the of biological chf8try vol. 263, no. 21, of july 25 ...the journal of biological chf"8try 0...

THE JOURNAL OF BIOLOGICAL CHF"8TRY 0 1988 by The American Society for Biochemistry and Moleculsl Biology, Inc.

Vol. 263, No. 21, Issue of July 25, pp. 10104-10110,1988 Printed in U.S.A.

Isoprenoid Synthesis during the Cell Cycle STUDIES OF 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A SYNTHASE AND REDUCTASE AND ISOPRENOID LABELING IN CELLS SYNCHRONIZED BY CENTRIFUGAL ELUTRIATION*

(Received for publication, January 29,1988)

William A. Maltese# and Kathleen M. Sheridan From the Department of Neurology, College of Physicians and Surgeons of Columbia University, New York, New York 10032 and the Weis Center for Research, the Geisinger Clinic, Danville, Pennsylvania I7822

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase were assayed in exponentially growing LM fibroblasts and Friend murine erythroleukemia cells isolated at various stages of the cell cycle by centrifugal elutriation. The activities of these enzymes were similar in all phases of the cell cycle, regardless of whether the cells were cultured in the presence or absence of serum. These observations were confirmed in murine erythroleukemia cells synchronized by recultivation of pure populations of G1 cells. The incorporation of [''Clacetate or 'HzO into sterols decreased by 3040% in later stages of the cell cycle, whereas the incorporation of ['"Clacetate into ubiquinone increased as the cells progressed toward mitosis. Similar changes in the labeling of sterols compared to ubiquinone and dolichol were observed when ['Hlmevalonate was used, suggesting that cell cycle- dependent alterations may occur in the flux of farnesyl pyrophosphate into the various branches of the isoprenoid pathway. Synchronized murine erythroleukemia cells incorporated [SH]mevalonate into protein- bound isoprenyl groups at all stages of the cell cycle, and there were no substantial changes in the electrophoretic profiles of these labeled polypeptides. The finding that the activities of the enzymes regulating mevalonate synthesis did not vary substantially during the cell cycle implies that changes in the endogenous mevalonate pool probably do not play a limiting role in regulating cell cycle traverse when cells are undergoing exponential growth. Although small cell cycle-dependent changes may occur in the relative activity of various post-mevalonate branches of the isoprenoid biosynthetic pathway, there is no evidence that synthesis of any major isoprenoid end product is confined exclusively to a specific phase of the cell cycle.

Mammalian cells synthesize cholesterol and various nonsterol isoprenoid compounds via a branched pathway in which mevalonate, the product of the reaction catalyzed by HMG'- CoA reductase, occupies a central position as a precursor for all end products (for reviews, see Refs. 1 and 2). In preparation

* This work was supported by United States Public Health Service Grant R01 CA 34569. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "oduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Weis Center for Research, Geisinger Clinic, Danville, PA 17822.

The abbreviations used are: HMG, 3-hydroxy-3-methylglutaryl; LPPS, lipoprotein-poor serum; PBS, phosphate-buffered saline solution; MEL, murine erythroleukemia; SDS, sodium dodecyl sulfate; FCS, fetal calf serum.

for mitosis, cells generally must replicate their complement of intracellular membranes and organelles along with the genomic material. Since cholesterol is a major membrane component in mammalian cells, it is logical to expect that an adequate supply of sterol, derived either from de m u 0 synthesis or lipoprotein uptake, would be needed to support cell proliferation. This hypothesis has been validated by studies showing that when cultured cells are grown in serum-free or lipoprotein-free medium and then exposed to oxysterols that suppress the activity of HMG-CoA reductase, arrest of cell growth ultimately occurs unless an exogenous source of cholesterol (e.g. low density lipoprotein) is added to the medium (3-6). More recent studies utilizing high concentrations of competitive inhibitors of HMG-CoA reductase, such as com- pactin (7) or mevinolin (8), have demonstrated that complete blockade of mevalonate synthesis results in arrest of cell cycling that cannot be prevented solely by sterol supplemen- tation (9-16). Therefore, it appears that even when the sterol requirement for cell cycling is met, cells need small amounts of mevalonate for the synthesis of nonsterol isoprenoid compounds that are essential for cell proliferation. Quesney- Huneeus et al. (17) originally proposed that isopentenylad- enine was the mevalonate derivative required for DNA synthesis, but studies in other laboratories (14,18-20) have failed to support this hypothesis. It remains possible that growth- related metabolic processes, such as mitochondrial replication or glycosylation of newly synthesized membrane proteins, create an increased demand for ubiquinone or dolichol, both of which are derived from mevalonate (21-23). Alternatively, recent evidence suggests that the mevalonate requirement for cell cycling may be related to its role as a precursor for unidentified isoprenyl chains that appear to be covalently attached to unique cellular polypeptides (19, 24, 25).

