Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[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 The utilization of labeled guanosine for the biosynthesis of RNA and DNA has been studied in cells cultured from the Novikoff ascites tumor of the rat. Guanosine contrib uted primarily to guanine moieties of RNA and DNA, whereas labeled adenosine contributed to both adenine and guanine moieties. The labeled ribose moiety of uniformly labeled guanosine-14C did not enter a pool of ribose phosphate intermediates, judging from lack of contribution to adenine, uracil, and cytosine nucleosides in RNA and DNA. The group of enzymatic activities that catalyze the conversion of guanine and guanosine to guanosine triphosphate (namely, guanosine kinase, purine nucleoside phosphorylase, purine nucleoside monophosphate kinase, nu cleoside diphosphate kinase, and purine nucleoside triphosphate phosphatase) have been prepared from an extract of Novikoff ascites cells in a single procedure by the use of diethylaminoethyl cellulose. Gel permeation chromatography on Sephadex G-150 was used to resolve these enzymes sufficiently to permit determination of their individual activities, substrate specificities, molecular weight, and other characteristics. New rapid assays were developed for purine nucleoside kinase and phosphorylase, utilizing la beled nucleosides with chromatography on diethylamino ethyl paper. These techniques were designed to be useful for the measurement of the individual enzymes of the guanosine salvage pathway in studies of nucleotide metabolism and therapeutic effects. Practical methods for culture in suspension of Novikoff ascites tumor cells and for determination of the rates of incorporation of guanosine (and other nucleic acid precur sors) into RNA and DNA are described. RNA and DNA are extracted with hot 2.5 M potassium acetate and sepa rated by use of alkali in a form convenient for resolution of individual nucleotides and bases by electrophoresis and chromatography. INTRODUCTION Although nucleic acid derivatives are not required in the diet, the utilization of bases and nucleosides may have 'This work has been supported by N IH Research Grant ÇA-10407to A. O. Development of the methods for cell culture and the "hot potassium acetate" procedures for extraction and separation of nucleic acids was 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 the Robert A. Welch Foundation. Received December 20, 1972; accepted June 8, 1973. OCTOBER considerable significance in the adult metazoan in which large amounts of nucleic acids are degraded hourly in the normal processes of destruction and renewal of cells. The life-span of neutrophils is estimated at 70 to 80 hr (8), and that of lymphocytes is estimated at 24 hr (7, 26). Erythrocytes are replaced at a rate of 0.83% every day (2), and 60 to 70% of the lining epithelium of the intestine is shed daily (11). rRNA and mRNA are also degraded and are presum ably utilized intracellularly. The possibility that these salvage processes may be regulated or coordinated with synthesis de novo of nucleotides has not received the proper attention. In this communication we report on the utilization of guanosine for biosynthesis of nucleic acids in Novikoff ascites rat tumor cells. These cells in tissue culture suspen sion readily utilize 14C-labeled guanosine and adenosine for nucleic acid biosynthesis. Here, we describe both the pattern of utilization of the nucleosides by living tumor cells and the partial isolation from these cells of a group of enzymes responsible for the conversion of guanosine to GTP. It may be useful that these enzymatic activities can be partially purified simultaneously. MATERIALS AND METHODS All the 14C-labeled nucleosides and nucleotides were purchased from Schwarz/Mann, Orangeburg, N. Y., or from Amersham/Searle Corp., Arlington Heights, 111.All the other nucleosides and nucleotides used were products of P-L Biochemicals, Milwaukee, Wis. Ribose 1-phosphate was purchased from Sigma Chemical Co., St. Louis, Mo. The Novikoff ascites tumor was originally supplied by Dr. Alex B. Novikoff. Whatman DEAE-cellulose DE 52 was purchased from H. Reeve Angel and Co., Inc., Clifton, N. J. Amino acids and vitamins were purchased from Sigma; bovine and calf serum was from Grand Island Biological Co., Grand Island, N. Y., Pluronic acid F68 was from Wyandotte Chemical Co., Wyandotte, Mich.; streptomycin sulfate was from Charles Pfizer and Co., Inc., New York, N. Y.; and neomycin was from the Upjohn Co., Kalamazoo, Mich. ; The Novikoff ascites cells were transplanted and grown for 5 to 6 days in the peritoneal cavity of young female Holtzman Sprague-Dawley rats (120 to 150 g). Cell Culture. For establishment of a suspension culture of tumor cells in vitro, rats displaying modest amounts of ascitic fluid were selected since tumor cells derived from 1973 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. 