Nucleic Acid Metabolism in Regenerating Rat Liver I. The Rate of Deoxyribonucleic Acid Synthesis in Vivo1

LISELOTTEI.HECHTANDVANR. POTTER

(McArdie Memorial Laboratory, University of Wisconsin, Madison 6, Wis.)

An ideal system for examining the relationship MATERIALS AND METHODS between ribonucleic acid (UNA) and deoxyribo- Partial hepatectomy was performed by the method of nucleic acid (DNA) metabolism in animals would Higgins and Anderson (18) on male albino rats1 weighing 180 be a tissue where synchronous cell division occurs. to 185 gm. The animals were fasted for 16-18 hours before the Isotopie tracer studies (9, 22, 25) and determina operation and fed ad libitum after the operation. At the indi cated times each animal received a single intraperitoneal in tion of the DNA content per nucleus in the liver jection of 1 mg. (5.75 pinoles) of orotic acid-6-C14which con cells of partially hepatectomized rats (25, 26, 83) tained 4.1 X 10* counts/min/mg. Following the injection, have suggested that in animals the most feasible some animals were kept in metabolism cages to facilitate col approach to this situation is by the use of re lection of urine and respiratory CO2. The animals were killed by decapitation. The liver was perfused in situ with ice-cold generating liver. The observations that incorpora 0.25 H sucrose containing 0.00018 M CaCl2, excised, and tion of isotopically labeled precursors into DNA weighed. is correlated with the occurrence of cell division Preparation of cellfractions.'—Theliver was forced through (5,17,30) and that isotopes are retained extensive a plastic mincer, and a 10 per cent homogenate of the liver was ly in the DNA of mitotically inactive and active prepared in 0.25 Msucrose + 0.00018 u CaCl2 (19) with the use cells (2, 4, 6,10-12,16, 31, 32) indicate that DNA of a Potter-Elvehjem glass homogenizer. The nuclear fraction was sedimented by centrifugation at 600 X g for 10 minutes at displays a high biochemical stability. Therefore, 2°-5°C.in an International Centrifuge, with a flat yoke No. it is assumed that the extent of the incorporation 269. The nuclear fraction was then washed once with 0.25 M of natural precursors into DNA can be taken as sucrose + 0.00018 MCaCl2 and by centrifugation of the nuclei at 600 X g for 10 minutes through a two-layer phase of a measure of the rate of DNA synthesis. This is 0.25 M and 0.84 u sucrose, both containing 0.00018 MCaClj supported by the reasonable agreement between (19). Cytoplasmic contamination was removed by exposing the incorporation of P32into DNA and the quan the nuclei to 2 per cent citric acid for 30 minutes 2 times and titative increase of DNA that has been demon by washing the nuclei twice with 2 per cent acetic acid, sedi- strated in regenerating liver (25). In the present menting the nuclei at each step by centrifugation at 600 X g for 10 minutes (3, 25). The combined supernatants comprised report the rates of DNA synthesis during the early the cytoplasmic fraction. period of liver regeneration, as studied with orotic Determination of radioactivity.'—Allradioactivity measure acid-6-C14in vivo,are described in detail to provide ments were made in internal gas-flow counters, and all the a time schedule for studies of the nucleic acid values were corrected for self-absorption.1 Urine samples were plated directly on aluminum discs inscribed with a circle 1.85 metabolism in a tissue which is changing its cm. in diameter, dried at room temperature, and their radio metabolism to meet the synthetic and energy re activity was measured. The respiratory CO2was collected in 6 N quirements of cell division. NaOH, precipitated as BaCOj, plated, and its radioactivity Orotic acid, a precursor with relatively few was measured (7). The values are expressed as per cent of the dose of radioactivity administered. alternative metabolic pathways, is extensively Extraction and radioactivity analyses of the acid-solublefrac converted to the acid-soluble uracil and cytosine tion, RNA, and DNA.