A full understanding of the nature of the mevalonate requirement for cell proliferation will require detailed informa- tion about the regulation of isoprenoid synthesis during the cell cycle. A major unanswered question is whether the synthesis of mevalonate and its isoprenoid derivatives proceeds continuously throughout the cell cycle or instead occurs primarily or exclusively during a specific phase, where it may constitute a permissive or regulatory event. Early experiments (26, 27) with lectin-stimulated lymphocytes revealed a sharp increase in the incorporation of [14C]acetate into sterols prior to the onset of DNA synthesis. These findings have been confirmed in quiescent smooth muscle cells (11) and fibroblasts (13) stimulated to enter the cell cycle by treatment with platelet-derived growth factor. However, because quiescent ( i e . Go) cells generally exhibit much lower rates of sterol synthesis than cells undergoing rapid proliferation (28-311, it is possible that the aforementioned increases in sterol synthesis are typical of cells that have been induced to resume

10104

Isoprenoid Synthesis during Cell Cycle 10105

cycling from a Go state, rather than cells undergoing continuous division, such as tumor cells or embryonic tissues.

Cytosolic HMG-CoA synthase catalyzes the first committed step in the de novo isoprenoid biosynthetic pathway (reviewed in Ref. 32), but no data are currently available concerning the activity of this enzyme during the cell cycle. The next enzyme in the pathway, HMG-CoA reductase, has been studied in synchronized cells, but these studies have produced conflict- ing data. In BHK-21 cells released from thymidine blockade, Quesney-Huneeus et al. (10) observed a major peak of HMG- CoA reductase activity in the late GI or early S phase, with the activity declining sharply to a minimal level in the G*/M phase. In contrast, Roussillon et al. (33), working with rat fibroblasts synchronized by thymidine blockade, observed only modest cyclic variations in reductase activity, with max- imal activities attained in the S and G*/M phases. Difficulty in the interpretation of these data may arise from the use of a chemical block to induce cell synchrony, with undefined consequences for lipoprotein uptake, enzyme synthesiddeg- radation, and isoprenoid metabolism.

The technique of centrifugal elutriation provides a means for isolating relatively large numbers of cells at specific stages of the cell cycle, starting with an asynchronous cell population (34-36). The procedure can be applied to actively cycling cell suspensions without removing the cells from their usual medium, thereby avoiding chemical or physical perturbations that could affect cell metabolism. In this study, we have used this methodology to establish cell cycle patterns for the activities of HMG-CoA synthase and reductase and to study the synthesis of major isoprenoid end products in continuously proliferating LM fibroblasts and Friend murine erythroleukemia cells.

EXPERIMENTAL PROCEDURES

Materials-[l-14C]Acetic acid (56 mCi/mmol) and sodium b ~ r o [ ~ H ] hydride (7.5 Ci/mmol) were purchased from Amersham Corp. 3H20 (1 Ci/ml) was purchased from Du Pont-New England Nuclear. Tissue culture medium and serum were obtained from GIBCO/Bethesda Research Laboratories. Plastic tissue culture flasks (Falcon Labware, Oxnard, CA) and roller bottles (Corning, Corning, NY), X-Omat AR film (Kodak), and Amplify fluorography enhancing solution (Amer- sham Corp.) were obtained from the designated sources. Mevaldic acid, lipid standards, and other chemicals were purchased from Sigma. (RS)-[5-3H]Mevalonolactone was prepared by reduction of mevaldic acid with sodium b~ro[~H]hydride as described by Keller (37). The purity of the tritium-labeled product was checked by thin-layer chromatography on Silica gel G plates (Brinkman Instruments) developed with acetone:toluene (l:l, v/v). At least 95% of the radioactivity cochromatographed with authentic [2-14C]mevalonolactone (Amer- sham Corp.). Lovastatin (mevinolin) was a gift from Alfred W. Alberts (Merck, Sharp & Dohme Research Laboratories). Prior to addition of lovastatin to the culture medium, the lactone was converted to the sodium salt as described previously (38).

Culture Collection (Rockville, MD). Murine erythroleukemia (MEL) Cell Culture-LM cells were obtained from the American Type

cells (line 745) were a gift from Dr. Charlotte Friend (Mt. Sinai School of Medicine). LM cells were seeded at 4 X lo' cells/cm2 and maintained in monolayer culture (175- or 75-cm2 flasks) a t 37 "C in Dulbecco's modified Eagle's medium without antibiotics, supplemented with 10% (v/v) fetal calf serum. The culture medium contained 2.2 g/liter sodium bicarbonate and was equilibrated with 5% COZ in air. In some experiments, LM cells were grown in serum-free Dulbecco's medium or medium supplemented with lipoprotein-poor serum (LPPS), prepared as described by Have1 et al. (39). When the LM cells reached confluency, they were subcultivated by trypsiniza- tion as described previously (25). MEL cells were grown under the same conditions as the LM cells, except that they were maintained in suspension culture in plastic 490-cm2 roller bottles containing 100- 200 ml of medium. Stock cultures were initiated at a density of 10' cells/ml, and subcultivation was performed twice each week by dilut- ing the cells in fresh medium. Cell numbers were determined with a Coulter Counter (Model ZBI).

