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 triphosphate 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 Ã‡A-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

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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 37Â°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 37Â°. 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 37Â°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 37Â°;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 2-4Â° 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. glycine, 75; L-serine, 263; L-alanine, 134; L-proline, 173; The suspension was homogenized in an Emanuel-Chaik- L-aspartic acid, 199; i.-glutamic acid, 221. It was stored off orifice-type homogenizer (Microchemical Specialties frozen. Co., Berkeley, Calif.) (5). In order to break most of the The antibiotic mixture contained (g/liter): phenol red cells, it was necessary to pass the suspension through the (sodium salt), 0.5; streptomycin sulfate, 5; and neomycin, 2. homogenizer twice. The homogenate was centrifuged at It was stored frozen. 20,000 x g for 20 min in a refrigerated International Model The complete medium should not be used after a 3-month B20 centrifuge. The supernatant was collected and cen period; the vitamin stock solution should be discarded after trifuged again for 2 hr at 147,000 x g in a refrigerated 6 months. International Model B35 centrifuge. The sediment was The cell cultures were grown as suspensions in 60- x 150-mm centrifuge bottles with flat bottoms and a 38-mm 2These culture conditions were developed by Dr. C. Vaughan, with the black plastic screw cap (Pyrex brand glass, serial 1261). The advice of Dr. M. Sheek. independently from those described tor long-term bottles were coated with silicone and baked overnight before culture of the Novikoff ascites tumor by Morse and Potter (16). The use. medium described here is more complete than that of Morse and Potter Subsequent Feeding of Cell Suspensions. At the end of the and, in direct comparison in our laboratory, gave consistently more favorable results in establishment of primary cultures when it was not de 24-hr period of incubation, following sterile procedures, sired to maintain cultures continually. We have found it useful in routine NaHCO3 (7.5% solution, w/v) was added dropwise until the preparation of Novikoff tumor RNA highly labeled with ribonucleosides color of the medium changed from yellow to orange-red or with phosphate-32?.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. GuarÃosmeAnabolism in Novikoff Ascites Cells discarded and the supernatant was called Fraction 1. origin. An average 1%of the counts remains on the origin at Protein concentration was determined by the method of 0 incubation time. This value may vary with the purity of Lowry et al. (13) and the concentration was then adjusted to the commercial preparations of the radioactive nucleosides 5.0 mg/ml with 0.02 MTris-Cl (pH 7.5). For each 1000 ml used. The nucleotide spots were located by UV illumination, of Fraction 1, 600 ml of DEAE-cellulose suspension in 0.01 cut out, and counted in a Nuclear-Chicago low-background MTris-Cl (pH 8.0): 10% glycerol (50:50, v/v)3 were added flow counter. The products were shown to be GMP and slowly with continuous stirring. After 2 hr of gentle stirring ADP by electrophoretic assays. The entire procedure takes the suspension was centrifuged and the supernatant was 2 hr. discarded. The sediment was then washed with 1000 ml of Purine Nucleoside Phosphorylase Assay. The standard H2O adjusted to make the solution pH 8.0 with NaOH. assay mixture (final volume of 60 ^1)contained 10AmÃ³lesof After centrifugation the supernatant was discarded, and Tris-Cl (pH 7.4), 50 nmoles of guanosine-U-14C,4 100 the sediment was washed again with 400 ml of 0.075 M nmoles of sodium phosphate buffer (pH 7.4), and suitable KC1:0.01 M Tris-Cl: 10% glycerol (pH 8.0). The superna amounts of enzyme. The incubation time was 15 min. The tant was discarded. Chromatographie system was the same as that used for the The sediment was then eluted by 2 hr of gentle stirring in nucleoside kinase assay. Since the guanosine used is labeled 500 ml of 0.3 M potassium phosphate buffer (pH 6.5). The both in the guanine and ribose, this allowed measurement of DEAE-cellulose was removed by centrifugation, and the the ribose 1-phosphate produced. Whereas the nucleoside volume of supernatant was reduced at least 6 times using and base moved with the solvent, the ribose 1-phosphate Centriflo ultrafiltration membrane (American Scientific produced by the phosphorylase remained at the origin. Systems Division, Lexington, Mass.). This was called Carrier guanosine and 5'-GMP were also added as de Fraction 2. scribed for nucleoside kinase. 