2265 M. Schaffer, R. B. Huribert, and A. Orengo large ascites volumes had a higher initial mortality when set up in suspension culture. A rat was anesthetized and the abdominal area thoroughly cleaned with 70% ethanol. Using a sterile disposable 10-ml syringe and 20-gauge needle, 4 to 5 ml of ascitic fluid were removed and diluted with an equal amount of culture medium previously warmed at 37°using sterile conditions. The cells were dispersed using a gentle rocking motion and centrifuged at 60 xg for about 5 to 6 min. The supernatant, which should contain most of the red cells, was carefully decanted. The sedimented cells were then dispersed using the same rocking motion in 5 ml of fresh medium at 37°. One-tenth ml of the cell suspension was diluted to 10 ml with culture medium and counted in an hemacytometer. Once the number of cells in the suspension was determined, aliquots containing 1.9 x 10' cells were transferred to culture bottles and culture medium warmed at 37°was added to a total volume of 25 ml. Sterile technqiues were used at all times. The bottles were capped well and placed in the roller drum at 37°;then they were incubated for 24 hr. The culture medium was prepared by mixing in order the following ingredients: a dry mixture of salts, 100 ml of a modified McCoy's amino acid (4, 15) 10 x stock solution, 20 ml of McCoy's vitamin 50 x stock solution (15), 10 ml of (neutral point of phenol red); 1.0 ml was withdrawn and the bottles were returned to the roller drum. The aliquot was then diluted with medium and counted as described above. The desired cell number after the start of the culture was 5 x IO5cells/ml of medium and this applied as long as the cell line was continued. Consequently, if the cell suspen sions were grown to a density of more than 5 x IO5cells/ ml, appropriate aliquots were withdrawn and discarded or cultured separately. The remainder was centrifuged for 5 min at 60 x g to remove the old medium and to replace it with 25 ml of fresh, warmed medium. At the end of any 48-hr period if the cell count had not doubled the suspension was discarded. On all subsequent feedings, the same proce dure was followed to keep the cell count at ca. 5 x IO5 cells/ml. The cells should have a rounded appearance with a sharp cell membrane. Cells that were very large and granular, developing swelling or vacuoles, were regarded as unlikely to survive and were not counted when taking a cell count. Cells were not subjected to heavy mechanical or thermal shock. They were not left for more than 24 hr without changing the medium. When NaHCO3 was added, the bottle was swirled to prevent a sudden localized massive pH change. The bottles were not shaken but merely tilted or 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 used.2 double glass-distilled water and sterilized by filtration Extraction and Initial Purification of Guanosine-metabothrough a Seitz filter which had been autoclaved using a size 6 filter sheet (Republic Seitz Filter Co., Newark, N. J.). It lizing Enzymes. For studies on the purification of the enzymatic activities, 20 to 50 tumor-bearing rats were was stored at 04. decapitated and the ascitic fluid was collected and diluted The dry mixture of salts was composed of: lactalbumin 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 the tumor cells were collected as a sediment. This sediment NaHCO3, 2.0 g. 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 M sucrose (pH 7.7) at a cell to buffer ratio of 393; t.-valine, 176; i.-threonine, 179; i.-asparagine, 450; 1:4. The suspension was homogenized in an Emanuel-Chaikglycine, 75; L-serine, 263; L-alanine, 134; L-proline, 173; L-aspartic acid, 199; i.-glutamic acid, 221. It was stored off orifice-type homogenizer (Microchemical Specialties Co., Berkeley, Calif.) (5). In order to break most of the frozen. 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 20,000 x g for 20 min in a refrigerated International Model It was stored frozen. 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 International Model B35 centrifuge. The sediment was 6 months. 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 medium described here is more complete than that of Morse and Potter use. in direct comparison in our laboratory, gave consistently more Subsequent Feeding of Cell Suspensions. At the end of the and, 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?. 