Two major regulatory steps in cholesterol synthesis by human renal cancer cells

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 196, No. 2, September, pp. 574-580, 1979

Two Major Regulatory by Human

Steps in Cholesterol Renal Cancer Cells’

Synthesis

RICARDO GONZALEZ, JOHN P. CARLSON, AND MARY E. DEMPSEY The Departments of Urologic Surgery and Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455

Received January 23, 1979; revised May 2, 1979 Two major mechanisms regulating cholesterol biosynthesis exist in a human renal cancer cell line, Caki-1. Caki-1 is a newly established cell line whose characteristics of rapid growth and active cholesterol synthesis qualify it as a potentially valuable tool for elucidation of regulatory mechanism of cholesterol synthesis and transport. In the absence of exogenous cholesterol, cholesterol is the dominant sterol arising from labeled acetate and mevalonate. As expected, in the presence of exogenous cholesterol, the conversion of acetate to cholesterol and the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) is markedly reduced and this inhibition is released when cholesterol is removed from the medium. An unexpected and possibly unique finding is the inhibition of the conversion of mevalonate to cholesterol in the presence of exogenous cholesterol. This second major control process results in the accumulation of squalene and may involve additional late steps in cholesterol biosynthesis or metabolism. The occurrence of two major mechanisms regulating cholesterol synthesis may be a unique property of renal cancer cells or a previously unrecognized characteristic of a variety of cultured cells.

The major limiting step in cholesterol synthesis is generally accepted to be the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCOA)~ to mevalonic acid, catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating) EC 1.1.1.341 (e.g., 1, 2). The activity of this enzyme in liver decreases with cholesterol feeding and also responds to other physiological and nonphysiological manipulations affecting sterol synthesis (e.g., 3). A parallel and well-documented finding is a decrease in the activity of the reductase in cultured cells when cholesterol is included in the medium (e.g., 4, 5). The major regulatory role of HMG-CoA reductase is further supported by reports that sterol synthesis from mevalonate in cultured cells is not influenced by exogenous cholesterol while acetate

incorporation into sterols in markedly depressed (e.g., 6-8). However, other evidence suggests that there are additional mechanisms regulating cholesterol synthesis. For example, synthesis of cholesterol from mevalonate decreases in rat liver after prolonged cholesterol feeding, an observation that cannot be explained by postulating a change in the activity of HMG-CoA reductase (e.g., 2, 9, 10). A discrepancy in the rate of sterol synthesis and the activity of HMG-CoA reductase was also noted after exposure of rat liver to a lipid load in vivo (11). More recently, similar observations were made during studies with &oxygenated sterols in mouse L-cell cultures and liver cell preparations (12). The primary purpose of this communication is to present evidence that exogenous cholesterol not only decreases the activity of HMG-CoA reductase in cultured human I Supported by NIH Grants CA-18256 and HLB8634 and by a grant of the Minnesota Medical Founda- renal cancer cells but also inhibits the conversion of mevalonate to cholesterol, tion. 2 Abbreviation used: HMG-CoA, 3-hydroxy-3-methyl- resulting in the accumulation of squalene. glutaryl coenzyme A. Similar observations have not been previously 0003-9861/79/100574-07$02.00/O

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

574

CHOLESTEROL

SYNTHESIS

reported for human cells in culture. The corollary purpose of this communication is to describe specific properties of a new, human cell line offering advantages for the study of sterol synthesis and transport. Also, study of cholesterol synthesis in human renal cancer cell lines is of interest because these tumors, which originate in the epithelium of the proximal convoluted tubule of the kidney (13), tend to accumulate cholesterol in the cytoplasm (14), and studies in viva suggest that this cholesterol is the product of intracellular synthesis by the tumor (15). MATERIALS