5'-GMP was added as a UV Gel Permeation Chromatography. Fraction 2 was cen marker for the position of the ribose 1-phosphate with no trifuged briefly to remove any precipitate. Of the resulting UV absorbance. The spots remaining at the origin were cut solution, 20 ml containing approximately 200 mg of protein out and counted in a Nuclear-Chicago low-background flow were applied in 0.3 M potassium phosphate buffer (pH 6.5) counter. on a Sephadex G-150 column (3 x 52 cm). The flow rate In pilot experiments the location of ribose 1-phosphate at was 15 ml/hr and fractions of 4 ml were collected. The the origin was established by chromatography of ribose proteins were eluted with 0.01 M Tris (pH 7.7) and the 1-phosphate and spraying with aniline :acetic :orthophos- fractions were read at 280 nM to follow the protein elution. phoric acid reagent (6:200:20) (22). The enzymatic activities were determined with the radioac Assays for Nucleoside Monophosphate and Nucleoside tive assays described below. Diphosphate Kinases and for Purine Nucleoside Assays of Enzymes. All enzymatic activities were deter Triphosphatase. The standard assay mixture (final volume, mined by radiochemical assays. All assays were done at 37Â° 50 n\) contained 5 AmÃ³lesof Tris-Cl buffer (pH 7.7), 300 and initial velocities were measured (no more than 10% nmoles of ATP, 700 nmoles of MgCl2, and 50 nmoles of conversion allowed). In this range, the measured activities GMP-8-14C or GDP-U-14C or GTP-8-l4C plus a suitable were proportional to enzyme concentration. The products amount of enzyme. The reactions were incubated at 37Â°for and substrates were separated either by chromatography on 10 min and stopped by cooling the mixture to near 0Â°.In a DEAE-paper (Whatman DE 81) or by high-voltage electro- volume of 30 /tl, 100 nmoles each of guanosine, GMP, phoresis on Whatman No. 3MM paper. GDP, and GTP were added. Aliquots of 5 or 10 ^1 were Nucleoside Kinases Assay. A new radiochemical assay spotted 13cm from the cathodic end of a sheet of Whatman was used which measures the conversion of labeled Ã±ucleo- No. 3MM â€¢¿'paperv(56".5'x '29:5 cm) in duplicate. The side to nucleotide by Chromatographie separation of the compounds were separated by paper electrophoresis on 0.05 reactants and products on DEAE-paper. The standard assay Mcitrate buffer (pH 3.5) on a flat-plate apparatus. The field mixture (final volume of 60 Â¿tl)contained 5 AmÃ³lesof strength of the system was 47 V/cm and the temperature Tris-Cl buffer (pH 7.4), 420 nmoles of MgCl2, 300 nmoles was kept between 5 and 7Â°.Each spot was localized by UV of ATP, 100 nmoles of nucleoside labeled with 14Cin the illumination, cut out, and counted in a Packard liquid purine or pyrimidine ring (1 /ÃCi//Â¿mole),and varying scintillation counter. Assays for the other nucleoside kinases amounts of enzyme. After 1 hr of incubation, 25-^1 ali- were conducted by substitution of appropriately labeled quots of the reaction mixture were pipetted in duplicate nucleotides for the guanosine nucleotides. The RI-TP, i.e., as spots 5 cm from 1end of DEAE paper strips 2.9 x 14cm relative migration with respect to UTP for the nucleotides, (previously spotted with 0.1 /Â¿moleofunlabeled nucleoside can be listed as follows: UMP, 0.548, UDP, 0.882; GMP, as carrier), dried, and subjected to descending chromatog 0.420; GDP, 0.741; GTP, 0.821; AMP, 0.252; ADP, 0.583; raphy. The solvents used were isobutyric acid:H2O:NH3 ATP, 0.735. (65.7:34:2:0.1) for the adenosine and guanosine and 80% One unit of enzyme was defined as the amount catalyzing ethanol for the uridine and cytidine. Whereas the nucleo- the conversion of l ^mole of substrate in 60 min under the sides move with the solvent, the nucleotides remain at the condition of the standard assay for all of the previously