2266 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. GuarÃ-osmeAnabolism in Novikoff Ascites Cells discarded and the supernatant was called Fraction 1. Protein concentration was determined by the method of Lowry et al. (13) and the concentration was then adjusted to 5.0 mg/ml with 0.02 MTris-Cl (pH 7.5). For each 1000 ml of Fraction 1, 600 ml of DEAE-cellulose suspension in 0.01 MTris-Cl (pH 8.0): 10% glycerol (50:50, v/v)3 were added slowly with continuous stirring. After 2 hr of gentle stirring the suspension was centrifuged and the supernatant was discarded. The sediment was then washed with 1000 ml of H2O adjusted to make the solution pH 8.0 with NaOH. After centrifugation the supernatant was discarded, and the sediment was washed again with 400 ml of 0.075 M KC1:0.01 M Tris-Cl: 10% glycerol (pH 8.0). The superna tant was discarded. The sediment was then eluted by 2 hr of gentle stirring in 500 ml of 0.3 M potassium phosphate buffer (pH 6.5). The DEAE-cellulose was removed by centrifugation, and the volume of supernatant was reduced at least 6 times using Centriflo ultrafiltration membrane (American Scientific Systems Division, Lexington, Mass.). This was called Fraction 2. Gel Permeation Chromatography. Fraction 2 was cen trifuged briefly to remove any precipitate. Of the resulting solution, 20 ml containing approximately 200 mg of protein were applied in 0.3 M potassium phosphate buffer (pH 6.5) on a Sephadex G-150 column (3 x 52 cm). The flow rate was 15 ml/hr and fractions of 4 ml were collected. The proteins were eluted with 0.01 M Tris (pH 7.7) and the fractions were read at 280 nM to follow the protein elution. The enzymatic activities were determined with the radioac tive assays described below. Assays of Enzymes. All enzymatic activities were deter mined by radiochemical assays. All assays were done at 37° and initial velocities were measured (no more than 10% conversion allowed). In this range, the measured activities were proportional to enzyme concentration. The products and substrates were separated either by chromatography on DEAE-paper (Whatman DE 81) or by high-voltage electrophoresis on Whatman No. 3MM paper. Nucleoside Kinases Assay. A new radiochemical assay was used which measures the conversion of labeled ñucleoside to nucleotide by Chromatographie separation of the reactants and products on DEAE-paper. The standard assay mixture (final volume of 60 ¿tl)contained 5 Amólesof Tris-Cl buffer (pH 7.4), 420 nmoles of MgCl2, 300 nmoles of ATP, 100 nmoles of nucleoside labeled with 14Cin the purine or pyrimidine ring (1 /Ã-Ci//¿mole),and varying amounts of enzyme. After 1 hr of incubation, 25-^1 aliquots of the reaction mixture were pipetted in duplicate as spots 5 cm from 1 end of DEAE paper strips 2.9 x 14cm (previously spotted with 0.1 /¿moleof unlabeled nucleoside as carrier), dried, and subjected to descending chromatog raphy. The solvents used were isobutyric acid:H2O:NH3 (65.7:34:2:0.1) for the adenosine and guanosine and 80% ethanol for the uridine and cytidine. Whereas the nucleosides move with the solvent, the nucleotides remain at the 3The volume ratio of DEAE-cellulose and solution was determined after centrifugation of an aliquot for 10 min at 1000 x g. origin. An average 1% of the counts remains on the origin at 0 incubation time. This value may vary with the purity of the commercial preparations of the radioactive nucleosides used. The nucleotide spots were located by UV illumination, cut out, and counted in a Nuclear-Chicago low-background flow counter. The products were shown to be GMP and ADP by electrophoretic assays. The entire procedure takes 2 hr. Purine Nucleoside Phosphorylase Assay. The standard assay mixture (final volume of 60 ^1)contained 10 Amólesof Tris-Cl (pH 7.4), 50 nmoles of guanosine-U-14C,4 100 nmoles of sodium phosphate buffer (pH 7.4), and suitable amounts of enzyme. The incubation time was 15 min. The Chromatographie system was the same as that used for the nucleoside kinase assay. Since the guanosine used is labeled both in the guanine and ribose, this allowed measurement of the ribose 1-phosphate produced. Whereas the nucleoside and base moved with the solvent, the ribose 1-phosphate produced by the phosphorylase remained at the origin. Carrier guanosine and 5'-GMP were also added as de scribed for nucleoside kinase. 