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Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff Ascites Rat Tumor Cells in Culture1

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Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff Ascites Rat Tumor Cells in Culture1 [CANCER RESEARCH 33, 2265-2272, October 1973] Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff Ascites Rat Tumor Cells in Culture1 Martin Schaffer, Robert B. Huribert, and Antonio Orengo The Department of Biochemistry, The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025 SUMMARY considerable significance in the adult metazoan in which large amounts of nucleic acids are degraded hourly in the The utilization of labeled guanosine for the biosynthesis normal processes of destruction and renewal of cells. The of RNA and DNA has been studied in cells cultured from life-span of neutrophils is estimated at 70 to 80 hr (8), and the Novikoff ascites tumor of the rat. Guanosine contrib that of lymphocytes is estimated at 24 hr (7, 26). Erythro- uted primarily to guanine moieties of RNA and DNA, whereas labeled adenosine contributed to both adenine and cytes are replaced at a rate of 0.83% every day (2), and 60 to guanine moieties. The labeled ribose moiety of uniformly 70% of the lining epithelium of the intestine is shed daily labeled guanosine-14C did not enter a pool of ribose (11). rRNA and mRNA are also degraded and are presum phosphate intermediates, judging from lack of contribution ably utilized intracellularly. The possibility that these salvage processes may be regulated or coordinated with to adenine, uracil, and cytosine nucleosides in RNA and synthesis de novo of nucleotides has not received the proper DNA. The group of enzymatic activities that catalyze the attention. conversion of guanine and guanosine to guanosine triphos- In this communication we report on the utilization of phate (namely, guanosine kinase, purine nucleoside phos- guanosine for biosynthesis of nucleic acids in Novikoff ascites rat tumor cells. These cells in tissue culture suspen phorylase, purine nucleoside monophosphate kinase, nu sion readily utilize 14C-labeled guanosine and adenosine for cleoside diphosphate kinase, and purine nucleoside triphos- phate phosphatase) have been prepared from an extract of nucleic acid biosynthesis. Here, we describe both the pattern of utilization of the nucleosides by living tumor cells and the Novikoff ascites cells in a single procedure by the use of diethylaminoethyl cellulose. Gel permeation chromatogra- partial isolation from these cells of a group of enzymes phy on Sephadex G-150 was used to resolve these enzymes responsible for the conversion of guanosine to GTP. It may sufficiently to permit determination of their individual be useful that these enzymatic activities can be partially activities, substrate specificities, molecular weight, and purified simultaneously. other characteristics. New rapid assays were developed for purine nucleoside kinase and phosphorylase, utilizing la beled nucleosides with chromatography on diethylamino MATERIALS AND METHODS ethyl paper. These techniques were designed to be useful for the measurement of the individual enzymes of the guanosine All the 14C-labeled nucleosides and nucleotides were salvage pathway in studies of nucleotide metabolism and purchased from Schwarz/Mann, Orangeburg, N. Y., or therapeutic effects. from Amersham/Searle Corp., Arlington Heights, 111.All Practical methods for culture in suspension of Novikoff the other nucleosides and nucleotides used were products of ascites tumor cells and for determination of the rates of P-L Biochemicals, Milwaukee, Wis. Ribose 1-phosphate incorporation of guanosine (and other nucleic acid precur was purchased from Sigma Chemical Co., St. Louis, Mo. sors) into RNA and DNA are described. RNA and DNA The Novikoff ascites tumor was originally supplied by Dr. are extracted with hot 2.5 M potassium acetate and sepa Alex B. Novikoff. Whatman DEAE-cellulose DE 52 was rated by use of alkali in a form convenient for resolution of purchased from H. Reeve Angel and Co., Inc., Clifton, N. individual nucleotides and bases by electrophoresis and J. Amino acids and vitamins were purchased from Sigma; chromatography. bovine and calf serum was from Grand Island Biological Co., Grand Island, N. Y., Pluronic acid F68 was from INTRODUCTION Wyandotte Chemical Co., Wyandotte, Mich.; streptomycin Although nucleic acid derivatives are not required in the sulfate was from Charles Pfizer and Co., Inc., New York, diet, the utilization of bases and nucleosides may have N. Y.; and neomycin was from the Upjohn Co., Kalama- zoo, Mich. ; 'This work has been supported by N IH Research Grant ÇA-10407to The Novikoff ascites cells were transplanted and grown A. O. Development of the methods for cell culture and the "hot potassium acetate" procedures for extraction and separation of nucleic acids was for 5 to 6 days in the peritoneal cavity of young female Holtzman Sprague-Dawley rats (120 