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[CANCER RESEARCH 30, 2876—2880,December 1970] Studies on Liver Plasma Membranes of Rats Fed N-2-Fluorenylacetamide' N. Chandrasekhara2 and K. Ananth Narayan3 TheBurnsidesResearchLaboratory, University oflllinois, Urbana,Illinois 61801 SUMMARY study was to investigate the lipid composition of membranes at 2 different stages of carcinogenesis. In this article, we report Plasma membranes were prepared from livers of normal and N-2-fluorenylacetamide-ingesting rats. Of the two marker enzymes studied, the activity of 5'-nucleotidase was markedly decreased, whereas that of Mg2@-Na3-r-ATPase was unaffected striking in the membranes from animals fed the carcinogen. normal liver. Unlike in preparations from normal livers, a large amount of floating fraction (d < 1.16) was invariably observed from preneoplastic and neoplastic livers. INTRODUCTION The probable involvement of the cell surface in the neo plastic process has been indicated by several studies. Among these may be cited the work of Abercrombie et aL (1) regard ing contact inhibition; studies on the altered electrophoretic mobility of tumor cells (2), the decreased intercellular com munication between cancer cells (15), and the decreased incidence of the tight junction (12); and the more recent ob servation by Burger (4) of a difference in the architecture of the surface membrane of virally transformed cells. As a result of the development of techniques for the isola tion of plasma membranes from rat liver (18, 23), hepatomas (13), mouse fibroblast cells (26), Ehrlich ascites carcinoma (25), and HeLa cells (3), it has been possible to study the properties of the cell surface more directly. Wallach (24) has hypothesized that neoplasms would appear as a result of alterations critically involving the plasma membrane. There has been no study regarding changes in lipid composi tion and the characterization of the proteins of rat liver plasma membranes during carcinogenesis. The main object of this 1 Supported by a grant from the American Cancer Society and Grant CA-01932 from the USPHS. A preliminary report has been published (6). 2 On leave from the Biochemistry Division, Central Food in the phospholipid:cholesterol ratios and in The per centage of phospholipids in the preneoplastic and neoplastic liver membranes was very much higher than in the controls, and the phospholipid :cholesterol ratio was also significantly enhanced. The percentage of polyunsaturated fatty acids was higher in these membranes than in the plasma membranes of in membrane preparations changes the relative levels of total phospholipids in membranes from both preneoplastic and neoplastic livers, in comparison with the ratios and levels in membranes from normal liver. MATERIALS AND METHODS One group of male weanling rats (60 g, Holtzman Co., Madison, Wis.) was fed a corn oil diet (10 g oil/lOO g diet), and 2 other groups were fed the corn oil diet, containing 0.05 and 0.3 g FAA4/kg (16) (Eastman Organic Chemicals, Rochester, N. Y.). At the end of 20 weeks on the diet, the livers of the rats fed the high level of FAA were composed of large tumors, whereas livers from the rats fed the lower level of the car cinogen were in the preneoplastic stage (1 6). The average liver weight of the rats in the present experiment was: normal, 11.3 ; low FAA, 14.2; and high FAA, 20.4. The animals (not fasted) were sacrificed under Nembutal anethesia and bled through the abdominal aorta. Sera from 2 animals were pooled and constituted 1 sample. Livers were similarly pooled, and 20 g were used for the preparation of plasma membranes. In brief, the procedure was as follows. The 1,500 X g pellet obtained according to the procedure of Neville (18) was homogenized with 3 to 4 strokes of the loose-fitting pestle of a Dounce homogenizer. The density of this homogenate was adjusted to 1.197 with sucrose of density 134. Twenty-eight to 30 ml of this solution formed the bottom layer in a centrifuge tube for the SW 25.2 rotor. Twenty ml of sucrose of density 1.18 and 10 ml ofsucrose ofdensity 1.16 were layered and centrifuged at 98,500 X g for 150 mm. After collection, the floating layer (d < 1.16) and the plasma membranes (d 1J6 to 1.18) Were diluted with 4 volumes of 1 mM NaHCO3 and centrifuged at 10,000 X g for 30 mm. The pellets were dispersed in 1 mM NaHCO3 . Lipid, phospholipid, and protein determinations; gas chro matographic analyses of fatty acids; and the assays for 5'-nucleotidase (EC 3.1 .3.5), ATPase (EC 3.6.1 .4), and glucose 6-phosphatase (EC 3.1 .3.9) were all carried out as reported before (5). Succinic dehydrogenase (EC 1.3 .99. 