5'-GMP was added as a UV marker for the position of the ribose 1-phosphate with no UV absorbance. The spots remaining at the origin were cut out and counted in a Nuclear-Chicago low-background flow counter. In pilot experiments the location of ribose 1-phosphate at the origin was established by chromatography of ribose 1-phosphate and spraying with aniline : acetic :orthophosphoric acid reagent (6:200:20) (22). Assays for Nucleoside Monophosphate and Nucleoside Diphosphate Kinases and for Purine Nucleoside Triphosphatase. The standard assay mixture (final volume, 50 n\) contained 5 Amólesof Tris-Cl buffer (pH 7.7), 300 nmoles of ATP, 700 nmoles of MgCl2, and 50 nmoles of GMP-8-14C or GDP-U-14C or GTP-8-l4C plus a suitable amount of enzyme. The reactions were incubated at 37°for 10 min and stopped by cooling the mixture to near 0°.In a volume of 30 /tl, 100 nmoles each of guanosine, GMP, GDP, and GTP were added. Aliquots of 5 or 10 ^1 were spotted 13 cm fromv(56".5' the cathodic of ainsheet of Whatman No. 3MM •¿'paper x '29:5endcm) duplicate. The compounds were separated by paper electrophoresis on 0.05 Mcitrate buffer (pH 3.5) on a flat-plate apparatus. The field strength of the system was 47 V/cm and the temperature was kept between 5 and 7°.Each spot was localized by UV illumination, cut out, and counted in a Packard liquid scintillation counter. Assays for the other nucleoside kinases were conducted by substitution of appropriately labeled nucleotides for the guanosine nucleotides. The RI-TP, i.e., relative migration with respect to UTP for the nucleotides, can be listed as follows: UMP, 0.548, UDP, 0.882; GMP, 0.420; GDP, 0.741; GTP, 0.821; AMP, 0.252; ADP, 0.583; ATP, 0.735. One unit of enzyme was defined as the amount catalyzing the conversion of l ^mole of substrate in 60 min under the condition of the standard assay for all of the previously ' U refers to the uniformly labeled compound. OCTOBER 1973 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. 2267 M. Schaffer, R. B. Huribert, and A. Orengo described assays. The counting efficiencies were 72% for the scintillation counter and 9.5% for the flow counter. Hot Potassium Acetate Procedures for Extraction and Analysis of RNA and DNA. Ten ml of a 5% trichloroacetic acid solution were added to 0.5 ml of packed ascites cells. After centrifugation the sediment was washed 2 or 3 times with 10 ml of 5% trichloroacetic acid and then with 10 ml of 95% ethanol, rinsing the entire inner surface of tube and breaking up the precipitate. After centrifugation, the super natant was discarded and the tube was drained well. Three ml of cold 2.5 M potassium acetate and 1 small drop of concentrated phenol red solution were added to the sediment. While still cold, the suspension was neutralized to pH 7.4 to 8.0 with 1.0 and 0.1 M KOH. The suspension was stirred well and allowed to stand for 10 min at room temperature to ensure equilibration and diffusion into particles. The pH was adjusted if necessary; if too alkaline, RNA will subsequently be lost; if too acid, both RNA and DNA will be lost. The tube was capped, heated with occasional stirring for 30 min in a boiling water bath, and chilled in ice. After centrifugation, the supernatant was removed by means of a disposable pipet and filtered through a small plug of glass wool to remove particles. The pellet was reextracted in the same way with 1 ml of 2.5 M potassium acetate, with heating for 5 to 10 min. The 2 supernatants were combined and 2 to 2.5 volumes of absolute ethanol were added. The suspension was stirred well and set in a deep freeze for several hr. The flocculent precipitate of potassium nucleates was centrifuged down and washed with 7 ml of cold 70 to 80% ethanol. After centrifugation the supernatant was removed as completely as possible with a disposable pipet. Recovery of DNA is about 90% and of RNA is about 80%, based on comparison with assays by the Schneider method (Ref. 19, cf. Ref. 9). The DNA loss is primarily due to the mechanical manipula tions and it is suspected that some tRNA is lost due to slight degradation and solubility in the ethanol precipita tion.5 In order to separate RNA from DNA, 1 ml of 0.2 N NaOH (carbonate free) was added to the sediment, and the solution was incubated at 37°for 16 to 18 hr in a capped tube. At the end of the incubation the tube was cooled and 0.1 ml of 4.4 N perchloric acid was added. The DNA precipitate was centrifuged down in the cold, and the RNA hydrolysate was removed by disposable pipet and trans ferred to another small tube. The DNA precipitate was washed without delay with several ml of cold 0.2 N perchloric acid. The supernatant was discarded and the tube was drained well. The DNA was redissolved in 1.0 ml of 0.2 N NaOH and reprecipitated with 5The extraction with 2.5 M potassium acetate is preferable to the extraction with 10%NaCI solution previously described (18) because of the more convenient buffering capacity of potassium acetate during neutraliza tion and its greater solubility in ethanol to reduce salt concentration in the electrophoretic analysis. The procedure is highly reproducible and conven ient for isotope determinations on components of RNA and DNA in small tissue samples, when relatively specific precursors of nucleic acids are used. Where lipids and phospholipids are a problem, washing of the precipitated tissue or of the potassium nucleates with ethanol:ether and ethanol is necessary. 