Proc. Natl. Acad. Sci. USA Vol. 75, No. 3, pp. 1247-1249, March 1978 Biochemistry
Defective regulation of cholesterol biosynthesis and plasma membrane fluidity in a Chinese hamster ovary cell mutant (electron spin resonance/25-hydroxycholesterol)
MICHAEL SINENSKY Eleanor Roosevelt Institute for Cancer Research, The Florence Sabin Laboratories for Developmental Medicine, and the Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80262
Communicated by Theodore T. Puck, January 5, 1978
A Chinese hamster ovary cell mutant resistant ABSTRACT to killing by 25-hydroxycholesterol is shown to be defective in the regulation of cholesterol synthesis by exogenous cholesterol. When grown with various cholesterol supplements in delipidized serum, the mutant cell exhibits a variation in cholesterol content and plasma membrane fluidity that is not observed in the parental cell type.
The development of new spectroscopic techniques has permitted a large number of studies on the physical chemical properties of lipids found in biological systems. Most of these studies have been performed in various liposomal models. The results of these studies coupled with recent advances in cell biology now make possible the investigation of the significance of lipid physical properties in the regulation of membrane lipid synthesis. I have recently described (1) the isolation of a Chinese hamster ovary cell mutant resistant to killing by 25-hydroxycholesterol in cholesterol-free medium. This mutant was shown also to be resistant to the inhibition of incorporation of acetate into cholesterol by 25-hydroxycholesterol. Furthermore, the mutant incorporated more acetate but not mevalonate into cholesterol than did the parental cell line in medium containing whole serum. Thus, it is concluded that the mutant was defective in the regulation of cholesterol synthesis. In this report we demonstrate that this defective regulation of cholesterol synthesis is accompanied by defective regulation of membrane physical properties in a fashion that is in complete agreement with results of earlier liposomal studies. MATERIALS AND METHODS Cells and Media. Cells were grown on medium F12 (2) supplemented with 8% fetal calf serum or 8% delipidized fetal calf serum prepared by the method of Chain and Knowles (3). Delipidized serum was dialyzed and lyophilized prior to use and then taken up in distilled water to a protein concentration identical to that of the extracted serum. The Chinese hamster ovary cell used was the CHO-K1 (4) provided by T. T. Puck. Biochemical Analysis. Cholesterol mass determinations were performed by gas/liquid chromatography with a coprostanol internal standard on a 3-foot (91-cm) SP-2250 (Supelco) column. Phospholipid phosphate determinations were performed by the Bartlett method (5). Protein determinations were by the Lowry method (6). Plasma Membrane Preparations. Plasma membranes were prepared by the two-phase polymer method of Brunette and Till (7) as described by Juliano and Behar-Bannelier (8) for the Chinese hamster ovary cell.
Electron Spin Resonance Spin Probe Analysis. The order parameter of 5-nitroxystearic acid has been shown by Smith and coworkers (9) to be responsive to changes in cholesterol concentration in phospholipid liposomes. Membranes were spin-labled by evaporating 0.3 ml of a 0.2 mM solution of 5nitroxystearic acid (Syva Corp) in a test tube, followed by addition of 2 mg of membrane protein in approximately 0.1 ml of phosphate-buffered saline. This mixture was then vortexed vigorously for 2 min. Spectra were taken at 370 in a Varian E-104A electron spin resonance spectrometer. Whole cells were spin-labeled by evaporating 0.3 ml of 0.2 mM 5-nitroxystearic acid in a test tube and adding 107 cells suspended in 0.1 ml of phosphate-buffered saline followed by gentle vortexing for 1 min. All samples were held in the electron spin resonance cavity in sealed 22.9-cm pasteur pipettes. Order parameters were determined as described by Hubbell and McConnell (10). Determination of Labeled Cholesterol Derived from [14CJAcetate. Cells at half-confluence on 100-mm Lux petri plates were exposed to labeled acetate and then harvested by trypsinization. After the cells were counted in a Coulter counter, lipids were extracted by the method of Bligh and Dyer (11). The total lipids were saponified in 0.2 ml of 5% ethanolic KOH at 52° for 1 hr. The reaction mixture was neutralized by the addition of 10 ,l of acetic acid and the solvent was evaporated to dryness under a stream of N2. The lipid residue was taken up in hexane containing cholesterol as a mass standard and applied to silicic acid thin-layer chromatography plates (Merck). Cholesterol was isolated by chromatography in petroleum ether/diethyl ether/acetic acid, 80:20:1 (vol/vol), followed by visualization with iodine vapor. After disappearance of the coloration, the bands were scraped from the plates