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Cholesterol content and metabolism in normal and polyoma virus-transformed hamster embryo fibroblasts
Printed in Sweden Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/79/020377-05$02.00/O
Experimental
CHOLESTEROL
Cell Research 118 (1979) 377-381
CONTENT
AND POLYOMA HAMSTER D. MARK-MALCHOFF, University
AND METABOLISM
IN NORMAL
VIRUS-TRANSFORMED
EMBRYO
FIBROBLASTS
G. V. MARINETTI,
J. D. HARE and A. MEISLER
of Rochester School of Medicine and Dentistry, Departments of Biochemistry, Microbiology rend Medicine, Rochester, NY 14642, USA
SUMMARY The total cholesterol content of normal hamster embryo tibroblasts and polyoma virustransformed hamster embryo fibroblasts were found to be similar. However, the free cholesterol : cholesterol ester ratio was 41.5 in normal cells as contrasted to I .8 in their transformed counterparts. This difference is due in part to an increase in cholesterol esterification and a decrease in the hydrolysis of cholesterol esters in the transformed cell.
Alterations have been found in the cell lipid composition induced by virus transformation [l-20]. Cholesterol is of particular interest because it influences membrane fluidity. It is reported to occur predominately in the free form in several normal and transformed cells. We report here a marked change in the free cholesterol : cholesterol ester ratio in hamster embryo fibroblasts transformed by polyoma virus as compared to normal fibroblasts. This change appears to be caused by alterations in cholesterol metabolism. METHODS AND MATERIALS Cell cultures of hamster embryo tibroblasts (HEF) and polyoma virus-transformed hamster embryo fibroblasts (HFT) were grown in Eagle’s minimum essential medium with Earle’s salts (Autopow, Flow Labs) supplemented with 5 % bovine fetal serum (BFS), penicillin (100 U/ml) and streptomycin (100 &ml). Approx. 25x lo6 cells were extracted three times with redistilled chloroform : methanol (CHCI, : MeOH) I : I, (v : v). The neutral lipids were analyzed by thin layer chromatography on silica gel plates (Merck and Darmstadt SG5763) using a solvent system of heptane :
ether: acetic acid (65 : 15 : 1, v : v : v). Cholesterol and cholesterol palmitate were run as standards to identify the free and esterified forms of cholesterol, resnectively. The appropriate lipid bands were scraped-out and eluted with MeOH. The MeOH eluants were evaporated to dryness under nitrogen and redissolved in 0.20 ml acetic acid. Cholesterol was ouantitated bv a modification of the Lieberman and Be&hard method f211. To each sample was added 6.0 ml of cold cholesterol reagent (acetic anhydride : acetic acid : sulfuric acid, 86 : 82 : 25, v : v : v). They were mixed and incubated at 37°C for I5 min. The absorbance of the resultinn blue-preen color was read on a Gilford spectrophotometer at 615 nm. A standard curve was obtained from samples of 100, 200 and 400 UP of cholesterol prepared from a stock solution of 4G fig cholesterol acetate per 0.20 ml acetic acid. Cholesterol acetate and free cholesterol give the same absorbance per pg using this calorimetric method. The uptake and metabolism of f’4Clcholesterol and [‘Qhoiesterol oleate were carried out by incubating 40~10~ cells for 15 and 30 min in 5.0 ml phosphatebuffered saline containing 2.0 $Zi [4,‘4C]choldsterol (spec. act. 55.5 mCi/mmole) or 2.0 &i [oleatel,‘F]cholesterol oleate (spec. act. 50 mCi/mmole). These radioactive compounds were purchased from New England Nuclear, Boston, Ma. The cells were centrifuged at 2000 rpm for 10 min. Lipids were extracted from the cell peilet and from the supematant fluid by the method of Bligh & Dyer [22]. Thin layer chromatography was performed as previously described. The lipid bands were scraped out and treated with 0.10 ml of MeOH in countinn vials for 60 min. After this time, 10.0ml of ACSTM, shntillationcocktail, were added and the radioactivity measured on a Packard Tri E.xp Cell Res I I8 ( 1979)
378
Mark-Malchoff et al.