2268 cold perchloric acid as before to ensure removal of the last traces of ribonucleotides. The pellet was then dissolved in 0.5 ml of 0.5 M NaCI, 1 drop of phenol red was added, and the solution was neutralized. Ethanol, 1.5 volumes, was added, and the mixture was set in the freezer for several hr. The precipitate was centrifuged and washed by suspension and centrifugation in 1 ml of ethanol. The precipitate was dried and then heated for 2 hr at 175°in 91% formic acid. The hydrolysis was carried out in a sealed pyrex tube. The hydrolysate was evaporated under reduced pressure to dryness and redissolved in 100 to 300 n\ of l N HC1. Aliquots ( 10 to 20 ¿il) taken for Chromatographie separation of the bases were applied on Whatman No. 1 paper and subjected to descending chromatography using the solvent system of Wyatt (25). In no case was radioactive uracil detected indicating that the DNA was free of RNA. The RNA hydrolysate [2'(3')-nucleotides] was prepared for paper electrophoresis as follows. The solution was adjusted to pH 3.5 with KOH, chilled near freezing to maximize precipitation of KC1O4, and centrifuged. Ali quots (20 /il) of the supernatant were applied as a line on a sheet of Whatman No. 3MM filter paper, and paper electrophoresis was carried out in 0.05 Mcitrate buffer (pH 3.5). The field strength of system was 47 V/cm and the temperature was kept between 5 and 7°.The nucleotides were separated in the following order going from the negative to the positive pole: cytosine, adenine, guanine, and uracil. The paper was dried and the bases or the nucleotides were located by UV and cut out. The paper spots were immersed in a scintillator solution (4 g of PPO and 50 mg of POPOP per liter of toluene) and counted in a liquid scintillation counter. RESULTS Table 1 shows that 14C-labeled nucleosides are readily incorporated into cultured Novikoff rat ascites cells. The patterns of incorporation into nucleic acids are distinctly different for adenosine and guanosine. When guanosine-814C is used as precursor of RNA and DNA, the label is found predominantly in guanine moieties, whereas adenosine-8-14C contributed strongly to both guanine and adenine moieties. From the experiments with guanosine-U-14C labeled in both guanine and ribose, it appears that the ribose of the guanosine did not contribute significantly to a common pool of ribose 1-phosphate or 5-phosphoribosylpyrophosphate for other nucleotide biosynthesis. One would expect more radioactivity in the pyrimidine nucleotides if the labeled ribose 1-phosphate derived from the action of guanosine phosphorylase were available. The ready utiliza tion of adenosine for the synthesis of both AMP and GMP suggest that adenosine may be utilized through conversion of its deamination product, inosine, to inosinic acid as well as interconversion of AMP and GMP via inosinic acid at the nucleotide level. These results are in line with those obtained by Williams and LePage (23, 24). These authors studied the in vivo and in vitro incorporation of preformed purines into nucleotides and polynucleotides using incuba tion periods varying from 5 to 60 min. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. Guanosine Anabolism in Novikoff Ascites Cells 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/¿Ã-mole), 0.5 ml, 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 37°and incubated 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. of labeled ofDNAA4.14.37.769.8C2.42.62.53.6T3.82.02.50.7 labeled -(^moles/5 added 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 the reticulocyte (3) and the erythrocyte of the rabbit (14). Again, guanine and guanosine-8-'"C was utilized extensively for GTP synthesis but to a very limited extent for the synthesis of ATP. In order to study and characterize the enzymatic machin ery responsible for these in vivo findings, it was necessary to assay for the guanosine-anabolizing enzymes. Fraction 2, as described, was the only fraction of the cell extract that metabolizes guanosine. It was observed to convert guano sine to a number of metabolites, and therefore it was not possible to assay specifically for the individual enzymes involved. The resolution of these enzymes was attempted and gel permeation chromatography on Sephadex G-150 was found to yield a sufficient resolution of the