[CANCER RESEARCH 38, 1258-1262, May 1978] Decrease in 5'-Nucleotidase Activity in Malignant Transformed and Normal Stimulated Cells

A. Raz,' J. G. Collard, and M. Inbar

Department of Membrane Research, The Weizmann Institute of Science, Rehovot, Israel [A. R., M. I.], ano Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands ¡J.G. C.]

ABSTRACT MATERIALS AND METHODS

Analysis of six different cell types of normal and trans Normal and Leukemic Lymphocytes. Normal lympho formed fibroblasts grown in vitro and of four different cell cytes were obtained from lymph node or thymus of A and types of normal and leukemic lymphocytes grown in vivo C57BL/6 mice. Cells were collected by teasing the tissue have shown a marked decrease of 3- to 30-fold in the apart and allowing the pieces to sediment (14). The malig specific activity of 5 -nucleotidase in the malignant cells nant lymphoma cells were obtained from an ascites form of as compared to their normal parental cells. The results Moloney virus-induced lymphoma growing in A mice and have also indicated that a serum stimulation of untrans- from an ascites form of a methylcholanthrene-induced formed or normal fibroblasts and a stimulation of normal lymphoma growing in C57BL/6 mice. Normal and leukemic lymphocytes by concanavalin A resulted in a significant lymphocytes were freshly obtained from mice, and cells decrease in the specific activity of 5 -nucleotidase of the were washed twice with 0.15 M NaCI and used immediately stimulated cultures as compared to the resting cells. In in the experiments. both the malignant cells and the stimulated normal cells, Normal and Transformed Fibroblasts. Secondary cul the decrease in 5 -nucleotidase activity was not accom tures of hamster embryo and rat embryo cells and a line of panied by a similar decrease in the specific activity of Swiss albino mouse (3T3)3 were used as normal and un- acid phosphatase, indicating a specific enzyme alteration transformed fibroblasts. Lines of 3T3/SV40, hamster cells in the surface membranes of the transformed and the transformed by polyoma, and rat cells tranformed by poly- normal stimulated cells. oma were used as transformed fibroblasts. Normal and transformed fibroblasts were grown in vitro in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf INTRODUCTION serum (15). For the experiments 2 x 105 cells in 2 ml It is now well recognized that malignant transformation medium with serum were seeded in 35-mm Falcon plastic of normal cells is accompanied by changes in the surface tissue culture dishes, and the cultures were grown for 24 hr in a CC-2 incubator at 37°.Cell monolayers were then properties of the transformed cell. A great deal of experi mental evidence has been accumulated in recent years in washed twice with 0.15 M NaCI and used immediately in the support of this basic notion, illustrating changes in cell-to- experiments. cell contact, agglutinability, permeability and transport, Serum Stimulation. For the serum stimulation experi lipid and protein composition, and dynamic behavior of ments, 3T3 cells and secondary cultures of chicken embryo membrane components (6, 8, 9, 13, 15, 20, 27). The overall fibroblasts were seeded at a density of 2 x 106cells/sq cm alterations in the structure of membrane constituents may in 50-mm Falcon plastic tissue culture dishes in 5 ml be well reflected in functional changes such as the specific medium supplemented with 4% serum and allowed to grow activity of membrane-associated enzymes (10). Numerous to confluency. Stimulation of the resting G,, cells was proteases (24, 26), galactosidases (5), collagenases (29), initiated by replacing the original medium by medium con and transferases (23) show changes in their specific activity taining 25 and 10% serum for the 3T3 and the chicken upon malignant transformation. In recent studies, it has fibroblasts, respectively (7). An incubation of these cultures been also proposed that the surface properties of normal in high serum for 20 hr at 37°resulted in 60 to 80% cycling cells stimulated for growth and differentiation are more cells (7). Therefore, cells were stimulated for 20 hr, washed related to the transformed cell surface than to the untrans- twice with 0.15 M NaCI, and used immediately in the formed resting cell surface (7). experiments. The following study was undertaker^for the determination Con A Stimulation. Mitogenic stimulation of rat (CR/RAR) of the specific activity of the plasma membrane-bound lymph node normal lymphocytes induced by Con A was enzyme, 5'-nucleotidase, in (a) normal and malignant trans determined by measuring incorporation of [3H]thymidine formed cells and (b) normal cells stimulated for growth and into the acid-precipitable material (4, 14). Lymphocytes differentiation. were washed twice with Dulbecco's phosphate-buffered saline, pH 7.2, and 5 x 106 cells in 0.5 ml medium were mixed with 0.5 ml medium containing 4 /J.QCon A in a 35- 1 Present address: Frederick Cancer Research Center, Frederick, Md. mm Retri dish. One ml of medium with fetal calf serum was 21701. 2 To whom requests for reprints should bft addressed. 3 The abbreviations used are: 3T3, untransformed mouse fibroblasts; Received October 28, 1977; accepted January 27, 1978. 3T3/SV40, SV40-transformed mouse fibroblasts; Con A, concanavalin A.