Table 1. Cholesterol content of HEF and HFT cells, expressed in t.~glIO”cells” HEF cells Total cholesterol* Free cholesterol Cholesterol esters Molar ratio of free cholesterol : cholesterol-esters
6.8+ 0.2 (6) 6.6+ 0.2 (6) 0.2f 0.01 (6) 41.5k22.0 (6)
HFT cells 6.6kO.3 (5) 4.2kO.3 (5) 2.4i0.1 (5) I .8* I .2 (5)
” Values represent the mean k S.D. of 5-6 experiments done in duplicate or triplicate. h Total cholesterol is the sum of the free and the esterified forms of cholesterol.
Carb Scintillation Counter. The corrected counts per minute were converted to picomoles of cholesterol or cholesterol oleate by comparison with the respective standard of [Ylcholesterol or [Wlcholesterol oleate.
RESULTS The analysis of the cholesterol ester and free cholesterol content of HEF and HFT cells are presented in table 1. The total cholesterol content of these cells did not differ. However, HEF cells exhibited a very high free cholesterol : cholesterol ratio, approx. 42 : 1. In contrast, HFT cells demonstrated a much lower free cholesterol : cholesterol ester ratio of about 2 : 1. In order to elucidate the difference in the ratio of free to esterified cholesterol in these
cells, we studied the uptake and metabolism of Y-labeled cholesterol and cholesterol oleate. The increased amount of cholesterol ester in HFT cells may be explained by (N) increased acylation of free cholesterol by acylating enzymes; (6) decreased hydrolysis of cholesterol esters by cholesterol esterases; (c) a combination of (a) and (b) or (d) an increase in uptake of cholesterol ester from the growth medium. Uptake of [‘“Clcholesterol and [14C]cholesterol oleate into HEF and HFT cells was measured by centrifuging the cells at different time intervals and determining the radioactivity lost from the supernatant and the radioactivity incorporated into the cell pellet. Metabolism was examined by ex-
Table 2. Uptake and metabolism of [14C]cholesterol and [‘“C]cholesterol oleate bv HEF and HFT cells a expressed in pmoles per IO6ceils [‘T]Cho/estero/ Cholesterol uptake Cholesterol esters % Esterification of cholesterol [‘JC]Cholesterol
HEF cells
HFT cells
995+29 I .37*0.4 0.14~0.04
625+ I6 4.1?1.2 0.66&O. I8
98tU44 18+0.2 I J&O.2
640+ 10 9+0.5 1.4f0.07
oleate
Cholesterol ester uptake Fatty acids % Hydrolysis of cholesterol oleate
” Cells were incubated at 37°C with 2 /.Ki (36 nmoles) [Ylcholesterol or 2 &i (40 nmoles) [r4C]cholesterol oleate for 60 min in Dulbecco’s phosphate-buffered saline, pH 7.4, containing 5% BFS. Lipids were extracted from the cells three times with CHCI, : MeOH, 1 : 1, and the extracts were run on a TLC plate in the solvent system heptane: ether: HAc, 65: 15: 1. Bands for cholesterol, cholesterol esters and free fatty acids were scraped off and measured for radioactivity.
