[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. 1258 CANCER RESEARCH VOL. 38 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.