CELL STRUCTURE AND FUNCTION 9, 117-123 (1984) C by Japan Society for Cell Biology

Cytidine Deaminase Levels in Cultured Mammalian Cell Lines Measured by the Growth Tests and Assays

Kazuhiro Ishii1, Hiromi Sakamoto*, Jun-ichi Furuyama* and Masao Hanaoka 1 Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606 and *Department of Genetics , Hyogo College of Medicine, Nishinomiya 663, Japan

ABSTRACT. We developed a test medium for deaminase in order to examine the distribution of this enzyme in cultured cell lines. The growth of various mammalian cell lines was tested in culture medium containing 2 ƒÊM pyrazofurin and 100 ƒÊM cytidine. Enzymological assays for the enzyme also were made spectrophotometrically with cell extracts. A good correlation was found between results of cell growth tests and the levels of enzyme activity. Twelve of twenty cell lines were killed in the test medium, but the remaining lines showed good growth. The levels of enzyme activities were lower in the former lines than in the latter. The critical level of enzyme activity required to support cell growth was approximately 30 units per mg

protein. These findings indicate that culture medium containing 2 ƒÊM py- razofurin and 100 ƒÊM cytidine serves as a test medium for cytidine deaminase. The possibility that the cytidine deaminase may be useful in determining the embryonic origin of cultured cell lines is discussed, based on the growth

properties of various cultured cell lines in the test medium.

Cytidine deaminase (EC 3.5.4.5) is a salvage enzyme, and utilizes both cytidine and deoxycytidine as substrates (1, 12). This enzyme is widely distributed in various tissues, but the levels of enzyme activity differs greatly among the tissues (8, 9). The distribution of the enzyme in cultured mammalian cells lines has not been studied enough. Therefore, we developed a test medium for it that contained pyrazofurin and cytidine in usua 1 culture medium. By assaying the ability of cell lines to grow in this medium, we could determine the levels of cytidine deaminase in the cells. From the experimental results, a possibility was proposed that the cytidine deaminase might prove useful in studies to identify the embryonic origin of cultured cell lines.

MATERIALS AND METHODS

Cell culture. The cell lines used here were as follows : 284 (human skin) (2), 3T3 (mouse embryo) (13), 3Y1 (rat fetus) (9), C3H2K (mouse kidney) (20), CH252 (Chinese hamster lung) (14), CHO-Kl (Chinese hamster ovary), CPAE (bovine pulmonary artery endothelial

1 To whom all correspondence and reprint requests should be addressed.

117 118 K. Ishii et al,

cells) (ATCC CCL-209), HaK (Syrian hamster kidney) (ATCC CCL-15), HeLa (human cervical carcinoma), HT1080 (human acetabulum fibrosarcoma) (ATCC CCL-121), HY1

(incompletely transformed 3Y1 cells) (15), IMR-32 (human neuroblastoma), KB (human oral carcinoma), MDCK (canine kidney), NRK (rat kidney) (3), RPMI-1846 (Syrian hamster melanoma), SA31 (SV40-transformed BALB-3T3-A31 cells) (7), SKH (porcine kidney), V79 (Chinese hamster lung) (5) and Vero (monkey kidney). Cells were grown in Dulbecco-Vogt's modification of Eagle's MEM medium supplemented with streptomycin

(50 mg/L), penicillin (50,000 units/L) and 10 % serum. Pyrazofurin was kindly supplied by Dr. M. J. Sweeney of Eli Lilly and Company. and cytidine were purchased from Kohjin Co., Ltd. (Japan).

Preparation of cell extracts. Subconfluent cultures were used to prepare cell extracts. For cell dissociation, the cell sheets were washed three times with 0.02 % EDTA and incubat- ed in the same solution at 37•Ž for 5-10 min. After being washed with phosphate-buffered saline, the cells were suspended in 10 mM Tris-HCl/pH 8.0 containing 1 mM dithiothreitol, and disrupted by sonication at 2-second intervals for 10-20 seconds. The cell homogenates were centrifuged at 10,000 g for 20 min, and the supernatants were collected. The protein content in the supernatants was determined by the method of Lowry et al. (11).

