Proc. Natl. Acad. Sci. USA Vol. 73, No. 8, pp. 2735-2739, August 1976
Biochemistry
Comparison of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in hepatoma cells grown in vivo and in vitro (hepatoma/cholesterol synthesis/malignant transformation)
OWEN Ross BEIRNE AND JOHN A. WATSON University of California at San Francisco, Department of Biochemistry and Biophysics and the Liver Center, San Francisco, California 94143
Communicated by John A. Clements, June 2,1976
Unlike the normal liver, numerous transABSTRACT plantable rodent and human hepatomas are unable to alter their rate of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] activity in response to a dietary cholesterol challenge. It has been suggested that this metabolic defect is linked to the process of malignant transformation. Hepatoma 7288C "lacks" feedback regulation of cholesterol synthesis when grown in vivo but expresses this regulatory property when grown in vitro (then called HTC). Therefore, it was used as a model system to answer whether an established hepatoma cell line that modulates its rate of cholesterol synthesis in vitro can express this property when grown in vivo, and whether cells reisolated from the tumor mass have the same regulatory phenotype as before transplantation. Our results show that long-term growth of hepatoma 7288C in tissue culture has not caused a biotransformation that permits feedback regulation of HMG-CoA reductase when the cells are transplanted back into host animals. In addition, HTC cells reisolated from the tumor mass and established in tissue culture continue to have the ability to regulate HMG-CoA reductase activity. Therefore, malignant transformation is not categorically linked to the loss of the cellular components necessary to regulate sterol synthesis and HMG-CoA reductase activity. The rate of hepatic cholesterol synthesis is less in normal animals fed a diet that contains cholesterol than in those given one that is devoid of this sterol. 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] is the rate-limiting enzyme and the primary site for dietary regulation of cholesterol synthesis (1, 2). It is not known whether this "feedback" regulation of reductase activity is caused by a direct interaction of effectors with the enzyme and/or by a change in enzyme concentration
(1,2).
Numerous transplantable rodent and human hepatomas have been shown to be unable to alter their rate of sterol synthesis (2, 3) and reductase activity (4) in response to a dietary cholesterol challenge. In addition, leukemic cells grown in vivo appear to lack the ability to regulate their rate of cholesterol biosynthesis in response to a dietary sterol challenge (3). Animals fed chemical hepatocarcinogens (i.e., aflatoxin) do not express an ability to regulate their rate of hepatic cholesterol synthesis. This apparent defect develops before any hepatomas are evident (2, 5). From these studies and those with transplantable tumors, it has been proposed that the "loss" of feedback control of cholesterol synthesis is a unique property of cancerous cells (2, 6, 7). Because this apparent "loss" is an early event in the conversion of a normal cell to a malignant one, it has been suggested that this metabolic defect is linked to the transformation process (2). However, recent studies with malignant tissue culture cells Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; TAT, tyrosine aminotransferase; HTC, hepatoma in tissue culture; LPPS, lipoprotein-poor serum; DTT, dithiothreitol. 2735
have shown that tumors can modulate their rate of cholesterol synthesis and reductase activity in response to exogenous cholesterol (serum lipoproteins) (8, 9). Low density and very low density lipoproteins are specific regulators of sterol synthesis in HeLa (10), mouse L-cells (11), and minimal deviation hepatoma 7288C (8, 9) grown in tissue culture. These are the same effectors that modulate the rate of cholesterol synthesis and reductase activity in normal fibroblasts (10, 12, 13), smooth muscle cells (14), and epithelial cells in culture (15). This regulation of cholesterol biosynthesis by malignant cells grown in vitro contrasts strongly with their apparent inability to express this property when grown in vio (within a host animal). Because of this difference, it is of interest to characterize the regulation of cholesterol synthesis with the same cells grown in an animal (in vivo) and in tissue culture (in vitro). Hepatoma 7288C (HTC cells) was chosen for this investigation because it shows a lack of regulation of cholesterol biosynthesis when grown in the Buffalo rat (7), but displays "feedback" control when grown in tissue culture (8, 9). The regulation of