Regular paper Sterol stringency of proliferation and cell cycle progression in human cells Yajaira Sua ´rez a,1 , Carlos Ferna ´ndez a , Beatriz Ledo a , Miguel Martı ´n a , Diego Go ´ mez-Coronado a , Miguel A. Lasuncio ´n a,b, T a Servicio de Bioquı ´mica-Investigacio ´n, Hospital Ramo ´n y Cajal, Ctra. de Colmenar, km 9, E-28034 Madrid, Spain b Departamento de Bioquı ´mica y Biologı ´a Molecular, Universidad de Alcala ´, Spain Received 21 January 2005; received in revised form 15 February 2005; accepted 15 February 2005 Available online 16 March 2005 Abstract Cholesterol is a major component of the plasma membrane in mammalian cells, where it acts as a modulator of bulk physical state and integrity. In addition to its structural role, cholesterol is essential for proliferation and other cell processes. The present study was undertaken to explore the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression. Comparisons were made between cholesterol and other sterol analogs that differ from cholesterol in three specific elements: the presence of a D 5 double bond in ring B, the hydroxyl group at C-3, and the presence of an aliphatic side chain. The human leukemia cells HL-60 and MOLT-4 were cultured in cholesterol-free medium and treated with different sterols in the presence or absence of SKF 104976, a competitive inhibitor of lanosterol 14a-demethylase that allows the synthesis of isoprenoid derivatives but not cholesterol. Our results show that the h-hydroxyl group at C-3 and the unsaturated bond at D 5 are necessary for cell proliferation and cell cycle progression. The sterol analog 5a-cholestan-3h-ol (dihydrocholesterol), which is saturated at D 5 and has an A/B ring junction in the trans configuration, was also able to support cell growth. However, 5h-cholestan-3h-ol and 5h-cholestan-3a-ol, both of which have an A/B ring junction in the cis configuration, were totally ineffective in supporting cell growth. Indeed, they produced an inhibition of cell proliferation and arrested the cell cycle specifically in the G2/M phase. These effects of 5h-cholestanols were abrogated by cholesterol in a concentration-dependent manner. Moreover, 5h- cholestanols potently inhibited cholesterol biosynthesis and transcription driven by the sterol response element. In addition to providing a description of the structural features of sterols associated with their supporting action on cell proliferation in mammalian cells, the present results demonstrate that selected cholesterol analogs may act as cytostatic agents, interrupting cell cycle progression specifically in the G2/M phase. D 2005 Elsevier B.V. All rights reserved. Keywords: Cell proliferation; Cell cycle; Cholesterol biosynthesis; Cholesterol analog 1. Introduction As a major component of mammalian cell membranes, cholesterol is essential for cell growth and division [1–3]. In addition to this structural requirement, cholesterol is important in other cell processes, such as embryonic development and synaptogenesis, as well as for bile acid and hormone synthesis [4,5]. Although the role of choles- terol in growth and division of mammalian cells has been known for many years [1,2,6,7], it remains to be established whether this is just a consequence of its use in membrane formation or whether it also plays a regulatory role in this process. We previously reported that cholesterol starvation produced by incubating the cells in a cholesterol-free medium and inhibiting cholesterol synthesis with SKF 104976, a competitive inhibitor of lanosterol 14a-demethy- lase, blocks cell proliferation and causes cell cycle arrest specifically in the G2/M phase [6,7]. This arrest can be prevented and fully reversed by cholesterol, suggesting that 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.02.003 T Corresponding author. Servicio de Bioquı ´mica-Investigacio ´ n, Hospital Ramo ´n y Cajal, Ctra. de Colmenar, km 9, E-28034 Madrid, Spain. Tel.: +34 91 3368077; fax: +34 91 3369016. E-mail address: miguel.a.lasuncion@hrc.es (M.A. Lasuncio ´n). 1 Present address: Instituto de Investigaciones Biome ´dicas, CSIC, C/ Arturo Duperier 4, Madrid 28029, Spain. Biochimica et Biophysica Acta 1734 (2005) 203 – 213 http://www.elsevier.com/locate/bba