 14 alpha-ethyl diol from the incubation medium. Incubation of the cells with the 14 alpha-ethyl diol had no effect on the levels of cAMP under the conditions studied. Incubation of the cells with miconazole (10 microM) or with lanosterol (10 microM) was also associated with the accumulation of C30 sterols and an elongation of the cells. 24,25-Dihydrolanosterol (10 microM) also induced similar changes in cellular morphology. The results presented herein demonstrate that marked changes in the sterol composition of CHO-K1 cells can be effected by incubation of the cells with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, miconazole, or lanosterol. In addition, the findings reported herein indicate an important role of sterols in the control of the shape of these cells.

Highlights

X-ray crystal analysis of an appropriate derivative [3], has been shown to be highly active in the inhibition of the incorporation of labeled acetate into digitonin-precipitable change in cell morphology
CHO-K1 cells were incubated in lipid-deficient medium in the presence and absence of the ethyl diol (0.1 pM) for varying periods of time
Whereas cholesterol was the major sterol in control cells, the cells incubated with the ethyl diol showed a marked accumulation of C30 sterols, with lanosterol and 24,25-dihydrolanosterol constituting 70% of total cellular sterols

Summary

Introduction

X-ray crystal analysis of an appropriate derivative [3], has been shown to be highly active in the inhibition of the incorporation of labeled acetate into digitonin-precipitable change in cell morphology. The ethyl diol lowered the levels of 3-hydroxy-3-methylglutarylcoenzyme A (HMG,-CoA) reductase activity in the same cells [1, 4]. IThe results presented demonstrate that marked changes in the sterol composition of CHO-K1 cells can be effected by incubaethyl diol, at a concentration of 1.0 p M , caused an almost complete inhibition of the incorporation of [3H]acetate into C2, sterols in the 10,000g supernatant fraction of a rat liver homogenate. This inhibition was associated with tion of the cells with 14a-ethyl-5a-cholest-7-ene-3@,15a-diol, miconazole, or lanosterol. The ethyl diol gle component on TLC (solvent system, n-butanol-acetic (0.5 p ~ al)so caused an inhibition of the synthesis of C27 acid-water 12:l:l; Rf 0.41). [3H]aceticanhydride (0.5 mCi sterols in CHO-Kl cells, which was accompanied by per mg) was purchased from New England Nuclear (Boston, a striking accumulation of labeled lanosterol and MA). (3RS)-[2-3H]mevalonolactone(176 mCi per mmol)

Methods

Results

Conclusion