Enzyme assays. The reaction mixtures contained 50 mM Tris-HCl buffer/pH 8.0, 100 M cytidine and the cell extracts (0.1-2.4 mg protein/ml). The mixtures were incubated ƒÊat 37•Ž for 15 min. The enzymatic reactions were linear for the time and concentrations of the cell proteins (unpublished). of the substrate was determined by measuring the decrease in absorbance at 282 nm. The specific activity was expressed as nmols of uridine

produced per 30 min per mg protein (units/mg). Orotate uptake. KB cells were cultivated for one day where the cell density was 5 •~ 105 cells/30 mm dish. The cultures were incubated with pyrazofurin for 1 h. Then, "C-orotate

(50 ƒÊCi/ml, 0.1 mM) was added and further incubated for 6 h. To count the incorporation of 14C-orotate into the intracellular macromolecules , the cells were washed with phosphate- buffered slaine and treated with 0.5 N PCA, after Which they were washed with 95 % ethanol and dried. Radioactivities Were counted in a Packard TRI-CARBR 460CK liquid scintillation counter.

RESULTS

Selection medium for cytidine deaminase. Pyrazofurin is a C-riboside antibiotic that blocks orotidylate decarboxylate (EC 4.1.1.23), one of the in de novo

pyrimidine synthesis (6, 16) so that, in the presence of the drug, cells are killed through pyrimidine starvation (19). If cells have uridine-cytidine kinase activity (18), the additon of uridine to the pyrazofurin medium supports cell growth because it supplies UMP. In contrast, cytidine is available for cell growth only when cells have activities of both cytidine deaminase and uridine-cytidine kinase because CMP is not converted to UMP in mammalian cells (4) (Fig. 1). Therefore, the test medium for cyditine deaminase contains pyrazofurin and cytidine and that for uridine-cytidine kinase pyrazofurin and uridine. To determine the concentrations of pyrazofurin in the test medium, we examined the effects of the drug on orotate uptakes in KB cells. In the presence of 2 ƒÊM py- razofurin, the incorporation of orotate into the macromolecules was only 4 % that of the control, indicating that de novo pyrimidine biosynthesis was almost completely blocked (Fig. 2). Cytidine Deaminase in Mammalian Cells 119

Fig. 1. De novo pyrimidine biosynthesis and salvage pathways (in part). 1. Orotate phosphoribo- syl-, 2. Orotidine 5'-monophosphate decarboxylase, 3. Uridine-cytidine kinase, 4. Cytidine deaminase. OMP: Orotidine 5'-monophosphate, UMP : Uridine 5'-monophosphate. Pyrazofurin inhibits OMP-decarboxylase activity.

Fig. 2. Inhibition of orotate uptake by pyrazofurin. KB cells were incubated with 14C-orotate

(50 ƒÊCi/ml, 0.1 mM) for 5 h in the presence of pyrazofurin. Incorporation of 14C-orotate into the intracellular macromolecules was measured. Radioactivities in the control cultures were 3182 cpm/dish.

Pyrazofurin toxicity was examined in HYl and KB cells. Both lines showed similar sensitivities to the drug (Fig. 3). Even at 0.2 ƒÊM of the drug, cell growth was inhibited to below 10 % that of the control. At 2 ƒÊM, growth was only 1-3 % that of the control in both lines. Growth inhibition of pyrazofurin, however, was completely prevented 120 K. Ishii et al.

Fig. 3. Sensitivity to pyrazofurin toxicity of the HY1 (a) and the KB cells (b). Cells were in- oculated at 105 cells per 30-mm dish in the media containing pyrazofurin alone (•›), pyrazofurin+ cytidine (100 ƒÊM) (•œ) or pyrazofurin+uridine (100 ƒÊM) (•œ). After cultivation for 4-5 days, cells were harvested and counted. At harvest, the cell numbers in cultures in regular medium were 3.5 •œ 106 cells per dish for HY1 cells and 2 •œ 106 cells per dish for KB cells.

by the simultaneous addition of uridine in the cultures of both HYI and KB cells when the pyrazofurin concentrations were below 10ƒÊM (Fig. 3). In contrast, cytidine showed preventive effects in cultures of KB cells (Fig. 3b) but not in those of HY1 cells although a weak preventive effect was present in medium containing 0.2 ƒÊM pyrazofurin (Fig. 3a). Therefore, media containing 2 ƒÊM pyrazofurin and 100 ƒÊM cytidine or uridine were used as the test media for cytidine deaminase or uridine- cytidine kinase.

Growth tests in the test media. To examine the presence of uridine-cytidine kinase, we cultivated cells in medium containing 2µƒÊM pyrazofurin and 100 ƒÊM uridine. Each of the twenty cell lines, except one (MDCK), grew well in the test medium (data not shown), indicating that those lines had enough uridine-cytidine kinase activity to support cell growth.