to 150 g). supported by The American Cancer Society Research Grant P-146 to R. B. H. Additional support was provided by Grants G-460 and G-447 from Cell Culture. For establishment of a suspension culture of the Robert A. Welch Foundation. tumor cells in vitro, rats displaying modest amounts of Received December 20, 1972; accepted June 8, 1973. ascitic fluid were selected since tumor cells derived from OCTOBER 1973 2265 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. M. Schaffer, R. B. Huribert, and A. Orengo large ascites volumes had a higher initial mortality when set (neutral point of phenol red); 1.0 ml was withdrawn and the up in suspension culture. A rat was anesthetized and the bottles were returned to the roller drum. The aliquot was abdominal area thoroughly cleaned with 70% ethanol. then diluted with medium and counted as described above. Using a sterile disposable 10-ml syringe and 20-gauge The desired cell number after the start of the culture was needle, 4 to 5 ml of ascitic fluid were removed and diluted 5 x IO5cells/ml of medium and this applied as long as the with an equal amount of culture medium previously warmed cell line was continued. Consequently, if the cell suspen at 37°using sterile conditions. The cells were dispersed sions were grown to a density of more than 5 x IO5cells/ using a gentle rocking motion and centrifuged at 60 xg for ml, appropriate aliquots were withdrawn and discarded or about 5 to 6 min. The supernatant, which should contain cultured separately. The remainder was centrifuged for 5 most of the red cells, was carefully decanted. The sedi- min at 60 x g to remove the old medium and to replace it mented cells were then dispersed using the same rocking with 25 ml of fresh, warmed medium. At the end of any motion in 5 ml of fresh medium at 37°. 48-hr period if the cell count had not doubled the suspension One-tenth ml of the cell suspension was diluted to 10 ml was discarded. On all subsequent feedings, the same proce with culture medium and counted in an hemacytometer. dure was followed to keep the cell count at ca. 5 x IO5 Once the number of cells in the suspension was determined, cells/ml. The cells should have a rounded appearance with a aliquots containing 1.9 x 10' cells were transferred to sharp cell membrane. Cells that were very large and culture bottles and culture medium warmed at 37°was granular, developing swelling or vacuoles, were regarded as added to a total volume of 25 ml. Sterile technqiues were unlikely to survive and were not counted when taking a cell used at all times. The bottles were capped well and placed in count. Cells were not subjected to heavy mechanical or the roller drum at 37°;thenthey were incubated for 24 hr. thermal shock. They were not left for more than 24 hr The culture medium was prepared by mixing in order the without changing the medium. When NaHCO3 was added, following ingredients: a dry mixture of salts, 100 ml of a the bottle was swirled to prevent a sudden localized massive modified McCoy's amino acid (4, 15) 10 x stock solution, pH change. The bottles were not shaken but merely tilted or 20 ml of McCoy's vitamin 50 x stock solution (15), 10ml of rocked back and forth. an antibiotic mixture, 50 ml of bovine serum, and 50 ml of For the incorporation studies described here only cultures fetal calf serum. The solution was diluted to 1 liter with that doubled their cell number for 3 consecutive days were double glass-distilled water and sterilized by filtration used.2 through a Seitz filter which had been autoclaved using a size Extraction and Initial Purification of Guanosine-metabo- 6 filter sheet (Republic Seitz Filter Co., Newark, N. J.). It lizing Enzymes. For studies on the purification of the was stored at 04. enzymatic activities, 20 to 50 tumor-bearing rats were The dry mixture of salts was composed of: lactalbumin decapitated and the ascitic fluid was collected and diluted hydrolysate, 5.0 g; Pluronic F68, 1.0 g; NaCl, 8.0 g; KC1, 1:2 with 0.25 Msucrose : 1 m.MMgCl2 and then cooled. The 0.4 g; MgSO4-7H2O, 0.2 g; Na2HPO4-2H2O, 0.060 g; material was maintained at 2-4° throughout the entire KH2PO4, 0.060 g; glucose, 3.0 g; glutamine, 0.219 g; and procedure. The fluid was then centrifuged at 200 x g, and NaHCO3, 2.0 g. the tumor cells were collected as a sediment. This sediment The modified 10 x McCoy's amino acid mixture con was repeatedly suspended in fresh sucrose solution and tained the following amino acids (mg/liter): i.-tryptophan, centrifuged at 200 x g to remove most of the erythrocytes. 31; i.-phenylalanine, 165; L-tyrosine, 181; L-arginine-HCl, Finally, the cells were packed by centrifugation at 1000 x g 421; i.-histidine-HCl-H2O, 209; L-lysine-HCl, 365; L-cys- to estimate their volume and then suspended in 0.01 M tine, 315; i.-methionine, 149; i.-isoleucine, 393; L-leucine, Tris-Cl:0.25 Msucrose (pH 7.7) at a cell to buffer ratio of 393; t.-valine, 176; i.-threonine, 179; i.-asparagine, 450; 1:4.
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