Collection of Cells a t Different Stages of Cell Cycle by Centrifugal Elutriation-The technique of centrifugal elutriation is based on the observation that cell volume generally increases as cells progress through the cell cycle (34, 40). Thus, a heterogenous (i.e. nonsyn- chronized) cell population can be separated into several subpopula- tions in different phases of the cell cycle by methods that sort cells according to their relative size. In centrifugal elutriation, cells are introduced into a rotor chamber, and equilibrium conditions are established so that a constant flow of culture medium through the chamber counterbalances the centrifugal force. By increasing the flow rate of the medium in a stepwise manner, separate cell populations with increasing median cell volumes are eluted from the rotor. Ali- quots of each cell population are then removed for determination of cell cycle stage (DNA histograms) by flow cytometry, and the remaining cells can be used for biochemical assays. For the studies described in this report, the elutriations were performed with a Beckman JE-GB elutriation rotor and chamber in a Beckman J-6B centrifuge, equipped with a stroboscope and an LKB Multiperpex peristaltic pump. Prior to each run, the rotor was disinfected by flushing with 70% ethanol, followed by several volumes of sterile phosphate-buffered saline solution (PBS). Only cells that were growing exponentially were used for elutriation. The proper time for harvesting the cells was established by preliminary studies in which growth curves were constructed for LM cells (cultured with whole serum, LPPS, or no serum) and MEL cells. Prior to elutriation, LM cells in 8-10 monolayer cultures (175 cm') were dispersed by trypsin- ization and suspended in 30 ml of medium. MEL cells growing in 200-ml suspension cultures were collected by centrifugation and resuspended in 30 ml of medium. Aliquots of the cell suspensions were stained with trypan blue (41) and examined in a hemocytometer to verify that the cells were intact and that the suspension was free of cell aggregates.

All elutriations were performed with Dulbecco's medium, with or without serum as indicated under "Results." The general procedure for elutriation of LM cells involved loading approximately 10' cells into the chamber at 2000 rpm and a flow rate of 16 ml/min. The temperature was maintained at 4 "C throughout the collection procedure to minimize cell aggregation and prevent cell cycle traverse during the sorting period. After collecting 200 ml of medium, which usually contained a few overflow cells, the flow rate was increased to 20 ml/min, and the separation was started. Routinely, 7-10 fractions were collected, each consisting of 200 ml. For each fraction, the flow rate was increased by 2 ml/min while the centrifuge speed was held constant a t 2000 rpm. Aliquots were removed from each fraction for determination of the median cell volume using a Coulter Counter (Model ZBI) equipped with a ClOOO Channelyzer, calibrated with latex microspheres of known diameter (42). Additional aliquots (lo6 cells) were removed for flow cytometry (see below). The remaining cells in each fraction were collected by centrifugation at 1000 X g for 5 min and were used for biochemical determinations. The cell pellets were washed twice with PBS and frozen at -80 'C. Similar procedures were used to elutriate MEL cells, except that the initial flow rate during loading was 10 ml/min, and collection of fractions began at 16 ml/min.

Synchronization of MEL Cells-In some experiments, the technique of centrifugal elutriation was used to collect a pure population of GI cells. These cells were then used to initiate a highly synchronous suspension culture. MEL cells were elutriated as described above, except that antibiotics were included in the medium (200 units/ml penicillin, 20 mg/ml streptomycin), and the entire procedure was carried out at room temperature with medium that had been prewarmed at 37 "C. The cell populations with the smallest median cell volumes (usually eluted at 20-22 ml/min) were pooled, concentrated by centrifugation, resuspended in 100 ml of fresh medium, and placed in an Erlenmeyer flask on an orbital shaker in the C02 incubator a t 37 "C. Aliquots of this cell suspension were removed at 2-h intervals for flow cytometry, enzyme assays, or labeling experiments.

Flow Cytornetry-For generation of DNA histograms, lo6 LM cells were fixed, treated with RNase, and stained with propidium iodide as described previously (14). Flow cytometry was performed with a Coulter Epics IV instrument equipped with a 488-nm argon ion laser and MDADS data reduction system. MEL cells were fixed in a 70% (v/v) solution of ethanol in Hanks' balanced salt solution and stored at 4 "C. Just prior to flow cytometry, the fixed cells were stained with a solution of 20 mg/ml chromomycin AB in 32 mM MgC12 for 45 min at room temperature. DNA histograms of the MEL cells were obtained with an FACS IV flow cytometer equipped with a 458-nm argon ion laser, a 520-nm long-pass filter, and a 550-nm wide-band

10106 Isoprenoid Synthesis during Cell Cycle filter. All DNA histograms were derived from samples of 20,000- 50,000 cells.

Enzyme Assays-Assays were performed on duplicate cell aliquots at each stage of the cell cycle. Total cellular activity of HMG-CoA reductase was determined radiochemically by measuring the NADPH-dependent conversion of [3H]HMG-CoA to [3H]mevalonate as described previously (29). One unit of enzyme activity is defined as the amount required to convert 1 pmol of HMG-CoA to mevalonate per min at 37 "C.

Cytosolic HMG-CoA synthase activity was measured by the standard radiochemical assay (29, 43), with 1 unit of activity defined as the amount required to convert 1 nmol of ["Clacetyl-CoA to nonvol- atile product per min at 30 "C.