—With samples high in radioactivity the ribonucleotides (21) and the pyrimidine nucleo- acid-soluble fraction was extracted from the homogenate or cell tides of UNA (20, 27, 35) in normal liver. In re fraction with cold trichloroacetic acid (TCA), final concentra tion 5 per cent. The acid-insoluble precipitate was washed once generating liver it has been demonstrated that and the combined extracts comprised the acid-soluble fraction orotic acid also serves as a precursor for the Aliquot nucleotides of DNA (27, 34). 1Obtained from the Holtzman Rat Company, Madison * This work was supported by a grant (No. C-646) from the Wis. National Cancer Institute, National Institutes of Health, United States Public Health Service. Preliminary reports of 1Most of the radioactivity counting was done by Mrs. this work were given previously (13, 14). Dorothy McManus and Mrs. Lynn Gilboe under the super vision of Dr. Charles Heidelberger. Their assistance is grate Received for publication July 16, 1956. fully acknowledged.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1956 American Association for Cancer Research. HECHT AND POTTER—Rate of DNA Synthesis in Regenerating Liver 989 tralimi on the plate with NH4OH, dried, and the radioactiv RESULTS AND DISCUSSION ity was determined. The acid-soluble fraction of tissues con Metabolism of orotic add-6-Clt in the rat 24-hours taining low amounts of radioactivity was prepared by direct extraction of the tissue with two volumes of 0.6 N HC1O4,and after partial hepatectomy.—Theutilization of orotic the precipitate was washed twice with 0.2 N HC104. The acid- acid for the synthesis of acid-soluble components, soluble extract was neutralized with KOH, KC1O4was centri- RNA, and DNA in the regenerating liver was fuged off after the solution was cooled in ice, and the sample was concentrated to a small volume in a desiccator, plated, and examined 24 hours postoperatively. The animals the radioactivity was measured. received a single injection of 1 mg. of orotic acid- The RNA and DNA content of the tissue was determined 6-C14 24 hours after the operation and were quantitatively in an aliquot of the homogenate or cell fraction sacrificed at times varying between 15 minutes by the hot TCA extraction procedure of Schneider (29). The and 100 days after the injection. The animals extracts were analyzed for RNA and DNA by the orcinol (23) and diphenylamine (8) reactions, respectively. excreted an average of 40 per cent of the dose of For the determination of the specific activity, the nucleic radioactivity in the urine during the first 2-4-hour acids were extracted with 10 per cent NaCl (1, 20). The acid- period after the injection, and very little C14ap insoluble fraction was washed 4-5 times with 0.2 N HC1O4and peared in the urine at later times. The absolute 3 times with 95 per cent ethanol, and the lipids were extracted twice with ethanol :ether (3:1) at 50°C.for 10 minutes. Three quantity of respiratory C14O2exhaled varied from volumes of 10 per cent NaCl were added to the acid-insoluble animal to animal; the individual time curves are precipitate, the pH was adjusted to pH 7-7.4 by addition of shown in Chart 1. NaOH (with the use of phenol red as internal indicator), and this pH range was maintained throughout the extraction at 100°C.for 30 minutes. The precipitate was re-extracted with 1.5 volumes of 10 per cent NaCl at 100°C.for 5 minutes. The tn sodium nucleates were precipitated from the combined NaCl §20 extracts with 2.5 volumes of cold 95 per cent ethanol. The precipitate was sedimented after 6-12-hour standing at "- Ie,ID —¿10°C.,washed twice with cold 95 per cent ethanol, and dis solved in 0.1 N NH4OH. The specific activity of the sodium ribonucleates of the cytoplasmic fraction was determined by Lu direct plating and RNA analyses (23) of aliquot s of this sample. °5 ce The nuclear RNA and DNA were isolated as sodium nucleates, tlJ „¿ and the RNA was degraded to the acid-soluble mononucleo- a. 0 tides with 0.1 NNaOH at 37°C.for 18-24 hours (28). The solu 4 8 12 16 20 24 28 32 tion was chilled, and the DNA was precipitated by addition of HOURS AFTER INJECTION 0.1 volume of 2 N HC1. The specific activity of the RNA was CHABT1.