AND METHODS

[12-W]Acetate (50 mCi/mmol), Dr&3H]mevalonate (500 mCi/mmol), 3H,0 (1 mCi/g), and DL-3-hydroxy3-methyl-[3-*4C]glutaryl CoA (25 mWmmo1) were purchased from New England Nuclear Corporation. NADPH, glucose &phosphate, and glucose-&phosphate dehydrogenase were obtained from Calbiochem; 3-hydroxy-3-methylglutaryl CoA, from P-L Biochemicals Inc., and Kyro EOB was a gift from Procter and Gamble Company. Cholesterol was purchased from ICN Pharmaceuticals Inc. and purified by passage through the dibromide derivative and crystallization (16, 17). It was stored under nitrogen prior to use. Squalene was purchased from Sigma and purified by passage through the thiorurea clathrate (18). Caki-1, the cell line3 used for these studies, was established from a kidney clear cell carcinoma metastatic to skin (19). By both light and electron microscopy these cells exhibit epithelii characteristics, their doubling time is as short as 24 h (dependent on growth conditions), and they produce tumors in the immunosuppressed hamster and nude mouse (J. Fogh, personal communication). The Caki-1 line used in these studies was maintained in culture for more than 40 passages. Stock cells were grown in 75-cm* vessels (Falcon) with tight caps at 37°C in a medium composed of RPM1 1640 (GIBCO) and 20% fetal calf serum (Reheis) supplemented with penicillin (300 units/ml) and streptomycin (300 pg/ml). Cells were harvested using a trypsin (0.25%)-Versene (0.1%) solution and inoculated (1 x lo6 cells) into 25cm2 vessels (Falcon) containing the growth medium (3 ml) just described. When the cells were confluent (3 days after subculture), the medium was replaced by a test medium containing the desired lipid composition. Cholesterol-free medium was RPM1 1640supplemented with 20% fetal calf lipoprotein-poor serum (20); 3 Gift of Dr. Jergen Fogh, Sloan-Kettering Institute for Cancer Research.

IN HUMAN RENAL CANCER

575

final protein concentration in the medium was 8 mg/ml. Cholesterol-containing medium was RPM1 1640 supplemented with 20% fetal calf serum (final cholesterol concentration 30 pg/ml) or RPM1 1640with 20% fetal calf lipoprotein-poor serum supplemented with free cholesterol (dissolved in ethanol) to produce a final concentration, again, of 30 pg/ml. In some experiments protein and lipid sources in the medium were human lipoprotein fractions and human lipoproteinpoor serum (21) (see Results and Discussion). For all experiments concerned with cholesterol biosynthesis from labeled precursors the final volume of growth medium was 1 ml. Incubation reactions were stopped by adding ethanol (1 ml) and KOH (450 mg). After saponification for 24 h at 25°C the nonsaponifiable fraction was isolated by threefold extraction with 4 ml petroleum ether (bp 30-60°C). Control experiments showed this procedure recovered greater than 90% of the labeled cholesterol in the incubations. Labeled cholesterol in the nonsaponifiable extracts was quantitated by passage through the dibromide derivative (16, 17). The results of the dibromide procedure were verified by silicic and acid chromatography (using benzene as eluent) of the nonsaponifiable fractions in the presence of unlabeled cholesterol (22). The two methods agree within 22%. The latter technique was also used to demonstrate the accumulation of labeled squalene by Caki-1 cells grown in the presence of cholesterol (see Results and Discussion). The occurrence of labeled squalene was further verified by gas-liquid chromatography (23) and by passage of the squalene and containing fractions (obtained by silicic acid chromatography) through the thiourea clathrate (18). For the HMG-CoA reductase studies (Fig. 1) Caki-1 cells were incubated in test media and harvested as just described. The cells were washed with 50 mM Tris-Cl, pH 7.4, containing 0.15 mM KCl, stored until used at -196”C, and broken with Kyro EOB as described in detail by Brown et al. (24). The solubilized cell extracts were assayed for HMG-CoA reductase by the method of Goldfarb and Pitot (25); specific details are given in the legend with Table I. The protein content of the cell extract was determined by the method of Lowry et al. (26) after trichloroacetic acid precipitation. RESULTS AND DISCUSSION

Characteristics of Caki-1 cells pertinent to their use in studies on regulatory mechanisms of cholesterol synthesis and transport are (a) their human origin (see Materials and Methods) and (b) their rapid growth, accompanied by production of cholesterol as the major product of the sterol synthesis (cf. Tables II and III). In this