Incorporation of Labeled Precursors into Isoprenoid Compounds- Cells were incubated at 37 "C in 3-5 ml of culture medium containing [1-"Clacetate (4 pCi/ml), 3Hz0 (10 mCi/ml), or [5-3H]mevalonolactone (200 pCi/ml) for 2-4 h as indicated for specific experiments described under "Results." At the end of the incubation period, cell cultures were immediately placed on ice and collected by centrifugation at 4 "C. Cell pellets were washed three times with ice-cold PBS and stored at -80 "C prior to analysis. Incorporation of radioactivity into digitonin-precipitable sterols was performed as described by Maltese et al. (29). Extraction of total lipids and two-dimensional thin-layer chromatography of isoprenoids were performed as described previously, after addition of appropriate standards to the cell pellets (14,44). Radioactivity in each of the zones scraped from the thin-layer plates was quantitated by counting in a liquid scintillation spectrometer, and all results were normalized to the amount of cell protein extracted. Protein was quantitated in cell suspensions by the method of Lowry et al. (45) or in cell extracts by a microbiuret method (46) using bovine serum albumin as the standard.

Electrophoresis of Isoprenylated Proteins-Cells collected by centrifugation were washed three times with PBS and dissolved by heating at 100 "C for 5 min in 200-300 pl of a sample buffer containing 8 M urea, 2% (w/v) SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.05% (w/v) bromphenol blue, and 0.0625 M Tris-HC1, pH 6.8. Aliquots were removed for protein determination (451, and samples were subjected to SDS-polyacrylamide gel electrophoresis as described by Laemmli (47) using a 4% polyacrylamide stacking gel and a 10% polyacrylamide separating gel. After staining with Coomassie Blue, gels were permeated with fluorographic enhancer (Amplify), dried, and exposed to preflashed X-Omat AR film for 10 days at -80 "C using a Du Pont-New England Nuclear Cronex intensifying screen.

In separate experiments, the total protein-bound isoprenoid radioactivity was determined by lysing cells in 40 mM Tris-HC1, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride, 0.1 p M pepstatin, and 50 p~ leupeptin. After incubation with RNase I (Sigma; 20 pg/ ml) at 37 "C for 1 h, the protein was precipitated by addition of trichloroacetic acid (lo%, w/v), and the precipitate was collected by centrifugation at 10,000 rpm in an Eppendorf microcentrifuge. Non- covalently bound lipid was removed by sequential extraction of the precipitate with organic solvents as described previously (24, 25), and the insoluble material was dissolved in 0.5 M KOH. Aliquots of the extract were removed for protein determination (46), and the remain- der was neutralized with HC1 and counted in a liquid scintillation spectrometer.

RESULTS

Activity of HMG-CoA Reductase in Cells Sorted by Elutria- tion-Monolayer cultures of LM cells were subjected to centrifugal elutriation after being grown in medium without serum, medium supplemented with LPPS, or medium containing whole fetal calf serum. Flow cytometric analysis of the eluted cell populations of increasing median cell volume (fractions 1-7) indicated that the earliest fractions, 1 and 2, consisted almost exclusively of cells in the Gl phase of the cell cycle (Fig. 1). The later fractions contained increasing proportions of cells in the S and G2/M phases (Fig. 1). When these cell populations were assayed for HMG-CoA reductase (Table I), there was no evidence of a major peak of enzyme activity during a specific phase of the cell cycle, comparable to that previously reported for cell lines released from thymidine blockade (10,33). This was true regardless of whether the cells had been cultured under conditions that promoted

I 2 3 4 5 6 7

4 lLPPSl

LY celk (10% FCSJ

FIG. 1. DNA histograms generated by flow cytometric analysis of cell populations with increasing median cell volumes (fractions 1-7) collected by centrifugal elutriation and assayed for HMG-CoA reductase activity. For each panel, the ordinate represents the relative number of cells, whereas the abscissa represents relative fluorescence intensity, which is proportional to the cellular DNA content. After removal of aliquots for flow cytometry, the remaining cells in each fraction were collected by centrifugation and assayed for HMG-CoA reductase activity (see Table I).

TABLE I Activity of HMG-CoA reductase in cells isolated at various stages of

ceU cycle by centrifugal elutriation See Fig. 1 for DNA histograms of cell populations in the elutriated

fractions. HMG-CoA reductase activity

Cell line conditions Assay in fractions:

1 2 3 4 5 6 7 pmol/min/mgprotein

LM (no serum) -F 79 84 89 72 68 72 70 LM (LPPS) -F 107 92 84 92 88 81 85 LM (10% FCS) -F 20 23 15 17 21 17 25

MEL (10% FCS) +F 16 17 16 21 19 15 16 MEL (10% FCS) -F 40 53 56 41 49 45 44

full expression of reductase activity (without serum or with LPPS) or that suppressed the activity of the enzyme (whole serum) (Table I).

To test the possibility that the foregoing observations were peculiar to the LM cell line, similar studies were carried out with MEL cells grown in suspension culture. As shown in Fig. 1, elutriation of the MEL cells yielded fractions that were enriched in GI cells (fractions 1 and 2), S cells (fractions 3- 5), and G2/M cells (fractions 6 and 7). However, there were no major changes in reductase activity that could be correlated with cell cycle progression (Table I).

