ICANCER RESEARCH 51. 3602-3609. July 1. 1991] Synchronization of Tumor and Normal Cells from G, to Multiple Cell Cycles by Lovastatin1 Khandan Keyomarsi,2 Larue Sandoval, Minia Band, and Arthur B. Pardee Divisions of Cell Growth and Regulation [K. K., L. S., A. B. P.I and Cancer Genetics [\. B.], Dana Farber Cancer Institute, Boston, Massachusetts 02115 ABSTRACT nization by serum-starvation or contact inhibition is often used to arrest the growth of nontumorigenic diploid fibroblast cells Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in in Go or quiescence. In tumor or transformed cells, however, all types of cells, through several cycles and with minimal overall proliferation is relatively independent of the presence of serum metabolic perturbations, has not yet been available. We describe a or growth factors (5, 6). As a result, neoplastic cells, for the procedure for synchronizing normal as well as tumor cells reversibly in most part, do not cease proliferation upon serum deprivation, the GI phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy- nor do they display stringent density-dependent G0 arrest (7). 3-methylglutaryl-coenzyme A reducíase. This method of synchronization Furthermore, although synchronization by serum starvation was successful with all cell lines tested, including normal and tumor cells may be an appropriate method for studying the events that of mouse, hamster, and human origins. For example, when MCF-7 human occur during the transition from the resting to the growing breast cancer cells were synchronized with Lovastatin and released by state, it also subjects the cultures under study to long periods the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reducíase), 3 phases of accel of incubation in the absence of essential nutrients and growth erated ih\rumino incorporation into DNA corresponding to 3 S phases of factors. As a result, the cells are metabolically imbalanced and the cell cycle occurred during a 90-h period of cell replication. Thymidine out of cycle, thus inappropriate for analyzing the cell cycle incorporation was decreased to •-4'; during the initial lag of 18 h before events in continually dividing cells. These cells are generally the first S phase, and maximum incorporation was then achieved after poorly synchronized after the initial cycle. only 6 h. The antibody Ki-67, which detects a nuclear antigen associated To develop a procedure effective for synchronizing normal with proliferation, was present in cells arrested with Lovastatin. This and neoplastic populations, the following criteria should be fact, together with the lack of thymidine incorporation during the initial met: (a) both normal and tumor cells should be arrested at the lag time, indicates that the cells were arrested in the d and not in the Co phase of the cell cycle. Furthermore, in synchronized tumor-derived same, specific cell cycle phase; (b) the synchronization must be reversible and noncytotoxic; (c) the metabolic block should human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant target a specific reaction and must be reversible; (d) large synthesis of DNA and histone H4 RNA expression could be observed quantities of synchronous cells should be obtained; (e) the for 2 cycles. Minimal perturbations of general metabolic functions oc synchronization must be density and medium independent; and curred since the rate of RNA, protein, and initial DNA synthesis were (/) synchrony should be for more than one cycle, rendering it unaffected by Lovastatin, as evidenced by |'H|uridine, ['HJIeiidne, and particularly useful for studying the biochemical processes that initial | "II|ih> micline incorporation. Finally, while the Lovastatin-induced occur in cycling cells as well as immediately after the initial synchronization was overcome by mevalonic acid, addition of squalene or arrest. cholesterol-ethanol had no such effect. Thus, Lovastatin appears to In this paper, we report that these criteria for synchronization prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early (., phase. are met using Lovastatin to arrest normal and tumor cells of epithelial as well as fibroblast origin reversibly in the GI phase of the cell cycle. Lovastatin, an antihyperlipodemic agent, is INTRODUCTION widely used for the treatment of hypercholesterolemia. This agent competitively inhibits HMG-CoA' reducíase,the enzyme The study of cellular, biochemical, and molecular mecha nisms that regulate cell division in normal versus tumor cells is required for conversion of HMG-CoA to mevalonic acid (8). In a major topic of cancer research. For this purpose, large quan addition, inhibition of mevalonate synthesis by Lovastatin or tities of highly synchronized populations of both