1) was assayed according to the method of Shelton and Rice (22). All sub Techno logical Research Institute, Mysore, India. 3 Recipient of a Research Career Development 31,063 from the National Cancer Institute. Received June 29, 1970; accepted August 21, 1970. 2876 Award, 5K3-CA 4The abbreviations used are: FAA,N-2-fluorenylacetamide;ATPase, Mg@-Na@-K@-adenosine triphophatase, all 3 ions included in the incuba tion medium; EFA, essential fatty acid. CANCER RESEARCH VOL. 30 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1970 American Association for Cancer Research. Hepatoma Plasma Membranes strates were purchased from Sigma Chemical Co., St. Louis, among Mo. All enzyme assays were done at 2 enzyme concentrations, and the incubation time and concentration of enzyme were adjusted to conform to zero-order kinetics. the 3 groups of livers in the present experiments with respect to the ease ofhomogenization. The yield was about 5 mg membrane protein from 20 g normal liver and nearly twice as much from livers of animals fed the carcinogen. As indicated in Table 1, the specific activities of 5'-nucleotidase and ATPase were lower in liver homogenates of FAA-fed rats than in normal liver homogenates. The specific activities of these 2 marker enzymes in membranes from RESULTS normal livers were enhanced about 15- and 5-fold as compared with the homogenate. These enzyme activities were in the Unlike EFA-deficient livers, which were more easily homogenizable than normal livers (5), there was no difference range reported in the literature (5, 13). An important observa tion in the present experiments was that membranes from both preneoplastic 5'-nucleotidase Table 1 However, Enzyme activities ofliver and plasmamembranes Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats).Inorganic Sample LH° 3.1 ±0.6 7.4 ±1.3 2.3 ± 0.4― 5.9 ± 0.3― 0.6cControl High FAA LH 2.0 ±O.3@ 5.5 ± PM 45.1 ±6.1 36.7 ±7.9 Low FAA PM 26.9 ± 6.3'@ 37.0 ±3.6 High FAA PM 19.6 ±7•6d 31.8 ±6.9 PM, plasma membrane. and high FAA, 0.05 and 0.3 g FAA/kg diet, there was no change in the ATPase activity. Similar animals (5), succinic dehydrogenase activity of membranes from animals fed FAA was higher than that of membranes S'-Nucleotidase LH b i.ow lower from normal livers. suggested that there was very little contamination with micro somes. As in the case of membranes from EFA-deficient Low FAAb a LH, liver homogenate; livers had a markedly changes in enzyme activities were observed by Emmelot and Bos (13) in hepatoma membranes. Glucose 6-phosphatase activity was quite low in membranes from all groups, and this phosphate formed (jsmoles/mg protein/hr) ATPaseControl Diet and neoplastic activity than membranes respectively. from normal animals. On the basis of experiments with iso lated mitochondria under identical conditions, such con tamination was estimated to be less than 10% (N. Chandra sekhara and K. A. Narayan, manuscript in preparation). As shown in Table 2, the lipid composition of plasma membranes from normal liver was similar to the values re ported by Dod and Gray. (1 1), Pfleger et a!. (20), and Ray et a!. (21). One of the significant fmdings of this study was a “p <0.05. dp <0.005. marked increase membranes in the percentage of total from both preneoplastic phospholipids in and neoplastic livers in Table 2 Lipid composition ofserum, liver, and plasmamembranes Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats), except in the case of serum from the high FAA group, in which the results were obtained from 4 preparations. as ratiomg/mg % of total lipidTotal lipidPhospholipids DietSampleTotal [email protected] 0.1Low o.2eHigh FAAbPM0.45 ±0.0359.3 ±0.04―71.4 [email protected] FAAPM0.53 ±0.1679.3 0.5Low ±0.0357.7 0.2cHigh FAALH0.21 ±ControlSerummg/100 FAALH0.22 1.5High FAASerum361.6 FAASerum499.2 a PM, plasma membrane; b Low and high FAA, Cp @ ±2.414.4 ±2•3d14.2 ±65d13.7 ±1.42.1 ±1.12.5 ±2.53.0 ±4.65.5 ±2.26.8 ± ± o.ole60.3 ±0.02e66.5 ±[email protected] ml 289.7 ±32.741.2 1.6Low LH, 0.05 liver cholesterol, cholesterol as mole % of total lipidPhospholipid: ± ± ± ± ±2.324.7 ± ±[email protected] ±1.9e22.7 ± ±291d41.6 ±0.227.4 ±2.1 homogenate. and 0.3 g FAA/kg diet, respectively. <0.01. d@ < ep < 0.05. DECEMBER 1970 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1970 American Association for Cancer Research. 