Aminopeptidase Activities on the Surface of Mammalian Cells and Their Alterations Associated with Transformation1

[CANCER RESEARCH 38, 3505-3508. October 1978] 0008-5472/78/0038-OOOOS02.00 Aminopeptidase Activities on the Surface of Mammalian Cells and Their Alterations Associated with Transformation1

Takaaki Aoyagi, Machiko Nagai, Minoru Iwabuchi, Wen-Shing Liaw,2 Toshiwo Andoh,2 and Hamao Umezawa Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141, Japan

ABSTRACT such as aminopeptidases, alkaline phosphatase, esterase, and others on the cellular surface. Activities of various hydrolytic enzymes were deter In the present paper we studied the enzyme activities on mined in rat organ homogenates and on the surface of the surface of cultured cells and also in the homogenates cells from various sources, i.e., tumor cell strains, primary of rat organs. In primary cultured or normal cells, the cultured cells, normal cells, and their transformants. Ala- activity of APA was higher than that of APB, whereas in nine, leucine, methionine, phenylalanine, and glycyl-pro- tumor cells the situation was reversed. A drastic decrease line aminopeptidases and esterase showed relatively high in APA among other peptidases was observed when the activities in all these organs and cells. In the kidney cells were transformed by wild-type or fs A mutant of SV40 homogenate the aminopeptidase A activity was higher and maintained at permissive temperature. However, when than that of aminopeptidase B. The situation was reversed the transformed cells were grown at the restrictive temper in other organs; i.e., the aminopeptidase A activity was ature, the ratio approached that of normal cells. Further lower than that of aminopeptidase B. Normal cells derived more, patterns of aminopeptidase activities in various or from kidneys showed the kidney-type pattern of amino gans are discussed in terms of dysdifferentiation of tumor peptidases A and B on the surface of cells, whereas tumor cells. cells from various origins were of another organ type. When cultured mouse fibroblast strain C3H2K and rat fibroblast strain 3Y1 cells were transformed by SV40 or by MATERIALS AND METHODS a fs A mutant and maintained at permissive temperature, Cells. Primary monkey kidney cells were prepared by aminopeptidase A activity was drastically decreased, and trypsinization and were grown in Earle's balanced salt the ratio of aminopeptidase A to aminopeptidase B was solution containing 0.5% lactalbumin hydrolysate (5). A reduced to the levels of tumor cells. If the fs A mutant- continuous line of canine kidney cells (MDCK) was grown transformed cells were grown at the restrictive tempera in Eagle's Medium F11. Mouse L- and FM3A cells were ture, the ratio approached that of normal cells. In normal grown in Eagle's minimal essential medium (3, 19). The cells, however, cultivation at high or low temperature did C3H2K cell line was established by Yoshikura ef al. (24) not cause any change of the activities. from kidney of a newborn C3H/He mouse. The 3Y1-B cell line was established by Kimura ef al. (14) from Fischer rat INTRODUCTION embryo. Clonal lines of these fibroblasts, C3H2K-C4 and 3Y1-B clone 1-6, were transformed respectively, by wild- Previously, we have shown that activities of aminopepti type SV40 strain 777 (W-2K-11) and its fs A mutant fs Â¿900 dases, alkaline phosphatase, esterase, and glycosidases (900-3Y-11) (17). All of these normal and transformed cell were located on the surface of mammalian cells (3, 4). We lines were generously supplied by Dr. K. Oda, Institute of have succeeded in obtaining inhibitors against these en Medical Science, University of Tokyo, Tokyo, Japan. Ehrlich zymes from culture filtrates of the actinomycetes, bestatin, ascites cells, Gardner lymphosarcoma cells, AH66 cells, amastatin, forphenicine, and esterastin (6, 7, 21, 22). These and AH66F cells were collected from the ascitic fluids of inhibitors proved to bind to the cellular surface and modi dd/Y mice and Donryu rats inoculated previously with the fied the fractions of immunoresponsive cells. Bestatin, corresponding cells. The L-cell was established from con found by the study of an inhibitor of APB,3 enhanced nective tissues of the C3H mouse; the FM3A cell was delayed-type hypersensitivity, and amastatin, found as an established from mammary tumor of mouse strain C3H/He. inhibitor of APA, increased the number of antibody-forming Organs, macrophages, and polymorphonuclear leuko cells (1, 20). Furthermore, these inhibitors are presently cytes induced by casein were taken from a 5-week-old under clinical evaluation in cancer Â¡mmunochemotherapy. female Wistar rat. Spleen lymphocytes of mice were ob These data suggest the importance of hydrolytic enzymes tained after removal of adherent cells in plastic Petri dishes according to the method described previously (4). Macro phages, polymorphonuclear leukocytes, AH66 cells, and ' This work was supported partly by National Cancer Institute Contract AH66F hepatoma cells were kindly provided by Dr. A. NO1-CM-57009 from the Division of Cancer Treatment and by the Ministry of Education, Science and Culture of Japan. Matsuda and Dr. O. Yoshioka, Nippon Kayaku Co., Ltd., * Present address: Institute of Medical Science. University of Tokyo. Tokyo,Japan. Shirokanedai, Minato-ku, Tokyo 108. Japan. Commercial Source of Substrates. L-u-Glutamic acid NA 3 The abbreviations used are: APB, aminopeptidase B; APA, aminopepti dase A; NA, 0-naphthylamide hydrochloride. and arginine NA were used for assay of APA and APB. Received February 13. 1978; accepted July 11. 1978. These compounds and L-methionine NA were obtained