Cholesterol
content
and metnbolism
tracting the cell pellet with CHCI,: MeOH and analyzing the various lipids by TLC. These results are given in table 2. With HEF cells [14C]cholesterol uptake was 995 pmoles/106 cells/h compared to 625 pmoles/ lo6 cells/h for HFT cells. Thus normal cells take up more cholesterol than their transformed counterparts. The same was observed for the uptake of [14C]cholesterol oleate. It is also noteworthy that the rate of uptake of free cholesterol was equivalent to the rate of uptake of cholesterol oleate for each respective cell type. However, the esterification of cholesterol was higher and the hydrolysis of cholesterol oleate was lower in HFT cells. These observations offer an explanation for the increased amounts of cholesterol ester in the transformed cell, namely an increased production of cholesterol esters and a decreased hydrolysis of these esters. It is also apparent that the extent of cholesterol ester hydrolysis is greater than the extent of cholesterol esterifications, especially in the HEF cell. This explains the higher ratio free cholesterol : cholesterol ester in HEF cells but presents an enigma with HFT cells where the free cholesterol : cholesterol ratio is low. DISCUSSION Cholesterol plays a role in the permeability and stability of the cell membrane by affecting its fluidity [17, 23-2.51. An increase in cholesterol causes the membrane to become less permeable. Cholesterol produces a tighter packing of the phospholipids within the lipid bilayer of the cell membrane by interaction with the fatty acid chains and the polar head groups of the phospholipids. This effect is dependent upon the nature of the fatty acids. However, the role of cholesterol esters in cell membranes is not well
in hamster
embryo
fibroblasts
379
understood. Cholesterol esters are usually minor constituents in cell membranes. Whereas cholesterol decreases the fluidity of cell membranes, cholesterol esters, especially those having polyunsaturated fatty acid chains, can be expected to increase the fluidity of the membrane. Although HEF and HFT cells have the same total cholesterol content on a cell basis, they differ markedly in the amount of free and esterified cholesterol present. HEF cells contain predominately free cholesterol and very little cholesterol esters while HFT cells contain appreciable amounts of both forms. This finding suggests a different arrangement between cholesterol and phospholipids within the membranes of normal and transformed cells. A feature of some transformed cells is an increase in unsaturated fatty acids in their phospholipids [26-281 or a decrease in their cholesterol content [29, 301. Since cholesterol would be expected to decrease the fluidity of the membrane lipid bilayer and cholesterol esters might enhance the fluidity, a change in either of these parameters might influence the permeability properties of membranes of transformed cells. However, the nature of the fatty acids found in the phospholipids and in the cholesterol esters of HEF and HFT cells were not examined. Moreover, our studies were carried out on intact cells so it is not known which specific membrane of the cell has an altered lipid content. It has been demonstrated that some hepatomas as compared with normal liver cells exhibit an increase in the cholesterol content of their subcellular organelles as well as the plasma membrane [31]. The changes in free and esterified cholesterol in HEF and HFT cells may be associated with differences in the rates of cholesterol esterification and cholesterol ester hydrolysis. These reactions are car&p Cdl/?e.\I/8 ,197YJ
380
Mark-Malchoffet
al.
ried out by a cholesterol-0acyl transferase and a cholesterol esterase, respectively. These enzymes have been identified in various cells, especially human skin fibroblasts [32-351. In the uptake and metabolism studies of [‘“C]cholesterol and [‘4C]cholesterol oleate presented, HFT cells have an enhanced cholesterol ester formation and a decrease in cholesterol ester hydrolysis as compared with HEF cells. These changes may appear to be small but may account for the difference in the free and esterified cholesterol contents of normal and transformed cells. The prolonged period of cell culturing must be considered. Over the 6 days in culture the increase in cholesterol esterification and decrease in this ester hydrolysis in the transformed cell is cumulative and may explain the changes in cholesterol and cholesterol ester in HFT cells. The cells studied were confluent and removed at high density. Confluent normal cells are in a maintenance or steady state, while confluent transformed cells are still growing and rapidly dividing. Cells at low density may differ from those at high density. It would be desirable to study both cell types in their exponential growth phase or before they attain confluency. The differences in cholesterol esterification and cholesterol ester hydrolysis under these circumstances might better reflect the changes in free and esterified cholesterol in the transformed state. Studies by Brown & Goldstein [36] have shown that normal fibroblasts contain receptors for LDL serum lipoproteins. Uptake and metabolism of cholesterol esters contained in LDL is dependent on initial binding of LDL to membrane receptors. This is followed by endocytosis of LDL which are entrapped in small membrane vesicles. These fuse with lysosomes where
the LDL are degraded. The cholesterol esters are hydrolyzed to free cholesterol which somehow gets into the cell cytoplasm and regulates HMG CoA reductase and cholesterol-acyl CoA transferase. The level of LDL receptors appear to be regulated by the LDL present. In our studies, the fibroblasts were grown in medium containing serum and hence were exposed to serum lipoproteins. How these may influence the lipid composition of the fibroblasts remains to be investigated. The significance of the altered lipid composition of HFT cells may be related to the enhanced uptake of nutrients by these cells possibly conferring on them an advantage for growth. In this regard, HFT cells are reported to have an increased ability to take up amino acids [37] and nucleosides [38, 391. It remains to be determined whether the change in cholesterol : cholesterol ester occurs in the plasma membrane of HFT cells and whether this accounts in part for the enhanced permeability of nutrients. This work has been supported in part by contract no. I-CP-45611, NCI. and grant HLB 02063, Heart and Lung Institute.
