The dual role of mevalonate in the cell cycle.

It is well established that either exogenous or endogenous cholesterol is required for both cell growth and proliferation. This laboratory has recently discovered that, in baby hamster kidney-21 cells, independent of its role as a cholesterol precursor, mevalonic acid plays an essential role in S phase DNA replication. It was later shown that isopentenyl adenine, a known product of mevalonate in prokaryotes and lower eukaryotes, is 100 to 200 times more effective than mevalonate in restoring DNA replication in cells in which mevalonic acid synthesis is blocked with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor, compactin. The present study was designed to determine the relationship in the cell cycle between the known requirement for cholesterol and the newly discovered effect of mevalonic acid and isopentenyl adenine on S phase DNA synthesis. Employing cells arrested by serum depletion, it was shown that the cholesterol requirement is limited to the early and mid-G1 phases, whereas the isopentenyl effect is required at the late G1-S interphase of the cell cycle. The evidence supporting these conclusions involves: first, in serum-arrested cells blocked early in G1 by compactin, only the combination of cholesterol added in early G1 and either mevalonate or isopentenyl adenine in late G1 permitted progression through the G1 and S phase DNA synthesis. Neither isopentenyl adenine added early in G1 nor cholesterol in late G1 was capable of restoring DNA synthesis in this system. Second, in accord with the above formulation, inhibition of cholesterol synthesis with the oxidosqualene cyclase inhibitor, dl-4,4,10 beta-trimethyl-trans-decal-3 beta-ol, affected only the early G1 phase of the cell cycle, but had no late G1 effect on DNA replication.

It is well established that either exogenous or endogenous cholesterol is required for both cell growth and proliferation. This laboratory has recently discovered that, in baby hamster kidney-21 cells, independent of its role as a cholesterol precursor, mevalonic acid plays an essential role in S phase DNA replication. It w a s later shown that isopentenyl adenine, a known product of mevalonate in prokaryotes and lower eukaryotes, is 100 to 200 times more effective than mevalonate in restoring DNA replication in cells in which mevalonic acid synthesis is blocked with the P-hydroxy-/I-methylglutaryl-CoA reductase inhibitor, compactin.
The present study was designed to determine the relationship in the cell cycle between the known requirement for cholesterol and the newly discovered effect of mevalonic acid and isopentenyl adenine on S phase DNA synthesis. Employing cells arrested b y serum depletion, it was shown that the cholesterol requirement is limited to the early and mid-G1 phases, whereas the isopentenyl effect is required at the late GI-S interphase of the cell cycle. The evidence supporting these conclusions involves: first, in serum-arrested cells blocked early in GI by compactin, only the combination of cholesterol added in early GI and either mevalonate or isopentenyl adenine in late G1 permitted progression through the GI and S phase DNA synthesis. Neither isopentenyl adenine added early in GI nor cholesterol in late GI was capable of restoring DNA synthesis in this system. Second, in accord with the above formulation, inhibition of cholesterol synthesis with the oxidosqualene cyclase inhibitor, dl-4,4,10P-trimethyl-trmns-decal-3~-ol, affected only the early GI phase of the cell cycle, but had n o late GI effect o n DNA replication.
This laboratory has recently reported that mevalonic acid, independent of its well known function as a cholesterol pre-* These studies were supported by the Veterans Administration and by Grant CA-15979 from the National Cancer Institute and Grant HL-23083 from the National Heart Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cursor, plays an essential role in DNA replication which characterizes the S phase of the cell cycle (1)(2)(3). This conclusion was based on the finding that baby hamster kidney cells synchronized by the double thymidine block procedure undergo a 5-to 10-fold increase in the activity of HMG-CoA' reductase, the enzyme catalyzing mevalonate synthesis, at or just prior to the S phase of the cell cycle. Inhibition of this pulse of HMG-CoA reductase activity by the competitive inhibitor, compactin, totally prevents DNA replication, but has no detectable effect on nonreplicative DNA synthesis, and finally, mevalonate, but not cholesterol, completely restores DNA replication in compactin-treated cells (I). Habenicht et al. (4) and Perkins et a2. (5) have confirmed this independent requirement for mevalonate in DNA synthesis. We have subsequently demonstrated that isopentenyl adenine, a known product of mevalonate both in prokaryotic and in lower eukaryotic cells, is more than 100 times more effective than mevalonate in restoring DNA synthesis in cells in which HMG-CoA reductase, and hence DNA replication, is inhibited by compactin (2,3). Most important, a marked effect, of both mevalonate and isopentenyl adenine on DNA replication was observed within minutes of their addition to the cell culture, suggesting that mevalonate, perhaps through the synthesis of isopentenyl adenine or a related compound, may play an initiative role in DNA replication.