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. Membrane Enzymes then added to yield a final concentration of Con A, 2 Table 1 and 10% serum. Control cultures were incubated with the Specific activities of 5'-nucleotidase and acid phosphatase in same concentration of Con A that was pretreated with 0.1 M hamster, rat, and mouse normal and untransformed fibroblasts a-methyl-D-mannopyranoside to saturate all the specific and in polyoma and SV40-transformed fibroblasts carbohydrate-binding sites of the mitogen before incuba -Nucleotidase phosphatase tion with lymphocytes (14). Cultures were incubated for 48 activity (nmol P/mg activity (nmol P,/ CellsHamster protein)203 mgprotein)175.0 hr in a CO2 incubator, and cells were then labeled with ± 9.5" [3H]thymidine by adding 0.2 ¿iCiin 0.1 ml medium to each normal ± 8.1 HamsterpolyomaRat 8.42192.670.2 ± 119.3 ±8.9859.0 plate for 24 hr. After 72 hr of total incubation, cells were washed and used for measurements of both [3H]thymidine normal ±133.7 ±73.9 incorporation and enzyme activities. Ratpolyoma3T33T3/SV405 69.6 ±8.6751 417.0 ±26.5117.4 Determination of Enzymes. Monolayers of normal and .8 ± 44.7 ±14.6 transformed fibroblasts and suspensions of normal and 81 .0 ± 2.4Acid 216.7 ±13.5 leukemic lymphocytes were washed 3 times each with 5 ml " Mean ±S.E., computed from triplicate samples of 3 independ 0.15 M NaCI to remove all phosphate contaminations. The ent experiments. washed cells were-lysed with 0.6 ml of glass-distilled water and left to freeze at -24° for 24 hr. After thawing, triplicate pared to their normal parental cells. Decreases of 3- and 30- samples were used for the determination of acid phospha- fold in the specific activity of 5'-nucleotidase were observed tase and 5'-nucleotidase. For determination of acid phos- in the hamster and rat systems, respectively. For the deter phatase, a volume of 0.6 ml 0.2 M sodium acetate buffer, mination of whether or not the decrease in 5'-nucleotidase pH 5, containing 0.1 M /3-glycerophosphate was added to activity is also associated with a similar decrease in cyto- each sample, and the reaction mixture was then incubated for 60 min at 37°.The reaction was terminated by the plasmic enzyme activities, cultures of the same experiments were also assayed for the lysosomal enzyme marker, acid addition of 0.4 ml cold 20% trichloroacetic acid (12). The phosphatase. The results have shown that acid phospha reaction mixture was then centrifuged, and the supernatant tase shows almost the same specific activity in normal and was analyzed for P¡released according to the method of malignant transformed hamster cells, and in the rat system Ames and Dubin (1). The activity of 5'-nucleotidase was under conditions of 30-fold difference in 5'-nucleotidase determined essentially by the same procedure as that of the acid phosphatase shows only a 2-fold difference be acid phosphatase. The activity of 5'-nucleotidase was deter tween normal and transformed cells (Table 1). mined by monitoring the amount of P¡released from aden- As a test of the possibility that the monitored differences osine 5'-monophosphoric acid (28). The reaction mixture at are not due to the fact that the normal cells were obtained a volume of 1.2 ml contained cells, buffer (100 mM Tris-HCI from secondary cultures of normal embryos whereas the and 100 mw MgCI2, pH 8.5), and substrate, 5 HIM adenosine transformed cells were obtained from in vitro grown lines, 5'-monophosphoric acid. The measurements of the specific similar experiments were also carried out with 2 lines of 3T3 activity of the 2 enzymes were corrected by blanks for free and 3T3/SV40. The experiments have indicated a similar P¡in cells and blanks for nonenzymatic hydrolysis. behavior (Table 1). The decrease in the specific activity of Protein concentrations of cell homogenates were deter 5'-nucleotidase was found to be 9-fold in 3T3/SV40 as mined by the method of Lowry et al. (19), and the specific compared to 3T3. However, as in the hamster and rat activities of both enzymes are given as nmol P¡released per systems, this reduction of 5'-nucleotidase in the trans mg cell protein. formed cell was not accompanied by a similar reduction in the specific activity of acid phosphatase. Results have RESULTS shown even a slight increase in this enzyme in the 3T3/ SV40 as compared to 3T3 (Table 1). Normal and Transformed Fibroblasts. For the determi Normal and Leukemic Lymphocytes. Several recent ob nation of possible changes in the enzymatic activity of the servations have suggested that some differences in the cell surface membrane, normal and malignant transformed dynamic behavior of the plasma membrane show opposite fibroblasts were analyzed for the specific activity of the directions in the fibroblast system as compared to the plasma membrane enzyme marker, 5'-nucleotidase, and for lymphoid system (25). Therefore, experiments were under the specific activity of the lysosomal enzyme marker, acid taken for the determination of whether or not a similar phosphatase. The specific activity of both enzymes was reduction in the specific activity of 5'-nucleotidase is also monitored as nmol P¡released from the appropriate sub associated with mouse leukemia. Essentially, the same strate (see "Materials and Methods") per mg protein of the pattern as in the fibroblast system was recorded for the cell homogenate. The analyzed normal cells were second lymphoid system. The results have shown a marked reduc ary cultures of hamster and rat embryo fibroblasts, and the tion in the specific activity of 5'-nucleotidase in 2 independ malignant cells were hamster and rat fibroblast cell lines ent lymphoma tumors as compared to their normal parental transformed by polyoma virus. All cell types were obtained lymphocytes. The results summarized in Table 2 have from growing cultures, and all 4 cell cultures were analyzed shown a 4-fold difference between Maloney's virus-induced at similar cell density conditions. The results summarized lymphoma cells and normal lymphocytes obtained from A in Table 1 have shown a marked reduction in the specific mice and an 11-fold difference between methylcholan- activity of 5'-nucleotidase in the malignant cells as corn- threne-induced lymphoma cells and normal lymphocytes