REFERENCES 1. Bailey, J M, Proc sot exp biol med 107 (1961) 30. 2. Rothblatt, G M, HartzelI, R W, Mailhe, H & Kritchevsky, D, Biochim biophys acta 116 (1966) 113. 3. Hakomori, S & Murakami, W T, Proc natl acad sci US 59 (1968) 254. 4. Rothblatt, G H, Buckho, M K & Kritchevsky, D, Biochim biophys acta 164(1968) 327. 5. Peterson, J A & Rubin, M, Exp cell res 58 (1969) 365. 6. Bergelson, J S, Dyatlovitskaya, D V, Torkhovskaya, T I, Sorkina, I B & Gorkova, N P, Biochim biophys acta 619 (1970) 287. 7. Cunningham, D D, J biol them 247 (1970) 2464. 8. Pastemak, G A & Friedricks, B, Biochem j 119 (1970) 473. 9. Peterson, J A & Rubin, M, Exp cell res 60 (1970) 383. 10. Harry, D S, Morris, H P & McIntyre, J, J lipid res 17 (1971) 313. 11. Lucas, D D, Shohet, S F & Merler, E, J immunol 106 (1971) 768.
Cholesterol content and metabolism in hamster embryo fibroblasts 12. Quigley, J P, Rifkin, D P & Reich, E, Virology 46 (1971) 106. 13. Rytter, D J & Comatzer, W E, Lipids 7 (1972) 142. 14. Hirschberg, C B & Robbins, P W, Virology 61 (1974) 602. 15. Hakomori, S, Biochim biophys acta (1975) 55. 16. Wahach, D F H, Membrane molecular biology of neoplastic cells, p. 141. Elsevier Scientific Publishing Co., Amsterdam (1975). 17. -Ibid, p. 217 (1975). 18. Fishman, P H & Brady, R 0, Science 194 (1976) 906. 19. Itaya, K, Hakomori, S & Lein, G, Proc natl acad sci US 73 (1976) 1568. 20. Mark-Malchoff, D, Marinetti, G V, Hare, J D & Meisler, A, Biochem biophys res commun 75 (1977) 589. 21. Stadtman, T C, Methods in enzymology (ed S P Colwick & N 0 Kaplan) vol. 3, p. 392. Academic Press, New York (1957). 22. Bligh, E G & Dyer, W J, Can j biochem physiol 37 (1959)911. 23. Oldfield, E & Chapman, D, FEBS lett 23 (1970) 285. 24. Feinstein, M B, Fernandez, S M & Sha’afi, R I, Biochim biophys acta 413 (1975) 354. 25. Jain, M K, Current topics in membrane and transport 6 (1975) 1.