In addition to this newly discovered function in DNA replication, mevalonate is well known to serve as a precursor for the cholesterol that is required for the growth of all living cells. Invertebrates, which are incapable of de nouo cholesterogenesis, require an exogenous source of cholesterol in order to grow and divide (6). Further, studies using either oxygenated cholesterol derivatives or compactin to inhibit mevalonate and hence cholesterol synthesis have demonstrated that after long term treatment, both cell growth (6-10) and DNA synthesis (8, 10,12) cease; in several cases these effects could be reversed with both cholesterol and its precursor, mevalonate (7-9, 11, 12). Kaneko et al. (13) have also reported that such long term inhibition of HMG-CoA reductase by compactin results in an inhibition of cell growth but they failed to find an effect on DNA synthesis. Finally, it has been repeatedly shown (14-17) that blockage of cholesterol synthesis with 25-hydroxycholesterol for 1-3 days prevents the typical lectininduced stimulation of DNA synthesis in lymphocytes. There is no question, therefore, that cholesterol itself a n d perhaps other isoprenoids, such as coenzyme Q, are required for cells to complete the cell cycle and to synthesize DNA.
In view of our finding that mevalonate, independent of its function as a cholesterol precursor, causes the prompt restoration of DNA replication in the S phase of the cell cycle (1-3), we have begun studies employing baby hamster kidney cells synchronized by the serum deprivation technique to determine where within the cell cycle the cholesterogenic function of mevalonate is required for cell growth and DNA synthesis. The resulting studies demonstrate that the need for cholesterol, either synthesized from mevalonate or derived exogenousIy, occurs in the cell cycle early in GI, many hours before DNA replication takes place. Mevalonate in early GI presumably provides structural cholesterol which permits the cell to pass through GI and reach the S phase of t h e cell cycle.
However, the present findings also confirm our earlier conclusions that, in order to advance from late G I into S phase, the synthesis of mevalonate is required to provide the isopentenyl adenine or related isoprenes which are required for DNA replication.
These findings indicate that cholesterol, while necessary in early GI for the passage of cells through GI, has no direct effect on S phase DNA replication. By contrast, quite independent of its function in cholesterogenesis, mevalonic acid probably by means of its isopentenyl adenine or similar isoprene effect, is required in late GI for cells to carry out S phase DNA replication.
Lipoprotein-Human LDL (density 1.019 to 1.063 g/ml) was obtained from the plasma of a type I1 individual and prepared by differential centrifugation as described (20, 21). Cell Culture-BHK-21 cells were obtained from the Cell Culture Facility of the University of California, San Francisco. Stock cultures of these cells were grown as monola-yers in 150-cm2 flasks maintained in a 5% CO, atmosphere at 37 "C in a humidified incubator. The stock cells were maintained by subculture every 4 days at a split ratio of 1:lO.
Experiments for DNA synthesis were carried out in 6-well trays (Linbro Division, Flow Laboratories). For the HMG-CoA reductase assay and [I4C]acetate incorporation into cholesterol, Petri dishes (60 X 15 mm) were used. Cells were plated at a concentration of 3 x lo" cells/dish.
Cell Synchronization by Serum-deprivation Repletion-Cells were arrested by serum deprivation (22). They were incubated for 48 h in Dulbecco's modified Eagle's media supplemented with 0.1% fetal calf serum. After 48 h Dulbecco's modified Eagle media supplemented with 5% fetal calf serum was added. Following the 5% fetal calf serum addition, cells were studied under the incubation conditions described in the individual experiments. It is recognized that cells are not absolutely synchronized by this procedure but, as indicated by Figs. DNA Synthesis-Cells were incubated with [methyl-"H]dThd (2 pCi/ml) for 30 min. They were then washed once with phosphatebuffered saline and then cells were maintained at 4 "C in 5% trichloroacetic acid for 30 min. Cells were then washed 2 times with 5% trichloroacetic acid and two times with cold 90% ethanol. Once the cell precipitate was d r y , 1 ml of 2 mM sodium dodecyl sulfate was added to disrupt the cell membranes. A 200-pi aliquot was used for protein determinations and a 0.5-ml aliquot for DNA determination.