2877 N. Chandrasekhara and K. Ananth Narayan comparison with the percentage in membranes from normal livers. However, the percentages of phospholipid in livers of animals fed the carcinogen were only nominally increased in comparison with those in animals with normal livers. On the other hand, there was no change in the percentage of choles terol in the membranes during carcinogenesis, although there was an increase in the percentage of cholesterol in the liver. The above changes were also reflected in a higher mole ratio of phospholipid:cholesterol in these membranes. The lipid: protein ratio decreased significantly in the livers of animals fed the carcinogen. There was a marked increase in the total lipids of serum in FAA-fed animals, but little change was seen in the percentage of phospholipid or cholesterol. The fatty acid composition of the total lipids of plasma membranes, livers, and sera is given in Table 3. No marked difference was observed in the fatty acid composition of livers from the 3 groups of animals. However, there was a significant increase (p < 0.05) in the total polyunsaturated fatty acids and a corresponding decrease in total saturated fatty acids in mem branes from FAA-fed rats in comparison with the fatty acid composition in membranes from normal rats. An increase in 18: 1 and 18:2 acids and a decrease in 18:0 and 20:4 acids were observed in the sera of animals receiving the carcinogen. However, there was no difference between the sera of the control and experimental groups of animals when the satu rated and the polyunsaturated acids were taken into considera tion. In studies with plasma membranes from EFA-deficient rats Table 3 Fatty acid composition ofthe total lipids ofserum, liver, and plasmamembranes Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats), except in the case of serum from preparations.DietSampleFatty the high FAAgroup, in which the results wereobtained from4 total16:016:118:018:118:220:4Control 1.8Control High FAAPM@ PM PM25.4 ±4.0 ±1.5 ±0.8 ±1.4 ±0.8 ±3.0 25.5 ± 18.9 ±2.5 2.7 ±O.8@ 18.8 ±1.3 11.3 ±0.8 20.7 ±0.6c 21.8 ±[email protected] 2.3 ±0•8d19.416.8 ±[email protected] 11.1 ±1.517.6 21.8 ±2.0'@20.323.7 ± ±1.2 ±0.5 Low FAA LH 22.3 ± 2.5 3.8 ±1.6 2.9Control High FAALH LH23.0 21.7 ±3.03.3 3.2 ±0.711.6 Low FAA High FAASerum Serum Serum19.0 ±1.7 19.4 ±0.8 18.8 ±1.02.8 ±0.5 3.5 ±O.S@ 2.2 ±0.48.2 Low FAAb @ acid as% of a PM, plasma membrane; b Low and high FAA, Cp ±1.8 12.3 ±0.6 13.6 ±0.914.8 ±2.0 21.3 ±4.0 16.4 ±0.8―25.224.2 ±2.320.5 ±1.1 ±2.0 16.5 ±1.3 20.8 ±4.3 ±1.4 ±1.3 ±0.6 16.4 ±0.8'@ 32.2 ±2.l@ 6.9 ±O.6@ 1.2e26.6 6.8 ±0.le13.4 15.5 ±[email protected] 19.1 ± ±3.1 20.7 ±13d 22.3 ±1.8 LH, liver homogenate. 0.05 and 0.3 g FAA/kg diet, respectively. <0.05. dp < 0.01. ep < 0.001. Table 4 Propertiesof the floating fraction Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats). The results for the floating fraction from EFA-deficient rats were obtained from 5 preparations. formedSample(jsmoles/mgInorganic phosphate protein/hr)Total lipid ATPasePlasma 0.10fedmembranes from ± 5.2 34.5 ±3.80.59 ±2.3 20.9 ±1.51.4 20.9 ±7.9 39.7 ±9.10.64 rats21.2 as as cholesterol, % of total lipidPhospholipid: % of total lipidCholesterol mole (mg/mg protein)Phospholipid ratio5'-Nucleotidase ±0.0653.9 ± 6.718.3 ±2.31.5 ± ± 2.416.5 ±1.70.5 ± diet(17 0.3 g FAA/kg diet)Floating weeks on 0.10fed fraction from rats15.0 diet(17 0.3 g FAA/kg diet)Floating weeks on 0.77deficient fraction from EFA rats'@ a These data are from a previous experiment 2878 ± 0.1016.5 ± 0.0732.3 ±1 1.77.3 ±1.02.2 ± (5). CANCER RESEARCH VOL. 30 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1970 American Association for Cancer Research. Hepatoma Plasma Membranes (5) as well as in the a considerable present amount experiments, of material it was observed floated that on top of sucrose with a density of 1.16. In comparison, 5 to 10 mg protein collected as float in the case of preparations from livers of both EFA-deficient and FAA-fed rats, whereas about 1 mg protein floated at this density in normal preparations. The properties of the floating fraction (obtained in a subsequent experiment) are summarized in Table 4. The floating layer was characterized by fairly high activities of 5'-nucleotidase and ATPase and low activities of glucose 6-phophatase and succinic dehydrogenase. However, the lipid:protein ratio was much higher, and the percentage of phospholipid was much lower in this fraction than in the corresponding plasma membranes. The increased yield of plasma membranes and the presence fraction in preparations from preneoplastic and neoplastic livers, in comparison with the yields in preparations from normal livers, may perhaps be due to the presence of increased