Induction of tyrosine alpha-ketoglutarate transaminase by steroid hormones in a newly established tissue culture cell line.

Our attention was drawn to the possibility of studying enzyme induction in tissue culture by a brief report from Pitot et al.1 that the Reuber hepatoma in tissue culture showed an increase in tyrosine transaminase activity in response to treatment with hydrocortisone.' Through the courtesy of Dr. H. P. Morris of the National Cancer Institute, we obtained primary cultures from rats containing two lines of hepatomas in the ascites form. Each of these resulted in a permanent tissue culture line. In this paper we describe the characteristics of one of these lines in which glucocorticoids induce a rapid, substantial increase in the activity of tyrosine a-ketoglutarate transaminase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5). While being carried in serial transfer for over a year, this cell line (designated HTC for hepatoma tissue culture) has remained stable as to growth and inducibility. Furthermore, from single cells of the original line, clones have been isolated which continue to show the same characteristics. Materials and Methods.-Sera were obtained from Microbiological Associates; the NIH medie unit prepared Swim's medium. Merck, Inc., very kindly supplied the dexamethasone phosphata (Dx). Porcine kidney p-hydroxyphenyl pyruvate keto-enol tautomerase (4.3 K units/ml) was purchased from Sigma Chemical Co., centrifuged at 20,000 g for 10 min, the pellet discarded, and the soluble fraction stored frozen. The cell line described here came from an ascites tumor2 which in turn had been derived from a solid hepatoma (#7288c) originally induced by feeding male Buffalo rats a diet containing 0.04% N,N '-2,7-fluorenylenebis-2,2,2-trifluoroacetamide for 12.4 months.3 4 Primary culture was carried out by sterile peritoneal puncture and withdrawal of 0.1 ml ascitic fluid which was placed in a T30 culture flask to which 5 ml growth medium was at once added. After an initial lag of a few days, a layer of epithelioid cells grew out. For the first 8 months, growth was maintained in tightly stoppered bottles in a standard laboratory incubator, but since then a humidified CO2 incubator running with 3% CO2-97% air has been used with the bottles stoppered loosely. The growth medium was Swim's medium 77 (S77). S77 has the same composition as S103 described by Swim and Barker,5 except that hydroxyproline was omitted, the serine concentration was 0.2 mM, and choline bitartrate was substituted for choline chloride. S77, when used to support growth, was supplemented with 20% bovine serum and 5% fetal bovine serum. Penicillin G, 106 units/ml, streptomycin sulfate, 12.5 ,g/ml, were added for routine culturing; however, periodically they have been omitted for several days and the medium was then cultured for bacterial contamination. Checks for PPLO contamination also have been carried out by plating on "mycoplasma agar" as described by Hayflick.6 No mycoplasmas have been found. Periodically, cells have been frozen in 5% glycerol-95% growth medium by standard techniques7 and stored in liquid nitrogen. Upon thawing after as much as a year of such storage, HTC cells exhibited the same growth and induction as the original line. Chromosome preparations, stained with GCiemsa, were carried out by a slight modification of the method of Tjio and Puck.8 For enzyme induction studies, growth medium was replaced by serum-free S77; then the cells were gently shaken free, pooled, and apportioned as needed for a given experiment. Tyrosine transaminase was assayed as follows: Each aliquot of cells was centrifuged from the induction medium at 600 g for 5 min at 00, twice washed and recentrifuged with aliquots of 0.15 AI sodium phosphate, p11 7.9 at 0)-4, and the resulting pellet frozen. To the frozen pellet 0.5 il