OCTOBER 1978 3505

from Schwarz/Mann, Orangeburg, N. Y. Glycine NA, L- reaction the mixture was processed as described previously alanine NA, L-serine NA, L-proline NA, L-valine NA, L-leucine (2, 7, 21, 22), and the absorbance of the final mixture was NA, L-phenylalanine NA, glycyl-L-proline NA, and glycyl-L- determined as follows: 525 nm for the assay of aminopepti prolyl-L-leucine NA were obtained from Bachern Feinchem- dase, 550 nm for the assay of glycosidase, 420 nm for the ikalien AG, Switzerland. p-Nitrophenyl-a-D-glucopyrano- assay of phosphatase, and 400 nm for the assay of esterase. side and p-nitrophenyl-a-D-mannopyranoside were ob Kinetic studies on the enzymes located on the cell surface tained from Calbiochem, San Diego, Calif. p-Nitrophenyl- were shown previously (3, 4). ÃŸ-D-galactopyranoside, p-nitrophenyl-A/-acetyl-/3-D-glucos- Protein Determination. The method of Lowry ef al. (15) aminide, and p-nitrophenyl phosphate were obtained from was used, with bovine serum albumin as the standard. BDH Pharmaceuticals Ltd., Poole, England. p-Nitrophenyl acetate was obtained from Tokyo Kasei Co., Tokyo, Japan. RESULTS Determination of Enzyme Activities. The method was principally the same as that described previously (3). In Comparison of the Enzyme Activities in Homogenates monkey kidney, canine kidney, C3H2K-C4, W-2K-11, 3Y1-B of Rat Organs and Those Located on the Surface of clone 1-6, and 900-3Y-11 cells, enzyme activities were Various Cells. Activities of various kinds of hydrolytic assayed on the monolayer cultures grown in 30-mm Petri enzymes were determined in homogenates of rat organs dishes (2.5 x 10s cells). Culture medium was replaced with and compared with those located on the surface of various 1.5 ml of Hanks' medium without phenol red (pH 7.2), cultured cells (Table 1). containing the respective substrates. In L-, FM3A, lympho On the whole, esterase and certain kinds of aminopepti- cyte, and tumor cells and in rat organ homogenates, en dases, i.e., alanine, leucine, methionine, phenylalanine, zyme activities were assayed in suspension in test tubes and glycyl-proline aminopeptidases, showed rather high (1.5 x 10 cm; 2.5 x 105 cells) with 1.5 ml of the same activities. Although aminopeptidase, phosphatase, and es medium containing the respective substrates. In macro terase activities were always detected, glycosidase activities phage and polymorphonuclear leukocyte cells, enzyme were not detected or were present only at low levels. The activities were assayed in siliconized test tubes (1.5 x 10 homogenate from kidney had rather high activities of various cm; 2.5 x 105 cells). The dishes and test tubes were enzymes, compared to the homogenates from other incubated for 1 hr at 37Â°.After the reaction the supernatant sources. Enzyme activities located on the cell surface were was withdrawn and centrifuged (3000 rpm for 10 min) for rather low in comparison with those detected in the homog further measurements. For aminopeptidase assay the reac enates of rat organs, except for esterase. tion mixture included 0.25 ml of 0.5 rriM /3-naphthylamide It is noteworthy that, in kidney and lung, APA activity was derivatives. For glycosidase assay the reaction mixture higher than APB activity. The higher activity of APA to included 0.05 ml of 50 ITIMp-nitrophenyl derivatives. For APB held for the homogenates of kidney and lung from phosphatase and esterase assay, the reaction mixture in 5-week-old rats and also for the homogenate of kidney cluded 0.03 ml of 50 mw p-nitrophenyl derivatives. After the from 32-week-old rats. The lung homogenate from 32-week-