enzymatic activities responsible for the incorporation of guanosine into guano sine nucleotides. The enzymes were not in all cases com pletely separated from each other but in every case a specific assay was possible. For example, in the case of purine nucleoside phosphorylase and nucleoside diphosphate kinase the enzymes were not resolved but the specificity of the assays distinguish them. The former is assayed with labeled guanosine and P,, the latter with labeled GDP and ATP. The cluster of enzymatic activities included guanosine kinase, purine nucleoside phosphorylase, purine nucleoside monophosphate kinase, nucleoside diphosphate kinase, and purine nucleoside triphosphate phosphatase (Charts 1 and 2). Chart 3 shows the apparent molecular weights of these enzymatic activities as obtained by the gel permeation chromatography method to be: purine nucleoside monophosphate kinase, 1.9 x IO4,purine nucleoside triphosphate phosphatase, 3.2 x 10*; nucleoside diphosphate kinase, 6.2 x IO4and guanosine phosphorylase, 6.8 x 10*.No apparent molecular weight could be determined for guanosine kinase since the enzymatic activity was eluted with the void volume. It was not possible to determine amounts of individual enzymes in the initial extract or Fraction 2 because substrates and products would be acted upon by the other enzymes in the sequence. Hence, we cannot present information on recoveries. Substrate Specificity Studies. The center tube of the peak labeled guanosine kinase was shown to convert guanosine only to GMP when the products were analyzed by the electrophoretic analysis. This fraction was tested for activity as adenosine kinase with no conversion to nucleotides. Uridine kinase activity was present in the guanosine kinase preparation here described but the possibility that I enzyme was responsible for both activities was excluded on the finding that (a) highly purified uridine kinase from Novikoff tumor cells (17) was tested with this assay and did not exhibit guanosine kinase activity (The lower limit of detectability for guanosine kinase in the conditions of our assay is 0.01 unit/ml. A 90-fold purified preparation of uridine kinase (32 units/ml) shows no guanosine kinase activity.) and (¿>) unlabeled uridine (120 nmoles) does not compete with 14C-labeled guanosine for the kinase. The nucleoside phosphorylase appears to be specific for guanosine since uridine and adenosine could not be used as substrates. Table 2 shows that the peak labeled NMK in Chart 2 catalyzes the phosphorylation of GMP to GDP but does not phosphorylate UMP. AMP is phosphorylated to ADP to a minor extent. This may be due to contamination by adenylate kinase or may be a property of the guanylate kinase itself; we have not explored this point further. However, Entner and Gonzales (6) reported that the reproductive tract of Ascaris lumbricoides contains a high level of AMP and GMP kinase. They also presented evidence of a partial separation of the guanylate from the adenylate kinase. More recently, Shimono and Sugino (21) have reported the purification of an enzyme that catalyzes transphosphorylation between ATP, GMP, and dGMP. The enzyme has been purified 1000-fold from extracts of calf thymus and exhibits a strict specificity for these nucleotides, which contain guanine as the base component. Nucleoside diphosphate kinase is present in very high levels in Novikoff ascites rat tumor cells. We find little or no substrate specificity in this activity (Table 3) as is the case with the enzyme prepared from other sources (1, 10, 18). The nucleoside triphosphatase activity observed had no activity on pyrimidine nucleotides (CTP, UTP, or TTP) and OCTOBER 1973 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. 2269 M. Schaffer, R. B. Huribert, and A. Orengo 30 40 50 60 FRACTION 70 80 90 100 110 NUMBER Chart I. Elution of guanosine (G/î)kinase andguanosine phosphorylase from a Sephadex G-150 column. An aliquot öl"Fraction 2 was applied to a Sephadex G-150 column (3 x 52 cm) equilibrated 7.7. The same buffer Fractions of 4 ml enzymatic activities. radiochemical assays with 0.01 MTris-CI, pH was used to elute the enzymes at a rate of 15 ml/hr. were collected, read at 280 nm, and assayed for The standard conditions described in the text for were used. Va, void volume. converted GTP to GDP 1.75 times faster than ATP to ADP. Considerable amounts of nucleoside triphosphatase activity were removed by the pH 8.0 water wash. 