The presence of cytidine deaminase also was examined in growth tests. Eight of the twenty lines showed good growth in the test medium containing 2 ƒÊM pyrazofurin and 100 ƒÊM cytidine, but the remaining twelve lines did not (Fig. 4). These findings indicate that the levels of cytidine deaminase activity differ greatly among the various cell lines.

Cytidine deaminase activities. It was examined how the growth properties of cells in the test medium are correlated to the levels of cytidine deaminase activity. Cell Cytidine Deaminase in Mammalian Cells 121

Fig. 4. Correlation between cell growth in the test medium and the levels of cytidine deaminase activity. Cells were cultivated for 3-5 days in the medium containing 2 ƒÊM pyrazofurin and 100 ƒÊM cytidine. Various cells showed relative growth rates above (•›) or below (•œ) 50 % of the control. extracts were prepared from the various cultured cell lines and used for spectrophoto- metric measurement of enzyme activity. There was good correlation between the level of cytidine deaminase activity and the ability of cells to grow in the test medium (Fig. 4) . The critical level of enzyme activity required to support cell growth was 30 nmols per 30 min per mg protein as inferred from Fig. 4. SKH cells grew well in the test medium, in spite of the fact that these cells had lower level of the enzyme than the critical ones. This may be due to the low sensitivity to pyrazofurin of SKH cells. HY1 cells had a lower level of the enzyme, in comparison to the parental cells (3Yl). This may be due to the changes in expression or to clonal selection of cells through cell transformation. The different levels of cytidine deaminase activity found among the various cell lines were not due to the presence of enzyme inhibitors. When extracts of HY1 cells were mixed with those of KB cells in the enzyme assays, the level of enzyme activity was cumulative (data not shown). The differences in enzyme activity among the cell lines may be due to the different enzyme contents. 122 K. Ishii et al.

DISCUSSION

We have developed two kinds of test media ; one for cytidine deaminase and the other for uridine-cytidine kinase. These two test media are required for surveys of cytidine deaminase levels in various cultured cell lines because cytidine is converted to uridine by the deaminase and then phosphorylated to UMP by the kinase. The enzymological assays of the cytidine deaminase levels revealed that the critical level for the support of the cell growth in the test medium was 30 nmol/30 min/mg protein. Cytidine deaminase levels differed greatly among the cultured cell lines tested, but some tendencies can be seen. First, neuroblastoma (IMR-32) and melanoma cells (RPMI-1846) had very low levels of enzyme activity. Both lines are derived embryo- logically from neural crests of ectodermal origin. In our preliminary experiments, murine epidermal cells (PAM) (21) did not grow in the test medium for cytidine deaminase although they grew well in the test medium for uridine-cytidine kinase (unpublished). This suggests that PAM cells have cytidine deaminase activity below the critical level. These findings suggest that cultured cells of ectodermal origin have low cytidine deaminase levels. Second, two (MDCK and HaK) of four kidney-derived epithelial-like cell lines showed low enzyme levels, whereas two other lines (SKH and NRK) had higher levels. MDCK cells are derived from the distal tubules of kidneys and express the differentiation phenotype, "dome formation" (17). HaK cells also have ability to form the "dome" (unpublished), suggesting that this line also is derived from the distal tubules of kidneys. In contrast, SKH and NRK cells did not show the phenotype of dome formation, suggesting that the two lines came from other regions of the kidneys. Third, in seven of the nine fibroblast lines examined so far, the level of cytidine deaminase activity was below the critical level, but in two lines (3Yl and V79) the level was above the critical. This suggests that fibroblast lines can be classified in two groups according to their cytidine deaminase levels. Because fibroblastic cells are known to be derived embryologically from mesenchymal cells, cytidine deaminase may be useful in embryological studies of mesenchymal cells.

Acknowledgements. We are grateful to Dr. K. Toda for useful discussion, to Dr. H. Green for supplying the 284 cells, to Dr. A. Ishihama for MDCK, to Dr. M. Kohno for NRK, to Dr. T. Kusano for V79, to Dr. T. Shibuya for CH252, to Dr. Y. Sokawa for SKH, to Dr. K. Takeda for IMR-32 and Vero, to Dr. S. Toyama for KB and to Dr. M. Takigawa for PAM.