381
26. Howard, B V & Kritchevsky, D, Int j cancer 4 (1969) 393. 27. Yau, T M & Weber, M J, Biochem biophys res commun 49 (1972) 114. 28. Howard, B V & Howard, W J, 3 prog biochem pharmacol 10 (1975) 135. 29. Chao, F, Eng, L W & Griffin, A, Biochim biophys acta 260 (1972) 197. 30. Gotffried, E L, J lipid res 81 (1967) 321. 31. Van Hoeven, R P & Emmelot, P, J membrane biol 9 (1972) 105. 32. Brown, M S & Goldstein, J L, Proc natl acad sci US 71 (1974) 2925. * 33. Brown, M S, Dana, S E&Goldstein, J L, Proc natl acad sci US 72 (1975) 2925. 34. Goldstein, J L & Brown, M S, J biol them 249 (1974) 5153. 35. Goldstein, J L, Dana, S E&Brown, M S, Proc natl acad sci US 71 (1974) 4288. 36. Brown, M S & Goldstein, J L, Science 191 (1976) 150. 37. Hare, J D, Cancer res 27 (1967) 2357. 38. - Ibid 30 (1970) 684. 39. Lemkin, J A & Hare, J D, Biochim biophys acta 318 (1973) 113. Received August 15, 1978 Accepted September 7, 1978
Exp Cell Res 118 (1979)
Experimental
CHOLESTEROL
Cell Research 118 (1979) 377-381
CONTENT
AND POLYOMA HAMSTER D. MARK-MALCHOFF, University
AND METABOLISM
IN NORMAL
VIRUS-TRANSFORMED
EMBRYO
FIBROBLASTS
G. V. MARINETTI,
J. D. HARE and A. MEISLER
of Rochester School of Medicine and Dentistry, Departments of Biochemistry, Microbiology rend Medicine, Rochester, NY 14642, USA
SUMMARY The total cholesterol content of normal hamster embryo tibroblasts and polyoma virustransformed hamster embryo fibroblasts were found to be similar. However, the free cholesterol : cholesterol ester ratio was 41.5 in normal cells as contrasted to I .8 in their transformed counterparts. This difference is due in part to an increase in cholesterol esterification and a decrease in the hydrolysis of cholesterol esters in the transformed cell.
Alterations have been found in the cell lipid composition induced by virus transformation [l-20]. Cholesterol is of particular interest because it influences membrane fluidity. It is reported to occur predominately in the free form in several normal and transformed cells. We report here a marked change in the free cholesterol : cholesterol ester ratio in hamster embryo fibroblasts transformed by polyoma virus as compared to normal fibroblasts. This change appears to be caused by alterations in cholesterol metabolism. METHODS AND MATERIALS Cell cultures of hamster embryo tibroblasts (HEF) and polyoma virus-transformed hamster embryo fibroblasts (HFT) were grown in Eagle’s minimum essential medium with Earle’s salts (Autopow, Flow Labs) supplemented with 5 % bovine fetal serum (BFS), penicillin (100 U/ml) and streptomycin (100 &ml). Approx. 25x lo6 cells were extracted three times with redistilled chloroform : methanol (CHCI, : MeOH) I : I, (v : v). The neutral lipids were analyzed by thin layer chromatography on silica gel plates (Merck and Darmstadt SG5763) using a solvent system of heptane :
ether: acetic acid (65 : 15 : 1, v : v : v). Cholesterol and cholesterol palmitate were run as standards to identify the free and esterified forms of cholesterol, resnectively. The appropriate lipid bands were scraped-out and eluted with MeOH. The MeOH eluants were evaporated to dryness under nitrogen and redissolved in 0.20 ml acetic acid. Cholesterol was ouantitated bv a modification of the Lieberman and Be&hard method f211. To each sample was added 6.0 ml of cold cholesterol reagent (acetic anhydride : acetic acid : sulfuric acid, 86 : 82 : 25, v : v : v). They were mixed and incubated at 37°C for I5 min. The absorbance of the resultinn blue-preen color was read on a Gilford spectrophotometer at 615 nm. A standard curve was obtained from samples of 100, 200 and 400 UP of cholesterol prepared from a stock solution of 4G fig cholesterol acetate per 0.20 ml acetic acid. Cholesterol acetate and free cholesterol give the same absorbance per pg using this calorimetric method. The uptake and metabolism of f’4Clcholesterol and [‘Qhoiesterol oleate were carried out by incubating 40~10~ cells for 15 and 30 min in 5.0 ml phosphatebuffered saline containing 2.0 $Zi [4,‘4C]choldsterol (spec. act. 55.5 mCi/mmole) or 2.0 &i [oleatel,‘F]cholesterol oleate (spec. act. 50 mCi/mmole). These radioactive compounds were purchased from New England Nuclear, Boston, Ma. The cells were centrifuged at 2000 rpm for 10 min. Lipids were extracted from the cell peilet and from the supematant fluid by the method of Bligh & Dyer [22]. Thin layer chromatography was performed as previously described. The lipid bands were scraped out and treated with 0.10 ml of MeOH in countinn vials for 60 min. After this time, 10.0ml of ACSTM, shntillationcocktail, were added and the radioactivity measured on a Packard Tri E.xp Cell Res I I8 ( 1979)
378
Mark-Malchoff et al.