Table 1 Comparison of the enzyme activities in homogenates of rat organs and those located on the surface of various cells The enzyme activities in the organ homogenates of a 5-week-old female Wistar rat were compared. The homogenates were prepared in a Dounce homogenizer with 200 strokes. Enzyme activities (nmol/min/mg protein)

Rat organs Cells

EnzymesAPAAPBGlycine kidney0.70.20.74.50.40.70.83.66.25.70.91.000002.2113.5Caninekidney0.40.23.04.01.10.51.33.86.65.13.62.300001.365.8FM3A0.32.72.59.31.71.30.26.41.46.50.20.70.200.20.23.715.8

aminopeptidaseAlanine aminopeptidaseSerine aminopeptidaseProline aminopeptidaseValine aminopeptidaseLeucine aminopeptidaseMethionine aminopeptidasePhenylalanine aminopeptidaseGlycylproline aminopeptidaseGlycylprolylleucineaminopeptidasea-D-Glucosidasej3-D-GalactosidaseÂ«-D-MannosidaseA/-Acetyl-0-D-glucosaminidasePhosphataseEsteraseKidney49.1Â°15.919.7107.84.10.81.658.6135.1102.1168.130.70.91.60.61.64.9413.8Lung12.69.11.310.20.40.60.47.210.98.527.03.20.30.10.20.41.8303.1Liver3.56.90.96.90.50.75.05.412.96.815.13.50.60.10.40.51.1406.1Spleen0.28.30.35.20.10.52.24.36.84.111.51.90.30.20.30.51.623.9Pancreas1.38.60.98.00.10.80.13.66.56.32.90.50.10.10.20.10.4122.9Monkey

" Each figure represents the average of triplicate experiments.