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 of the effects of certain chemotherapeutic agents on the biosynthesis of RNA-guanine in sensitive and resistant strains of tumor. We expect that different tissues and tumors will utilize purines with different metabolic patterns; therefore, these patterns should be specifically determined for each system. It is also conceivable that the coordinated and normal functioning of salvage pathways may exercise reciprocal controls on the de novo synthesis of purines. The LeschNyhan (12) syndrome seems to substantiate such an infer ence. The syndrome is an X-linked neurological disorder consisting of mental retardation, choreoathetoses, cerebral palsy, and a typical compulsive biting of fingers and lips DISCUSSION In this paper we have described convenient methodology for culture of Novikoff ascites tumor cells and a practical and reliable method for determination of the rates of incorporation of guanosine (and other nucleosides or nucleic acid precursors) into RNA and DNA. These methods have a wide range of applicability in addition to the specific studies here. In addition, the group of enzymatic activities that catalyze the conversion of guanosine to GTP have been prepared from an extract of these same cells in a single procedure by use of DEAE-cellulose, and methods are presented for sufficient resolution of these enzymes by gel permeation chromatography to permit study of their activi ties and characteristics. 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 studies interest us inasmuch as they offer an accessible system in order to study (a) details of specificities of substrates and phospho-donors in assimilation of guanine in a single cell type and (b) the biosynthesis of GTP from 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 regulatory mechanisms of allosteric nature could be investi gated in a controllable model system accessible by relatively simple analytical techniques. Bases and nucleosides are not synthesized de novo in the 2270 130 NOK 120 100 '- 90 80 r_ 70 E to t 60 3 2.0 I Õ 50>PNl * 40 - 30 I.OK 20 - CO tr o IO - 20 30 40 50 60 FRACTION 70 80 NUMBER 90 100 110 Chart 2. Elution of purine nucleoside monophosphate kinase (NMK), nucleoside diphosphokinase (NDK), and GTPase from a Sephadex G-150 column. The conditions are identical to the ones described in Chart 1. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. Guanosine Anabolism in Novikoff Ascites Cells Table 3 Substrate specificity of nucleoside diphosphokinase from Novikoff rat tumor asciles cells The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7), 0.7 /jmole of MgCl2, 0.3 /imole of ATP, the labeled nucleoside triphosphates in the concentrations listed below, and 2.45 /ig of enzyme (Frac tion 63 from the Sephadex column) in a total volume of 60 ¡i\.The reac tion mixtures were incubated for IOmin at 37°.At the end of the incuba tion time, 75 nmoles each of the appropriate mono-, di-, and nucleoside 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 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. pH 7.0. Each spot was localized by illumination with UV, cut out, and counted in a Packard scintillation counter. 2.5CYTOCHROME NMK 19x10 CHYMOTRYPSINOGEN' i GTPose 3.2 x IO4 2.0- OVALBUMIN NDK 62 ALBUMIN t IO ' > GR Fhosphorylose (bovln«)«\6.8 x IO4 •¿X 1.5y GLOBULIN (human) y GLOBULIN V activity(cpm/nmole)436550561.61239.0Amount ofXTP tration(mM)0.8160.8681.2000.918Radio inIO formed SubstrateGDP-U-'K:ADP-U-"CUDP-U-14CCDP-2-"CConcenmin(nmoles)33.137.039.035.3 DIMER (?) 1.0- a! (o A 1 00. z z •¿-—¿â€”¿I —¿I 0.5 IO3 IO4 IO5 Molecular Weight —¿I with consequent mutilations. An enzyme defect associated with the syndrome is indeed a lack of hypoxanthine (guanine) 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. IO6- 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 mg of protein (Fraction 2) in a volume of 16 ml were filtered through the column. The enzymatic activities were determined by the radiochemical assays as described in the text. Table 2 Substrate specificity of purine nucleoside monophosphokinase from Novikoff ral tumor osciles cells The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7), 0.7 //mole of MgCl2, 0.3 /mióleof ATP. and 53.8 nmoles of AMP-8"C (980 cpm/nmole), 48.0 nmoles of UMP-2- "C ( 1144 cpm/nmole). or 38.8 nmoles of GMP-8-"C (1114 cpm/nmole), and 1.9 //g of enzyme (Fraction 82 from the Sephadex column) and was incubated in a total vol ume of 50 /il for 20 min at 37°. Adenosine, AMP, ADP, and ATP, 150 nmoles each, or uridine, UMP, U DP, and UTP. or guanosine, GMP, and GTP, 150 nmoles each, in a volume of 30 /¿I were added to the reaction mixture which was immediately frozen in a bath of Dry Ice and acetone. Aliquots of 10 //I were spotted on 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 illumination with UV, cut out, and counted in a Packard scintillation counter. Amount formed in 20 min (nmoles) Substrate UDP UTP ADP ATP GDP GTP 0.0AMP-8-14CGMP-8-"C0.04.2 UMP-2- "C 2.819.7 5.7 ACKNOWLEDGMENTS We wish to thank Dr. Caroline Vaughan for developing the optimal conditions for cell culture, Cynthia K. Parks for assistance in establishing the "hot potassium acetate procedure," and Frances J. Estes for excellent technical assistance. REFERENCES 1. Berg, P.. and Joklik, W. K. Transphosphorylation between Nucleoside Polyphosphates. Nature, 172: 1008 1009. 