576

GONZALEZ, CARLSON, AND DEMPSEY

of HMG-CoA to mevalonic acid. Similarly, in Caki-1 cells exogenous cholesterol decreased the activity of HMG-CoA reductase threefold (Table I). This difference was observed whether or not the medium contained serum lipoproteins, i.e., lipoprotein-poor serum plus cholesterol was as effective as fetal calf serum (Table I). Conversely, when cholesterol is removed from the medium a rapid increase in the activity of HMG-CoA reductase occurs (Fig. 1). These findings were then correlated

0, 0

TABLE I 4

I3

12

16

20

24

Time of Incubation with Test Medium (hours)

FIG. 1. Influence of exogenous cholesterol on activity of HMG-CoA reductase in Caki-1 cells. Cells (1 x 106) were inoculated into 25cm* flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5 (time 0) the medium was replaced with one of three test media: (a) RPM1 1640 with 20% fetal calf lipoprotein-poor serum (O), (2) RPM1 1640 with 20% fetal calf serum (final cholesterol concentration 30 fig/ml) (0), or (3) RPM1 1640 with 20% fetal calf lipoprotein-poor serum to which cholesterol (see Materials and Methods) was added in ethanol to a final concentration of 30 kg/ml (A). Final ethanol concentration in all media was 0.5% (v/v). At the time periods indicated in the figure, cell extracts were prepared for assay of HMG-CoA reductase as described in Materials and Methods (25). Conditions of incubation for the assay of HMGCoA reductase were identical to those described in Table I. Results are the mean of duplicate flasks which varied by less than 25%.

regard, many of the current studies on regulation of sterol synthesis in cultured cells have been performed with cells from lower mammalian species (e.g., mouse fibroblasts) (6) or with cells from peripheral human tissue sources (e.g., skin fibroblasts) (4). Some of these cells accumulate sterol precursors of cholesterol rather than cholesterol itself (6, 2’7). Earlier reports from this laboratory showed that the presence of cholesterol in the cufture medium decreases the rate of cholesterol synthesis by both normal kidney (28) and early passage renal cell cancer (ZO), probably by inhibiting the conversion

INFLUENCE OF EXOGENOUS CHOLESTEROL ON THE ACTIVITY OF HMG-CoA REDUCTASE IN C&i-l CELLS'"

HMG-CoA reductase activity (pmol/min/mg) Additions to growth medium Lipoprotein-poor serum (1) Fetal calf serum (2) Lipoprotein-poor serum with cholesterol (3)

270 2 14 92t 4 82 r 13

u Cells (1 x 1O6)were inoculated into 25cm* flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5 (time 0) the medium was replaced with one of three test media: (1) RPM1 1640 with 20% fetal calf lipoprotein-poor serum; (2) RPM1 1640 with 20% fetal calf serum (final cholesterol concentration 30 mg/ml), or (3) RPM1 1640 with 20% fetal calf lipoprotein-poor serum to which cholesterol (see Materials and Methods) was added to a final concentration of 30 pg/ml. Final ethanol concentration in all media was 0.5% (v/v). After 72 h of incubation in the test media, cell extracts were prepared for assay of HMG-CoA reductase as described in Materials and Methods (24). Two aliquots of each extract (20 and 40 fig of protein) were incubated in duplicate for 60 min at 37°C in a final volume of 0.2 ml containing 0.1 M potassium phosphate, pH 7.5; 20 mM glucose-6-phosphate; 2.5 mM NADPH; 0.7unit glucoseB-phosphate dehydrogenase; 5 mM dithiothreitol; and 5 ELMDL-[3-‘4C]HMG-CoA (5 pCi/mmol). Reactions were stopped by addition of 20 ~1 of 6 N HCl. Fifteen micromoles of [5-3H]mevalonolactone (58.8 &i/mole) was added as internal standard and the mixture incubated at 37°C for 15 min. Mevalonolactone was isolated by thin-layer chromatography on silica gel (24, 25). Reactions were linear with time and protein concentration. Results are the mean of four determinations t standard error of the mean.