cholesterol synthesis in HTC cells grown in tissue culture is similar to that seen in normal human fibroblasts (12, 13) and other tissue culture systems (14, 15). HTC cells were transplanted into the host animals (Buffalo rats) to determine if there had been any change in them during their 7 years of growth in tissue culture that would now allow the tumor mass to modulate its HMG-CoA reductase activity in response to a dietary cholesterol challenge. In addition, HTC cells were reisolated from the rat and grown in vitro to determine if their capacity to regulate reductase in the presence of low density lipoproteins was altered by their growth in the animal. Our results suggest that the regulation of sterol synthesis in HTC cells is not due to a permanent transformation or adaptation to long term growth in tissue culture. METHODS Biochemicals and Reagents. DL-3-Hydroxy-3-methyl[3'4C]glutaryl coenzyme A and DL-[5-3H]mevalonic acid (dibenzylethylenediamine salt) were purchased from New England Nuclear. Glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Boehringer Mannheim Corp. NADP and dithiothreitol (DTT) were purchased from Calbiochem. Fetal calf and calf sera, and Swim's 77 and Dulbecco's modified Eagle's media were from Grand Island Biological Co. All other reagents used were of analytical grade. Cell Culture and Cell Transfer. Cells were maintained in suspension culture in Swim's 77 medium which contained lipoprotein-poor serum (LPPS) or unfractionated serum as described previously (9). A cell density of 4 to 6 X 105 cells per ml was used for all the experiments in this report. Preparation of HTC Cell Homogenate. HTC cells were harvested by centrifugation at 600 X g for 4 min and washed
2736
Biochemistry:
Beirne and Watson
Proc. Natl. Acad. Sci. USA 73 (1976)
twice in cold phosphate-buffered saline, pH 7.6. The cell pellet was incubated at 40 for 10 min in 0.5 ml of hypotonic buffer (10 mM Tris-HCl at pH 7.6, 10 mM KCl, 5 mM MgCl2) and
homogenized by 25 passes in a Dounce homogenizer with a tight-fitting pestle. An 0.25 ml portion of buffer (90 mM EDTA, 30 mM 2-mercaptoethanol, 210 mM KCl, 150 mM KH2PO4, pH 7.4) was added and the suspension was rehomogenized with 10 more passes of the pestle. This homogenate was assayed immediately for HMG-CoA reductase activity. Preparation of Microsomes. Livers or transplanted tumors from two animals (four hind legs) were pooled, minced, and washed several times in buffer (100 mM sucrose, 50 mM KCI, 5 mM DTT, 40 mM KH2PO4, pH 7.4,30 mM EDTA) (16). Two grams of the minced liver or tumor were homogenized in 6 ml of buffer in a Dounce homogenizer with 20 passes of a loosefitting pestle. The homogenate was centrifuged twice at 10,000 X g for 15 min and the pellets from each were discarded. The second 10,000 X g supernatant fluid was centrifuged at 40 for 1 hr at 100,000 X g. The 100,000 X g pellet was resuspended into the homogenization buffer and assayed immediately for HMG-CoA reductase activity. HMG-CoA Reductase Assay. The assay used was a modification of the micro method described by Shapiro et al. (17). Reactions were carried out in a total volume of 0.155 ml which contained (,umol), EDTA, (3.0); KCI, (7.0); glucose 6-phosphate, (1.0); NADP, (0.54); DTT (2.6); KH2PO4, pH 7.4, (5.0); 0.14 units of glucose-6-phosphate dehydrogenase, and 100-500 Mg of protein. The reaction was started by the addition of 5 ,l of DL-3-hydroxy-3-methyl[3-14C]glutaryl coenzyme A (200 nmol; 1000-2000 cpm/nmol). The reactions were incubated for 15-30 min, and terminated by the addition of 20 Ml of concentrated HCI. Carrier [3H]mevalonic acid (24,000 cpm, 200 Mg) was added to each assay tube for recovery. After 30 min of lactonization at 370, the samples were centrifuged for 4 min in a Beckman 152 Microfuge and 100 Ml were spotted on a 0.25 Mm silica gel plate (Brinkmann). The mevalonolactone was separated and quantitated as described previously (9). This assay procedure was used for tumor and liver microsomes. However, the reaction mix contained 1.02 Mmol of 2-mercaptoethanol in addition to DTT used in the measurement of reductase activity in HTC cells grown in vitro. Tyrosine Aminotransferase Assay. HTC cells, grown in Swim's 77 medium which contained intact serum, were split into two flasks. One flask received dexamethasone (0.01 ml of mM dexamethasone in 95% ethanol per 100 ml of culture medium) and the other flask received only ethanol. The cells were grown for 20 hr, centrifuged, and washed twice with phosphate-buffered saline. The washed cell pellet was resuspended into buffer containing 50 mM KH2PO4 (pH 7.6), 0.2 mM pyridoxal, 1 mM EDTA, 5 mM a-ketoglutarate, and sonicated (Brinkmann sonicator) at 20% maximum