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Cholesterol starvation decreases P34^cdc2 kinase activity and arrests the cell cycle at G2

Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, 28034 Madrid; and Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá de Henares, Spain

¹Correspondence: Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar, km 9, 28034 Madrid, Spain. E-mail: miguel.a.lasuncion{at}hrs.es

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT-4 and HL-60 cell lines were cultured in a cholesterol-deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14- demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [¹⁴C]-acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC₅₀ = 0.1 µM), as assessed by both [³H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34^cdc2 and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34^cdc2 immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34^cdc2 phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34^cdc2 and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34^cdc2 activity.—Martínez-Botas, J., Suárez, Y., Ferruelo, A. J., Gómez-Coronado, D., Lasunción, M. A. Cholesterol starvation decreases P34^cdc2 kinase activity and arrests the cell cycle at G2.

Key Words: SKF 104976 • cholesterol synthesis • cell proliferation • low density lipoprotein • caffeine

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