and quantitated by liquid scintillation spectrometry. In a separate experiment, the rates of conversion of acetate to cholesterol were determined at various concentrations but constant radioactivity of exogenous acetate in an effort to correct for pool variations. Under these conditions of variable specific activity, the counts incorporated should be proportional to the exogenous specific activity with the proportionality constant being the absolute rate constant for conversion. These rate constants were determined by least squares fit of 12 data points. RESULTS Previous studies (1) had given preliminary indications that CR1 did not vary its rate of cholesterol synthesis in response to cholesterol in the growth medium. To verify this, the following experiment was performed. CHO-K1 and CR1 were grown in F12 medium supplemented with fetal calf serum. The serum supplement was then changed to delipidized fetal calf serum either with or without cholesterol at 7.5 jig/ml. After 4 hr, labeled acetate was added. The cells were harvested and incor-
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. §1734 solely to indicate this fact. 1247
Biochemistry: Sinensky
1248
100 c A
Proc. Nati. Acad. Sci. USA 75 (1978) 0.700r
B
0
0
0.690p
Co 0 %_
50
cn 0.6801
0.
0
5
10
15
5 10 0 15 20 Time, hr FIG. 1. Synthesis of cholesterol from labeled acetate in the presence (e) and absence (0) of cholesterol in CHO-K1 (A) and CR1 (B). The absolute rates of incorporation in CHO-K1 and CR1 were 2.77 ± 0.89 and 2.90 + 1.18 pmol/106 cells per hr, respectively.
poration of label into cholesterol was determined every 2 hr after that. The results (Fig. 1) indicate that, under conditions in which cholesterol represses the rate of acetate incorporation into cholesterol by a factor of 3.5 in CHO-K1, there is no effect in CR1. These results led us to examine the cholesterol content of CR1 grown in the presence of various concentrations of cholesterol in delipidized serum. The results (Table 1) indicate that the cholesterol content of CR1 varies with the cholesterol supplement of the medium. Subsequent experiments on plasma membrane factions of CR1 revealed that, as would be expected, the variation in cholesterol content of the cell is correlated with variation in plasma membrane cholesterol content relative to plasma membrane phospholipid. As reported by Smith and coworkers (9), the immobilizing effect of cholesterol on phospholipid acetyl chains can be detected in synthetic lipid vesicles by measurement of the order parameter of the spin-probe 5-nitroxystearic acid. Spin-label spectra of CR1 plasma membranes prepared from cells grown on various cholesterol supplements were taken and the order parameters were determined. By using these same preparations, cholesterol and phospholipid levels were measured and the mole fraction of cholesterol in these membranes was determined. The results reported by Smith and coworkers (9) on liposomes show a linear relationship between-order parameter and cholesterol mole fraction. Similar results (Fig. 2) were found in our plasma membrane preparations from CR1. Because all plasma membrane preparations are open to question in regard to purity, the order parameter of 5-nitroxysteric acid added to intact cells also was measured. It has been reported that this probe will only be incorporated into the plasma membranes of such cells (12). For five different cholesterol supplements, the order parameter measured for the intact cells was the same as that measured for plasma membranes prepared from cells grown on the same supplement. Table 1. Cholesterol content of CR1 grown in various concentrations of cholesterol
Cholesterol supplement,
0.670p I
1.0
Assuming that the cholesterol content of the plasma membrane is porportional to that of the whole cell, one would expect, on the basis of the plasma membrane and liposome results, a linear relationship between the reciprocal of the order parameter and the protein-to-cholesterol ratio. The results shown in Fig. 3 indicate the expected linear relationship and further demonstrate that spin-labeling of whole cells with 5-nitroxystearic acid can be used to obtain data on plasma membrane fluidity. When the cholesterol content and fluidity of the CR1 parent cell, CHO-K1, grown on various cholesterol supplements were examined, the cholesterol content and order parameter of whole CHO-K1 cells were not altered by growth on various cholesterol supplements in delipidized serum. The cholesterol content of CHO-K1 cells at all supplements tried was about 10 ag/mg of protein and the order parameter was 0.657. In an initial experiment to examine the role of cholesterol or fluidity variation in determining physiological properties of the cell, the growth rate of CR1 with various cholesterol supplements was examined. As shown in Table 2 there clearly was an optimal cholesterol supplement for growth, 5 ,g/ml.