Previous studies (48-51) have demonstrated that the meas- urable activity of HMG-CoA reductase may be substantially lower when cells are homogenized and assayed in the presence of a phosphatase inhibitor such as sodium fluoride. This has been interpreted to mean that some of the enzyme in the intact cell may exist in a latent phosphorylated form and that measurements of total activity in the absence of phosphatase inhibitor may not necessarily reflect the physiological level of enzyme activity. To determine whether our routine assay conditions (without fluoride) prevented the detection of changes in the ratio of active to latent enzyme during the cell cycle, MEL cells were elutriated and assayed for HMG-CoA reductase in the presence of 50 mM NaF (Table I). Under these conditions, all of the enzyme activities were decreased by 50-70% compared to the same cells assayed without fluo-


ride. However, again there was no evidence of a major change in HMG-CoA reductase activity occurring during a specific phase of the cell cycle.

Activity of HMG-CoA Synthase in Cells Sorted by Elutria- tion-Although HMG-CoA reductase is generally regarded as the major rate-controlling enzyme in the biosynthesis of sterols, several studies have suggested that cytosolic HMG-CoA synthase may play a regulatory role in certain circumstances. For example, conditions that suppress the activity of the reductase in liver (52), adrenal gland (43), Chinese hamster ovary cells (54, 55), and LM cells (29, 30) also produce coordinate suppression of HMG-CoA synthase activity. In cases where sterol synthesis in cultured cells was suppressed by treatment with glucocorticoids (56) or linoleic acid (57), evidence suggests that the synthase, rather than the reductase, may be the primary site of regulation. The aforementioned studies, coupled with the absence of data concerning HMG- CoA synthase during the cell cycle, prompted us to assay this enzyme in LM and MEL cells isolated at various stages of the cell cycle. Fig. 2 depicts the DNA histograms of the elutriated cell populations used for the synthase assays. As in the studies of the reductase, fractions enriched in G1, S, and G2/M cells were obtained. The data in Table I1 indicate that, like the activity of HMG-CoA reductase, the activity of HMG- CoA synthase remained relatively constant throughout the cell cycle.

Incorporation of Labeled Precursors into Sterols in Cells Sorted by Elutriation-In view of earlier evidence demonstrat- ing a marked increase in sterol synthesis during the late GI or early S phase in cells synchronized by stimulation with growth factors (11, 13) or lectins (26, 27), we questioned whether the relatively constant activities of HMG-CoA reductase and synthase in the elutriated cell populations were

Frocnm No I 2 3 4 5 6 7 I I I I I I I I

LM cdb fLPPSI

Relolire DNA Conlenf lF luor~rcenre lntrnsqj

FIG. 2. DNA histograms generated by flow cytometric analysis of cell populations with increasing median cell volumes (fractions 1-7) collected by centrifugal elutriation and assayed for HMG-CoA synthase (see Table 11).

TABLE I1 Activity of HMG-CoA synthase in cells isolated at various stages of

cell cycle by centrifugal elutriation See Fig. 2 for DNA histograms of cell populations in the elutriated

fractions.

Cell line HMG-CoA synthase activity in fractions:

1 2 3 4 5 6 7 nrnol/rnin/rng protein

LM (no serum) 3.5 4.0 3.7 3.7 4.2 3.0 2.5 LM (LPPS) 6.0 5.2 6.0 6.0 5.3 4.6 4.2 LM (10% FCS) 1.8 1.7 1.7 1.5 1.4 1.8 1.0 MEL (10% FCS) 0.8 0.8 0.8 0.9 0.8 0.8 1.0

providing a true indication of the relative rate of sterol synthesis. To address this issue, exponentially growing populations of LM cells were pulse-labeled with [14C]acetate or 3H20 for 30 min. The cells were then washed, chilled to 4 "C, and sorted immediately by centrifugal elutriation. When the radioactivity incorporated into digitonin-precipitable sterols was compared in each of the elutriated fractions (consisting of cells that were in different stages of the cell cycle at the time of labeling), we found that the incorporation of ["C] acetate in the early fractions (GI cells) was slightly higher than in the later fractions, which were composed primarily of S and G2/M cells (Table 111). Similar results were noted when 3H20 was used as the labeled precursor, suggesting that this observation probably was not related to cell cycle-dependent variations in acetate uptake or endogenous acetate pool size.

Enzyme Studies and Isoprenoid Synthesis in Synchronized MEL Cells-In the preceding studies, the earliest elutriated fractions consisted of pure populations of GI cells. However, the later fractions, although enriched in S or G2/M cells, were not homogeneous. It also was necessary to sort the cells at 4 "C to prevent cell cycle traverse during the approximate 90- min period that it took to complete the elutriation. Although preliminary studies indicated that cooling the cells to 4 "C for 2 h had little or no effect on the enzyme activities ultimately measured in frozen cell pellets, we felt that it was important to confirm the foregoing findings in synchronized cells maintained at 37 "C. Since it was relatively easy to obtain pure Gl cells by elutriation, we devised an alternative approach for studying cell cycle progression, taking advantage of the short generation time of the MEL cells (approximately 8-10 h).