CHOLESTEROL

SYNTHESIS

with the rate of cholesterol synthesis from three labeled precursors (water, acetate, and mevalonate). Cholesterol either as fetal calf serum or added to the medium with lipoprotein-poor serum decreased the incorporation into cholesterol of all three precursors three- to fivefold relative to the maximum incorporation observed with lipoprotein-poor serum (Table II). Although the inhibition of cholesterol synthesis from acetate and water could be explained by the observed decrease in the TABLE

II

INFLUENCE OF EXOGENOUSCHOLESTEROLON CHOLESTEROLSYNTHESISFROMWATER, ACETATE, AND MEVALONATE ON Caki-1 CELLS” Cholesterol synthesis (dpm per lo6 cells)

Labeled precursor 3Hz0 [2-14C]Acetate DL+VH]Mevalonate

Medium without cholesterol

Medium with cholesterol

400 12,200 35,400

80 4,400 6,600

u Cells (1 x 106)were inoculated into 25cm2 flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5 the medium in all flasks was replaced with RPM1 1640 containing 20% fetal calf lipoprotein-poor serum. Twenty-four hours later (time 0) the medium was replaced with 1 ml of three test media. Medium without cholesterol was RPM1 1640 with 20% fetal calf lipoprotein-poor serum (8 mg protein/ml). In the experiments with 3H,0, medium with cholesterol was RPM1 1640 with 20% fetal calf serum (final concentration 30 pg/ml). In the experiments with labeled acetate and mevalonate, the medium with cholesterol was RPM1 1640with 20% fetal calf lipoprotein-poor serum to which cholesterol was added (see Materials and Methods) (final concentration of cholesterol 30 pg/ml). Final ethanol level in all media was 0.5% (v/v). Incubation of the cells with 3H20 (3.26 mCi) were for 48 h; those with [2-14C]acetate (0.5 &i; 50 mCi/mmol) and DL-[~-~H]mevalonate (10 &i; 500 mCi/mmol) were for 72 h. Reactions were stopped by addition of ethanol and KOH; the level of cholesterol present in the nonsaponifiable extract was determined by the dibromide method (see Materials and Methods). Results (mean of duplicate incubations) reflect the total labeled cholesterol in cells and medium.

IN HUMAN

RENAL

CANCER

577

activity of HMG-CoA reductase, this hypothesis cannot explain the reduced synthesis of cholesterol from mevalonate (cf. Table II). To further verify the data in Table II, cholesterol synthesis was measured from the two labeled precursors, acetate and mevalonate, simultaneously. Again, exogenous cholesterol reduced cholesterol synthesis from both labeled precursors (Table III). In addition, mevalonate conversion to nonsaponifiable lipids was independent of the cholesterol content in the growth medium; strong evidence that the observed decrease in cholesterol synthesis from mevalonate did not result from diminished cellular uptake of this labeled precursor. The possibility existed that the decrease in cholesterol synthesis from mevalonate reflected exhaustion of medium mevalonate during the incubation periods (Table III). This possibility was excluded by studies showing that there was no effect on the observed decrease in cholesterol synthesis when medium mevalonate levels were increased (0.02 to 1 pmol) (results not presented). Also, as is described in the following paragraph, inhibition of cholesterol synthesis from mevalonate is observed at short as well as long incubation times. As would be expected from the findings in Table I, there was a decrease in the conversion of acetate to nonsaponifiable lipids in the presence of cholesterol (Table III). In contrast to nonsaponifiable lipid synthesis, cholesterol synthesis from mevalonate was sharply medium dependent (Table III) suggesting that the effect of exogenous cholesterol is at the level of squalene or postsqualene enzymatic step(s). Indeed, chromatography on silicic acid columns of the nonsaponifiable fractions from inhibited incubations showed accumulation with time of squalene paralleled by decreasing levels of cholesterol (cf. Table IV) (see Materials and Methods). The accumulation of squalene suggests inhibition at the level of squalene epoxidase. There was no accumulation of squalene-2,3oxide, of hydroxymethyl28-30 carbon-atom sterols, or monohydroxy 27 carbon-atom precursors of cholesterol. Findings similar to those just described (i.e., inhibition of both HMG-CoA reductase

578

GONZALEZ, CARLSON, AND DEMPSEY TABLE III INFLUENCE OF EXOGENOUS CHOLESTEROL ON THE INCORPORATION OF ACETATE AND MEVALONATE INTO NONSAPONIFIABLE LIPIDS AND CHOLESTEROLS