—Radioactivity content of respiratory COj follow determined in this acid-soluble ribonucleotide fraction by ing injection of orotic acid-6-C1424 hours postoperatively. Each direct plating of aliquota (neutralized with NH4OH on the curve represents the values obtained for one animal. plate) and RNA analyses of aliquota (23).»TheDNA precipi tate was washed with cold 0.05 N HC1. Three to four further extractions of the DNA with 0.1 N NaOH at 80°C.for 20 Within 30 minutes normal as well as partially minutes and removal of the remaining traces of acid-soluble hepatectomized animals were able to concentrate ribonucleotides yielded a sample of DNA free of detectable between 35 and 45 per cent of the administered RNA. The DNA was dissolved in 0.lNNH4OH, and the dose of radioactivity in the liver in the form of specific activity was determined by direct plating and DNA acid-soluble components and nucleic acids. The analyses (8) of aliquot s of this solution. The specific activity of the DNA remained constant after 2 further alkaline extrac rapid conversion of acid-soluble components to the tions and reprecipitations of the DNA. DNA isolated from 3- nucleic acids is shown in Chart 2. In normal liver hour regenerating liver, 2 hours after administration of 1 mg. of the labeling of DNA is too low to be plotted on a orotic acid-6-C14, contained no radioactivity after the nuclear graph of this type, but in 24-hour regenerating RNA, which had a specific activity of 60,000 counts/min/mg RNA, was separated from the DNA by this method. liver the incorporation into DNA is highly sig nificant, as shown in Chart 2. Moreover, a com *It was necessary to remove the salt present in the ribo parison of this curve with that for the normal liver nucleotide solution obtained from the nuclear RNA fraction (20) suggests that the acid-soluble components are of the liver of animals killed several weeks after the injection, converted to RNA at a more rapid rate than in the to permit the plating of a large enough aliquot. The ribonucleo normal liver.4 The specific activities and the total tide solution was neutralized with NaOH, diluted tenfold with water, and passed through a Dowex 1-xlo formate resin C14content of the nuclear and cytoplasmic RNA column (1 X 10 cm.). The column was washed with 20 ml. of fractions and of DNA are shown in Charts 3 and 4. water, and the mixture of mononucleotides held on the resin was In the regenerating liver, as in the normal liver eluted with 40-60 ml. of 6 N HCOOH. The eluates were dried (20), a marked difference in the rate of incor- in a desiccator over CaClj:NaOH (1:3). The nucleotides Were dissolved in water, and the specific activity of the RNA 4The changing rate of incorporation of C14 from orotic was determined by the plating of the major portion of the acid-6-C14 into the RNA of normal liver and regenerating sample and determination of the RNA content (23) in an liver at different times after the operation will be described in aliquot. a later publication.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1956 American Association for Cancer Research. 990 Cancer Research poration of this precursor into these two RNA nucleus or a fraction thereof, and also on the prob fractions was observed. Initially the nuclear RNA lem of whether the various RNA fractions are in had a much higher specific activity than the cyto- reversible equilibrium with the same acid-soluble plasmic UNA, but as the radioactivity content of constituents which enter into their synthesis. It is the acid-soluble fraction diminished the specific difficult to conceive a mechanism of cytoplasmic activity of the nuclear RNA fraction approached RNA synthesis in which the origin is entirely that of the cytoplasmic RNA. At later times (1, nuclear without an eventual time point at which the specific activity of the cytoplasmic RNA or a particular fraction thereof becomes greater than that of the nuclear RNA. The fact that the two

