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Synchronization of Tumor and Normal Cells from G₁ to Multiple Cell Cycles by Lovastatin¹

Divisions of Cell Growth and Regulation [K. K., L. S., A. B. P.] and Cancer Genetics [V. B.], Dana Farber Cancer Institute, Boston, Massachusetts 02115

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G₁ phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G₁ and not in the G₀ phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [³H]uridine, [³H]leucine, and initial [³H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G₁ phase.

¹ This work was supported in part by Grant CA 22427 (A. B. P.) and S07RR05526-27 (a Basic Research Support Grant to K. K.) from the NIH. K. K. is a recipient of National Research Service Award CA08949-01 from the NIH.

² To whom requests for reprints should be addressed, at Dana Farber Cancer Institute, Division of Cell Growth and Regulation, 44 Binney Street, Room D-810A, Boston, MA 02115.

Received 4/ 2/91. Accepted 4/17/91.

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