REFERENCES

1. CHABOT, G.G., J. BOUCHARD and R.L. MOMPARLER. Kinetics of deamination of 5-aza-2'-deoxy- cytidine and cytosine arabinoside by human liver cytidine deaminase and its inhibition by 3- deazauridine, thymidine or uracil arabinoside. Biochem. Pharmacol. 32, 1327-1328, 1983 2. CHAN, T.-S., K. ISHII, C. LONG and H. GREEN. Purine excretion by mammalian cells deficient in adenosine kinase. J. Cell. Physiol. 81, 315-322, 1973 3. DUC-NGUYEN, H., E.N. ROSENBLUM and R.F. ZEIGEL. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bact. 92, 1133-1140, 1966 4. ELLIMS, P.H., A.Y. KAO and B.A. CHABNER. Deoxycytidylate deaminase. Purification and some properties of the enzyme isolated from human spleen. J. Biol. Chem. 256, 6335-6340, 1981 5. FORD, D.K. and G. YERGANIAN. Observations on the of Chinese hamster cells in tissue culture. J. Natl. Cancer Inst. 21, 393-425, 1958 6. GUTOWSKI, G.E., M.J. SWEENEY, D.C. DELONG, R.L. HAMILL, K. GERZON and R.W. DYKE. Cytidine Deaminase in Mammalian Cells 123

Biochemistry and biological effects of the pyrazofurins (pyrazomycins): Initial clinical trial.

Ann. N.Y. Acad. Sci. 255, 544-551, 1975 7. HAKURA, A. Simian virus 40 facilitates multiplication of replication defective mutants of

polyoma virus in BALB/c 3T3 mouse cells. Nature 267, 528-529, 1977 8. Ho, D.H.W. Distribution of kinase and deaminase of 1-ƒÀ-D-arabino-furanosylcytosine in tissues of man and mouse. Cancer Res. 33, 2816-2820, 1973

9. KIMURA, G., A. ITAGAKI and J. SUMMERS. Rat cell line 3Y1 and its virogenic polyoma and SV40 transformed derivatives. Int. J. Cancer 15, 694-706, 1975

10. KRAJEWSKA, E., E. DE CLERCQ and D. SHUGAR. Nucleoside-catabolizing enzyme activities in

primary rabbit kidney cells and human skin fibroblasts. Biochem. Pharmacol. 27, 1421-1426, 1978 11. LOWRY, O.H., N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL. Protein measurement with

the Folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951 12. MALATHI, V.G. and R. SILBER. Effects of murine viral leukemia on spleen nucleoside deaminase: Purification and properties of the enzyme from leukemic spleen. Biochem. Biophys. Acta 238,

377-387, 1971 13. MATSUYA, Y. and H. GREEN. A somatic cell hybrid between the human established line D98

(presumptive HeLa) and 3T3. Science 163, 697-698, 1969 14. SEKIGUCHI, T. and F. SEKIGUCHI. Interallelic complementation in hybrid cells derived from Chinese hamster diploid clones deficient in hypoxanthine-guanine phosphoribosyl transferase

activity. Exp. Cell Res. 77, 391-403, 1973 15. SHIROKI, K., H. SHIMOJO, Y. SAWADA, Y. UEMIZU and K. FUJINAGA. Incomplete transformation

of rat cells by a small fragment of adenovirus 12 DNA. Virol. 95, 127-136, 1979 16. SUTTLE, D.P. and G.R. STARK. Coordinate overproduction of orotate phosphoribosyltransferase

and orotidine-5'-phosphate decarboxylase in hamster cells resistant to pyrazofurin and 6- azauridine. J. Biol. Chem. 254, 4602-4607, 1979

17. THOMAS, S.R., S.G. SCHULTZ and J.E. LEVER. Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation : Dissociation from effects on transepithelial resistance and cyclic AMP levels. J. Cell. Physiol. 113, 427-432, 1982

18. ULLMAN, B., B.B. LEVINSON, D.H. ULLMAN and D.W. MARTIN, JR. Isolation and characteri- zation of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase. J. Biol. Chem.

254, 8736-8739, 1979 19. WORZALLA, J.F. and M.J. SWEENEY. Pyrazofurin inhibition of purine biosynthesis via 5-

aminoimidazole-4-carboxamide-1-ƒÀ-D-ribofuranosyl 5' -monophosphate formyltransferase. Cancer Res. 40, 1482-1485, 1980

20. YOSHIKURA, H., Y. HIROKAWA and M. YAMADA. Synchronized cell division induced by medium change. Exp. Cell Res. 48, 226-228, 1976 21. YUSPA, S.H., P. HAWLEY-NELSON, B. KOEHLER and J.R. STANLEY. A survey of transformation

markers in differentiating epidermal cell lines in culture. Cancer Res. 40, 4694-4703, 1980

(Received for publication, May 1, 1984)