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2 RNA^A-j"^NUCLEAR o0(--^„ 02468 16 HOURS AFTER INJECTION i) 1 1 120 1 "If »-•64 24JL96INJECTION-][\\\\\ir CHART2.—Incorporation of C14into the acid-soluble frac 416HOURS 8 12 100DAYS tion, RNA, and DNA following injection of orotic acid-6-C1424 AFTERRNA hours after partial hepatectomy. The per cent of the dose of CHART4.—Total incorporation of C14into DNA and into administered C14present in DNA, RNA, and in the acid-soluble nuclear and cytoplasmic RNA at different times following in fraction is indicated by the space between each curve and the jection of orotic acid-6-C1424 hours postoperatively. next line below it.

QG TABLE 1 5O RNA•">! •¿NUCLEAR »CYTOPL.RNA.' RETENTIONOFRADIOACTIVITYINTHE „¿Â¡Ti DNA OFREGENERATINGLIVER '0i4LU DNA\>\ » DAYS AFTEB INJECTION 1 4 64 100 3o^'Q \l-\j• (counts/min/total liver DNA) **"--•,°'° 83,000 88,500 77,000 88,800 83,000 91,500 100,000 92,800 1*" 90,000 99,000 112,000 83,800 iXQ?-_9*"°/o t - --4-K 87,600 64,100 AV. 85,300 91,700 96,300 82,400 =«-)\ i i ir i i i "^p» 1 mg. of orotic acid-6-C14was injected 24 hours postoper 4 8 12 JUI 4 13 JL 64---4OIOC <* atively, and the animals were sacrificed at intervals after the DAYS 0 HOURS TIME AFTER INJECTION injection. Each figure represents data from one animal. CHART3.'—¿Specificactivities of the DNA, nuclear and cyto plasmic RNA at time intervals following injection of orotic spéculeactivitiestend to converge with time seems acid-6-C1424 hours postoperatively. more compatible with a mechanism in which both categories of RNA include fractions that are in 4, and 13 days) the specific activities of the nuclear reversible equilibrium with a pool of acid-soluble and cytoplasmic RNA fractions decreased at the precursors which are at least partly common to same rate, and a nearly complete loss of radio both. activity from the RNA had occurred 64 and 100 The DNA accumulated increasing amounts of days after the injection of orotic acid-6-C14 (the C14during the first 24-hour period after adminis specific activities were between 20 and 30 counts/ tration of orotic acid-6-C14, and thereafter the min/mg of nuclear or cytoplasmic RNA 100 days total C14content of the total liver DNA remained after the injection in three animals studied). at about the same high level. The individual values These data have a bearing on the question of for the total radioactivity present in the DNA at whether cytoplasmic RNA is independently syn different times after the injection are shown in thesized or whether part of it has its origin in the Table 1. Such long-term retention of tracers in the

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1956 American Association for Cancer Research. HECHTANDPOTTER—RateofDNA Synthesis in Regenerating Liver 991

DNA has been demonstrated in regenerating liver C14content of the DNA of the whole liver is shown following incorporation of P82 (2, 6) and of labeled in Chart 5. In agreement with the previous reports purine precursors (4,10,11) and in young, growing (20) in the normal rats the labeling of DNA was rats after administration of orotic acid-C14 (32) low. The total C14 incorporation into DNA was and CaC14O3 (31). Similarly, there was no loss of greatest when the precursor was given 24 hours radioactivity from the DNA, labeled with P32, of postoperatively and somewhat lower when the cells grown in tissue culture during an 8-day period orotic acid-6-C14was injected 30, 36, and 48 hours (12) and during bacterial growth (16). The concept after the operation. Administration of the precur of the biochemical stability of DNA is thus sup sor 1 and 12 hours postoperatively also resulted in ported by the findings of isotope retention in the greatly increased DNA labeling as compared with DNA phosphate, purines, and pyrimidines. that in the normal rat.6 These data summarize the On the basis of the above findings it was as extent of the over-all DNA synthesis and indicate sumed that incorporation of orotic acid-6-C14 into a maximum rate at about 24 hours postopera tively, but, in order to measure the exact tim

KwiO>5tx5 LIVER1'*\.i ing of the beginning and maximum rate of DNA synthesis during the early period of liver re generation, the extent of DNA labeling over short time intervals was determined, as shown in Chart •¿â€¢ •¿*\ 6. Groups of animals received a single injec- ' -—_—*]