Lipoproteinpoor serum

Fetal calf serum

Lipoprotein-poor serum + cholesterol

[Z-W]Acetate incorporation into Nonsaponifiable Cholesterol Ratio: CholesteroUNonsaponifiable

48,500 f 11,700 7,500 2 650 0.16

21,800 ? 6,000 1,100 z? 300 0.05

3,300 + 800 1,200 + 900 0.04

DL-[5-3H]Mevalonate incorporation into Nonsaponifiable Cholesterol Ratio: Cholesterol/Nonsaponifiable

4,700 A 360 1,500 k 30 0.32

3,600 ‘- 1,100 560 + 90 0.16

5,860 2 560 370 ” 180 0.06

a Conditions were the same as given in Table II except that cells (1 x 10s) were incubated with both [12J4C]acetate (0.5 &i; 50 mCi/mmol) and DL-[5-3H]mevalonate (10 @i; 500 mCi/mmol) for 24, 48, and 96 h. Results reflect total synthesis in cells and medium and are expressed as mean incorporation ? standard error for three experiments. Lipid synthesis was linear with time. The results are expressed as dpm x 24 h-l per lo6 cells.

activity and conversion of mevalonate to cholesterol) were obtained with the low density lipoprotein fraction of human serum. For example, human low density lipoprotein (SO-ZOOpg cholesterol/ml, final concentration in the medium) in four incubations reduced cholesterol synthesis from mevalonate lo-fold relative to that observed without low density lipoprotein. Other human serum lipoprotein fractions were not effective at any level tested in reducing cholesterol synthesis from either acetate or mevalonate.

An intriguing aspect of the data in Table III is the indication that the inhibition of cholesterol synthesis from both acetate and mevalonate in Caki-1 follows a similar time course. To further verify this point the effects of exogenous cholesterol on mevalonate conversion to cholesterol were examined at early times (e.g. 4 h) rather than after long incubation times (e.g. 24-96 h, cf. Table III). These short-term studies were essential because, as mentioned previously, inhibition of mevalonate con-

TABLE IV INFLUENCE OF EXOGENOUS CHOLESTEROL ON THE COMPOSITION OF THE NONSAWNIFIABLE LIPIDS SYNTHESIZED BY C&i-la

Nonsaponifiable compounds

Lipoproteinpoor serum

Fetal calf serum

Lipoprotein-poor serum + cholesterol

Squalene 30 Carbon-atom sterols 28-29 Carbon-atom sterols 27 Carbon-atom sterols Cholesterol

13 5 1 7 74

56 9 2 1 32

67 12 5 9 7

a Cells (1 x 10’) were inoculated into 75-cm* flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5 the medium was replaced by one of three test media (see legend to Table I). On Day 6 the medium was replaced by one of identical composition with [2-W]acetate (0.5 &i; 50 mWmmo1) and incubated for an additional 96 h. Reactions were stopped by addition of ethanol and KOH. Silicic acid-Super Cel column chromatography of the nonsaponiiiable fraction was performed as described by Frantz (23-29). The results are expressed as the percentage of total radioactivity.

CHOLESTEROL

SYNTHESIS

version to cholesterol by cholesterol feeding is a delayed phenomenon in viva, secondary to rapid inhibition of mevalonate synthesis (9, 10). The influence of exogenous cholesterol on the conversion of mevalonate to cholesterol was examined using cells grown previously in either cholesterol-deficient medium (Fig. 2) or in the presence of cholesterol (Fig. 3). The data of Fig. 2 demonstrate that in Caki-1 cells there is a marked reduction in conversion of mevalonate to cholesterol as early as 4 h following incubation with exogenous cholesterol. Similarly, the data of Fig. 3 show that there is also a rapid stimulation or release of I

01 0

I 4

I 8

I 12

Time of lncubotlon

/ 16

1 20

I 24

with Test Medium

(hours)