Incorporation of [I4C/Acetate into Cholesterol-Monolayers of BHK-21 were incubated for 2 h with ['%]acetate (2.5 pCi/ml) at 37 "C in 2 ml of phosphate-buffered saline. After incubation the medium from each Petri dish was transferred into 50-ml screwtop glass tubes; the cells were dissolved in 1 ml of 0.1 M NaOH and added to the medium from the corresponding Petri dish. To each 4 ml of this mixture 7 ml of ethanol/90% KOH/H?O, 5:1:2 (v/v) and lo5 cpm of [1,2-:iH]cholesterol as an internal standard were added. Total B P-hydroxy sterols were isolated as the digitonide as described (23).
Autoradiography-Cells were incubated with [methyl-:'H)thymidine (2 pCi/ml) for 30 min. They were then washed twice with phosphate-buffered saline and fixed for 30 min in Carnoy's fixative, rinsed with distilled water ten times, and air-dried. The cells were overlayed with NTB-3 emulsion (Eastman Kodak) and developed 7 days later.
Protein Determinations-Protein concentrations were measured by the method of Lowry et al. (24) with bovine serum albumin as a standard.
HMG-CoA Reductuse-The rate of conversion of [3-I4C]HMC-CoA to 114Clmevalonate was measured in detergent-solubilized extracts oftheBHK-21 cells as described by Brown and Goldstein (21) and modified by Avigan et al. (25) and Cohen et al.

Synchronization of BHK Cells by Serum Deprivation-Re-
pletion-The method employed for cell synchronization throughout this study involves, as noted above, the use of serum deprivation for 48 h to produce cell arrest in GI. When the media of such cells is supplemented with 5% fetal calf serum, the cells re-enter GI in a relatively synchronized manner with first order kinetics. To determine the extent of synchronization by this procedure, preliminary studies were carried out using both tritiated thymidine incorporation into DNA and the percentage of cells whose nuclei were labeled with [3H]thymidine as determined by autoradiography.
The results presented in Table I demonstrate that at 6 h after supplementation of the media with 5% fetal calf serum, both the level of incorporation of thymidine into DNA and the percentage of labeled nuclei remain unchanged from that at zero time. Between 8 and 12 h, there is a rapid increase of number of cells entering S phase by both criteria, with thymidine incorporation at 12 h increasing 12-fold above the zero hour value and the percentage of cells with labeled nuclei increasing at least 14-fold. At 14 h, there is no further increase in cells entering S phase by either criteria.  inhibit DNA replication in cells synchronized by the serum deprivation method. As indicated by the results in Fig. 1, the control cells arrested by serum deprivation entered S phase approximately 8 h after the addition of fresh media containing 5% fetal calf serum, and DNA synthesis reaches a maximum between 12 and 14 h. The addition of 20 /LM compactin (a concentration that resulted in at least a 99% inhibition of mevalonate synthesis) during late GI caused a marked inhibition of DNA replication throughout the S phase of the cell cycle. The addition of meValonate (0.4 mM) 2 h before the initiation of the normal S phase completely reversed this compactin induced inhibition of DNA replication (Fig. 1).

Role of Meualonate in Cell Cycle
To determine whether the compactin-induced block of DNA replication might be the result of cholesterol depletion rather than the absence of mevalonate itself, the ability of LDL cholesterol (100 pg/ml) to reverse this inhibition of DNA replication was next evaluated. When added during late GI at the same time as compactin, cholesterol at a final concentration of 100 pg/ml was clearly unable to overcome the inhibition of DNA synthesis produced by compactin. Lower concen- trations of cholesterol were also ineffective." There is no question in this experiment that the LDL-cholesterol was being taken up and metabolized by the cells, since the addition of the sterol produced a marked inhibition of HMG-CoA reductase activity, i.e. from 90 pmol/min/mg of protein to 25 pmol/min/mg of protein.