MAY 1978 1259

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. A. flaz ef al. obtained from C57BL/6 mice. Under the same experimental (Table 3). In both cellular systems the specific activity of conditions (same cell concentration) as those used for acid phosphatase was of the same order of magnitude in determination of 5'-nucleotidase, no acid phosphatase ac the stimulated and nonstimulated cultures (Table 3). tivity could be determined in normal lymphocytes. Mitogenic stimulation of normal lymphocytes into blast Normal Cells Stimulated for Growth and Differentiation. cells is induced by binding of a mitogen to membrane The cell surface has been found to participate in or even to receptors (11, 14). One of the mitogens that can induce play a leading role in growth control of normal cells. mitogenic stimulation of lymphocytes is the plant lectin, Moreover, it has been also suggested that the surface Con A (3, 4, 14). Therefore the effect of this growth and properties of normal cells stimulated for growth are more differentiation stimulus was analyzed in relation to the specific activity of the plasma membrane enzyme, 5'-nucle- related to the malignant cell surface than to the normal cell membranes (7). For the test of this assumption in view of otidase. The results summarized in Table 3 have shown a these results, the specific activity of 5'-nucleotidase was significant decrease in the specific activity of 5'-nucleotid- determined in normal cells stimulated for growth. The ase in the Con A-stimulated cultures as compared to con cellular systems that were analyzed were normal chicken trols. A 25% reduction of 5'-nucleotidase in the stimulated fibroblasts stimulated by 10% serum and 3T3 stimulated by cultures was associated with about 25% of lymphocytes 25% serum. The results summarized in Table 3 have shown that were transformed into blast cells, and therefore it will a marked decrease in the specific activity of 5'-nucleotidase be of interest to determine the decrease of 5'-nucleotidase in the stimulated cultures as compared to the nonstimulated activity of only the blast cells. No significant differences in cells. The data show a 4-fold difference in 5'-nucleotidase the total cellular protein content were observed in the activity in the chicken system when stimulated cells were stimulated cultures. Concomitantly, with the decrease in 5'- compared to resting cultures. Moreover in 3T3 system the nucleotidase activity in the stimulated lymphocyte cultures, decrease in 5'-nucleotidase activity was such that this acid phosphatase activity appears (Table 3). It is possible to enzyme could not be detected after 20 hr of stimulation assume that binding of Con A to the cell surface receptors

Table 2 Specific activity of 5'-nucleotidase in mouse normal and leukemia lymphocytes grown in vivo Under the same experimental conditions as for determination of 5'-nucleotidase (5 x 10" cells incubated with substrate for 30 min at 37°)acid phosphatase was not detectable in normal lymphocytes.

activity (nmol P¡/ MouseA mgprotein)224.4 ±30.1a virus-induced lymphocytes lymphomaMethylcholanthrene- LeukemiclymphocytesNormal 52.3 ±3.9280.0

C57BL/6TumorMoloney lymphocytes ±17.2 induced lymphomaCellsNormalLeukemic lymphocytes5'-Nucleotidase24.7 ± 2.0 " Mean ±S.E., computed from triplicate samples of 3 independent experiments.