output with three 10-sec bursts (18). The cell sonicate was assayed for tyrosine aminotransferase (TAT) activity (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) as described by Hayashi et al. (18). Protein Determination. The protein contents of the microsomal suspensions, cell sonicates, and Dounce homogenates were determined according to the method of Lowry et al. (19). Crystalline bovine serum albumin was used as the protein standard. Those samples that contained DTT were precipitated with 10% trichloroacetic acid and the precipitate was dissolved in 0.1 M NaOH before assay for their protein content. Transplantation and Reisolation of HTC Cells. HTC cells maintained in medium that contained unfractionated serum were isolated by centrifugation and washed twice in warm,
Table 1. HMG-CoA reductase activity in rats bearing HTC cells (7288t) Diet - Cholesterol +
Cholesterol
Tumor Liver nmol/hr per mg of protein
12.8 ± 3.5 (4) 3.2 ± 1.5 (3)
14.9 ± 7.7 (4) 10.0 ± 2.3 (3)
Liver and tumor microsomes were isolated and assayed as described in Methods. The number of pooled samples is indicated in the parentheses. Each sample was assayed at three different microsomal protein concentrations and averaged. The data are reported as the mean i the standard error of the mean. A t-test of reductase activities shows inhibition (P < 0.01) for the liver and no inhibition (P > 0.2) for the tumor.
sterile phosphate-buffered saline. The cells were resuspended in the buffered saline to a density of 10 X 107 cells per ml and 0.5 ml was injected into each hind leg of ether-anesthesized rats. Each injection site developed a 2- to 3-cm tumor in 4 weeks. Animals were fed Berkeley rat chow (Foodstuffs Co.) ad lib. during this period of time. HTC cells were reisolated by a modification of a previously described method (20). The tumor mass from the hind legs of a rat was aseptically removed and placed in cold sterile Dulbecco's modified Eagle's medium that contained 10% fetal calf serum, minced, and filtered through sterile gauze. Cells and debris from the gauze filtrate were allowed to settle by gravity for 10 min. The supernatant fluid was carefully decanted and the pellet was washed twice in fresh medium by centrifugation (600 X g). These washed cells were resuspended in medium and 10 ml was added to 100 mm petri dishes and grown at 370 in a humidified 95% air, 5% CO2 incubator. After 72 hr incubation, viable cells were isolated from the culture medium by centrifugation. These cells were resuspended in Swim's 77 medium that contained unfractionated serum and maintained in suspension culture until use (20 days after isolation). Animal Feeding. Buffalo rats, inoculated with HTC cells, (described above) were fed Berkeley rat chow ad lib. for 4 weeks and then fasted for 24 hr. After the 24 hr fast, the tumor-bearing rats were fed ad lib. a synthetic cholesterol-free diet for 3 days (21). On day four, the rats were divided into two groups; one of the groups (six rats) continued on the low-cholesterol diet and the other (eight rats) was given the same diet supplemented with 3% (wt/wt) cholesterol (the cholesterol had been purified by three recrystallizations from ethanol). On the third day of the dietary sterol challenge, the rats were sacrificed (9 a.m. to 12 noon) by a blow to the head followed by decapitation. The livers and tumors were removed and processed for assay of reductase activity as described above.
RESULTS HTC cells were transplanted into their original host (Buffalo rats) as described in Methods and allowed to grow for 28 days. After this period, the rats were fed an experimental diet that contained no added sterol or 3% cholesterol for 3 days. Microsomal HMG-CoA reductase activity in the tumor and host liver was measured as described in Methods. Table 1 shows that reductase activity in the tumor did not change significantly in response to a dietary cholesterol challenge; however, the host liver from rats fed cholesterol showed a 75% suppression in reductase activity. This is consistent with published observations that the rate of sterol synthesis from [14C]acetate in hepatoma 7288C was not suppressed by dietary cholesterol (7).
Biochemistry:
Beirne and Watson
Proc. Natl. Acad. Sci. USA 73 (1976)
2737
Table 3. Tyrosine aminotransferase activity in pre- and posttransplanted HTC cells c
Pre-
LPPS
Condition
3.5
Dexamethasone + Dexamethasone
-
E
3.0
16 125
11 114
Data for pretransplanted cells were obtained from Tomkins et al. (22). Reisolated cells were assayed for TAT activity as described in Methods and the data are the average of two assays from cells grown in Swim's 77 media containing whole serum in the presence or absence of dexamethasone.
