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

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Nucleic Acid Metabolism in Regenerating Rat Liver I. the Rate of Deoxyribonucleic Acid Synthesis in Vivo1 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 <of this fraction were plated on aluminum discs, neu pyrimidine 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 "-ID Ie, —¿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.
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