3506 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Surface Aminopeptidase Activities in Transformants old rats showed a higher activity of APB to APA. In other tured at permissive temperature, there was a drastic de organs the effect of age on the ratio of APA activity to APB crease in APA activity in comparison with their parental activity was not observed. cells. On the surface of monkey and canine kidney cells, APA Furthermore, it is of particular interest that the activity of activity was higher than APB activity, which seemed to APA in cells transformed by the ts A mutant and maintained reflect the activities in the kidney tissue from which these at the restrictive temperature exhibited a relatively high cells were obtained. In contrast, APA activity in FM3A was level approaching that of untransformed cells, compared lower than was APB activity. with the low level at the permissive temperature. When the mutant-transformed cells were passaged twice at 40.5Â°and Comparison of APA and APB Activities Located on the Cell Surface. Next, we focused our attention on the ratio of then plated at 40.5Â°,the cells in the dishes showed relatively APA activity to APB activity and surveyed the surface of high activity of APA, as shown in Table 3. When the cells various cells (Table 2). It is noteworthy that, in L-, lympho were passaged only once, intermediate value was obtained. cyte, macrophage, and polymorphonuclear leukocyte cells In contrast, normal cells showed a high APA activity and in all of the tumor cell lines tested, APB activities were whether the cells were maintained at high or low tempera always higher than were APA activities. This relation tures, and the relationship of APA and APB remained ship of APA and APB generally held in all kidney-type cells; constant. it would be interesting to know the effect of viral transfor mation on the relationship. Viral transformation is known to DISCUSSION cause several changes in the cellular physiology. Comparison of Enzyme Activities on the Cell Surface of Cell transformation by tumor viruses causes changes in Normal and Transformed Cells. We tested the relationship the pattern of growth, morphology, and other physiological of APA and APB in cultured normal cells and transformed characteristics. Also, there have been many reports de derivatives. Table 3 shows enzyme activities on the cell scribing a change in enzyme activity or the amount of surface of normal C3H2K cells, 3Y1 cells, and their SV40 specific protein on the surface of cells in association with transformants. The pattern of enzyme activities in normal transformation (10-12, 16, 18, 23). These studies led one to C3H2K-C4 and 3Y1-B clone 1-6 cells was the same as that consider that alteration of cell surface properties may of monkey and canine kidney cells, whether the cells were account for the altered behavior of the transformed cells. cultured at 35 or 37Â°,and the relationship of high APA and Previously, we reported that appreciable amounts of low APB again held in these cells. When the cells were aminopeptidases, phosphatase, and esterase with a variety transformed by wild-type SV40 or by is A mutant and cul- of specificities are localized on the surface membrane of a variety of mammalian cells (3, 4). In the present paper we Table 2 surveyed the enzyme activities in the homogenate of organs Comparison of APA and APB located on the cell surface of young rats (5 weeks old) and noticed that, in kidney and Specific activity (nmol/min/mg lung, APA activity is higher than is APB activity. The reverse protein) is the case with other organs. The ratio of APA activity to APB activity in kidney was constant without regard to the CellsLymphocyte Â±0.01" age of rats. In contrast, in lung of older rats (32 weeks old), APB activity was higher than APA activity, as was ob Macrophage 0.8 Â±0.03 4.8 0.21 Polymorphonuclear leukocyte 0.2 Â±0.01 3.6 0.30 served with other organs of young or old rats. Therefore EhrlichGardner 0.6 Â±0.030.4 14.72.30.460.24 kidney seems to have a distinguishing characteristic from lymphosarcoma Â±0.02 other organs. The same relationship holds for cultured cells AH66AH66FMonkey 3.5 Â±0.300.5 8.46.50.20.310.280.02from monkey and canine kidney. Â±0.090.7 kidney Â±0.04 All of the tumor cells derived from various organs and of Canine kidney 0.4 Â±0.02 0.22.10.020.02 various characteristics were found to possess APA activity L-cellFM3AAPA0.1 0.3 Â±0.020.3 0.252.7 that was lower than APB activity. Remarkably, C3H2K and Â±0.02APB0.7 0.21 3Y1 cells established from mouse kidney and rat embryo " Average Â±S.D. of 6 separate experiments. showed a higher APA activity than APB activity that was of

Table 3 Comparison of enzyme activities on the cell surface of normal and transformed cells Enzyme activities (nmol/min/mg protein)

C3H2K 3Y1 1-635Â°1.8-Belone "35Â°0.2 1 (37Â°)1.0 (37Â°)0.1 APA Â±0.14* Â±0.02 Â±0.10 Â±0.16 Â±0.02 Â±0.18 APBC3H2K-C40.6 Â±0.03W-2K-110.3 Â±0.023Y1 0.4 Â±0.0540.5Â°1.60.4 Â±0.05900-3Y-10.5 Â±0.0340.5Â°0.70.4 Â±0.03 " 900-3Y-11 cells were passaged twice at 40.5Â°and plated at 40.5Â°before determining the activities. '' Average Â±S.D. of 6 separate experiments.