2.5 U.'
Biochemistry
Comparison of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in hepatoma cells grown in vivo and in vitro (hepatoma/cholesterol synthesis/malignant transformation)
OWEN Ross BEIRNE AND JOHN A. WATSON University of California at San Francisco, Department of Biochemistry and Biophysics and the Liver Center, San Francisco, California 94143
Communicated by John A. Clements, June 2,1976
Unlike the normal liver, numerous transABSTRACT plantable rodent and human hepatomas are unable to alter their rate of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] activity in response to a dietary cholesterol challenge. It has been suggested that this metabolic defect is linked to the process of malignant transformation. Hepatoma 7288C "lacks" feedback regulation of cholesterol synthesis when grown in vivo but expresses this regulatory property when grown in vitro (then called HTC). Therefore, it was used as a model system to answer whether an established hepatoma cell line that modulates its rate of cholesterol synthesis in vitro can express this property when grown in vivo, and whether cells reisolated from the tumor mass have the same regulatory phenotype as before transplantation. Our results show that long-term growth of hepatoma 7288C in tissue culture has not caused a biotransformation that permits feedback regulation of HMG-CoA reductase when the cells are transplanted back into host animals. In addition, HTC cells reisolated from the tumor mass and established in tissue culture continue to have the ability to regulate HMG-CoA reductase activity. Therefore, malignant transformation is not categorically linked to the loss of the cellular components necessary to regulate sterol synthesis and HMG-CoA reductase activity. The rate of hepatic cholesterol synthesis is less in normal animals fed a diet that contains cholesterol than in those given one that is devoid of this sterol. 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] is the rate-limiting enzyme and the primary site for dietary regulation of cholesterol synthesis (1, 2). It is not known whether this "feedback" regulation of reductase activity is caused by a direct interaction of effectors with the enzyme and/or by a change in enzyme concentration
(1,2).
Numerous transplantable rodent and human hepatomas have been shown to be unable to alter their rate of sterol synthesis (2, 3) and reductase activity (4) in response to a dietary cholesterol challenge. In addition, leukemic cells grown in vivo appear to lack the ability to regulate their rate of cholesterol biosynthesis in response to a dietary sterol challenge (3). Animals fed chemical hepatocarcinogens (i.e., aflatoxin) do not express an ability to regulate their rate of hepatic cholesterol synthesis. This apparent defect develops before any hepatomas are evident (2, 5). From these studies and those with transplantable tumors, it has been proposed that the "loss" of feedback control of cholesterol synthesis is a unique property of cancerous cells (2, 6, 7). Because this apparent "loss" is an early event in the conversion of a normal cell to a malignant one, it has been suggested that this metabolic defect is linked to the transformation process (2). However, recent studies with malignant tissue culture cells Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; TAT, tyrosine aminotransferase; HTC, hepatoma in tissue culture; LPPS, lipoprotein-poor serum; DTT, dithiothreitol. 2735
have shown that tumors can modulate their rate of cholesterol synthesis and reductase activity in response to exogenous cholesterol (serum lipoproteins) (8, 9). Low density and very low density lipoproteins are specific regulators of sterol synthesis in HeLa (10), mouse L-cells (11), and minimal deviation hepatoma 7288C (8, 9) grown in tissue culture. These are the same effectors that modulate the rate of cholesterol synthesis and reductase activity in normal fibroblasts (10, 12, 13), smooth muscle cells (14), and epithelial cells in culture (15). This regulation of cholesterol biosynthesis by malignant cells grown in vitro contrasts strongly with their apparent inability to express this property when grown in vio (within a host animal). Because of this difference, it is of interest to characterize the regulation of cholesterol synthesis with the same cells grown in an animal (in vivo) and in tissue culture (in vitro). Hepatoma 7288C (HTC cells) was chosen for this investigation because it shows a lack of regulation of cholesterol biosynthesis when grown in the Buffalo rat (7), but displays "feedback" control when grown in tissue culture (8, 9). The regulation of cholesterol synthesis in HTC cells grown in