In view of our previous finding that isopentenyl adenine is at least 100 times more effective than mevalonate in restoring DNA replication in thymidine-synchronized cells treated with compactin (2, 3), we carried out a similar study in BHK cells arrested by the serum deprivation technique. As also indicated in Fig. 1, at 14 h but not at 12 h, the addition of isopentenyl adenine in a concentration of only 5 PM caused a significant stimulation of DNA synthesis in the compactin-inhibited cells. At the dose of isopentenyl adenine used, DNA synthesis in the compactin-IPA-treated cells did not reach the peak values observed in the control cells.
Effect of IPA Concentration upon DNA Synthesis in Late G1-The effect on DNA synthesis of increasing concentration of isopentenyl adenine added in late GI was next determined.
In these studies, 20 PM compactin and various concentrations of IPA were added 6 h after reversal of serum depletion, and thymidine incorporation into DNA was evaluated 12 h later, i.e. at the peak of S phase DNA synthesis in this experiment. As indicated by the results in Fig. 2, a significant stimulation of DNA synthesis was observed at an IPA concentration of 10 p~, and the effect of isopentenyl adenine increased to reach a maximum at between 20 and 40 PM. No further stimulation of DNA synthesis was observed at a concentration of 100 pM. This study demonstrates that IPA at maximum concentrations restored DNA synthesis to a level 80% that of control. Under these conditions, therefore, IPA can largely, if not completely, restore DNA synthesis to control levels in mevalonate-depleted cells.
Effect cycle. The effect of compactin added in early GI was therefore examined. As indicated in Fig. 3, when mevalonate synthesis was prevented throughout GI, no significant incorporation of thymidine into DNA above the basal level occurred during the expected S phase of the normal cell cycle. The effect of compactin added at zero time, i.e. in early GI, is more pronounced than when added a t 6 h, i.e. in late GI. As also indicated in Fig. 3, mevalonic acid, when added during early GI, reversed the compactin-induced inhibition of DNA replication. Once again, LDL-cholesterol added in early GI was totally ineffective in overcoming the inhibition of DNA synthesis. The major finding in this experiment, however, was that isopentenyl adenine, which as noted above, when added late in GI, would largely reverse the compactin inhibition of DNA replication, was completely incapable of restoring DNA synthesis to compactin-treated cells when added in early GI.

Effect on DNA Replication of Cholesterol Added in Early
GI and Meualonate or IPA Added in Late GI to Compactintreated Cells--In order to distinguish the distinct roles of mevalonate in the cell cycle which were implied by the previous experiments, cells were again treated with compactin throughout GI, and LDL-cholesterol and mevalonate were added sequentially, i.e. the cholesterol in early GI and the mevalonate in late GI. As indicated by the results in Fig. 4, this combination of cholesterol and mevalonate caused total rescue of DNA synthesis in compactin-treated cells.
By contrast, if this experiment is repeated, omitting the cholesterol treatment in early GI and adding mevalonate only in late GI, the peak of DNA synthesis is delayed by about 4 h, i.e. from 14 h to approximately 18 h after removal of the serum block. This finding would suggest that when cells are deprived early in GI of both cholesterol and its precursor, mevalonate, passage from early to late GI is retarded until mevalonate is added t,o the compactin-treated cells, thereby providing cholesterol that is required for optimum cell growth. When such cells proceed to the delayed late GI, the added mevalonate, presumably by providing isopentenyl adenine or related isoprenes, permits the initiation of DNA replicat,ion.
These studies were extended in a separate experiment in which the ability of cholesterol, mevalonate, and, in particular, isopentenyl adenine to reverse the compactin inhibition of S phase DNA synthesis was further determined in cells treated with compactin at zero hour. As indicated by the data in Table 11, the peak of S phase DNA synthesis occurred in the control cells a t 12 h. The degree of thymidine incorporation into DNA was not significantly affected in these cells by the addition of mevalonate, LDL-cholesterol, or cholesterol in ethanol. While not included in our study, we repeatedly find that, as expected, IPA added a t zero time to control cells has no effect on DNA synthesis. The addition of compactin a t zero time led to a marked decrease in S phase DNA synthesis which was most apparent

TABLE I1
Reversal of compactin inhibition of DNA replication by addition of cholesterol in early GI a n d meculonate or IPA in lute G I Cells were arrested by serum deprivation and after 48 h were stimulated to grow by adding media supplemented with 5%) fetal calf serum. The following treatments were at 0 h: compactin, 20 p~; and at 0 or 6 h as indicated: MVA, 0.4 mM; LDL-cholesterol, 100 pg/ml; cholesterol-ethanol 25

Dual Role
of Mevalonate in Cell Cycle at the 8th and 12th h after release of the serum arrest. Neither cholesterol nor IPA added at zero time had a significant effect upon DNA replication, however, in conformity with the results shown in Fig. 1, mevalonate added at zero time caused a significant stimulation of DNA synthesis, which in this experiment was most apparent a t 16 h. The major aim of this experiment was to determine whether cholesterol added either as lipoprotein bound or dissolved in ethanol, plus mevalonate or isopentenyl adenine added at 6 h would reverse the compactin inhibition of S phase DNA synthesis. The results in Table I1 demonstrate that, as compared to the insignificant effect of added cholesterol alone, the further addition of mevalonate or of IPA a t 6 h caused a highly significant. increase in DNA synthesis both at 12 and 16 h after removal of the serum arrest.