1953. 2. Callender, S. T., Powell, E. O., and Witts, L. J. Life-span of Red Cell in Man. J. Pathol. Bacterio!., 57: 129 139, 1945. 3. Cook, J. L., and Viber. M. The Utilization of Purines and Their Ribosyl Derivatives for the Formation of Adenosine Triphosphate and Guanosine Triphosphate in the Rabbit Reticulocyte. J. Biol. Chem.. 241: 158 160, 1966. 4. Eagle. H. Specific Amino Acid Requirements of Mammalian Cell (Strain L) in Tissue Culture. J. Biol. Chem., 214: 839 852, 1955. 5. Emanuel, C. F., and Chaikoff, I. L. A Hydraulic Homogenizer for the Controlled Release of Cellular Components from Various Tissues. Biochim. Biophys. Acta, 24: 254-261, 1957. 6. Entner, N., and Gonzalez, C. Nucleoside Mono- and Diphosphate Kinase of Ascaris lumhricoides. Biochim. Biophys. Acta, 47: 52 60, 1961. 7. Farr, R. S. Experiments on Fake of Lymphocyte. Anat. Record. 109: 515 533. 1951. 8. Jeanneret, H., and Fischer, R. Durée de la Vie des Polynucléaires OCTOBER 1973 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research. 2271 M. Schaffer, R. B. Huribert, and A. Orengo Neutrophiles et Easinophiles dans l'Organisme Humain. Schweiz. 9. 10. 11. 12. 13. 14. 15. 16. 17. Med. Wochschr., 71: 204 205, 1941. Kämmen,H. O., and Huribert. R. B. The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro. Cancer Res., 19: 654 663, 1959. Krebs, H. A., and Hems, R. Some Reactions of Adenosine and Inosine Phosphates in Animal Tissues. Biochim. Biophys. Acta, 12: 172 180. 1953. Leblond, C. P., and Stevens, C. E. Constant Renewal of Intestinal Epithelium in Albino Rat. Anal. Record. 100: 357 377, 1948. Lesch, M., and Nyhan. W. L. A Familial Disorder of Uric Acid Metabolism and Central Nervous System Function. Am. J. Med., 36: 561 570, 1964. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265-275, 1951. Lowy, B. A., Williams, M. K., and London, I. M. The Utilization of Purines and Their Ribosyl Derivatives lor the Formation of Adenosine Triphosphate and Guanosine Triphosphate in the Mature Rabbit Erythrocyte. J. Biol. Chem., 236: 1439 1441, 1961. McCoy, T. A., and Neuman, R. E. Cultivation of Walker Carcinosarcoma 256 in Vitro from Cell Suspension. J. Nati. Cancer Inst., 16: 1221-1229, 1956. Morse, P. A., Jr., and Potter, V. R. Pyrimidine Metabolism in Tissue Culture Cells Derived from Rat Hepalomas. I. Suspension Cell Cultures Derived from the Novikoff Hepatoma. Cancer Res., 25: 499 508, 1965. Orengo, A. Regulation of Enzymic Acitivity by Metabolites. I. 2272 18. 19. 20. 21. 22. 23. 24. 25. 26. Uridine-Cytidine Kinase of Novikoff Ascites Tumor. J. Biol. Chem., 244: 2204 2209, 1969. Ratliff, R. L., Weaver. R. H.. Lardy, H. A., and Kuby, S. A. Nucleoside Triphosphate-Nucleoside Diphosphate Transphosphorylase (Nucleoside Diphosphokinase). J. Biol. Chem., 239: 301-309, 1964. Schneider, W. C. Phosphorus Compounds in Animal Tissues. I. Extraction and Estimation of Desoxypentose Nucleic Acid and of Pentose Nucleic acid. J. Biol. Chem.. 161: 293-303, 1945. Seegmiller, J. E.. Rosenbloom, F. M.. and Kelley, W. N. Enzyme Defect Associated with a Sex-linked Human Neurological Disorder and Excessive Purine Synthesis. Science, 155: 1682 1684, 1967. Shimono, H., and Sugino, Y. Metabolism of Deoxyribonucleotides. Purification and Properties of Deoxyguanosine Monophosphokinase of Calf Thymus. European J. Biochem., /9: 256 263, 1971. Walborg, E. F., Jr., and Christensson, L. A Colorimetrie Method for the Quantitative Determination of Monosaccharides. Anal. Biochem.. 13: 186 193. 1965. Williams, A. M., and LePage, G. A. Purine Metabolism in Mouse Ascites Tumor Cells. II. In Vitro Incorporation of Preformed Purines into Nucleotides and Polynucleotides. Cancer Res., 18: 554 561, 1958. Williams, A. M., and LePage, G. A. Purine Metabolism in Mouse Ascites tumor Cells. III. In Vivo Incorporation of Preformed Purines into Nucleotides and Polynucleotides. Cancer Res., 18:562 568, 1958. Wyatt, H. R. The Purine and Pyrimidine Composition of Deoxypentose Nucleic Acids. Biochem. J., 4W;584 590, 1951. Yoffey, J. M. Quantitative Study of Lymphocyte Production. J. Anat., 67:250 262, 1933. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/10/2265 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.