tissue culture is similar to that seen in normal human fibroblasts (12, 13) and other tissue culture systems (14, 15). HTC cells were transplanted into the host animals (Buffalo rats) to determine if there had been any change in them during their 7 years of growth in tissue culture that would now allow the tumor mass to modulate its HMG-CoA reductase activity in response to a dietary cholesterol challenge. In addition, HTC cells were reisolated from the rat and grown in vitro to determine if their capacity to regulate reductase in the presence of low density lipoproteins was altered by their growth in the animal. Our results suggest that the regulation of sterol synthesis in HTC cells is not due to a permanent transformation or adaptation to long term growth in tissue culture. METHODS Biochemicals and Reagents. DL-3-Hydroxy-3-methyl[3'4C]glutaryl coenzyme A and DL-[5-3H]mevalonic acid (dibenzylethylenediamine salt) were purchased from New England Nuclear. Glucose 6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Boehringer Mannheim Corp. NADP and dithiothreitol (DTT) were purchased from Calbiochem. Fetal calf and calf sera, and Swim's 77 and Dulbecco's modified Eagle's media were from Grand Island Biological Co. All other reagents used were of analytical grade. Cell Culture and Cell Transfer. Cells were maintained in suspension culture in Swim's 77 medium which contained lipoprotein-poor serum (LPPS) or unfractionated serum as described previously (9). A cell density of 4 to 6 X 105 cells per ml was used for all the experiments in this report. Preparation of HTC Cell Homogenate. HTC cells were harvested by centrifugation at 600 X g for 4 min and washed
2736
Biochemistry:
Beirne and Watson
Proc. Natl. Acad. Sci. USA 73 (1976)
twice in cold phosphate-buffered saline, pH 7.6. The cell pellet was incubated at 40 for 10 min in 0.5 ml of hypotonic buffer (10 mM Tris-HCl at pH 7.6, 10 mM KCl, 5 mM MgCl2) and
homogenized by 25 passes in a Dounce homogenizer with a tight-fitting pestle. An 0.25 ml portion of buffer (90 mM EDTA, 30 mM 2-mercaptoethanol, 210 mM KCl, 150 mM KH2PO4, pH 7.4) was added and the suspension was rehomogenized with 10 more passes of the pestle. This homogenate was assayed immediately for HMG-CoA reductase activity. Preparation of Microsomes. Livers or transplanted tumors from two animals (four hind legs) were pooled, minced, and washed several times in buffer (100 mM sucrose, 50 mM KCI, 5 mM DTT, 40 mM KH2PO4, pH 7.4,30 mM EDTA) (16). Two grams of the minced liver or tumor were homogenized in 6 ml of buffer in a Dounce homogenizer with 20 passes of a loosefitting pestle. The homogenate was centrifuged twice at 10,000 X g for 15 min and the pellets from each were discarded. The second 10,000 X g supernatant fluid was centrifuged at 40 for 1 hr at 100,000 X g. The 100,000 X g pellet was resuspended into the homogenization buffer and assayed immediately for HMG-CoA reductase activity. HMG-CoA Reductase Assay. The assay used was a modification of the micro method described by Shapiro et al. (17). Reactions were carried out in a total volume of 0.155 ml which contained (,umol), EDTA, (3.0); KCI, (7.0); glucose 6-phosphate, (1.0); NADP, (0.54); DTT (2.6); KH2PO4, pH 7.4, (5.0); 0.14 units of glucose-6-phosphate dehydrogenase, and 100-500 Mg of protein. The reaction was started by the addition of 5 ,l of DL-3-hydroxy-3-methyl[3-14C]glutaryl coenzyme A (200 nmol; 1000-2000 cpm/nmol). The reactions were incubated for 15-30 min, and terminated by the addition of 20 Ml of concentrated HCI. Carrier [3H]mevalonic acid (24,000 cpm, 200 Mg) was added to each assay tube for recovery. After 30 min of lactonization at 370, the samples were centrifuged for 4 min in a Beckman 152 Microfuge and 100 Ml were spotted on a 0.25 Mm silica gel plate (Brinkmann). The mevalonolactone was separated and quantitated as described previously (9). This assay procedure was used for tumor and liver microsomes. However, the reaction mix contained 1.02 Mmol of 2-mercaptoethanol in addition to DTT used in the measurement of reductase activity in HTC cells grown in vitro. Tyrosine Aminotransferase Assay. HTC cells, grown in Swim's 77 medium which contained intact serum, were split into two flasks. One flask received dexamethasone (0.01 ml of mM dexamethasone in 95% ethanol per 100 ml of culture medium) and the other flask received only ethanol. The cells were grown for 20 hr, centrifuged, and washed twice with phosphate-buffered saline. The washed cell pellet was resuspended into buffer containing 50 mM KH2PO4 (pH 7.6), 0.2 mM pyridoxal, 1 mM EDTA, 5 mM a-ketoglutarate, and sonicated (Brinkmann sonicator) at 20% maximum output with three 10-sec