Table 1. Cholesterol content of HEF and HFT cells, expressed in t.~glIO”cells” HEF cells Total cholesterol* Free cholesterol Cholesterol esters Molar ratio of free cholesterol : cholesterol-esters
6.8+ 0.2 (6) 6.6+ 0.2 (6) 0.2f 0.01 (6) 41.5k22.0 (6)
HFT cells 6.6kO.3 (5) 4.2kO.3 (5) 2.4i0.1 (5) I .8* I .2 (5)
” Values represent the mean k S.D. of 5-6 experiments done in duplicate or triplicate. h Total cholesterol is the sum of the free and the esterified forms of cholesterol.
Carb Scintillation Counter. The corrected counts per minute were converted to picomoles of cholesterol or cholesterol oleate by comparison with the respective standard of [Ylcholesterol or [Wlcholesterol oleate.
RESULTS The analysis of the cholesterol ester and free cholesterol content of HEF and HFT cells are presented in table 1. The total cholesterol content of these cells did not differ. However, HEF cells exhibited a very high free cholesterol : cholesterol ratio, approx. 42 : 1. In contrast, HFT cells demonstrated a much lower free cholesterol : cholesterol ester ratio of about 2 : 1. In order to elucidate the difference in the ratio of free to esterified cholesterol in these
cells, we studied the uptake and metabolism of Y-labeled cholesterol and cholesterol oleate. The increased amount of cholesterol ester in HFT cells may be explained by (N) increased acylation of free cholesterol by acylating enzymes; (6) decreased hydrolysis of cholesterol esters by cholesterol esterases; (c) a combination of (a) and (b) or (d) an increase in uptake of cholesterol ester from the growth medium. Uptake of [‘“Clcholesterol and [14C]cholesterol oleate into HEF and HFT cells was measured by centrifuging the cells at different time intervals and determining the radioactivity lost from the supernatant and the radioactivity incorporated into the cell pellet. Metabolism was examined by ex-
Table 2. Uptake and metabolism of [14C]cholesterol and [‘“C]cholesterol oleate bv HEF and HFT cells a expressed in pmoles per IO6ceils [‘T]Cho/estero/ Cholesterol uptake Cholesterol esters % Esterification of cholesterol [‘JC]Cholesterol
HEF cells
HFT cells
995+29 I .37*0.4 0.14~0.04
625+ I6 4.1?1.2 0.66&O. I8
98tU44 18+0.2 I J&O.2
640+ 10 9+0.5 1.4f0.07
oleate
Cholesterol ester uptake Fatty acids % Hydrolysis of cholesterol oleate
” Cells were incubated at 37°C with 2 /.Ki (36 nmoles) [Ylcholesterol or 2 &i (40 nmoles) [r4C]cholesterol oleate for 60 min in Dulbecco’s phosphate-buffered saline, pH 7.4, containing 5% BFS. Lipids were extracted from the cells three times with CHCI, : MeOH, 1 : 1, and the extracts were run on a TLC plate in the solvent system heptane: ether: HAc, 65: 15: 1. Bands for cholesterol, cholesterol esters and free fatty acids were scraped off and measured for radioactivity.