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2<~i OJ n-_NORMAL i ili i HOURSO 12 24 30 36 O TIME OF OROTIC ACID INJECTION POST OP. x C'HABT5.—Incorporationof C14into DNA of normal and re O. generating livers during a period of 4-5 days. Orotic acid-6-C" o was administered to partially hepatectomized animals at differ ent times postoperatively, and the animals were killed 5 days af i ter the operation; orotic acid-6-C" was injected into four nor 5 10 ¡5 20 25 30 35 40 48 72 144 mal rats, and they were sacrificed 4 days after the injection. HOURS AFTER PARTIAL HEPATECTOMY The points represent the C" content of the DNA of the whole CHART6.—Timecurve of the rate of DNA synthesis in re liver of individual animals. generating liver. The bars represent the C" content of the DNA of the whole regenerating liver 2 hours after injection of orotic the DNA pyrimidines reflects the extent of DNA acid-6-C14at different times after partial hepatectomy. synthesis in the tissue under the conditions em ployed in these experiments. tion of 1 mg. of orotic-acid-6-C14 at different Studies of the rate of DNA synthesis at various times postoperatively, and all animals were killed times after partial hepatectomy.—A preliminary 2 hours after the injection.6 The total amount study of the labeling of DNA at various times of radioactivity incorporated into the DNA of postoperatively was carried out during a period the individual livers during the 2-hour period is which was long enough to include a substantial indicated. No labeled DNA was formed dur fraction of the period of DNA synthesis and the ing the initial 18-hour period of regeneration. A major part of the time period during which maximum rate of DNA synthesis was observed labeled DNA precursors remained available. In 24-30 hours after the operation. Although the rate the previous experiment and in subsequent ex at specific times after this period changed marked periments (15) it was shown that the latter period ly, a continued high over-all incorporation of C14 was about 24 hours following the injection of into DNA was demonstrated (cf. Chart 5). These orotic acid-6-C14. Four normal rats and groups of partially hepatectomized rats taken at different 1Administration of the precursor 14 hours or 4, 8, or 12 days before the operation also resulted in an increase in the times postoperatively were given a single injec C14 content of the DNA during the 5-day period after the tion of 1 mg. of orotic acid-6-C14. All animals which operation (15, 32). had been operated upon were killed 5 days (cf. •¿Allthe animals injected between 18 and 144 hours after Chart 4) after the operation, and the normal ani the operation and killed 2 hours after the injection were mals were sacrificed 4 days after the injection. The partially hepatectomized between 8:30 and 10:30 A.M.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1956 American Association for Cancer Research. 992 Cancer Research findings suggest that a maximum number of cells tion studies, since quantitative increases of the synthesize DNA after a given interval following DNA per liver, shown in Chart 7, the variable the operation, and thereafter DNA synthesis con values obtained for the DNA content per nucleus tinues but fewer cells synthesize DNA at a given (25, 26, 33) or per gram (24), and the over-all C14 time. The time points are insufficient to present a incorporation into DNA of regenerating liver comparison with the data described by Barnum (Chart 5) cannot provide such definitive informa (2). The timing of the period of most active DNA tion. These studies thus provide a basis for the synthesis is in agreement with the findings made timing of studies on the intermediates in DNA with P32 (22, 25) and labeled glycine (9). Since all synthesis and for studies with slices to be reported these precursors are actively incorporated into subsequently. the DNA at a specific time postoperatively, it is assumed that the incorporation of these precursors SUMMARY into the DNA represents de novo synthesis of 1. A single injection of orotic acid-6-C14 was DNA. administered to partially hepatectomized rats 24 From the combined information provided by hours after the operation. An average of 40 per the above studies it is possible to obtain a fairly cent of the dose of radioactivity was excreted in the urine during the first 4 hours, and an average 25ce of 20 per cent of the dose appeared in the respira tory COs within 20 hours. £20\15«IOoo 2. The liver contained 35-45 per cent of the dose of C1430 minutes after the injection. Initially the labeled acid-soluble intermediates were incor porated into nuclear RNA at a rate greatly exceed ing the appearance of C14in the cytoplasmic RNA. ^^ZM! w' A•: •¿â€¢¿tiri v Twelve hours after the injection, the specific 520(-I-••Ç^[ activities of the nuclear and cytoplasmic RNA were nearly the same, and a parallel decrease in iD i i i i ir the specific activity of the two RNA fractions was 120—-•--HOURSIO 20 30 40 50 JL observed thereafter. Sixty-four and 100 days after AFTER PARTIAL HEPATECTOfvT injection the RNA fractions contained very low CHART7.—TotalDNA content of regenerating livers at dif amounts of C14. ferent times after partial hepatectomy. 3. The isotope content of the DNA continued to increase during the first 24-hour period, and definitive picture of the course of DNA synthesis throughout the subsequent 100-day period the in regenerating rat liver. For the first 18 hours isotope level of the total liver DNA remained there is essentially no DNA synthesis (Charts 6 about the same. and 7). At about 18 hours there is a sudden 4. A time curve of the rate of DNA synthesis appearance of DNA synthesis, which rapidly during the early period of liver regeneration has rises to a maximum rate at about 24-30 hours been described. No DNA synthesis occurred dur (Chart 6). This increase in rate represents the ing the first 18-hour period postoperatively, a occurrence of DNA synthesis in a relatively maximum rate of DNA synthesis was observed small fraction of the total cell population, and on 24-30 hours after the operation, and incorporation the basis of Chart 7 and other data (25) this rate of C14 into DNA continued extensively for the is probably under 10 per cent per hour. Following subsequent 2-day period. this period, there is a decrease in the per cent of cells that are synthesizing DNA (Charts 5 and 6), REFERENCES but the rate continues at a lower level for the next 1. ItuiM M, C. P., and HUSBBT, R. A. The Intracellular few days. During this time the factors involved in Heterogeneity of Pentose Nucleic Acid as Evidenced by the Incorporation of Radiophosphorus. Arch. Biocbem., diurnal variation (2) probably are active and lead 29:7-26, 1950. to variable rates of DNA synthesis according to 2. BARNUM,C. P.; JARDETZKT,C. D.; and HALBEHO,F. the time of day studied if short periods of isotope Nucleic Acid Synthesis in Regenerating Liver. Texas Rep. incorporation are used as in Chart 6, and less Biol. & Med. (in press). variable rates if longer periods of incorporation are 3. BAKNUM,C.P.; NASH,C. W.; JENNINGS,E.; NTOAARD,O.; and VERMUND,H.The Separation of Pentose and Desoxy- studied as in Chart 5. The beginning and the pentose Nucleic Acids from Isolated Mouse Liver Cell changing rate of DNA synthesis could be evalu Nuclei. Arch. Biochem., 25:376-83, 1950. ated only with the short-time isotopie incorpora 4. BARTON, A. D. Evidence for the Biochemical Stability of