bursts (18). The cell sonicate was assayed for tyrosine aminotransferase (TAT) activity (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) as described by Hayashi et al. (18). Protein Determination. The protein contents of the microsomal suspensions, cell sonicates, and Dounce homogenates were determined according to the method of Lowry et al. (19). Crystalline bovine serum albumin was used as the protein standard. Those samples that contained DTT were precipitated with 10% trichloroacetic acid and the precipitate was dissolved in 0.1 M NaOH before assay for their protein content. Transplantation and Reisolation of HTC Cells. HTC cells maintained in medium that contained unfractionated serum were isolated by centrifugation and washed twice in warm,
Table 1. HMG-CoA reductase activity in rats bearing HTC cells (7288t) Diet - Cholesterol +
Cholesterol
Tumor Liver nmol/hr per mg of protein
12.8 ± 3.5 (4) 3.2 ± 1.5 (3)
14.9 ± 7.7 (4) 10.0 ± 2.3 (3)
Liver and tumor microsomes were isolated and assayed as described in Methods. The number of pooled samples is indicated in the parentheses. Each sample was assayed at three different microsomal protein concentrations and averaged. The data are reported as the mean i the standard error of the mean. A t-test of reductase activities shows inhibition (P < 0.01) for the liver and no inhibition (P > 0.2) for the tumor.
sterile phosphate-buffered saline. The cells were resuspended in the buffered saline to a density of 10 X 107 cells per ml and 0.5 ml was injected into each hind leg of ether-anesthesized rats. Each injection site developed a 2- to 3-cm tumor in 4 weeks. Animals were fed Berkeley rat chow (Foodstuffs Co.) ad lib. during this period of time. HTC cells were reisolated by a modification of a previously described method (20). The tumor mass from the hind legs of a rat was aseptically removed and placed in cold sterile Dulbecco's modified Eagle's medium that contained 10% fetal calf serum, minced, and filtered through sterile gauze. Cells and debris from the gauze filtrate were allowed to settle by gravity for 10 min. The supernatant fluid was carefully decanted and the pellet was washed twice in fresh medium by centrifugation (600 X g). These washed cells were resuspended in medium and 10 ml was added to 100 mm petri dishes and grown at 370 in a humidified 95% air, 5% CO2 incubator. After 72 hr incubation, viable cells were isolated from the culture medium by centrifugation. These cells were resuspended in Swim's 77 medium that contained unfractionated serum and maintained in suspension culture until use (20 days after isolation). Animal Feeding. Buffalo rats, inoculated with HTC cells, (described above) were fed Berkeley rat chow ad lib. for 4 weeks and then fasted for 24 hr. After the 24 hr fast, the tumor-bearing rats were fed ad lib. a synthetic cholesterol-free diet for 3 days (21). On day four, the rats were divided into two groups; one of the groups (six rats) continued on the low-cholesterol diet and the other (eight rats) was given the same diet supplemented with 3% (wt/wt) cholesterol (the cholesterol had been purified by three recrystallizations from ethanol). On the third day of the dietary sterol challenge, the rats were sacrificed (9 a.m. to 12 noon) by a blow to the head followed by decapitation. The livers and tumors were removed and processed for assay of reductase activity as described above.
RESULTS HTC cells were transplanted into their original host (Buffalo rats) as described in Methods and allowed to grow for 28 days. After this period, the rats were fed an experimental diet that contained no added sterol or 3% cholesterol for 3 days. Microsomal HMG-CoA reductase activity in the tumor and host liver was measured as described in Methods. Table 1 shows that reductase activity in the tumor did not change significantly in response to a dietary cholesterol challenge; however, the host liver from rats fed cholesterol showed a 75% suppression in reductase activity. This is consistent with published observations that the rate of sterol synthesis from [14C]acetate in hepatoma 7288C was not suppressed by dietary cholesterol (7).
Biochemistry:
Beirne and Watson
Proc. Natl. Acad. Sci. USA 73 (1976)
2737
Table 3. Tyrosine aminotransferase activity in pre- and posttransplanted HTC cells c
Pre-
LPPS
Condition
3.5
Dexamethasone + Dexamethasone
-
E
3.0
16 125
11 114
Data for pretransplanted cells were obtained from Tomkins et al. (22). Reisolated cells were assayed for TAT activity as described in Methods and the data are the average of two assays from cells grown in Swim's 77 media containing whole serum in the presence or absence of dexamethasone.
2.5 U.'