Cholesterol
content
and metnbolism
tracting the cell pellet with CHCI,: MeOH and analyzing the various lipids by TLC. These results are given in table 2. With HEF cells [14C]cholesterol uptake was 995 pmoles/106 cells/h compared to 625 pmoles/ lo6 cells/h for HFT cells. Thus normal cells take up more cholesterol than their transformed counterparts. The same was observed for the uptake of [14C]cholesterol oleate. It is also noteworthy that the rate of uptake of free cholesterol was equivalent to the rate of uptake of cholesterol oleate for each respective cell type. However, the esterification of cholesterol was higher and the hydrolysis of cholesterol oleate was lower in HFT cells. These observations offer an explanation for the increased amounts of cholesterol ester in the transformed cell, namely an increased production of cholesterol esters and a decreased hydrolysis of these esters. It is also apparent that the extent of cholesterol ester hydrolysis is greater than the extent of cholesterol esterifications, especially in the HEF cell. This explains the higher ratio free cholesterol : cholesterol ester in HEF cells but presents an enigma with HFT cells where the free cholesterol : cholesterol ratio is low. DISCUSSION Cholesterol plays a role in the permeability and stability of the cell membrane by affecting its fluidity [17, 23-2.51. An increase in cholesterol causes the membrane to become less permeable. Cholesterol produces a tighter packing of the phospholipids within the lipid bilayer of the cell membrane by interaction with the fatty acid chains and the polar head groups of the phospholipids. This effect is dependent upon the nature of the fatty acids. However, the role of cholesterol esters in cell membranes is not well
in hamster
embryo
fibroblasts
379
understood. Cholesterol esters are usually minor constituents in cell membranes. Whereas cholesterol decreases the fluidity of cell membranes, cholesterol esters, especially those having polyunsaturated fatty acid chains, can be expected to increase the fluidity of the membrane. Although HEF and HFT cells have the same total cholesterol content on a cell basis, they differ markedly in the amount of free and esterified cholesterol present. HEF cells contain predominately free cholesterol and very little cholesterol esters while HFT cells contain appreciable amounts of both forms. This finding suggests a different arrangement between cholesterol and phospholipids within the membranes of normal and transformed cells. A feature of some transformed cells is an increase in unsaturated fatty acids in their phospholipids [26-281 or a decrease in their cholesterol content [29, 301. Since cholesterol would be expected to decrease the fluidity of the membrane lipid bilayer and cholesterol esters might enhance the fluidity, a change in either of these parameters might influence the permeability properties of membranes of transformed cells. However, the nature of the fatty acids found in the phospholipids and in the cholesterol esters of HEF and HFT cells were not examined. Moreover, our studies were carried out on intact cells so it is not known which specific membrane of the cell has an altered lipid content. It has been demonstrated that some hepatomas as compared with normal liver cells exhibit an increase in the cholesterol content of their subcellular organelles as well as the plasma membrane [31]. The changes in free and esterified cholesterol in HEF and HFT cells may be associated with differences in the rates of cholesterol esterification and cholesterol ester hydrolysis. These reactions are car&p Cdl/?e.\I/8 ,197YJ
380
Mark-Malchoffet
al.