cell types are compactin, a related fungal toxin (9), also inhibits DNA repli necessary to localize sites of proliferation control. Cell prolif cation (10-15). DNA replication is restored by the addition of eration is stringently regulated in normal cells but is deranged mevalonate, the product of the reaction catalyzed by HMG- to varying degrees in cancer cells. Regulation occurs upon CoA reducíase. escape from quiescence and in the G, phase of the cell cycle We report here that inhibition of mevalonate synthesis by before a special restriction point event, which occurs a few Lovastatin synchronizes a wide array of normal and tumor cell hours before the onset of DNA replication (1,2). lines of mouse, hamster, and human origins in early G,. This Although a number of procedures have been developed for synchronization is noncytotoxic and fully reversible when mev establishing synchronous cultures of mammalian cells (3, 4), alonate is replenished. Rales of RNA and protein synthesis are many of these protocols have not been successfully adapted for not significantly altered by Lovastatin treatment. In addition, use with tumor as well as normal cells. For example, synchro- the synchrony can be followed for at least one full cell cycle and in some cases up to 3 cell cycles. Ki-67 antigen is present Received4/2/91;accepted4/17/91. The costs of publication of this article were defrayed in part by the payment in cells synchronized by Lovastalin bui noi in cells synchronized of page charges. This article must therefore be hereby marked advertisement in by serum slarvalion, suggesling a G, and noi a G0 arrest. This accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Grant CA 22427 (A. B. P.) and method of cell synchrony provides a high yield of synchronous 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. 'The abbreviations used are: HMG-CoA, .Vhydroxy-.Vmethylglutaryl coen- 2To whom requests for reprints should be addressed, at Dana Farber Cancer zyme A; PBS. phosphate-buffered saline; FCS, fetal calf serum; MEM, minimal Institute. Division of Cell Growth and Regulation. 44 Binney Street. Room D- essential medium: HEPES, ,V-[2-hydroxyethyl]piperazine-A"-[2-ethanesulfonic acid); DMEM, Dulbecco's modified Eagle's medium. 810A. Boston. MA 02115. 3602 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. SYNCHRONIZATION IN G, BY LOVASTATIN populations of cells, so that comparative studies between nor fibroblast cells were obtained from American Type Culture Collection mal and tumor cells can be performed. and grown in «-MEMsupplemented with 10% FCS, 2 mM glutamine. 0.1 m.Mnonessential amino acids, and 10 mM HEPES buffer. All cells were grown at 37°Cin a humidified incubator containing 6.5% CO^. MATERIALS AND METHODS except A31 and BPA/31, which require 10% COi. All cultures were maintained free of Mycoplasma as determined by the MycoTect Kit Materials (Gibco). (nipf/i.r/-['H]Thymidine (81 Ci/mmol), L-[4,5-'H)leucine (53 Ci/ mmol), [5-'H]uridine (27 Ci/mmol), and [«-'2P]CTP(3000 Ci/mmol) Determination of Population Doubling Time were purchased from New England Nuclear, Boston, MA. Lovastatin Approximately 5 x IO4 cells were plated per 35-mm dish (Falcon) was kindly provided by A. W. Alberts of Merck. Sharp and Dohme and grown at 37°Cin a humidified incubator. At 24-h intervals, cells Research Pharmaceuticals, Rahway, NJ. Before addition to cultures. from triplicate dishes were washed once with PBS, trypsinized, and Lovastatin was converted from its inactive lactone prodrug form to its counted in a Coulter Counter. The population doubling times were active dihydroxy open acid form by first dissolving 52 mg of the estimated from the linear portion of the growth curve as described compound in 1.04 ml of ethanol (95%) and then adding 813 ftl of l N previously (22). NaOH. The resulting solution was neutralized with l N HC1 to a pH of 7.2 and brought up to a volume of 13 ml with either distilled water or medium, and stored in multiple aliquots at -20°C until use. The Cell Synchronization final concentration was 10 HIM. Mevalonic acid lactone, squalene, Cells were plated in 24-well plates (Falcon) at 1 x 10" cells/well, and cholesterol, low density lipoprotein, and serum were purchased from 3 types of protocols were then followed: Sigma Chemical Co., St. Louis, MO. and cell culture medium was from Synchronization by Lovastatin Treatment. This method was applied Gibco Laboratories (Grand Island, NY). All other chemicals used were to all cell lines used in this study. Medium was removed 36-48 h after of reagent grade. The scintillation cocktail used was Budget-Solve from the initial plating and replaced with fresh medium containing Lovas Research Products International (Mount Prospect, IL).