OCTOBER 1978 3507

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. T. Aoyagi et al. kidney type but, on transformation by SV40, the APA activity ase Activities in Liver Cells of Developing Chicken Embryos. Mol. was drastically reduced and became another organ type. Cellular Biochem., 10: 81-95. 1976. 9. Fishman, P. H., McFarland, V. W., Mora, P. T., and Brady, R. O. Furthermore, it is of particular interest to note that the Ganglioside Biosynthesis in Mouse Cells: Glycosyltransferase Activities activity of APA in cells transformed by fs A mutant exhibited in Normal and Virally-transformed Lines. Biochem. Biophys. Res. Com mun. .48: 48-57, 1972. a low level at the permissive temperature, which turned to a 10. Goldberg, A. R., Wolf. B. A., and Lefebvre, P. A. Plasminogen Activators higher level after a few passages at the restrictive tempera of Transformed and Normal Cells. In: E. Reich, D. B. Rifkin, and E. Shaw ture. This finding suggests that the gene A function of the (eds.), Proteases and Biological Control, pp. 857-868. Cold Spring Har bor. N. Y.: Cold Spring Harbor Laboratory, 1975. virus may lead to the alteration of the surface enzyme. 11. Hynes, R. 0. Cell Surface Proteins and Malignant Transformation. Many reports have described a loss or a decrease of Biochim. Biophys. Acta, 458. 73-107, 1976. specific proteins or enzymes on the surface of cells in 12. Karasaki, S., and Okigaki. T. Surface Membrane Nucleoside Triphospha- tase Activity and Tumorigenicity of Cultured Liver Epithelial Cells. association with transformation, i.e., a large external trans Cancer Res., 36. 4491-4499, 1976. formation-sensitive protein (11), a protein with a molecular 13. Kijimoto, S., and Hakomori, S. Enhanced Glycolipid: Galactosyltransfer- ase Activity in Contact-inhibited Hamster Cells, and Loss of This Re weight of 142,000 to 145,000 (23), and glycosyltransferases sponse in Polyoma Transformants. Biochem. Biophys. Res. Commun.. (9, 13). APA as shown in the present paper seems to belong 44:557-563,1971. 14. Kimura, G . Itagaki, A., and Summers. J. Rat Cell Line 3Y1 and Its to this category of enzymes. On the other hand some Virogenic Polyoma- and SV40-transformed Derivatives. Intern. J. Can surface proteins or enzymes are reported to increase upon cer, 75:694-706, 1975. transformation (12, 16, 18). Similar transformation-associ 15. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L.. and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 793: 265- ated changes have often been observed with a variety of 275,1951. intracellular enzymes in tumor cells and have been re 16. Onodera, K.. Yamaguchi, N., Kuchino, T., and Aoi. Y. Alterations in garded as dysdifferentiation of tumor cells. Surface Glycoproteins and Level of Sialyltransferase of Cells Trans formed by a Temperature-sensitive Mutant of Simian Virus 40. Proc. Nati. Acad. Sei. U. S., 73: 4090-4094, 1976. REFERENCES 17. Segawa. K.. Yamaguchi, N., and Oda, K. Simian Virus 40 Gene A Regulates the Association between a Highly Phosphorylated Protein and 1. Aoyagi, T., Ishizuka, M., Takeuchi, T., and Umezawa, H. Enzyme Chromatin and Ribosomes in Simian Virus 40-transformed Cells. J. Inhibitors in Relation to Cancer Therapy. Japan. J. Antibiotics, 30 Virol.,22: 679-693, 1977. (Suppl.): 121-132, 1977. 