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Desoxyribosenucleic Acid (DNA). Fed. Proc., 13:422, 20. HUHLBERT,R. B., and POTTER,V. R. A Survey of the 1954. Metabolism of Orotic Acid in the Rat. J. Biol. Chem., 5. BBOWN,G. B., and ROLL, P. M. Biosynthesis of Nucleic 196:257-70, 1952. Acids. In: The Nucleic Acids, 2:341-92. New York: Aca 21. . Nucleotide Metabolism. I. The Conversion of demic Press Inc., 1955. Orotic Acid-6-C" to Uridine Nucleotides. Ibid., 209:1-21, 6. BBITES,A. M.; TRACT,M. M.; and COHN,W. E. Nucleic 1954. Acids of Rat Liver and Hepatoma: Their Metabolic Turn 22. JOHNSON,R. M., and ALBERT,S. The Uptake of Radio over in Relation to Growth. J. Biol. Chem., 166:619-83, active Phosphorus by Rat Liver Following Partial Hepa- 1944. tectomy. Arch. Biochim. & Biophys., 36:340-45, 1952. 7. CALVIN,M.; HEIDELBERGER,C.; REID, J. C.; TOLBEKT, 23. MEJBAUM, W. Über die Bestimmung kleiner Pento- B. M.; and YANKWICH,P. E. Isotopie Carbon. New semengen insbesondere in Derivaten der Adenylsäure. York: John Wiley & Sons, 1949. Ztschr. f. physiol. Chem., 268:117-20, 1939. 8. DISCHE, Z. Übereinige neue charakteristische Farben 24. NOVIKOFF,A.B., and POTTER,V. R. Biochemical Studies reaktionen der Thymonukleinsäure und eine Mikro- on Regenerating Rat Liver. J. Biol. Chem., 173:223-32, methode zur Bestimmung derselben in tierischen Organen 1948. mit Hilfe dieser Reaktionen. Mikrochemie, 8:4-32, 1930. 25. NTGAARD,O.,and RUSCH,H. P. Incorporation of Radio 9. ELIASSON, N. A.; HAMMAHBTEN,E.; REICHAHD,F.; active Phosphate into Nucleic Acids of Regenerating Rat ÂQUVIST,S.;THORELL,B.;and EHRENSVARD,G.Turnover Liver. Cancer Research, 15:240-45, 1955. Rates during Formation of Proteins and Polynucleotidcs 26. PRICE, J. M., and LAIRD, A. K. A Comparison of the in Regenerating Tissues. Acta Chem. Scandinav., 6:431- Intracellular Composition of Regenerating Liver and In 44, 1951. duced Liver Tumors. Cancer Research, 10:650-58,1950. 10. FRESCO,J. R.; BENDICH,A.;and RUSSELL,P. J. Metabolie 27. REICHARD,P. On the Turnover of Purines and Pyrimi- Stability of Deoxyribonucleic Acid in the Non-growing dines from Polynucleotides in the Rat Determined with Tissue. Fed. Proc., 14:214, 1955. N". Acta Chem. Scandinav., 3:422-32, 1949. 11. FÜRST,S.S., and BROWN,G.B. On the Role of Glycine and 28. SCHMIDT,G.,and THANNHAUSEB,S.J. A Method for the Adenine as Precursors of Nucleic Acid Purines. J. Biol. Determination of Desoxyribonucleic Acid, Ribonucleic Chem., 191:289-47, 1951. Acid, and Phosphoproteins in Animal Tissues. J. Biol. 12. HEALT, G. M.; SIMINOVITCHL.; PARKER, R. C.; and Chem., 161:83-89, 1945. GRAHAM,A. F. Conservation of