quantities, of membrane precursors. Whether similar increased yields would be obtained with rapidly growing normal tissue, like the regenerating liver, needs investigation. It is fairly well established that 5'-nucleotidase is a useful plasma membrane marker (12, 13). Although the total activity in the membrane isolated under our experimental conditions is only a small percentage of the total 5'-nucleotidase activity of the liver, its presence at the cell surface is important in view of the regulatory role of AMP in key enzyme systems. The ob served decrease in 5'-nucleotidase activity of membranes from FAA-fed rats may therefore be of greater significance than is recognized at the present time. In agreement with the earlier observations of Narayan (17), we observed an increase in the total lipid content of serum in the FFA-fed animals. However, the lipid content of preneo plastic and neoplastic livers was significantly less than that of normal liver. We also observed a marked change in the phos pholipid: cholesterol ratio and a shift towards polyunsaturated fatty acids in the total lipids of membranes from livers of rats fed FAA. It has been shown that the permeability of lipo somes is influenced by the cholesterol content (7, 8) and the degree of unsaturation of the fatty acid moiety of the lecithin (9). The importance of cholesterol and phospholipids in mem branes has also been demonstrated in studies concerning the mode of action of polyene antibiotics (10). Therefore, it is possible that the changes observed in the lipid composition of hepatoma membranes may have a bearing on the increased leakiness of the plasma membranes of cancer cells (24). Emmelot and Bos (13) reported that hepatoma plasma membranes had a lower density than normal liver plasma mem branes, but we have observed that membranes from livers of FAA-fed rats, as well as those from normal rats, floated be tween d 1.16 andd 1.18. However, a floating fraction(d< 1.16) was always observed in preparations from animals fed the car cinogen. On the basis of the density of this fraction (d < 1.16), the reported density of 1.13 for the outer mito chondrial membrane (19), and our similar results from the DECEMBER total lipid:protein and a considerably lower content distinguished the floating fraction membranes. Recently, pholipid plasma ratio, Evans (14) 1970 reported phos from the sub fractionation of normal plasma membranes (d 1.18) into a low-density (d 1.12) vesicular fraction and a high-density (d 1.2 1) sheet-like fraction, which differed from each other in chemical composition and enzyme activities. Whether plasma membranes from livers of FAA-fed rats are more susceptible to subfractionation is worthy of consideration. In conclusion, it may be stated that important alterations take place in the lipid composition DISCUSSION of the floating preparation of plasma membranes from EFA-deficient rats (5), the possibility that the fraction was the outer mitochondrial membrane was considered. Assay for monoamine oxidase ruled out this suggestion . A lower ATPase activity, a higher of rat liver plasma mem branes during carcinogenesis, and these lead to subtle changes in the properties of the plasma membrane. Whether membrane proteins are also similarly altered in neoplasia deserves to be investigated. REFERENCES 1. Abercrombie, M., Heaysman, J. E. M., and Karthauser, H. M. Social Behaviour of Cells in Tissue Culture. III. Mutual Influence of Sarcoma Cells and Fibroblasts. Exptl. Cell Res., 13: 276—291, 1957. 2. Ambrose, E. J., James, A. M., and Lowick, J. H. B. Differences between the Electrical Charge Carried by Normal and Homologous Tumor Cells.Nature, 177: 576—577,1956. 3. Bosmann, H. B., Hagopian, A., and Eylar, E. H. Cellular Mem branes: The Isolation and Characterization of the Plasma and Smooth Membranes of HeLa Cells. Arch. Biochem. Biophys., 128: 51—69,1968. 4. Burger, M. M. A Difference in the Architecture of the Surface Membrane of Normal and Virally Transformed Cells. Proc. Natl. Acad. Sci. U. S., 62: 994—1001, 1969. 5. Chand.rasekhara, N., and Narayan, K. A. Rat Liver Plasma Mem branes in Essential Fatty Acid Deficiency. J. Nutr., 100: 477—480, 1970. 6. Chandrasekhara, N., and Narayan, K. A. Rat Liver Plasma Mem branes in Chemical Carcinogenesis. Federation Proc., 29: 865, 1970. 7. De Gier, J., Mandersloot, J. G., and Van Deenen, L. L. M. Lipid Composition and Permeability of Liposomes. Biochim. Biophys. Acta, 150: 666—675,1968. 8. Dc Gier, J., Mandersloot, J. G., and Van Deenen, L. L. M. 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