MEL cells were subjected to rapid elutriation in prewarmed (37 "C) medium, and the fractions with the smallest median cell volume (early Gl phase) were pooled and returned immediately to suspension culture. This procedure took only 30 min; and, as shown in Fig. 3, the GI cells proceeded synchro- nously through the cell cycle during the next 8 h. When aliquots of the suspension were withdrawn at 2-h intervals, we were able to collect fractions composed almost entirely of cells in the G1, S, or G2/M phase of the cell cycle. This pattern of synchrony was extremely reproducible, provided that the starting G1 population was derived from an exponentially growing stock culture. Similar profiles were observed for cells used in all of the following experiments.

Table IV lists the activities of HMG-CoA reductase and synthase determined in separate experiments in which MEL cells were synchronized as described above. The results confirmed the absence of major cell cycle-specific variations in the activity of HMG-CoA reductase observed in the earlier elutriation experiments (Tables I and 11) and revealed only a small (-35%) differential in the activity of HMG-CoA synthase in the G1 cells compared to the late S or G2/M cells.

Studies of MEL cells incubated with [14C]acetate for 2-h intervals during the cell cycle showed an approximate 30% decline in labeling of cholesterol as the cells progressed from GI to G2/M (Table V). These findings were similar to those observed previously when G1-enriched cells were compared to S- and Ga/M-enriched cell fractions sorted by elutriation after labeling with [14C]acetate or 3H20 (Table 111). In contrast to sterols, the labeling of ubiquinone, another isoprenoid end product, increased by approximately 40% as the cells entered the later stages of the cell cycle (Table V). The changes in labeling of cholesterol and ubiquinone did not appear to reflect general changes in acetate uptake or its conversion to acetyl- CoA since the labeling of triglycerides with [14C]acetate was relatively constant at all stages of the cell cycle in the same samples (Table V).

10108 Isoprenoid Synthesis during Cell Cycle

TABLE I11 Incorporation of labeled precursors into sterols in cells isolated at various stages of cell cycle

by centrifugal elutriation DNA histograms were essentially the same as those depicted in Figs. 1 and 2 for LM cells with whole serum or

T.PPS.

Cell line Precursor Radioactivity incorporated into sterols in fractions:

1 2 3 4 5 6 I dprnlmg protein

LM (+serum) [l-”C]Acetate 571 708 504 537 460 509 360 LM (LPPS) 3 ~ 2 0 1056 1531 861 722 872 793 749

11,,4_ Unboctionoled !,,lk;i;; “-6 hrs &I

L w 0

5 .- a, 4 /$\ - -0 P

(Fluorescence IntensityJ Aelolive DNA Cmfent -

FIG. 3. DNA histograms generated by flow cytometric analysis of MEL cells prior to centrifugal elutriation (unfrac- tionated) and at 2-h intervals after reintroduction of pure GI cells into suspension culture.

TABLE IV Activities of HMG-CoA reductase and synthase in erythroleukemia

cells synchronized by culturing elutriated G1 cells

Time (cell cycle HMG-CoA reductase HMG-CoA synthase:

Exp. 3 stage) Exp. 1 Exp.2

pmollrninlmg protein

nmollrninlmg protein

0 (GI) 25.1 33.4 0.47 2 (Gl/S) 22.1 38.7 0.40 4 (S/Gz) 21.1 34.3 0.34 6 (Gz) 21.2 32.2 0.31

~

h

8 (GJGI) 23.0 30.4 0.34

TABLE V Incorporation of [“C]acetate and ~H]mevalonolactone into

isoprenoid compounds in erythrobukemia cells synchronized by culturing elutriated GI cells

All lipids were separated by two-dimensional thin-layer chromatography. Values are means of determinations performed on duplicate cell samples.

[“CIAcetate incorporation [3H]Mevalonate incorporation

Time Choles- Ubiqui- Triglyc- Choles- Ubiqui- Dolichol terol none eride terol none

h dprnlmg protein dprnlrng protein 0-2 20,932 2,564 26,788 ’ 123,758 20,194 1,588 2-4 17,935 3,856 27,257 74,856 21,857 1,195 4-6 14,919 4,322 29,386 61,874 30,934 2,569 6-8 16,889 4,158 28,729 66.269 30,878 2,323

The modest reciprocal changes in acetate incorporation into cholesterol versus ubiquinone during the cell cycle suggested that changes in the flux of mevalonate into various branches

TABLE VI Incorporation of ~H]mevalomlactone into protein-bound isoprenyl

groups in erythroleukemia cells synchronized by culturing elutriated GI cells

cate cell samples incubated with [3H]mevalonolactone (100 pCi/ml) Values are means of separate determinations performed on dupli-

and 25 ~ L M lovastatin. Cell extracts were treated with RNase, and trichloroacetic acid precipitates were extracted with organic solvents as described under “Experimental Procedures.”