DISCUSSION Although the simultaneous deficit in two functions always has a certain ambiguity in interpretation as to cause and effect, it is intriguing to speculate that, at least in part, the role of the 1.51 1.50
1.49
1.48 1.47
C,> 1.46 1.45
Cholesterol content,
1.44
Ag/mg
1.43
/Ag/ml
cell protein
0 2.5 5.0 7.5 10.0
8.7 12.6 17.9 33.6 40.0
Cells were grown on medium F12 supplemented with 8% delipidated serum and cholesterol as shown.
-
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 Cholesterol, mol % FIG. 2. Variation of the order parameter (S) of 5-nitroxystearic acid embedded in CR1 plasma membranes with various cholesterol contents. .0
1.42 1.41 1.40 0
0.05 0.10 0.15 Protein/cholesterol, mg/pg FIG. 3. Dependence of the order parameter (S) of 5-nitroxystearic acid spin-labeled CR1 cells on the cellular cholesterol content.
Biochemistry: Sinensky
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 2. Generation times of CR1 grown on various concentrations of cholesterol
Cholesterol supplement, Ig/ml
Generation time, hr
0 2 5 8 10
50-100 25+1 16k 1 21 1 22 1
Cells were grown on medium F12 supplemented with 8% delipidated serum and cholesterol as shown. Growth curves were obtained by sequentially harvesting and counting cells from 60-mm plates inoculated at an initial titer of 2 X 104 cells per plate. Cells were counted with a Coulter counter, and each time value is the mean (+SD) of four determinations. By comparison, CR1 exhibits a generation time of 16 hr when grown in F12 supplemented with 8% whole fetal calf serum. CHO-K1 has a generation time of 12 hr on this medium.
cholesterol-synthesizing enzymes of the mammalian fibroblast is to regulate membrane fluidity. Such fluidity regulation is well known in microorganisms in response to temperature (13) and could be important in mammalian cells as a response to variation in composition of dietary lipids. It would not seem that cholesterol synthesis in these cells is required simply to supply the cholesterol requirement for the cells. Under normal conditions the diet and the cholesterogenic tissues (liver, gastrointestinal tract, and skin) should meet the total body cholesterol requirement, considering that 97% of all body cholesterol is synthesized in these tissues operating at less than 50% of maximal synthetic capacity (14). This probable dependence of peripheral tissues upon exogenous cholesterol for growth is further demonstrated by the finding that primary fibroblasts exhibit a cholesterol requirement for growth in culture (15), despite the observation that all mammalian cells studied are capable of cholesterol synthesis (14). A reasonable hypothesis explaining these observations is that cholesterol biosynthesis exists in fibroblasts as a fine-tune control on lipid composition, thus regulating membrane fluidity. It is hoped that the isolation of cells that are defective in the regulation of cholesterol synthesis can provide models of
1249
pathologically produced cells. Both atherosclerotic cells (16) and malignant cells (17) have been suggested to be defective in the regulation of cholesterol synthesis. Defects in the regulation of cholesterol synthesis that can produce variation in membrane fluidity, as occurs in our mutant, should produce pleiotropic variation in membrane function. Such effects are illustrated by the variation in growth of the mutant with variation in cholesterol content but more specific variation in membrane enzyme function and transport should also be demonstrable with this system. The author thanks Ms. Barbara Hughes for her technical assistance in this project. This research was supported by Grant BC-219 from the American Cancer Society and Grants CA-20810 and HD-02080 from the National Institutes of Health. This is publication no. 256 from the Eleanor Roosevelt Institute. 1. Sinensky, M. (1977) Biochem. Biophys. Res. Commun. 78, 863-867. 2. Ham, R. G. (1965) Proc. Nati. Acad. Sci. USA 53,288-293. 3. Cham, B. E. & Knowles, B. R. (1976) J. Lipid Res. 17,176181. 