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[CANCER RESEARCH 34, 1813—1815,August 1974] Synthesis of a-Fetoprotein by Membrane-bound Polysomes of Rat Ascites Hepatoma Cells1 Koichi Kanai,2 Yasuo Endo, Toshitsugu Oda, and Naomi Tanaka The First Department of Medicine, Faculty of Medicine, University of Tokyo, Hongo 7-3-I, Bunkyo-ku, Tokyo, Japan by hemolysis. Less than 5% of the cells were stainable by trypan blue after hemolysis, and the tumor cells comprised Free and membrane-bound polysomes prepared from rat about 98% of the total cell population. Chemicals. Sodium deoxycholate and Freund's adjuvant ascites hepatoma cells (AH66) incorporated leucine-14C into a-fetoprotein in vitro. The amount of radioactivity were purchased from Difco Laboratories, Inc., Detroit, incorporated into antiserum-precipitable a-fetoprotein frac Mich.; ATP, GTP, phosphoenolpyruvate, and PK were tion by membrane-bound polysomes was 20 to 90 times from Boehringer Mannheim, Inc., Mannheim, Germany; and EDTA and leucine-14C (240 mCi/mmole, uniformly higher than that by free polysomes, indicating that a-feto protein is mainly synthesized on membrane-bound poly labeled) were the products of Daiichi Pure Chemicals Co., Tokyo, Japan. The 2,5-bis[2-(5-/er/-butylbenzoxazolyl)]somes in rat ascites hepatoma cells. thiopene was obtained from Packard Instrument Co., Downers Grove, 111. INTRODUCTION Antisera. Antiserum against rat AFP was prepared by immunizing rabbits with purified rat AFP (a gift of Dr. Since Abelev et al. (2) reported its appearance in sera of of Hokkaido University) emulsified in complete hepatoma-bearing mice, detection of AFP3 has become an Watabe Freund's adjuvant. Four injections were made at 2-week important aid to the diagnosis of primary liver cancer (1,3, intervals. The total amount of rat AFP used for immuniza 5, 19). Although there are several reports concerning the tion was 0.5 mg. Rabbits were bled 10 days after the last production of AFP in hepatoma cells (2, 6, 8, 13, 15, 20), immunization. little is known about the precise mode of AFP synthesis, its Anti-rat AFP serum contained no detectable antibodies regulation mechanism, or the biological function of AFP. to adult rat serum by double diffusion in agar and gave a In mammalian cells there are 2 types of polysomes, free single precipitation line against fetal rat serum and amand membrane-bound. Although the functional differences nionic fluid, as well as purified rat AFP. Immunoelectrobetween these 2 polysomes are not yet completely eluci phoresis compared with the precipitation in an adjacent dated, increasing evidence suggests that free polysomes trough produced by an antiserum against rat serum revealed mainly synthesize proteins that are utilized within the cells; the antiserum reacted with a protein that had the membrane-bound polysomes, on the other hand, synthesize that mobility of an a-globulin. proteins for export (7, 9, 12, 14, 16, 17, 18, 21). Antisera against rat serum was prepared by the same In the present study, AFP-synthesizing ability of free and method, normal rat serum as an antigen. Normal membrane-bound polysomes of rat ascites hepatoma AH66 rabbit IgGwith was prepared by salt fractionation and DEAEwas investigated in an attempt to determine the intracellular cellulose column chromatography (11). Goat antiserum site of AFP synthesis and to obtain some information as to against rabbit IgG was purchased from Dainabot Laborato its biological function. ries, Inc., Tokyo, Japan. To determine the maximum precipitation between rat AFP and its antiserum, 10 to 200 /ig of rat AFP were mixed MATERIALS AND METHODS with increasing amount of antiserum. After the mixture was Animals and Hepatoma Cells. AH-66 rat ascites hepatoma left overnight at 4°,the amount of precipitate was mea cells that produce AFP were transplanted i.p. to male sured. The maximum precipitation was obtained when 0.4 Donryu rats weighing 100 to 150 g. Ascites cells were ml of antiserum was added to 50 /ig of rat AFP. Preparation of Polysomes. Free and membrane-bound collected by peritoneal puncture 7 days after inoculation of polysomes were prepared from the tumor cells as follows. the tumor cells. Contaminating erythrocytes were removed Ten to 15 g of packed cells obtained from 20 tumor-bearing 1This work was partly supported by a Grant-in-Aid from the Ministry rats were homogenized with 3 volumes of Medium A containing 50 mM Tris-HCl (pH 7.6), 50 mM KC1, 5 mM of Education, Japan. 