FIG. 2. Influence of exogenous cholesterol on the incorporation of mevalonate into cholesterol by Caki-1 cells previously grown in cholesterol-deficient medium. Cells (1 x 10s)were inoculated into 25-cm* flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5, the medium was replaced with RPM1 1640 containing 20% fetal calf lipoprotein-poor serum. Twenty-four hours later (time 0) the medium was replaced with 1 ml of one of three test media: (1) RPM1 1640 with 20% fetal calf lipoprotein poor serum (0), (2) RPM1 1640 with 20% fetal calf serum (final cholesterol concentration 30 pg/ml) (O), and (3) RPM1 1640with 20% fetal calf lipoprotein poor serum to which cholesterol (see Materials and Methods) was added in ethanol to a fmal concentration of 30 pg/ml (A). Final ethanol concentration in all media was 0.5% (v/v). At the time periods indicated in the figure, 10 &i of DI&5-3H]mevalonate (500 mCi/ mmol) was added to all flasks. Two hours later reactions were stopped by addition of ethanol and KOH; labeled cholesterol was determined in the nonsaponitlable extracts by the dibromide method (see Materials and Methods). Results reflect total synthesis in cells and media and are the average values of duplicate flasks.

579

IN HUMAN RENAL CANCER

OoVL

48

Time of lncubotlon

with Test Medium

(hours)

FIG. 3. Influence of exogenous cholesterol on the incorporation of mevalonate into cholesterol using Caki-1 cells previously grown in medium containing cholesterol. Cells (1 x 106)were inoculated into 25-cm* flasks containing RPM1 1640 with 20% fetal calf serum and allowed to reach confluence (5 days). On Day 5 (time 0) media in all flasks were replaced with 1 ml of one of the three test media: (1) RPM1 1640 with 20% fetal calf lipoprotein-poor serum (0), (2) RPM1 1640 with 20% fetal calf serum, final concentration 30 pg/ml (O), and (3) RPM1 1640 with 20% fetal calf lipoprotein-poor serum to which cholesterol (see Materials and Methods) was added in ethanol to a final concentration of 30 pg/ml (A). Final ethanol concentration was 0.5%. At the time periods indicated in the figure, 10 &i of DL-[5-3H]mevalonate (500 mCi/mmol) was added to all flasks. Two hours later reactions were stopped by addition of ethanol and KOH; labeled cholesterol was determined in the nonsaponifiable extracts by the dibromide method (see Materials and Methods). Results reflect total synthesis in cells and media and are the average values of duplicate flasks.

inhibition of cholesterol synthesis when cholesterol is removed from the medium. Furthermore, the stimulation of cholesterol synthesis (Fig. 2) appears to occur simultaneously with the increase of HMG-CoA reductase activity when cholesterol is removed from the medium (cf. Fig. 1). Another finding of possible significance in understanding regulation of cholesterol synthesis is that cholesterol in the presence of lipoprotein-free serum was nearly as effective as lipoprotein-bound cholesterol in lowering HMG-CoA reductase activity (Table I) and blocking the conversion of acetate and mevalonate to cholesterol (Tables II and III, Figs. 2 and 3). With mouse and human fibroblasts, inhibition of HMG-CoA reductase by cholesterol in the

580

GONZALEZ,

CARLSON,

absence of lipoproteins is weak and requires long incubation times, probably leading to the production of oxidized cholesterol derivatives which are powerful inhibitors of HMG-CoA reductase (7, 29). Although in the studies reported here, incubations were performed with highly purified and freshly prepared cholesterol (see Materials and Methods), the oxidation of cholesterol during incubations cannot be excluded. Finally, these results conflict with several reports indicating that synthesis of digitonin precipitable sterols from mevalonate by cultured cells is not influenced by exogenous cholesterol (e.g., 6, 8). However since the digitonin technique does not distinguish among or quantitate all sterol intermediates equally well, effects of exogenous cholesterol at specific late stages of cholesterol synthesis may have been overlooked. Thus, our finding of a second major regulatory point may be a previously unrecognized, more universal characteristic of cultured cells. In this regard, accumulation of squalene, as reported here, would cause a decrease in the digitonin-precipitable radioactivity in the nonsaponifiable fractions. It is therefore possible that our findings are a unique characteristic of renal cancer cells or a general property of many cells in culture. Further studies are required to determine the mechanism of postmevalonate regulation of cholesterol biosynthesis in Caki-1 cells and whether or not similar processes occur in other cultured cell types. ACKNOWLEDGMENT We thank Mr. Kenneth Chang for his excellent technical assistance. REFERENCES 1. RODWELL, V. W., NORDSTROM, J. L., AND MITSCHELEN, J. J. (1976) Adw. Lipid Res. 14, l-74. 2. DEMPSEY, M. E. (1974) Ann. Rev. Biochem. 43, 967-990. 3. SIPERSTEIN, M. D. (1970) Curr. Top. Cell Regul. 2, 65-100. 4. BROWN, M. S., DANA, S. E., AND GOLDSTEIN, J. L. (1973) Proc. Nat. Acad. Sci. USA 70, 2162-2166. 5. WATSON, J. A. (1973) Tumor Lipids, Biochemistry and Metabolism (Wood, R., ed.), pp. 34-53, Am. Oil Chem. Sot., Chicago.