4. Kao, F. T. & Puck, T. T. (1969) J. Cell. Physiol. 74,245-258. 5. Bartlett, G. R. (1959) J. Biol. Chem. 234,466-471. 6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 7. Brunette, D. M. & Till, J. E. (1971) J. Membr. Biol. 5, 215224. 8. Juliano, R. L. & Behar-Bannelier, M. (1975) Biochemistry 14, 3816-3824. 9. Schreir-Mucillo, S., Marsh, D., Dugas, H., Schneider, H. & Smith, I. C. P. (1973) Chem. Phys. Lipids 10, 11-27. 10. Hubbell, W. L. & McConnell, H. M. (1971) J. Am. Chem. Soc. 93,314-326. 11. Bligh, E. G. & Dyer, W. H. (1959) Can. J. Biochem. Phys. 37, 911-917. 12. Landsberger, F. R. & Compans, R. W. (1976) Biochemistry 15, 2356-2360. 13. Sinensky, M. (1974) Proc. Natl. Acad. Sci. USA 71,522-525. 14. Dietschy, J. M. & Wilson, J. D. (1970) N. Engl. J. Med. 282, 1128-1138. 15. Holmes, R. H., Helms, J. & Mercer, G. (1969) J. Cell Biol. 42, 262-271. 16. Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, 897-930. 17. Siperstein, M. D. (1970) Curr. Top. Cell Regul. 2,65-100.
Defective regulation of cholesterol biosynthesis and plasma membrane fluidity in a Chinese hamster ovary cell mutant (electron spin resonance/25-hydroxycholesterol)
MICHAEL SINENSKY Eleanor Roosevelt Institute for Cancer Research, The Florence Sabin Laboratories for Developmental Medicine, and the Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80262
Communicated by Theodore T. Puck, January 5, 1978
A Chinese hamster ovary cell mutant resistant ABSTRACT to killing by 25-hydroxycholesterol is shown to be defective in the regulation of cholesterol synthesis by exogenous cholesterol. When grown with various cholesterol supplements in delipidized serum, the mutant cell exhibits a variation in cholesterol content and plasma membrane fluidity that is not observed in the parental cell type.
The development of new spectroscopic techniques has permitted a large number of studies on the physical chemical properties of lipids found in biological systems. Most of these studies have been performed in various liposomal models. The results of these studies coupled with recent advances in cell biology now make possible the investigation of the significance of lipid physical properties in the regulation of membrane lipid synthesis. I have recently described (1) the isolation of a Chinese hamster ovary cell mutant resistant to killing by 25-hydroxycholesterol in cholesterol-free medium. This mutant was shown also to be resistant to the inhibition of incorporation of acetate into cholesterol by 25-hydroxycholesterol. Furthermore, the mutant incorporated more acetate but not mevalonate into cholesterol than did the parental cell line in medium containing whole serum. Thus, it is concluded that the mutant was defective in the regulation of cholesterol synthesis. In this report we demonstrate that this defective regulation of cholesterol synthesis is accompanied by defective regulation of membrane physical properties in a fashion that is in complete agreement with results of earlier liposomal studies. MATERIALS AND METHODS Cells and Media. Cells were grown on medium F12 (2) supplemented with 8% fetal calf serum or 8% delipidized fetal calf serum prepared by the method of Chain and Knowles (3). Delipidized serum was dialyzed and lyophilized prior to use and then taken up in distilled water to a protein concentration identical to that of the extracted serum. The Chinese hamster ovary cell used was the CHO-K1 (4) provided by T. T. Puck. Biochemical Analysis. Cholesterol mass determinations were performed by gas/liquid chromatography with a coprostanol internal standard on a 3-foot (91-cm) SP-2250 (Supelco) column. Phospholipid phosphate determinations were performed by the Bartlett method (5). Protein determinations were by the Lowry method (6). Plasma Membrane Preparations. Plasma membranes were prepared by the two-phase polymer method of Brunette and Till (7) as described by Juliano and Behar-Bannelier (8) for the Chinese hamster ovary cell.