2To whom requests for reprints and communications should be MgCl2, and 5 mM mercaptoethanol in 0.25 Msucrose. After addressed. centrifugation at 10,000 x g for 15 min, 2 ml of the 3The abbreviations used are: AFP, a-fetoprotein; PK, pyruvate kinase; postmitochondrial supernatant were layered on a top of TCA, trichloroacetic acid. Received October 2, 1973; accepted March 6, 1974. discontinuous sucrose gradient consisting of 1.5 ml of 2 M SUMMARY AUGUST 1974 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research. 1813 K. Kanai el al. sucrose in Medium A and 1.5 ml of 0.5 M sucrose in Medium A in a 6-ml centrifuge tube. Centrifugation was performed in a Hitachi Model 55 centrifuge at 160,000 x g for 3.5 hr at 4°.Free polysomes were precipitated as clear pellets at the bottom of the tubes, and membrane-bound polysomes were sedimented as a turbid zone between 2 and 0.5 M sucrose at the end of centrifugation. The fraction containing membrane-bound polysomes was collected and 5% sodium deoxycholate was added to the fraction to a final concentration of 1.3% to remove membrane fraction from the polysomes. Four ml of the fraction treated with sodium deoxycholate were placed on 1 ml of l Msucrose in Medium A, and the 2nd centrifugation at 160,000 x g for 60 min was carried out to precipitate bound polysomes. Free polysomes were used without further purification. The A260/A280 ratio of these 2 polysome preparations ranged between 1.75 and 1.82. The supernatant fraction was prepared from normal rat liver according to the method of Kashiwagi et al. (IO). Cell-free Protein-synthesizing System. Free and mem brane-bound polysomes were tested for their protein-synthe sizing ability in a cell-free system containing 1 mM ATP, 0.25 mM GTP, 10 mM phosphoenolpyruvate, 100 ¡igof PK, 6 mg of supernatant proteins, 2.5 ¿¿Ci of leucine-14C, and 1 to 2 mg of polysomes in Medium A. The total volume of the reaction mixture was 2 ml. Incubation was carried out at 37°for the period as indicated. In some experiments, EDTA was added to the reaction mixture after incubation, and the incubation was continued for an additional 10 min. The reaction mixture was then centrifuged at 160,000 x g for 60 min and the supernatant was analyzed for radio activity. Measurement of Radioactivity. Nonspecific precipitates were first removed from the supernatant 3 times with rabbit IgG and its antiserum. The final supernatant was divided into 2 equal parts to which were added, to Part A, 50 ßg of rat AFP and 0.4 ml of its antiserum and, to Part B, 50 jig of rabbit IgG and 0.4 ml of its antiserum. They were left overnight at 4°after 30 min of incubation at 37°.The amount of precipitates generated in these 2 parts was about the same. The precipitated proteins were washed 5 times with cold 0.9% NaCl solution and dissolved in 0.3 ml of 88% formic acid. After adding 10 ml of scintillation fluid containing 0.5% 2,5-bis[2-(5-fm-butylbenzoxazolyl)]thiopene in 50% toluene-50% methylcellusolve, radioactivity was measured or? a Packard Tri-Carb Model 3320 liquid scintillation counter. To calculate the radioactivity of AFP, the count, Part A, was subtracted from the count, Part B, in order to exclude the possible contamination by nonspecific coprecipitation, expressed as the radioactivity of AFP. For the determination of