18. Stone, K. R., Smith, R. E., and Joklik, W. K. Changes in Membrane 2. Aoyagi. T., Nerome, K., Suzuki, J., Takeuchi, T., and Umezawa, H. Polypeptides That Occur When Chick Embryo Fibroblasts and NRK Cells Change of Enzyme Activities during the Early Stage of Influenza Virus Are Transformed with Avian Sarcoma Viruses. Virology, 58: 86-100, Infection. Biochem. Biophys. Res. Commun., 60: 1178-1184, 1974. 1974. 3. Aoyagi, T., Suda, H., Nagai, M.. Ogawa. K.. Suzuki, J.. Takeuchi, T., and 19. Tobita, K., Sugiura, A., Enomoto, C.. and Furuyama, M. Plaque Assay Umezawa, H. Aminopeptidase Activities on the Surface of Mammalian and Primary Isolation of Influenza A Viruses in an Established Line of Cells. Biochim. Biophys. Acta, 452: 131-143, 1976. Canine Kidney Cells (MDCK) in the Presence of Trypsin. Med. Microbiol. 4. Aoyagi, T., Suda, H., Nagai, M., Tobe, H., Suzuki, J., Takeuchi, T., and Immunol., 762: 9-14,1975. Umezawa, H. Release of a Plasma Membrane-bound Triaminopeptidase 20. Umezawa, H. Recent Advances in Bioactive Microbial Secondary Metab Activity from Mammalian Cells by Thermolysin. Biochem. Biophys. Res. olites. Japan. J. Antibiotics, 30 (Suppl.): 138-163, 1977. Commun., 80. 435-442, 1978. 21. Umezawa. H., Aoyagi, T., Hazato, T., Uotani, K., Kojima, F., Hamada, 5. Aoyagi. T., Suzuki. J., Nerome, K.. Nishizawa, R., Takeuchi, T., and M., and Takeuchi, T. Esterastin, an Inhibitor of Esterase, Produced by Umezawa. H. Sialic Acid Residues Exposed on Mammalian Cell Surface: Actinomycetes. J. Antibiotics, 3Ã•: 639-641, 1978. The Effect of Adsorption of Denatured Virus Particles. Biochem. Bio 22. Umezawa, H., Aoyagi. T., Suda. H., Hamada, M., and Takeuchi, T. phys. Res. Commun.. 57: 271-278, 1974. Bestatin, an Inhibitor of Aminopeptidase B, Produced by Actinomycetes. 6. Aoyagi. T., Tobe, H., Kojima, F., Mamada, M., Takeuchi, T., and J. Antibiotics. 29: 97-99, 1976. Umezawa, H. Amastatin. an Inhibitor of Aminopeptidase A Produced by 23. Vahaeri, A., and Rouslahti. E. Disappearance of a Major Cell-Type Actinomycetes. J. Antibiotics, 31: 636-638, 1978. Specific Surface Glycoprotein Antigen (SF) after Transformation of 7. Aoyagi, T.. Yamamoto. T., Kojiri, K., Kojima, F., Mamada, M., Takeuchi, Fibroblasts by Rous Sarcoma Virus. Intern. J. Cancer. 73: 579-586, T., and Umezawa. H. Forphenicine, an Inhibitor of Alkaline Phosphatase 1974. Produced by Actinomycetes. J. Antibiotics, 37: 244;en246, 1978. 24. Yoshikura, H., Hirokawa, Y.. and Yamada, M. Synchronized Cell Division 8. Arnold. D.. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Aminopeptidase Activities on the Surface of Mammalian Cells and Their Alterations Associated with Transformation

Takaaki Aoyagi, Machiko Nagai, Minoru Iwabuchi, et al.

Cancer Res 1978;38:3505-3508.

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