AND DEMPSEY

6. SOKOLOFF, L., AND ROTHBLATT, G. H. (1972) Biochim. Biophys. Acta 280, 172-181. 7. KANDUTSCH, A. A., AND CHEN, H. W. (1973) J. Biol. Chem. 251, 8408-8417. 8. BELL, J. J., SARGEANT, T. E., AND WATSON, J. A. (1976) J. Biol. Chem. 251, 1745-1758. 9. GOULD, G., AND SWRYD, E. A. (1966) J. Lipid Res. 7, 698-707. 10. SLAKEY, L. L., CRAIG, M. C., BEYTIA, E., BRIEDIS, A., FELDBRUEGGE, D. H., DUGAN, R. E., QUERESHI, A. A., SUBBARAYAN, C., AND PORTER, J. W. (1972) J. Biol. Chem. 247, 3014-3022. 11. NERVI, F. O., CARRELLA, J. M., AND DIETSCHY, J. M. (1976) J. Biol. Chem. 251, 3831-3833. 12. SCHROEPFER, G. J., PARISH, E. J., CHEN, H. W., AND KANDUTSCH, A. A. (1977) J. Biol. Chem. 252, 8975-8980. 13. BENNINGTON, J. L., (1973) Cancer 32,1017-1029. 14. LINDLAR, F. (1961) Verb. Deut. Ges. Pathol. 45, 144- 147. 15. GONZALEZ, R., AND GOLDBERG, M. E. (1977) Lancet 1, 912. 16. DEMPSEY, M. E. (1969)in Methods in Enzymology (Clayton, R. B., ed.), Vol. 15, pp. 501-514, Academic Press, New York. 17. DEMPSEY, M. E. (1975) in Handbook of Experimental Pharmacology, Pharmacology of Hypolipidemic Agents (Kritchevsky, D., ed.), Vol. 41, pp. l-28, Springer-Verlag, Berlin/New York. 18. POPJAK, G. (1969) in Methods in Enzymology (Clayton, R. B., ed.), Vol. 15, p. 442, Academic Press, New York. 19. FOGH, J., AND TREMPE, G. (1975) in Human Tumor Cells in Vitro (Fogh, J., ed.), pp. 115-151, Plenum, New York. 20. GONZALEZ, R., AND DEMPSEY, M. E. (1977) J. Ural. 117, 708-711. 21. HAVEL, R. J., EDER, H. A., ANDBRAGDON, J. H. (1955) J. Clin. Invest. 34, 1345-1353. 22. FRANTZ, I. D., JR. (1963)J. LipidRes. 4,176-178. 23. EDMOND, J., POPJAK, G., WONG, S., AND WILLIAMS, V. P. (1971) J. Biol. Chem. 246, 62546271. 24. BROWN, M. S., DANA, S. E., AND GOLDSTEIN, J. L. (1974) J. Biol. Chem. 249, 789-796. 25. GOLDFARB, S., AND PITOT, H. C. (1971) J. Lipid Res. 12, 512-515. 26. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 27. HOLMES, R. J. (1967) J. Cell Biol. 32, 297. 28. GONZALEZ, R., DEMPSEY, M. E., ELLIOTT, A. Y., AND FRALEY, E. E. (1974) Exp. Cell Res. 87, 152-157. 29. BROWN, M. S., AND GOLDSTEIN, J. L. (1974) J. Biol. Chem. 249, 7306-7314.