Electron Spin Resonance Spin Probe Analysis. The order parameter of 5-nitroxystearic acid has been shown by Smith and coworkers (9) to be responsive to changes in cholesterol concentration in phospholipid liposomes. Membranes were spin-labled by evaporating 0.3 ml of a 0.2 mM solution of 5nitroxystearic acid (Syva Corp) in a test tube, followed by addition of 2 mg of membrane protein in approximately 0.1 ml of phosphate-buffered saline. This mixture was then vortexed vigorously for 2 min. Spectra were taken at 370 in a Varian E-104A electron spin resonance spectrometer. Whole cells were spin-labeled by evaporating 0.3 ml of 0.2 mM 5-nitroxystearic acid in a test tube and adding 107 cells suspended in 0.1 ml of phosphate-buffered saline followed by gentle vortexing for 1 min. All samples were held in the electron spin resonance cavity in sealed 22.9-cm pasteur pipettes. Order parameters were determined as described by Hubbell and McConnell (10). Determination of Labeled Cholesterol Derived from [14CJAcetate. Cells at half-confluence on 100-mm Lux petri plates were exposed to labeled acetate and then harvested by trypsinization. After the cells were counted in a Coulter counter, lipids were extracted by the method of Bligh and Dyer (11). The total lipids were saponified in 0.2 ml of 5% ethanolic KOH at 52° for 1 hr. The reaction mixture was neutralized by the addition of 10 ,l of acetic acid and the solvent was evaporated to dryness under a stream of N2. The lipid residue was taken up in hexane containing cholesterol as a mass standard and applied to silicic acid thin-layer chromatography plates (Merck). Cholesterol was isolated by chromatography in petroleum ether/diethyl ether/acetic acid, 80:20:1 (vol/vol), followed by visualization with iodine vapor. After disappearance of the coloration, the bands were scraped from the plates and quantitated by liquid scintillation spectrometry. In a separate experiment, the rates of conversion of acetate to cholesterol were determined at various concentrations but constant radioactivity of exogenous acetate in an effort to correct for pool variations. Under these conditions of variable specific activity, the counts incorporated should be proportional to the exogenous specific activity with the proportionality constant being the absolute rate constant for conversion. These rate constants were determined by least squares fit of 12 data points. RESULTS Previous studies (1) had given preliminary indications that CR1 did not vary its rate of cholesterol synthesis in response to cholesterol in the growth medium. To verify this, the following experiment was performed. CHO-K1 and CR1 were grown in F12 medium supplemented with fetal calf serum. The serum supplement was then changed to delipidized fetal calf serum either with or without cholesterol at 7.5 jig/ml. After 4 hr, labeled acetate was added. The cells were harvested and incor-
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. §1734 solely to indicate this fact. 1247
Biochemistry: Sinensky
1248
100 c A
Proc. Nati. Acad. Sci. USA 75 (1978) 0.700r
B
0
0
0.690p
Co 0 %_
50
cn 0.6801
0.