As shown in Table 111, the ability of cholesterol added at early G I plus IPA added at late GI to restore DNA replication in compactin-treated cells is confirmed by autoradiographic assessment of the percentage of nuclei labeled by ["Hlthymidine.
The results in Table I1 also show that, as expected, the simultaneous addition of IPA or LDL-cholesterol to the compactin-treated cells did not result in a stimulation of DNA synthesis. Finally, if LDL and mevalonate are both added at 6 h, a stimulation of DNA synthesis occurs; however, the maximum thymidine incorporation into DNA is observed at 16, rather than 12, h. This lag, as noted above, probably represents the delay in initiation of GI until the time that LDL-cholesterol is added. In conformity with this assumption, the addition of mevalonate alone at 6 h likewise stimulated DNA synthesis to a somewhat lesser extent, with a comparable shift in the maximum DNA synthesis. Perhaps due to redistribution of isoprene precursors (see "Discussion") in this experiment, the addition of LDL-cholesterol at zero time resulted in a delayed, partial restoration of DNA synthesis a t 16 h. Deprivation of Cholesterol during Arrest of Cell Growth-Studies were next carried out to determine the effect of depriving cells of endogenous as well as of exogenous cholesterol during the period of serum depletion arrest. For this study, cells were placed in media containing 0.1% fetal calf serum with a total cholesterol concentration of 0.42 pg/ml. After 24 h, while still subject to such serum deprivation, the experimental cells were treated for an additional 24 h with 2.5 p~ compactin. At the end of this 24-h treatment with com-  pactin, the serum-deficient media was removed and cell growth initiated by addition of fresh media containing 5% fetal calf serum. Incorporation of ['Hlthymidine into DNA was determined at 8, 12, and 16 h after initiation of cell growth.
As indicated in Table IV, treatment, with compactin during the last 24 h of growth arrest resulted in marked inhibition of DNA synthesis at both the 8-and 12-h periods after reinitiation of cell growth. By 16 h, some recovery of DNA synthesis was noted; however, the degree of thymidine incorporation into DNA was still far less than in control cells not treated with compactin.
Supplementation of the compactin-containing media wit,h mevalonic acid during the last 24 h of serum deprivation caused complete restoration of S phase DNA replication. More significant, however, the addition of LDL-cholesterol during the 24 h of cornpactin treatment likewise led to normal progression into S phase, with the peak of DNA synthesis occurring at 12 h, coincident with that observed in the untreated control cells.
These data indicat,e that compactin treatment, even during the 24 h prior to being stimulated to enter the GI phase, prevents the cells from progressing normally into the S phase of the cell cycle, with at least an 8-h delay in the initiation of normal DNA synthesis. The restoration of normal progression to S phase by LDL-cholesterol even in the presence of compactin, demonstrates that cholesterol, either endogenous or exogenous, is required even during the growth arrest period for cells to enter and pass through GI.