3The volume ratio of DEAE-cellulose and solution was determined after centrifugation of an aliquot for 10 min at 1000 x g. ' U refers to the uniformly labeled compound.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. M. Schaffer, R. B. Huribert, and A. Orengo described assays. The counting efficiencies were 72% for the cold perchloric acid as before to ensure removal of the last scintillation counter and 9.5% for the flow counter. traces of ribonucleotides. The pellet was then dissolved in Hot Potassium Acetate Procedures for Extraction and 0.5 ml of 0.5 MNaCI, 1 drop of phenol red was added, and Analysis of RNA and DNA. Ten ml of a 5% trichloroacetic the solution was neutralized. Ethanol, 1.5 volumes, was acid solution were added to 0.5 ml of packed ascites cells. added, and the mixture was set in the freezer for several hr. After centrifugation the sediment was washed 2 or 3 times The precipitate was centrifuged and washed by suspension with 10 ml of 5% trichloroacetic acid and then with 10 ml of and centrifugation in 1 ml of ethanol. The precipitate was 95% ethanol, rinsing the entire inner surface of tube and dried and then heated for 2 hr at 175Â°in91% formic acid. breaking up the precipitate. After centrifugation, the super The hydrolysis was carried out in a sealed pyrex tube. The natant was discarded and the tube was drained well. hydrolysate was evaporated under reduced pressure to Three ml of cold 2.5 M potassium acetate and 1 small dryness and redissolved in 100 to 300 n\ of l N HC1. drop of concentrated phenol red solution were added to the Aliquots ( 10to 20 Â¿il)takenfor Chromatographie separation sediment. While still cold, the suspension was neutralized to of the bases were applied on Whatman No. 1 paper and pH 7.4 to 8.0 with 1.0 and 0.1 M KOH. The suspension was subjected to descending chromatography using the solvent stirred well and allowed to stand for 10 min at room system of Wyatt (25). In no case was radioactive uracil temperature to ensure equilibration and diffusion into detected indicating that the DNA was free of RNA. particles. The pH was adjusted if necessary; if too alkaline, The RNA hydrolysate [2'(3')-nucleotides] was prepared RNA will subsequently be lost; if too acid, both RNA and for paper electrophoresis as follows. The solution was DNA will be lost. The tube was capped, heated with adjusted to pH 3.5 with KOH, chilled near freezing to occasional stirring for 30 min in a boiling water bath, and maximize precipitation of KC1O4, and centrifuged. Ali chilled in ice. After centrifugation, the supernatant was quots (20 /il) of the supernatant were applied as a line on a removed by means of a disposable pipet and filtered through sheet of Whatman No. 3MM filter paper, and paper a small plug of glass wool to remove particles. The pellet electrophoresis was carried out in 0.05 Mcitrate buffer (pH was reextracted in the same way with 1 ml of 2.5 M 3.5). The field strength of system was 47 V/cm and the potassium acetate, with heating for 5 to 10 min. The 2 temperature was kept between 5 and 7Â°.The nucleotides supernatants were combined and 2 to 2.5 volumes of were separated in the following order going from the absolute ethanol were added. The suspension was stirred negative to the positive pole: cytosine, adenine, guanine, and well and set in a deep freeze for several hr. The flocculent uracil. precipitate of potassium nucleates was centrifuged down The paper was dried and the bases or the nucleotides were and washed with 7 ml of cold 70 to 80% ethanol. After located by UV and cut out. The paper spots were immersed centrifugation the supernatant was removed as completely in a scintillator solution (4 g of PPO and 50 mg of POPOP as possible with a disposable pipet. Recovery of DNA is per liter of toluene) and counted in a liquid scintillation about 90% and of RNA is about 80%, based on comparison counter. with assays by the Schneider method (Ref. 19, cf. Ref. 9). The DNA loss is primarily due to the mechanical manipula RESULTS tions and it is suspected that some tRNA is lost due to Table 1 shows that 14C-labeled nucleosides are readily slight degradation and solubility in the ethanol precipita tion.5 incorporated into cultured Novikoff rat ascites cells. The patterns of incorporation into nucleic acids are distinctly In order to separate RNA from DNA, 1 ml of 0.2 N different for adenosine and guanosine. When guanosine-8- NaOH (carbonate free) was added to the sediment, and the 14C is used as precursor of RNA and DNA, the label is solution was incubated at 37Â°for 16 to 18 hr in a capped found predominantly in guanine moieties, whereas adeno- tube. At the end of the incubation the tube was cooled and sine-8-14C contributed strongly to both guanine and adenine 0.1 ml of 4.4 N perchloric acid was added. The DNA moieties. From the experiments with guanosine-U-14C precipitate was centrifuged down in the cold, and the RNA labeled in both guanine and ribose, it appears that the ribose hydrolysate was removed by disposable pipet and trans of the guanosine did not contribute significantly to a ferred to another small tube. common pool of ribose 1-phosphate or 5-phosphoribosyl- The DNA precipitate was washed without delay with pyrophosphate for other nucleotide biosynthesis. One would several ml of cold 0.2 N perchloric acid. The supernatant expect more radioactivity in the pyrimidine nucleotides if was discarded and the tube was drained well. The DNA was the labeled ribose 1-phosphate derived from the action of redissolved in 1.0 ml of 0.2 N NaOH and reprecipitated with guanosine phosphorylase were available. The ready utiliza 5The extraction with 2.5 M potassium acetate is preferable to the tion of adenosine for the synthesis of both AMP and GMP extraction with 10%NaCI solution previously described (18) because of the suggest that adenosine may be utilized through conversion more convenient buffering capacity of potassium acetate during neutraliza of its deamination product, inosine, to inosinic acid as well tion and its greater solubility in ethanol to reduce salt concentration in the as interconversion of AMP and GMP via inosinic acid at electrophoretic analysis. The procedure is highly reproducible and conven the nucleotide level. These results are in line with those ient for isotope determinations on components of RNA and DNA in small obtained by Williams and LePage (23, 24). These authors tissue samples, when relatively specific precursors of nucleic acids are used. Where lipids and phospholipids are a problem, washing of the precipitated studied the in vivo and in vitro incorporation of preformed tissue or of the potassium nucleates with ethanol:ether and ethanol is purines into nucleotides and polynucleotides using incuba necessary. tion periods varying from 5 to 60 min.