Radioactivity incorporated

precipitable fraction Time into trichloroacetic acid-

h dprnlmgprotein 0-2 226,581 2-4 213,167 4-6 213,210 6-8 236,537

FIG. 4. Isoprenylated proteins in MEL cells at various stages of cell cycle. Duplicate aliquots of cell suspension were incubated with [3H]mevalonolactone (200 pCi/ml of medium) for 2- h intervals following reintroduction of Gl cells into suspension culture (see Fig. 3 for representative DNA histograms). Total cell protein was subjected to SDS-polyacrylamide gel electrophoresis and fluorography as described under “Experimental Procedures.” Each lane contained 200 pg of protein from cells incubated with [3H]mevalonolactone during the indicated periods of time: 0-2 h (lanes a and e), 2- 4 h (lanes b and f), 4-6 h (lanes c andg), and 6-8 h (lanes d and h). “C-Labeled protein molecular mass standards, run on the end lanes, were phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and lysozyme (14.3 kDa) (Amersham Corp.). The position of the tracking dye is marked with an arrow.

of the isoprenoid pathway might be occurring. To test this possibility, cells were incubated with [3H]mevalonolactone instead of [14C]acetate, and the incorporation of radioactivity into cholesterol, ubiquinone, and free dolichol was measured during 2-h intervals of the cell cycle (Table V). The data demonstrate a 50% decrease in labeling of cholesterol and an opposite increase in labeling of ubiquinone as the cells moved from GI through S and into G2/M. The labeling of dolichol


with [3H]mevalonate underwent an approximate 40% increase, comparable to that observed for ubiquinone.

Incorporation of pH]Meualonate into Protein-bound Iso- prenyl Groups during Cell Cycle-Recent studies in several laboratories (19, 24, 25, 58) have demonstrated that cultured cells can incorporate [3H]mevalonate into isoprenyl groups that are bound to unique polypeptides. Moreover, a decreased concentration of prelabeled protein-bound isoprenyl groups appears to correlate with arrest of cell proliferation when cells are deprived of mevalonate (19, 25). Thus, it was of considerable interest to determine whether cells were capable of incorporating mevalonate into protein-bound isoprenyl groups during all phases of the cell cycle and whether the electrophoretic profile of the isoprenylated proteins, which are localized in different subcellular compartments (25), might change in conjunction with cell cycle progression. TO optimize the incorporation of exogenous [3H]mevalonate into cell proteins during brief incubation periods, it was necessary to block endogenous mevalonate synthesis with inhibitors of HMG-CoA reductase (24, 25). Therefore, we included 25 PM lovastatin (mevinolin) in the medium during each 2-h incubation with [3H]mevalonolactone. This brief exposure to lovastatin had no effect on the usual pattern of synchronous cell cycle traverse depicted in Fig. 3, consistent with our earlier studies showing that blockade of mevalonate synthesis did not affect cell cycling for at least 8 h (14, 25). The data in Table VI indicate that the total amount of [3H]mevalonate- derived radioactivity incorporated into protein-bound isoprenyl groups did not change during the cell cycle. The electrophoretic profile of these proteins on SDS gels was the same as that described previously for MEL cells (25), and it did not vary as the cells progressed from one phase to the next (Fig. 4). Specifically, there was no enhancement or diminution of radioactivity in any particular polypeptide relative to the others, and there were no major changes in the molecular weights of the labeled proteins.

DISCUSSION

The association of high HMG-CoA reductase activity with proliferative activity of tissues in vivo has been known for many years. Among the examples of this association are the induction of HMG-CoA reductase during the postnatal “growth spurt” of the developing brain and its subsequent decline in the mature tissue (59, 60), the marked increase in reductase activity in subcutaneous tumors during the period when growth is most active (61), and the increase in HMG- CoA reductase activity during the early stages of liver regen- eration (53). Studies with LM cells have confirmed these correlations in vitro. For example, the activities of HMG-CoA synthase and reductase and the rate of sterol synthesis have been shown to be 4-5-fold higher in exponentially growing cultures compared to quiescent, density-inhibited cultures (30, 31). Similarly, LM or glioma cells induced to enter quiescent states by isoleucine deprivation or serum starvation showed sharp declines in their rates of sterol synthesis and activities of enzymes catalyzing mevalonate formation compared to their proliferating counterparts (28, 29). In this study, we asked whether the synthesis of mevalonate and its isoprenoid derivatives is confined to a specific phase of the cell cycle in cells that are undergoing continuous proliferation.