Table 3 Specific activity of 5'-nucleotidase and acid phosphatase in normal and untransformed fibroblasts stimulated for growth by high concentrations of serum and in normal lymphocytes stimulated for differentiation by Con A 5'-Nucleotidase Acid phosphatase activity (nmol P,/ activity (nmol Pt/mg Cells Treatment mg protein) protein) Chick embryo fi ±20.2 17.3± broblasts3T3 serum)Stimulated (10%serum)Nonstimulated(4%±44.5 45.2±

fibroblastsRat ±0*242 70.5± serum)Stimulated (25%serum)NonstimulatedStimulated 53.7±

lymphocytes0Nonstimulated(4% ±195

¿igCon (2 ±2.9"5.67.628.915.5239.6222.77506310»55.4±4.0 A/ml)77.2 " Mean ±S.E., computed from triplicate samples of 3 independent experiments. * No enzymatic activity could be detected under the experimental conditions. c [3H]Thymidine incorporation was found to be 112 ±25 and 10,174 ±175 cpm/plate for the nonstimulated and stimulated cultures, respectively.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. Membrane Enzymes is associated with internalization of Con A molecules into human leukemia have indicated that actively growing leu the activated lymphocytes, followed by degradation of the kemic cells are associated with shedding off of plasma lectin molecules, which triggers the synthesis of lysosomal membrane vesicles. These vesicles can be isolated in large enzymes (12). amounts from the ascites fluid of mouse tumors (2, 27) as well as from pleural effusion and blood serum of leukemic patients." These vesicles, which are exfoliated from the cell DISCUSSION surface membrane of leukemic cells, have a specific lipid The adenosine-generating enzyme, 5'-nucleotidase, is (27) and protein (21) composition, and they contain a high well recognized to be a plasma membrane-bound enzyme specific activity of 5'-nucleotidase. Based on these recent of mammalian cells. Based on its specific location in the observations, it can be suggested that the decrease in the cells it is often used as a plasma membrane enzyme marker, specific activity of 5'-nucleotidase in intact malignant trans and in many studies the increase in its specific activity is formed cells and in intact stimulated normal cells may used as a measure for the degree of purification of plasma reflect indirectly exfoliation of membrane vesicles. How membrane fractions. These studies have shown a marked ever, it is important to note that the relation between 5'- decrease in the specific activity of 5'-nucleotidase in malig nucleotidase activity and exfoliation of plasma membrane nant transformed fibroblasts as compared to normal or vesicles from leukemic cells may not represent a general untransformed parental fibroblasts. A similar decrease in behavior of all malignant and stimulated cells, and there the specific activity of 5'-nucleotidase also was observed in fore any alternative explanation for the previously de leukemic lymphocytes when compared to normal lympho scribed phenomenon cannot be excluded at present. cytes. These results are in good agreement with previous reports indicating a decrease in 5'-nucleotidase in hepa- ACKNOWLEDGMENTS toma cells when compared to normal liver cells (2, 10, 17) and a significant decrease in 5'-nucleotidase observed in The excellent technical assistance of N. Harpaz is greatly appreciated. human chronic lymphatic leukemic lymphocytes when compared to normal human lymphocytes (18). Moreover, REFERENCES these results also have indicated a significant reduction of 5'-nucleotidase in normal fibroblasts stimulated for growth 1. Ames, B. N.. and Dublin. D. T. The Role of Polyamines in Neutralization of Bacteriophage Deoxyribonucleic Acid. J. Biol. Chem.. 235. 769-775, and in normal lymphocytes stimulated for differentiation. 1960. 2. Arima, T., Shivasaka, T., Druda, H. and Fujii, S. Studies on a Reciprocal These results again are in line with other reports in which a Relationship between Nucleoside Kinase and 5'-Nucleotidase. Biochim. decrease in 5'-nucleotidase was observed in generating Biophys. Acta, 277. 15-24, 1972. liver cells (17) and in activated macrophages (16, 22). 