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Table 1 Incorporation of "C-labeled guanosine and adenosine into RNA and DNA of Novikoff tumor cells maintained in suspension cell cultures l4C-Labeled nucleosides (0.5 ^Ci/Â¿Ãmole),0.5ml, were added to 100 ml of cell culture suspension (5 x 10s cells/ml). The bottles were then capped and placed in a roller drum at 37Â°andincubated with continuous motion for 12 or 24 hr. At the end of the incubation period the cells were harvested by centrifugation and washed twice with medium, and nucleic acids were extracted and analyzed as described in the text. Part of the cell culture suspension was maintained without radioactive nucleoside and used as a control to follow cell division. RNA was hydrolyzed to 2'(3')-nucleotides and DNA to the bases guanine, adenine, cytosine, and thymine, which were separated by electrophoresis and chromatography as described in the text. RNA2'(3')-CMP00.50.70.82'(3')-AMP2.52.61.353.12'(3')-GMP95.795.297.445.82'(3')-UMP1.81.70.60.3G89.791.087.326.0%of labeled of labeledDNAA4.14.37.769.8C2.42.62.53.6T3.82.02.50.7 -(^moles/5added x10' cells)1.861.861.801.36% Guanosine-U-"C12 hr24 hrGuanosine-8-"C24

hrAdenosine-8-14C24

hrAmount

A similar pattern of incorporation has been reported for Substrate Specificity Studies. The center tube of the peak the reticulocyte (3) and the erythrocyte of the rabbit (14). labeled guanosine kinase was shown to convert guanosine Again, guanine and guanosine-8-'"C was utilized extensively only to GMP when the products were analyzed by the for GTP synthesis but to a very limited extent for the electrophoretic analysis. This fraction was tested for activity synthesis of ATP. as adenosine kinase with no conversion to nucleotides. In order to study and characterize the enzymatic machin Uridine kinase activity was present in the guanosine kinase ery responsible for these in vivo findings, it was necessary to preparation here described but the possibility that I enzyme assay for the guanosine-anabolizing enzymes. Fraction 2, as was responsible for both activities was excluded on the described, was the only fraction of the cell extract that finding that (a) highly purified uridine kinase from Novikoff metabolizes guanosine. It was observed to convert guano tumor cells (17) was tested with this assay and did not sine to a number of metabolites, and therefore it was not exhibit guanosine kinase activity (The lower limit of possible to assay specifically for the individual enzymes detectability for guanosine kinase in the conditions of our involved. assay is 0.01 unit/ml. A 90-fold purified preparation of The resolution of these enzymes was attempted and gel uridine kinase (32 units/ml) shows no guanosine kinase permeation chromatography on Sephadex G-150 was found activity.) and (Â¿>)unlabeleduridine (120 nmoles) does not to yield a sufficient resolution of the enzymatic activities compete with 14C-labeled guanosine for the kinase. responsible for the incorporation of guanosine into guano The nucleoside phosphorylase appears to be specific for sine nucleotides. The enzymes were not in all cases com guanosine since uridine and adenosine could not be used as pletely separated from each other but in every case a specific substrates. assay was possible. For example, in the case of purine Table 2 shows that the peak labeled NMK in Chart 2 nucleoside phosphorylase and nucleoside diphosphate ki- catalyzes the phosphorylation of GMP to GDP but does not nase the enzymes were not resolved but the specificity of the phosphorylate UMP. AMP is phosphorylated to ADP to a assays distinguish them. The former is assayed with labeled minor extent. This may be due to contamination by guanosine and P,, the latter with labeled GDP and ATP. adenylate kinase or may be a property of the guanylate The cluster of enzymatic activities included guanosine kinase itself; we have not explored this point further. kinase, purine nucleoside phosphorylase, purine nucleoside However, Entner and Gonzales (6) reported that the