When proliferating LM and erythroleukemia cells were sorted by centrifugal elutriation, the activities of HMG-CoA synthase and reductase and the synthesis of major isoprenoid end products derived from mevalonate were relatively constant throughout the cell cycle. The incorporation of radiola- beled acetate or HZ0 into sterols appeared to be slightly higher

during the G1 phase in comparison to the later stages of the cell cycle; and the studies with labeled mevalonate suggested that as the MEL cells approached mitosis, the flux of mevalonate-derived carbon into nonsterol isoprenoid compounds underwent a small increase relative to the sterol branch of the pathway. Nevertheless, the magnitude of these changes was quite small relative to the changes observed in the previously cited studies in which quiescent cells were compared to proliferating cells. In particular, there was no evidence of a major peak of HMG-CoA reductase activity or isoprenoid synthetic activity as cells progressed through the GI phase and into the S phase of the cell cycle. The latter finding is noteworthy in light of earlier reports describing substantial inductions of sterol synthesis or HMG-CoA reductase activity prior to DNA synthesis in lectin-treated lymphocytes (26,27), platelet-derived growth factor-treated cells (11, 13), or thymidine-blocked cells (10) stimulated to resume cell cycling from a quiescent state. Based on the foregoing evidence, we suggest that a substantial induction of HMG-CoA reductase activity may be necessary to build up the cellular mevalonate and isoprenoid pools in cells that must undergo the transition to the S phase, starting from a quiescent (i.e. Go) state. However, once the enzyme activity has risen and the synthesis of essential isoprenoids has accelerated to a level sufficient to support cell replication, it appears that mevalonate and isoprenoid synthesis continue throughout the cell cycle, with minimal fluctuations, as long as the cells remain in a proliferative state. This proposal needs to be tested in additional cell lines, but we believe that for the present it represents a valid working hypothesis.

Our previous studies with neuroblastoma cells exposed to lovastatin (mevinolin) have shown that cells can tolerate >90% suppression of the synthesis of sterols, free dolichol, and ubiquinone for at least 8 h without incurring changes in sterol/phospholipid molar ratio, protein glycosylation, mitochondrial oxidative metabolism, protein synthesis, RNA synthesis, or cell cycling (14, 44). Thus, it is doubtful that the small changes in the incorporation of [14C]acetate or [3H] mevalonate into sterols or nonsterol isoprenoids observed in the elutriated or synchronized cells (Tables I11 and V) signify regulatory events that could affect the passage of cells from one phase of the cell cycle to the next.

The studies described herein have a direct bearing on the now well-established mevalonate requirement for cell cycling. As mentioned previously, endogenous mevalonate synthesis or an exogenous source of mevalonate is required in order for quiescent cells to undergo a proliferative response to platelet- derived growth factor (11,13). Moreover, cells that are already growing will cease proliferation if exposed to high concentrations of HMG-CoA reductase inhibitors for prolonged periods (9, 14, 15). In both instances, the primary site of cell cycle arrest appears to be at the Gl/S boundary. These observations are subject to several possible interpretations. One possibility is that mevalonate is synthesized primarily during a specific phase of the cell cycle. This appears unlikely since the relatively invariant activities of HMG-CoA synthase and reductase imply that a constant supply of mevalonate is available throughout the cell cycle in rapidly proliferating cells. A second possibility is that mevalonate serves as a precursor for a specific regulatory isoprenoid molecule that is formed only at a certain point in the cell cycle (e.g. late GI or early S phase), thereby triggering or permitting the DNA synthetic process to commence. If the synthesis of this mevalonate- derived compound is prevented (e.g. by treating cells with inhibitors of HMG-CoA reductase), cell cycling is arrested at the point where this regulatory isoprenoid normally would

10110 Isoprenoid Synthesis during Cell Cycle

have appeared. Based on our observations of the labeling of cholesterol, ubiquinone, free dolichol, and protein-bound isoprenyl groups, we see no evidence for major cell cycle-specific changes in isoprenoid synthesis in continuously cycling cells. However, it should be noted that the partitioning of mevalonate-derived radioactivity into several important isoprenoid classes (phosphate esters of dolichol, isopentenyl-tRNA, heme a) was not studied. Thus, the possibility of a major diversion of mevalonate into one of these products at a specific point in the cell cycle cannot be completely ruled out.

In considering the role of isoprenoid compounds during the cell cycle, the recently discovered isoprenylated proteins ex- cite considerable interest, particularly since we have observed that at least one of these proteins (66-69 kDa) is localized in the nuclear matrix (25). Whereas our studies provide no direct indication as to the turnover rate of the polypeptide portion of these unique macromolecules, they do show that the cellular "machinery" for synthesizing the isoprenyl chains and incorporating them into the proteins is active throughout the cell cycle. Earlier studies with mev-1 mutant Chinese hamster ovary cells (19) and murine neuroblastoma cells (25) demonstrated a close correlation between a decline in the intracellular concentration of mevalonate-derived label associated with the isoprenylated proteins and a decrease in the propor- tion of mevalonate-starved cells entering the S phase of the cell cycle. Thus, a final explanation for the mevalonate requirement for cell cycling may be that mevalonate serves as a precursor for certain protein-bound isoprenoid compounds whose synthesis occurs throughout the cell cycle, but whose function within the cell may be particularly critical during a specific phase, such as the GI to S transition. As more is learned about these novel mevalonate derivatives, the technique of centrifugal elutriation should prove invaluable for elucidating their potential roles in cell replication.

Acknowledgments-We are grateful to Frieda Karp, Nancy Good- speed, and Dr. Tom Delohery for performing the flow cytometry, Alfred Alberts for providing lovastatin, Dr. Charlotte Friend for providing the erythroleukemia cells, and Kathy Knarr for help in preparing the manuscript.

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