3. Barnett, R. E., Scott, R. E., Furcht. L. T., and Kersey, J. H. Evidence That Mitogenic Lectins Induce Changes in Lymphocyte Membrane The outline given previously strongly suggests that malig Fluidity. Nature, 249. 465-466, 1974. nant transformed cells and normal cells stimulated for 4. Betel, I., and Van-den-Berg, K. J. Interaction of Concanavalin A with Rat growth and differentiation possess a similar alteration in Lymphocytes. European J. Biochem., 30. 571-578, 1972. 5. Bosmann. H. B., and Hull, T. C. 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Ultrastructural Labeling of Cell Surface Lectin Receptors results also were obtained with plasma membranes isolated during the Cell Cycle. Exptl. Cell. Res., 94. 159-175, 1975. from normal and leukemic human lymphocytes." 12. Goldman, R., and Raz, A. Concanavalin A and the In-Vitro Induction in Macrophage of Vacuolation and Lysosomal Enzyme Synthesis. Exptl. Although the mechanism by which malignant trans Cell Res., 96: 393-405, 1975. formed cells and normal stimulated cells show a reduction 13. Inbar, M., and Shinitzky, M. Cholesterol as a Bioregulator in the in 5'-nucleotidase activity is not understood, several possi Development and Inhibition of Leukemia. Proc. Nati. Acad. Sei. U. S., 71: 4229-4231, 1974. bilities such as a decrease in biosynthesis, inhibitor factors, 14. Inbar, M., and Shinitzky. M. 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Normal Rat Liver Including Newborn, Regenerating and Adult Livers. 24. Schnebli, H. P.. and Burger, M. M. Selective Inhibition of Growth of Biochem. J., 79. 739-748, 1976. Transformed Cells by Protease Inhibitors. Proc. Nati. Acad. Sei. U. S., 18. Lopes, J., Zuker-Franklin, D., and Silber, R. Heterogeneity of 5'-Nucleo- 63: 3825-3827, 1972. tidase Activity in Lymphocytes in Chronic Lymphatic Leukemia. J. Clin. 25. Shinitzky, M., and Inbar, M. Microviscosity Parameters and Protein Invest., 52: 1297-1300, 1973. Mobility in Biological Membranes. Biochim. Biophys. Acta, 433: 133- 19. Lowry. O. H., Rosebrough. N. J.. Farr, A. L., and Randall, R. J. Protein 149, 1976. Measurement with the Polin Phenol Reagent. J. Biol. Chem., 793. 265- 26. Unkeless, J. C., Daño,K., Kellerman, G. M., and Reich, E. Fibrinolysis 275, 1951. Associated with Oncogenic Transformation. Partial Purification and 20. Nicolson, G. L. Trans-membrane Control of the Receptors on Normal Characterization of the Cell Factor, a Plasminogenic Activity. J. Biol. and Tumor Cells. II. Surface Changes Associated with Transformation Chem., 249: 4295-4305, 1974. and Malignancy. Biochim. Biophys. Acta, 458. 1-72, 1976. 27. Van-Blitterswijk, W. J., Emmelot, P., Hilkman. H. A., Oomen-Meule- 21. Raz, A., Goldman, R., Yuli, I., and Inbar, M. Isolation of Plasma mans, E. P. M., and Inbar, M. Differences in Lipid Fluidity among Membrane Fragments and Vesicles from Ascites Fluid of Lymphoma- Isolated Plasma Membranes of Normal and Leukemic Lymphocytes and Bearing Mice and Their Possible Role in the Escape Mechanism of Membranes Exfoliated from Their Cell Surface. Biochim. Biophys. Acta, Tumors from Host Immune Rejection. Cancer Immunol. Immunotherapy, 467: 309-320, 1977. in press, 1977. 28. Wildnell, C. C., and Unkeless, J. C. Partial Purification of Lipoprotein 22. Raz, A., Shahar, A., and Goldman, R. Characterization of an In-Vitro with 5'-Nucleotidase Activity from Membranes of Rat Liver Cells. Proc. Induced Peritoneal Macrophage Population following I. P. Injection of Nati. Acad. Sei. U. S., 67: 1050-1057, 1968. Concanavalin A. RES J. Reticulendothelial Soc., in press, 1977. 29. Yamanishi. T., Maegens, E., Dubbous, M. K., Ohyama, H., and Hashi 23. Roth, S., and White, D. Intercellular Contact and Cell-Surface Galactosyl moto, K. Collagenolytic Activity in Malignant Membranes: Physiochemi- Transferase Activity. Proc. Nati. Acad. Sei. U. S., 69. 485-489. 1972. cal Studies. Canrer Res., 33: 2507-2512, 1973.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. Decrease in 5′-Nucleotidase Activity in Malignant Transformed and Normal Stimulated Cells

A. Raz, J. G. Collard and M. Inbar

Cancer Res 1978;38:1258-1262.

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