monophosphate kinase, nucleoside diphosphate kinase, and reproductive tract of Ascaris lumbricoides contains a high purine nucleoside triphosphate phosphatase (Charts 1 and level of AMP and GMP kinase. They also presented 2). Chart 3 shows the apparent molecular weights of these evidence of a partial separation of the guanylate from the enzymatic activities as obtained by the gel permeation adenylate kinase. More recently, Shimono and Sugino (21) chromatography method to be: purine nucleoside mono- have reported the purification of an enzyme that catalyzes phosphate kinase, 1.9 x IO4,purine nucleoside triphosphate transphosphorylation between ATP, GMP, and dGMP. phosphatase, 3.2 x 10*;nucleoside diphosphate kinase, 6.2 The enzyme has been purified 1000-fold from extracts of x IO4and guanosine phosphorylase, 6.8 x 10*.No apparent calf thymus and exhibits a strict specificity for these molecular weight could be determined for guanosine kinase nucleotides, which contain guanine as the base component. since the enzymatic activity was eluted with the void Nucleoside diphosphate kinase is present in very high volume. It was not possible to determine amounts of levels in Novikoff ascites rat tumor cells. We find little or no individual enzymes in the initial extract or Fraction 2 substrate specificity in this activity (Table 3) as is the case because substrates and products would be acted upon by the with the enzyme prepared from other sources (1, 10, 18). other enzymes in the sequence. Hence, we cannot present The nucleoside triphosphatase activity observed had no information on recoveries. activity on pyrimidine nucleotides (CTP, UTP, or TTP) and

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. M. Schaffer, R. B. Huribert, and A. Orengo metazoan to any significant extent, but rather they are derived primarily by degradation of nucleic acids. They may be so reinserted into nucleic acids as nucleotides resulting from the actions of nucleotide pyrophosphorylases, nucleo side phosphorylases, and kinases. We believe that salvage pathways must have greater significance in the adult metazoan, which degrades large amounts of nucleic acids, than is generally considered. Tumor metabolism seems to depend to a great extent on salvage mechanisms, and these mechanisms must be considered in design and comprehen sion of chemotherapeutic approaches involving biosynthesis of nucleic acids. Rapid and convenient procedures for determining incorporation in vivo of guanine precursors into RNA and DNA, in conjunction with isolation, partial resolution, and measurement of activities of the enzymes involved in the incorporation, may be of interest in studies 30 40 50 60 70 80 90 100 110 of the effects of certain chemotherapeutic agents on the FRACTION NUMBER biosynthesis of RNA-guanine in sensitive and resistant Chart I. Elution of guanosine (G/Ã®)kinase andguanosine phosphorylase strains of tumor. We expect that different tissues and from a Sephadex G-150 column. An aliquot Ã¶l"Fraction 2 was applied to a tumors will utilize purines with different metabolic patterns; Sephadex G-150 column (3 x 52 cm) equilibrated with 0.01 MTris-CI, pH therefore, these patterns should be specifically determined 7.7. The same buffer was used to elute the enzymes at a rate of 15 ml/hr. for each system. Fractions of 4 ml were collected, read at 280 nm, and assayed for It is also conceivable that the coordinated and normal enzymatic activities. The standard conditions described in the text for functioning of salvage pathways may exercise reciprocal radiochemical assays were used. Va, void volume. controls on the de novo synthesis of purines. The Lesch- Nyhan (12) syndrome seems to substantiate such an infer converted GTP to GDP 1.75 times faster than ATP to ence. The syndrome is an X-linked neurological disorder ADP. Considerable amounts of nucleoside triphosphatase consisting of mental retardation, choreoathetoses, cerebral activity were removed by the pH 8.0 water wash. palsy, and a typical compulsive biting of fingers and lips