Desoxyribonucleic Acid 29. SCHNEIDER,W. C. Phosphorus Compounds in Animal Phosphorus in Animili Cells Propagated in Vitro. Biochem. Tissues. I. Extraction and Estimation of Desoxypentose & Biophys. Acta, 20:456-46, 1956. Nucleic Acid and of Pentose Nucleic Acid. J. Biol. Chem., 13. HECHT,L. I.; HUHLBERT,R.B.; and POTTER,V.R. Pyrim- 161:293-303,1945. idine Nucleotide Metabolism in Regenerating Rat Liver. 30. SMELLIE,R. M. S. The Metabolism of the Nucleic Acids, Proc. Am. Assoc. Cancer Research, 1:20,1954. in: The Nucleic Acids, 2:393-434. New York: Academic 14. HECHT,L. I., and POTTER,V. R. Deoxyribonucleic Acid Press, Inc., 1955. Synthesis in Regenerating Rat Liver. Proc. Am. Assoc. 31. SWICK,R. W.; KOCH,A. L.; and TARARA,D. M. Stability Cancer Research, 2:23, 1955. of DNA during Growth. Fed. Proc., 14:149, 1955. 15. . Nucleic Acid Metabolism in Regenerating Rat Liver. III. Intermediates in the Synthesis of DNA Pyrim- 32. TAKAGI,Y.; HECHT,L. I.; and POTTER,V.R. Nucleic Acid idine Nucleotides. Cancer Research, 16:999-1004, 1956. Metabolism in Regenerating Rat Liver. II. Studies on Growing Rats. Cancer Research, 16:994-98, 1956. 16. HERBHEY,A. D. Conservation of Nucleic Acids during Bacterial Growth. J. Gen. Physiol., 38:145-48, 1954. 33. THOMSON,R. Y.; HEAGT,F. C.; HUTCHISON,W.C.; and 17. HEVEST,G., and OTTEBEN,J.Rate of Formation of Nucleic DAVIDSON,J. N. The Deoxyribonucleic Acid Content of Acid in the Organs of the Rat. Acta Physiol. Scandinav., the Rat Cell Nucleus and Its Use in Expressing the Results 6:237-47, 1943. of Tissue Analysis, with Particular Reference to the 18. HIOOINS,G. M., and ANDERSON,R. M. Experimental Composition of Liver Tissue. Biochem. J., 63:460-74, Pathology of the Liver. Restoration of the Liver in the 1953. White Rat following Partial Surgical Removal. Arch. 34. WEED,L. L. The Incorporation of Radioactive Orotic Acid Path., 12:186-202, 1931. into the Nucleic Acid Pyrimidines of Animal and Human 19. HOGEBOOM,G. H.; SCHNEIDER,W. C.; and STHIEBICH, Tumors. Cancer Research, 11:470-73, 1961. M. J. Cytochemical Studies. V. On the Isolation and Bio 35. WEED, L. L., and WILSON,D. W. The Incorporation of chemical Properties of Liver Cell Nuclei. J. Biol. Chem., C14Orotic Acid into Nucleic Acid Pyrimidines in Vitro. 196:111-«0,1952. J. Biol. Chem., 189:435-42, 1951.

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1956 American Association for Cancer Research. Nucleic Acid Metabolism in Regenerating Rat Liver: I. The Rate of Deoxyribonucleic Acid Synthesis in Vivo

Liselotte I. Hecht and Van R. Potter

Cancer Res 1956;16:988-993.

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