Effect  . Studies, the data for which are not presented, have shown that under the conditions employed, TMD has no effect upon HMG-CoA reductase activity. The effect on DNA synthesis of inhibiting cholesterogenesis with T M D in either early or late GI was next determined. As indicated in Table V, when TMD was added at zero time, that is, at the beginning of GI, subsequent DNA replication is greatly inhibited. By contrast, when the TMD inhibition of cholesterogenesis is induced late in GI, DNA replication was totally unaffected. These results further support the conclusion that cholesterol is required presumably for the synthesis of structural membranes in the early and mid-GI phases of the cell cycle, but that cholesterol itself plays no direct role in S phase DNA replication. This conclusion is strongly supported by the finding (Table V)  terol, either as LDL or in ethanol, during early GI completely reversed the inhibition of S phase DNA synthesis caused by TMD. By contrast, as shown in Fig. 5 , mevalonate added to TMD-treated cells early in GI did not reverse the TMDinduced inhibition of DNA synthesis. Finally, as demonstrated in Fig. 6, if synchronized BHK cells are treated with TMD early in GI to inhibit cholesterol synthesis and with compactin in late GI to inhibit mevalonate synthesis, the resulting inhibition of DNA replication can be overcome by a combination of cholesterol added in early GI and mevalonate replacement in late GI. Neither cholesterol nor mevalonate alone was capable of reversing the inhibition of DNA replication produced by the double block with T M D and compactin.

DISCUSSION
HMG-CoA reductase serves at least two obligatory functions in the cell cycle. First, as shown by many previous studies, HMG-CoA reductase provides the mevalonate that is needed for the synthesis of cholesterol which, in turn, is required for the production of cell membranes and, hence, cell growth (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Secondly, however, we have recently demonstrated that mevalonate, quite independent of its function as a cholesterol precursor, plays an essential role in the initiation of DNA replication (1,3). We have further provided evidence that isopentenyl adenine, or a related isoprene purine, may mediate this effect of mevalonate in the cell cycle (2, 3).
The primary purpose of the present study has been to confirm and extend the evidence for this dual function of mevalonate in the cell cycle and to determine specifically where within this cycle the cholesterogenic function of mevalonate is required. The major finding of the present study is that cholesterol, either endogenously synthesized from mevalonate or supplied exogenously, is specifically required early in G, to permit the cell to undertake the growth phase that is characteristic of passage from early to late GI in preparation for DNA replication. By contrast, as we have previously shown ( l ) , cholesterol itself appears to play no direct function in initiation of DNA replication during the S phase of the cell cycle. The present results also confirm our finding that mevalonate, independent of its role of providing endogenous cholesterol for the structural requirements of the cell, serves a relatively rapid function in initiating S phase DNA synthesis.
Moreover, this latter role of mevalonate in DNA replication could largely be replaced by isopentenyl adenine.
Our previous studies of the role of mevalonate in the cell cycle made use of BHK cells synchronized by the double thymidine block procedure. This technique causes a block in cell replication in the GI-S interphase and, therefore, precludes studies of the entire GI phase of the cell cycle. In the present studies, we employed the serum-depletion technique, which arrests cells in early GI, thereby allowing a detailed examination of the role of mevalonate synthesis both in early and late GI, as well as in the S phase of the cell cycle.
Employing cells synchronized by the serum depletion-repletion method, we could fully confirm our earlier findings that mevalonate is required in late GI for cells to initiate S phase DNA replication. Additionally, in this experiment, isopentenyl adenine could once again largely replace mevalonate in initiating S phase DNA replication and proved again to be significantly more potent than mevalonate in stimulating DNA replication in cells in which mevalonate synthesis was blocked with compactin. By contrast, the addition of cholesterol in late GI had no effect on DNA replication in the compactintreated cells. This experiment once more documents the requirement for mevalonate but not cholesterol for the initiation of DNA replication in cells that have reached the late GI phase of the cell cycle.
Studies next were focused on the role of mevalonate in early GI. When mevalonate synthesis was inhibited throughout G I by the addition of compactin at the time of the removal of the serum depletion arrest, S phase DNA replication, which usually begins 8-10 h later, was cornpletely prevented. The presence of mevalonate in early GI fully reversed the compactin inhibition of S phase DNA replication; however, isopentenyl adenine was totally incapable of replacing this mevalonate requirement in early GI. This finding provided the first direct evidence that mevalonate plays a t least two roles in the cell cycle: one during early GI that cannot be replaced by isopentenyl adenine and another in late GI which can be largely reproduced by isopentenyl adenine at 1/200 the concentration of mevalonate. Direct evidence that the early G I function of mevalonate is to serve as a cholesterol precursor is provided by the finding that the addition of lipoprotein-cholesterol during early G I could completely replace the requirement for mevalonate, whereas in late G I , cholesterol is incapable of assuming the role of mevalonate in DNA replication.