0
5
10
15
5 10 0 15 20 Time, hr FIG. 1. Synthesis of cholesterol from labeled acetate in the presence (e) and absence (0) of cholesterol in CHO-K1 (A) and CR1 (B). The absolute rates of incorporation in CHO-K1 and CR1 were 2.77 ± 0.89 and 2.90 + 1.18 pmol/106 cells per hr, respectively.
poration of label into cholesterol was determined every 2 hr after that. The results (Fig. 1) indicate that, under conditions in which cholesterol represses the rate of acetate incorporation into cholesterol by a factor of 3.5 in CHO-K1, there is no effect in CR1. These results led us to examine the cholesterol content of CR1 grown in the presence of various concentrations of cholesterol in delipidized serum. The results (Table 1) indicate that the cholesterol content of CR1 varies with the cholesterol supplement of the medium. Subsequent experiments on plasma membrane factions of CR1 revealed that, as would be expected, the variation in cholesterol content of the cell is correlated with variation in plasma membrane cholesterol content relative to plasma membrane phospholipid. As reported by Smith and coworkers (9), the immobilizing effect of cholesterol on phospholipid acetyl chains can be detected in synthetic lipid vesicles by measurement of the order parameter of the spin-probe 5-nitroxystearic acid. Spin-label spectra of CR1 plasma membranes prepared from cells grown on various cholesterol supplements were taken and the order parameters were determined. By using these same preparations, cholesterol and phospholipid levels were measured and the mole fraction of cholesterol in these membranes was determined. The results reported by Smith and coworkers (9) on liposomes show a linear relationship between-order parameter and cholesterol mole fraction. Similar results (Fig. 2) were found in our plasma membrane preparations from CR1. Because all plasma membrane preparations are open to question in regard to purity, the order parameter of 5-nitroxysteric acid added to intact cells also was measured. It has been reported that this probe will only be incorporated into the plasma membranes of such cells (12). For five different cholesterol supplements, the order parameter measured for the intact cells was the same as that measured for plasma membranes prepared from cells grown on the same supplement. Table 1. Cholesterol content of CR1 grown in various concentrations of cholesterol
Cholesterol supplement,
0.670p I
1.0
Assuming that the cholesterol content of the plasma membrane is porportional to that of the whole cell, one would expect, on the basis of the plasma membrane and liposome results, a linear relationship between the reciprocal of the order parameter and the protein-to-cholesterol ratio. The results shown in Fig. 3 indicate the expected linear relationship and further demonstrate that spin-labeling of whole cells with 5-nitroxystearic acid can be used to obtain data on plasma membrane fluidity. When the cholesterol content and fluidity of the CR1 parent cell, CHO-K1, grown on various cholesterol supplements were examined, the cholesterol content and order parameter of whole CHO-K1 cells were not altered by growth on various cholesterol supplements in delipidized serum. The cholesterol content of CHO-K1 cells at all supplements tried was about 10 ag/mg of protein and the order parameter was 0.657. In an initial experiment to examine the role of cholesterol or fluidity variation in determining physiological properties of the cell, the growth rate of CR1 with various cholesterol supplements was examined. As shown in Table 2 there clearly was an optimal cholesterol supplement for growth, 5 ,g/ml.
DISCUSSION Although the simultaneous deficit in two functions always has a certain ambiguity in interpretation as to cause and effect, it is intriguing to speculate that, at least in part, the role of the 1.51 1.50
1.49
1.48 1.47
C,> 1.46 1.45
Cholesterol content,
1.44
Ag/mg
1.43
/Ag/ml
cell protein
0 2.5 5.0 7.5 10.0
8.7 12.6 17.9 33.6 40.0
Cells were grown on medium F12 supplemented with 8% delipidated serum and cholesterol as shown.
-
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 0.0 Cholesterol, mol % FIG. 2. Variation of the order parameter (S) of 5-nitroxystearic acid embedded in CR1 plasma membranes with various cholesterol contents. .0
1.42 1.41 1.40 0
0.05 0.10 0.15 Protein/cholesterol, mg/pg FIG. 3. Dependence of the order parameter (S) of 5-nitroxystearic acid spin-labeled CR1 cells on the cellular cholesterol content.