DISCUSSION 130 NOK In this paper we have described convenient methodology 120 - for culture of Novikoff ascites tumor cells and a practical 100 '- and reliable method for determination of the rates of incorporation of guanosine (and other nucleosides or nucleic 90 - acid precursors) into RNA and DNA. These methods have a wide range of applicability in addition to the specific 80 - studies here. In addition, the group of enzymatic activities that catalyze the conversion of guanosine to GTP have been r_ 70 - prepared from an extract of these same cells in a single E to procedure by use of DEAE-cellulose, and methods are t 60 - presented for sufficient resolution of these enzymes by gel 3 permeation chromatography to permit study of their activi Ã• 50- 2.0 I >- ties and characteristics. PNl * 40 - Although the enzymatic reactions of guanosine metabo lism here described have been known for some time to occur in certain types of cells and/or bacteria, the results of these 30 studies interest us inasmuch as they offer an accessible I.OK 20 - system in order to study (a) details of specificities of CO tr substrates and phospho-donors in assimilation of guanine in o a single cell type and (b) the biosynthesis of GTP from IO - guanine or guanosine by a multienzyme system recon structed in vitro. As a result, interrelated phenomena such as competition for substrates, rate-limiting steps, and 20 30 40 50 60 70 80 90 100 110 regulatory mechanisms of allosteric nature could be investi FRACTION NUMBER gated in a controllable model system accessible by relatively Chart 2. Elution of purine nucleoside monophosphate kinase (NMK), simple analytical techniques. nucleoside diphosphokinase (NDK), and GTPase from a Sephadex G-150 Bases and nucleosides are not synthesized de novo in the column. The conditions are identical to the ones described in Chart 1.

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Table 3 2.5- Substrate specificity of nucleoside diphosphokinase from Novikoff CYTOCHROME rat tumor asciles cells NMK 19x10 The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7), CHYMOTRYPSINOGEN' 0.7 /jmole of MgCl2, 0.3 /imole of ATP, the labeled nucleoside triphos- i GTPose 3.2 x IO4 phates in the concentrations listed below, and 2.45 /ig of enzyme (Frac tion 63 from the Sephadex column) in a total volume of 60 Â¡i\.Thereac tion mixtures were incubated for IOmin at 37Â°.At the end of the incuba 2.0- OVALBUMIN tion time, 75 nmoles each of the appropriate mono-, di-, and nucleoside NDK 62 t IO ' triphosphates in a total volume of 20 /il were added to the reaction mix ture which was immediately frozen in a bath of Dry Ice and acetone. Ali > GR Fhosphorylose ALBUMIN (bovlnÂ«)Â«\6.8 x IO4 quots of 10 //I were spotted on Whatman No. 3MM paper and the com pounds were separated by paper electrophoresis in 0.05 M citrate buffer. â€¢¿X1.5- pH 7.0. Each spot was localized by illumination with UV, cut out, and counted in a Packard scintillation counter.

y GLOBULIN (human) V activity(cpm/nmole)436550561.61239.0AmountofXTP tration(mM)0.8160.8681.2000.918RadioinIO formed y GLOBULIN DIMER (?) SubstrateGDP-U-'K:ADP-U-"CUDP-U-14CCDP-2-"CConcenmin(nmoles)33.137.039.035.3 1.0-