Consistent with this conclusion was the observation that when cells treated with compactin in early GI were not rescued with mevalonate until late GI, the peak of DNA replication was delayed for approximately 4-6 h beyond its normal occurrence. Presumably under these circumstances, passage through GI was delayed until amounts of endogenous cholesterol adequate for cell growth were synthesized from the mevalonate. The added mevalonate was thereafter available to serve a second function, namely the initiation of DNA replication, which was maximal at the 18th instead of the normal 14th h after release of the serum block.
The dissociation of the two functions of mevalonate during the GI phase of cell replication was further documented by the use of a specific inhibitor of oxidosqualene cyclase, TMD, which prevents the synthesis of cholesterol but not of mevalonate (19). Consistent with a cholesterol requirement in early, but not in late, GI, the addition of TMD in early GI prevented subsequent DNA replication, whereas T M D was totally without effect on DNA replication when added in late GI. Moreover, cholesterol completely reversed the effect of TMD on DNA synthesis if the cholesterol was added in early GI, but could not reverse the effect of TMD if added in late GI. In view of the main site of action of TMD, mevalonate alone could not reverse the TMD effect. The dual effect of mevalonate on the cell cycle could be further confirmed by the combined use of TMD to inhibit cholesterol synthesis and compactin to inhibit HMG-CoA reductase and hence mevalonate production. As would be predicted, the presence of TMD early in GI and compactin in late GI results in an inhibition of DNA synthesis that can only be reversed by the combined addition of cholesterol in early G I and mevalonate in late GI.
Taken together, these findings strongly support our previous conclusion that the synthesis of mevalonate performs two distinct and essential functions in the cell cycle. First, mevalonate serves a relatively long term role as a precursor of the cholesterol that is necessary for membrane structure, for cell growth, and for the resulting progression of the cell through the GI phase of the cell cycle. Consistent with such a formulation, this function of mevalonate can be completely replaced by exogenous cholesterol. It is very likely that with prolonged cell propagation in the presence of compactin, deprivation of other structural and functional isoprenoids, such as dolichol and coenzyme Q, would become apparent.
The present findings indicate, however, that cholesterol, but not other isoprenes such as dolichol and coenzyme Q , is limiting for cell growth during the 8-10 h of the single GI phase characteristic of the BHK cell examined in these studies.
The experiment in Table IV further demonstrates that cholesterol is needed not only during the GI phase of cell growth, but is also required during the period of cell arrest (Go). In this experiment, when cells were deprived by compactin treatment of endogenous choiesterol during serum depletion arrest, restoration of cholesterogenesis by the addition of 5% fetal calf serum did not result in normal progression through GI; and S phase DNA synthesis was delayed by at least 8 h. Since the addition of either mevalonate or cholesterol to compactin-treated, growth-arrested cells completely restored normal GI progression, it is clear that cholesterol must serve an essential function either for cell growth or membrane replenishment, even during cell arrest or the GO phase of the cell cycle.
In addition to providing the cholesterol required for cell growth, this and our previous studies have demonstrated that mevalonate, completely independent of its function as a cholesterol precursor, is required in late GI to initiate DNA replication. In contrast to the relatively long term requirement for cholesterol, amounting to several hours in the cell cycle, mevalonate can, as shown in previous studies, initiate DNA replication in late GI within minutes after its addition.
Isopentenyl adenine can largely or completely replace the late GI-S phase functions of mevalonate; and it is likely, therefore, as we have previously suggested ( 2 , 3 ) , that isopentenyl adenine or a closely related purine may mediate the effect of mevalonate in initiating DNA replication. In certain of the experiments reported in the present study, isopentenyl adenine, while causing a marked stimulation of DNA replication in compactin-inhibited cells, did not result in full restoration of DNA replication. This finding raises the possibility that after deprivation of mevalonate for several hours, in addition to isopentenyl adenine, other isoprenes may play a role in achieving maximal DNA replication.
In this regard, Brown and Goldstein have shown that after maximum cholesterol-induced feedback inhibition of HMG-CoA reductase, the addition of mevalonate results in a significant further inhibition of this enzyme. On the basis of this finding, they have postulated that a nonsterol derivative of meValonate acts cumulatively with cholesterol to inhibit