total-protein radioactivity, proteins were precipitated from the supernatant by 10% TCA, heated at 95°for 20 min, then washed 5 times with 5% TCA. The precipitates were dissolved in 0.3 ml of formic acid, and radioactivity was counted as described above. RESULTS AND DISCUSSION The incorporation time of leucine-14C into total proteins of the tumor cells is presented in Chart 1. Radioactivity 1814 increased rapidly for the 1st 20 min and reached a plateau at about 40 min in both free and membrane-bound polysomes. It was also observed that the activity of free polysomes which were prepared without sodium deoxycholate treat ment was apparently higher than that of membrane-bound polysomes treated with sodium deoxycholate in agreement with the results of other investigators (4, 6). When EDTA was added, the radioactivity of the supernatant increased by 50% in both free and bound polysomes. The relative amount of AFP released from the polysomes was not altered by EDTA. It was also confirmed, in our preliminary experiment of a similar line, that membrane-bound polysomes prepared from rat liver incorporated leucine-14C into serum albumin, whereas free polysomes did not (unpublished observation). Synthesis of AFP by hepatoma cells has been reported (1, 15, 20), while the presence of AFP in hepatoma cells has also been confirmed morphologically (6, 8, 13). According to Nishioka et al. (13), AFP was most commonly found in the cytoplasm of tumor cells. However, the intracellular site r E lulu' 20 40 60 mm Chart 1. Incorporation of leucine-"C into proteins by free and mem brane-bound polysomes of rat ascites hepatoma cells. Two mg of free (O) or bound (•)polysomes were incubated in a cell-free system containing 1 mMATP, 0.25 m.MGTP, 10 mMphosphoenolpyruvate, 100jig of PK, 6 mg of supernatant proteins, and 2.5 /iCi of leucine-"C at 37°.Reaction was stopped by 5% TCA, and the radioactivity of the precipitated proteins was counted as described in "Materials and Methods." Table 1 Incorporation ofleucine-"C into AFP by free and bound polysomes of rat ascites hepatoma cells One to 2 mg of free or bound polysomes were incubated at 37°for 60 min in complete reaction mixture with 2.5 nCt of leucine-"C. After incubation, EDTA was added to a final concentration of 15 mM. The reaction mixture was centrifuged at 160,000 x g for 60 min, and the supernatant was analyzed for radioactivity of total proteins and AFP as described in "Materials and Methods." Radioactivity (cpm/mg rRNA) Experiment123PolysomesFree protein (%)0.032.770.08 proteins11,452 4,38412,571 12210 BoundFreeBoundFreeBoundTotal 9,82515,898 7,813AFP3 1527 1.540.04 101AFP/total 1.29 CANCER RESEARCH VOL. 34 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research. AFP Synthesis by Bound Poly somes of Rat Hepatoma of AFP synthesis has not yet been elucidated. As shown in Table 1, incorporation of leucine-14C into the AFP fraction by membrane-bound polysomes was about 20 to 90 times higher than that by free polysomes. These results indicate clearly that AFP is synthesized mainly on mem brane-bound polysomes in rat ascites hepatoma cells, suggesting that AFP in hepatomas belongs to "excretory proteins" such as serum albumin. We do not know, however, how the synthesis of AFP is regulated in hepatoma cells and this, as well as its biological function, should be the focus of further studies. ACKNOWLEDGMENTS The authors are grateful to Professor T. Higashi, Department of Biochemistry, Showa University, for helpful discussions during the course of the study; to Dr. H. Watabe, Department of Biochemistry, Hokkaido University, for supplying us with purified rat AFP, as well as AH-66 hepatoma cells; and to M. Tamoto for excellent technical assistance. REFERENCES 1. Abelev, G., Assecritova, I., Kraevsky, N., Kraevsky, A., Perova, S. D., and Perevodchikova, N. I. Embryonal Serum a-Globulin in Cancer Patients: Diagnostic Value. Intern. J. Cancer, 2: 551-557, 1967. 2. Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A., and Irlin, I. S. Production of Embryonal Alpha-Globulin by Transplantable Mouse Hepatoma. Transplantation, /: 174-180, 1963. 3. Alpert, M., Uriel, J., and de Nechaud, B. Alpha-1-Fetoglobulin in the Diagnosis of Human Hepatoma. New Engl. J. Med., 278: 984 986, 1968. 4. Bloemendal, H., Bont, W. S., de Vries, M., and Benedetti, E. L. Isolation and Properties of Polyribosotnes and Fragments of the Endoplasmic Reticulum from Rat Liver. Biochem. J., 103: 177-182, 1967. 5. Endo, Y., Kanai, K., lino, S., and Oda, T. Clinical Significance of a-Fetoprotein. Alpha-Fetoprotein and Hepatoma. Gann Monograph, 14: 67-78, 1973. 6. Engelgardt, N. V., Goussev, A. I., Shipova, L. J., and Abelev, G. I. Immunofluorescent Study of Alpha-fetoprotein (afp) in. Liver and Liver Tumours. I. Techniques of afp Localization in Tissue Sections. Intern. J. Cancer, 7: 198 206, 1971. AUGUST 7. Ganoza, C. M., and Williams, C. A. In Vitro Synthesis of Different Categories of Specific Protein by Membrane-bound and Free Ribosomes. Proc. Nati. Acad. Sei. U. S., 63: 1370-1376, 1969. 8. Goussev, A. L, Engelgardt, N. V., Masseyeff, R., Camain, R., and Basteris, B. Immunofluorescent Study of Alpha-fetoprotein (afp) in Liver and Liver Tumours. II. Localization of afp in the Tissues of Patients with Primary Liver Cancer. Intern. J. Cancer, 7: 207-217, 1971. 9. Hicks, S. J., Drysdale, J. W., and Munro, H. N. Preferential Synthesis of Ferritin and Albumin by Different Populations of Liver Polysomes. Science, 164: 584-585, 1969. 10. Kashiwagi, K., Tobe, T., and Higashi, T. Studies on Rat Liver Catalase. V. Incorporation of "C-leucine into Catalase by Isolated Rat Liver Ribosomes. J. Biochem., 70: 785 793, 1971. 11. Levy, H. B., and Sober, H. A. A Simple Chromatographie Method for Preparation of Gamma Globulin. Proc. Soc. Exptl. Biol. Med., 103: 250-252, I960. 12. Nihei, T. In Vitro Amino Acid Incorporation into Myosin by Free Polysomes of Rat Skeletal Muscle. Biochem. Biophys. Res. Com mun., 43: 1139-1144, 1971. 13. Nishioka, M., Ibata, T.. Okita, K., Harada, T., and Fujita, T. Localization of a-Fetoprotein in Hepatoma Tissues by Immunofluorescence. Cancer Res., 32: 162-166, 1972. 14. Redman, C. M. Biosynthesis of Serum Proteins and Ferritin by Free and Attached Ribosomes of Rat Liver. J. Biol. Chem., 244: 4308-4315, 1969. 15. Stanislawski-Birencwajg, M., Uriel, J., and Graber, P. Association of Embryonic Antigens with Experimentally Induced Hepatic Lesions in the Rat. Cancer Res., 27: (Part 1): 1990 1997, 1967. 16. Takagi, M., and Ogata, K. Direct Evidence of Albumin Biosynthesis by Membrane Bound Polysomes in Rat Liver. Biochem. Biophys. Res. Commun., 33: 55-60, 1968. 17. Takagi, M., Tanaka, T.. and Ogata. K. Functional Differences in Protein Synthesis between Free and Bound Polysomes of Rat Liver. Biochim. Biophys. Acta, 217: 148-158, 1970. 18. Tanaka, T., and Ogata, K. Preferential Synthesis of Arginase by Free Polysomes from Rat Liver. J. Biochem., 70: 693-697, 1971. 19. Tatarinov, Y. S. Detection of Embryospecific «-Globulinin the Blood Serum of a Patient with Primary Liver Cancer. Vop. Med. Khim., 10: 90 91. 1964. 20. Watabe, H., Hirai, H., and Satoh, H. a-Fetoprotein in Rats Trans planted with Ascites Hepatoma. Gann, 63: 189-199, 1972. 21. Woodward, W. R., Adamson, S. D., McQueen, H. M., Larson, J. W., Estvanik, S. M., Wilairat, P., and Herbert, E. Globin Synthesis on Reticulocyte Membrane-bound Ribosomes. J. Biol. Chem., 248: 1556-1561, 1973. 1974 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research. 1815 Synthesis of α-Fetoprotein by Membrane-bound Polysomes of Rat Ascites Hepatoma Cells Koichi Kanai, Yasuo Endo, Toshitsugu Oda, et al. Cancer Res 1974;34:1813-1815. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/34/8/1813 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.