a! A 1 (- 00. o z z 0.5 â€¢¿--â€”¿â€”¿I â€”¿I â€”¿I with consequent mutilations. An enzyme defect associated IO3 IO4 IO5 IO6- with the syndrome is indeed a lack of hypoxanthine (gua- MolecularWeight nine) phosphoribosyltransferase activity detectable in sev Chart 3. Determination of the molecular weights of guanosine phospho- eral tissues of the affected subjects (20). Since these patients rylase (GR phosphorylase), purine nucleoside monophosphokinase have a marked increase in the rate of purine biosynthesis de (NMK), nucleoside diphosphokinase (N[>K), and GTPase by filtration on novo, the deficiency of the salvage enzyme strongly indi Sephadex G-150. Mixtures of 10 mg bovine serum albumin and I mg cates a coordination between the 2 convergent pathways but cytochrome c, 8 mg chymotrypsinogen A and ovalbumin, and 5 mg human -y-globulin and I mg of cytochrome in volumes of 5 ml were filtered does not yet define the biochemical lesion responsible for through a column of Sephadex G-150 (3 x 52 cm) equilibrated with 0.01 M the physiological signs. Tris-Cl. pH 7.7, in 3 separate runs. Cytochrome c was determined by following the absorbance at 412 nm. All the other molecular markers were determined by following the absorbance at 280 nm. In a separate run 210 ACKNOWLEDGMENTS mg of protein (Fraction 2) in a volume of 16 ml were filtered through the column. The enzymatic activities were determined by the radiochemical We wish to thank Dr. Caroline Vaughan for developing the optimal conditions for cell culture, Cynthia K. Parks for assistance in establishing assays as described in the text. the "hot potassium acetate procedure," and Frances J. Estes for excellent technical assistance. Table 2 Substrate specificity of purine nucleoside monophosphokinase from Novikoff ral tumor osciles cells REFERENCES The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7), 0.7 //mole of MgCl2, 0.3 /miÃ³leof ATP. and 53.8 nmoles of AMP-8- "C (980 cpm/nmole), 48.0 nmoles of UMP-2- "C ( 1144 cpm/nmole). or 1. Berg, P.. and Joklik, W. K. Transphosphorylation between Nucleoside 38.8 nmoles of GMP-8-"C (1114 cpm/nmole), and 1.9 //g of enzyme Polyphosphates. Nature, 172: 1008 1009. 1953. (Fraction 82 from the Sephadex column) and was incubated in a total vol 2. Callender, S. T., Powell, E. O., and Witts, L. J. Life-span of Red Cell ume of 50 /il for 20 min at 37Â°. in Man. J. Pathol. Bacterio!., 57: 129 139, 1945. Adenosine, AMP, ADP, and ATP, 150 nmoles each, or uridine, UMP, 3. Cook, J. L., and Viber. M. The Utilization of Purines and Their U DP, and UTP. or guanosine, GMP, and GTP, 150 nmoles each, in a Ribosyl Derivatives for the Formation of Adenosine Triphosphate and volume of 30 /Â¿Iwereadded to the reaction mixture which was immediately Guanosine Triphosphate in the Rabbit Reticulocyte. J. Biol. Chem.. frozen in a bath of Dry Ice and acetone. Aliquots of 10//I were spotted on 241: 158 160, 1966. Whatman No. 3MM paper, and the compounds were separated by paper electrophoresis in 0.05 Mcitrate buffer, pH 3.4. Each spot was locali/ed by 4. Eagle. H. Specific Amino Acid Requirements of Mammalian Cell illumination with UV, cut out, and counted in a Packard scintillation (Strain L) in Tissue Culture. J. Biol. Chem., 214: 839 852, 1955. counter. 5. Emanuel, C. F., and Chaikoff, I. L. A Hydraulic Homogenizer for the Controlled Release of Cellular Components from Various Tissues. Amount formed in 20 min (nmoles) Biochim. Biophys. Acta, 24: 254-261, 1957. 6. Entner, N., and Gonzalez, C. Nucleoside Mono- and Diphosphate Substrate UDP UTP ADP ATP GDP GTP Kinase of Ascaris lumhricoides. Biochim. Biophys. Acta, 47: 52 60, 0.0AMP-8-14CGMP-8-"C0.04.2UMP-2- "C 1961. 7. Farr, R. S. Experiments on Fake of Lymphocyte. Anat. Record. 109: 2.819.7 515 533. 1951. 5.7 8. Jeanneret, H., and Fischer, R. DurÃ©edela Vie des PolynuclÃ©aires

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1973 American Association for Cancer Research. Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff Ascites Rat Tumor Cells in Culture

Martin Schaffer, Robert B. Hurlbert and Antonio Orengo

Cancer Res 1973;33:2265-2272.

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