Biochemistry: Sinensky
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 2. Generation times of CR1 grown on various concentrations of cholesterol
Cholesterol supplement, Ig/ml
Generation time, hr
0 2 5 8 10
50-100 25+1 16k 1 21 1 22 1
Cells were grown on medium F12 supplemented with 8% delipidated serum and cholesterol as shown. Growth curves were obtained by sequentially harvesting and counting cells from 60-mm plates inoculated at an initial titer of 2 X 104 cells per plate. Cells were counted with a Coulter counter, and each time value is the mean (+SD) of four determinations. By comparison, CR1 exhibits a generation time of 16 hr when grown in F12 supplemented with 8% whole fetal calf serum. CHO-K1 has a generation time of 12 hr on this medium.
cholesterol-synthesizing enzymes of the mammalian fibroblast is to regulate membrane fluidity. Such fluidity regulation is well known in microorganisms in response to temperature (13) and could be important in mammalian cells as a response to variation in composition of dietary lipids. It would not seem that cholesterol synthesis in these cells is required simply to supply the cholesterol requirement for the cells. Under normal conditions the diet and the cholesterogenic tissues (liver, gastrointestinal tract, and skin) should meet the total body cholesterol requirement, considering that 97% of all body cholesterol is synthesized in these tissues operating at less than 50% of maximal synthetic capacity (14). This probable dependence of peripheral tissues upon exogenous cholesterol for growth is further demonstrated by the finding that primary fibroblasts exhibit a cholesterol requirement for growth in culture (15), despite the observation that all mammalian cells studied are capable of cholesterol synthesis (14). A reasonable hypothesis explaining these observations is that cholesterol biosynthesis exists in fibroblasts as a fine-tune control on lipid composition, thus regulating membrane fluidity. It is hoped that the isolation of cells that are defective in the regulation of cholesterol synthesis can provide models of
1249
pathologically produced cells. Both atherosclerotic cells (16) and malignant cells (17) have been suggested to be defective in the regulation of cholesterol synthesis. Defects in the regulation of cholesterol synthesis that can produce variation in membrane fluidity, as occurs in our mutant, should produce pleiotropic variation in membrane function. Such effects are illustrated by the variation in growth of the mutant with variation in cholesterol content but more specific variation in membrane enzyme function and transport should also be demonstrable with this system. The author thanks Ms. Barbara Hughes for her technical assistance in this project. This research was supported by Grant BC-219 from the American Cancer Society and Grants CA-20810 and HD-02080 from the National Institutes of Health. This is publication no. 256 from the Eleanor Roosevelt Institute. 1. Sinensky, M. (1977) Biochem. Biophys. Res. Commun. 78, 863-867. 2. Ham, R. G. (1965) Proc. Nati. Acad. Sci. USA 53,288-293. 3. Cham, B. E. & Knowles, B. R. (1976) J. Lipid Res. 17,176181. 4. Kao, F. T. & Puck, T. T. (1969) J. Cell. Physiol. 74,245-258. 5. Bartlett, G. R. (1959) J. Biol. Chem. 234,466-471. 6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 7. Brunette, D. M. & Till, J. E. (1971) J. Membr. Biol. 5, 215224. 8. Juliano, R. L. & Behar-Bannelier, M. (1975) Biochemistry 14, 3816-3824. 9. Schreir-Mucillo, S., Marsh, D., Dugas, H., Schneider, H. & Smith, I. C. P. (1973) Chem. Phys. Lipids 10, 11-27. 10. Hubbell, W. L. & McConnell, H. M. (1971) J. Am. Chem. Soc. 93,314-326. 11. Bligh, E. G. & Dyer, W. H. (1959) Can. J. Biochem. Phys. 37, 911-917. 12. Landsberger, F. R. & Compans, R. W. (1976) Biochemistry 15, 2356-2360. 13. Sinensky, M. (1974) Proc. Natl. Acad. Sci. USA 71,522-525. 14. Dietschy, J. M. & Wilson, J. D. (1970) N. Engl. J. Med. 282, 1128-1138. 15. Holmes, R. H., Helms, J. & Mercer, G. (1969) J. Cell Biol. 42, 262-271. 16. Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, 897-930. 17. Siperstein, M. D. (1970) Curr. Top. Cell Regul. 2,65-100.