ried out by a cholesterol-0acyl transferase and a cholesterol esterase, respectively. These enzymes have been identified in various cells, especially human skin fibroblasts [32-351. In the uptake and metabolism studies of [‘“C]cholesterol and [‘4C]cholesterol oleate presented, HFT cells have an enhanced cholesterol ester formation and a decrease in cholesterol ester hydrolysis as compared with HEF cells. These changes may appear to be small but may account for the difference in the free and esterified cholesterol contents of normal and transformed cells. The prolonged period of cell culturing must be considered. Over the 6 days in culture the increase in cholesterol esterification and decrease in this ester hydrolysis in the transformed cell is cumulative and may explain the changes in cholesterol and cholesterol ester in HFT cells. The cells studied were confluent and removed at high density. Confluent normal cells are in a maintenance or steady state, while confluent transformed cells are still growing and rapidly dividing. Cells at low density may differ from those at high density. It would be desirable to study both cell types in their exponential growth phase or before they attain confluency. The differences in cholesterol esterification and cholesterol ester hydrolysis under these circumstances might better reflect the changes in free and esterified cholesterol in the transformed state. Studies by Brown & Goldstein [36] have shown that normal fibroblasts contain receptors for LDL serum lipoproteins. Uptake and metabolism of cholesterol esters contained in LDL is dependent on initial binding of LDL to membrane receptors. This is followed by endocytosis of LDL which are entrapped in small membrane vesicles. These fuse with lysosomes where
the LDL are degraded. The cholesterol esters are hydrolyzed to free cholesterol which somehow gets into the cell cytoplasm and regulates HMG CoA reductase and cholesterol-acyl CoA transferase. The level of LDL receptors appear to be regulated by the LDL present. In our studies, the fibroblasts were grown in medium containing serum and hence were exposed to serum lipoproteins. How these may influence the lipid composition of the fibroblasts remains to be investigated. The significance of the altered lipid composition of HFT cells may be related to the enhanced uptake of nutrients by these cells possibly conferring on them an advantage for growth. In this regard, HFT cells are reported to have an increased ability to take up amino acids [37] and nucleosides [38, 391. It remains to be determined whether the change in cholesterol : cholesterol ester occurs in the plasma membrane of HFT cells and whether this accounts in part for the enhanced permeability of nutrients. This work has been supported in part by contract no. I-CP-45611, NCI. and grant HLB 02063, Heart and Lung Institute.
REFERENCES 1. Bailey, J M, Proc sot exp biol med 107 (1961) 30. 2. Rothblatt, G M, HartzelI, R W, Mailhe, H & Kritchevsky, D, Biochim biophys acta 116 (1966) 113. 3. Hakomori, S & Murakami, W T, Proc natl acad sci US 59 (1968) 254. 4. Rothblatt, G H, Buckho, M K & Kritchevsky, D, Biochim biophys acta 164(1968) 327. 5. Peterson, J A & Rubin, M, Exp cell res 58 (1969) 365. 6. Bergelson, J S, Dyatlovitskaya, D V, Torkhovskaya, T I, Sorkina, I B & Gorkova, N P, Biochim biophys acta 619 (1970) 287. 7. Cunningham, D D, J biol them 247 (1970) 2464. 8. Pastemak, G A & Friedricks, B, Biochem j 119 (1970) 473. 9. Peterson, J A & Rubin, M, Exp cell res 60 (1970) 383. 10. Harry, D S, Morris, H P & McIntyre, J, J lipid res 17 (1971) 313. 11. Lucas, D D, Shohet, S F & Merler, E, J immunol 106 (1971) 768.
Cholesterol content and metabolism in hamster embryo fibroblasts 12. Quigley, J P, Rifkin, D P & Reich, E, Virology 46 (1971) 106. 13. Rytter, D J & Comatzer, W E, Lipids 7 (1972) 142. 14. Hirschberg, C B & Robbins, P W, Virology 61 (1974) 602. 15. Hakomori, S, Biochim biophys acta (1975) 55. 16. Wahach, D F H, Membrane molecular biology of neoplastic cells, p. 141. Elsevier Scientific Publishing Co., Amsterdam (1975). 17. -Ibid, p. 217 (1975). 18. Fishman, P H & Brady, R 0, Science 194 (1976) 906. 19. Itaya, K, Hakomori, S & Lein, G, Proc natl acad sci US 73 (1976) 1568. 20. Mark-Malchoff, D, Marinetti, G V, Hare, J D & Meisler, A, Biochem biophys res commun 75 (1977) 589. 21. Stadtman, T C, Methods in enzymology (ed S P Colwick & N 0 Kaplan) vol. 3, p. 392. Academic Press, New York (1957). 22. Bligh, E G & Dyer, W J, Can j biochem physiol 37 (1959)911. 23. Oldfield, E & Chapman, D, FEBS lett 23 (1970) 285. 24. Feinstein, M B, Fernandez, S M & Sha’afi, R I, Biochim biophys acta 413 (1975) 354. 25. Jain, M K, Current topics in membrane and transport 6 (1975) 1.
381
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Exp Cell Res 118 (1979)