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Pyrimidine Metabolism in Tissue Culture Cells Derived II. Thymidine

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Pyrimidine Metabolism in Tissue Culture Cells Derived II. Thymidine Pyrimidine Metabolism in Tissue Culture Cells Derived from Rat Hepatomas II. Thymidine Uptake in Suspension Cultures Derived from the Novikoff Hepatoma1 GLENN A. GENTRY,2 PAUL A. MORSE, JR.,2 DAVID H. IvEs,' RONALD GEBERT, AND V@ R. POTTER (McArdle Memorial L,aboratoryMedical Center, University of Wisconsin, Madison, Wisconsin) SUMMARY Novikoff tissue culture cells were made totally dependent on thymidine supplied via the medium, by blocking with amethopterin. Radioactive thymidine then was supplied in various molarities, and after incubation periods of varying length the cul tures were fractionated into medium, intracellular acid-soluble compounds, and de oxyribonucleic acid (DNA), and the fractions assayed for radioactivity. Several observations were made. a) The cells rapidly concentrated thymidine from the medium into the acid-soluble fraction, reaching equilibrium in less than iO min. b) Radiochromatographic studies showed that the intracellular thymidine was largely (80—90%) in the form of thymidine triphosphate, with some (iO—20%) thy midine diphosphate and little, if any, thymidine monophosphate or thymidine. c) Increasing the concentration of thymidine in the medium caused an increase in the intracellular concentration of thymidine compounds. d) For cell multiplication, the optimal intracellular concentration of thymidine triphosphate was roughly 5—iOX 10' M. This was inferred from growth rates in van ous external concentrations of thymidine, and the relationship of extracellular to in tracellular concentrations of thymidine and thymidine compounds. e) While the over-all rate (but not onset) of DNA synthesis of Novikoff tissue cul ture cells can be modified by limiting the intracellular thymidine tniphosphate con centration, the importance of this mechanism in vivoremains to be demonstrated. In the search for the mechanisms which control the onset being influenced by rather than influencing the rate of DNA and rate of DNA synthesis in the mammalian cell, the synthesis, some evidence is available to suggest that intracellular production of thymidine (Tdn) nucleotides alterations in the tissue and/or intracellular levels of Tdr has received some attention and has been discussed in a compounds may have an effect on the rate of DNA syn recent review article by O'Brien (31). Increased activity thesis. Greulich et at. (17) found that within 6 hr follow of the enzymes related to the production of Tdr nucleo ing an i.p. injection of 10 @gofTdr per adult male mouse tides and their incorporation into DNA has been shown there was a 29 % increase in the number of metaphase to occur in regenerating rat liver (3, 4, 5, 9, 25) as well as mitotic figures in the duodenal epithelium, suggesting in other growing tissues (26, 27, 34, 36, 41). Although alterations in the mitotic cycle and perhaps in the rate of these alterations have been interpreted as representing DNA synthesis. This leads to the question of the general changes in homeostatic mechanisms supplying the neces significance of Tdr kinase and the possibility of intra sary ingredients for DNA synthesis (8), and therefore cellular and/or circulating Tdr in the animal. Relative to this, the work of Rieke (39) is pertinent. Using adult 1 This work was supported in part by grant CA-00646 from the National Cancer Institute, NIH, USPHS. mice prelabeled with tritiated Tdr, he showed that host 2 Present address : Department of Microbiology, The University lymphocytes were able to contribute label to the DNA of of Mississippi Medical Center, Jackson, Miss. transplanted sarcomas; conversely, prelabeled sarcoma 3 Present address : Department of Biochemistry, The Ohio cells donated label to peripheral monocytes. Because the State University, Columbus, Ohio. Received for publication June 29, 1964; revised December ii, biochemical reactions involved in the donation of the 1964. labeled material were not elucidated, it is not known if the 509 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. 510 Cancer Research Vol.25,May 1965 labeled thymidylate (dTMP) in the recipient DNA was eral cell lines were employed, including the parent line, degraded to Tdr during the transfer, and if so, whether or Ni-Si, and a cloned line, N1-54. No significant differ not the Tdr was circulating in acid-soluble form. In a ences with respect to Tdr metabolism were noted between somewhat related study, Bryant (6) found that when the 2 lines, and most of the data to be presented were lymphoid cells labeled in vitro with tritiated Tdr were obtained with the Ni-S4 line. The composition of the injected i.p. into partially hepatectomized mice, the radio complete growth medium No. 71, which contains 5 % beef activity appeared in the liver parenchymal cell nuclei. serum, and of the buffered basal medium 5-77, has been Although the mechanisms of transfer again are not known, described (30). these works are important because they suggest a possible 180t0pe8.—Thymldlne-2-'4C, 25 mc/mmole, and thymi course of exogenous thymine by demonstrating that cells thne-3H of various initial specific activities were diluted may under certain conditions synthesize DNA, at least in with nonradioactive Tdr to the desired final specific ac part utilizing preformed thymine supplied from other parts tivity. While paper chromatography of the isotopes in of the body. Also pertinent to this question is the demon Fink's system No. 8 (12) revealed that the ‘@CTdrdid stration of Tdr in rat and in calf thymus (14, 35, 46) and in not contain significant impurities, it did show that several the Novikoff hepatoma (43) as well as regenerating rat batches of 3HTCITapparently contained moderate amounts liver (44), which indicates the existence in the mammal of of 8H thymine. For this reason, ‘4CTdrwas employed Tdr in the nucleoside form. exclusively in quantitative experiments, while 3HTdr was Although cells in tissue culture are removed from the restricted to autoradiography and chromatography of the controlling influence of the body, their growth can be intracellular acid-soluble compounds. In the latter case, inhibited by the addition of certain precursors of DNA to parallel experiments carried out with ‘@CTdrrevealed no the medium. It has been demonstrated for several types differences in either the type or the proportion of intra of mammalian cells that large concentrations of Tdr in the cellular Tdr compounds. The final specific activities of culture medium (in the range of i0@ M) will depress cell the ‘@CTdrsolutions were determined as follows: radio growth (28, 29, 30, 48), apparently by producing a defi activity was measured in the liquid scintillation counter, ciency in the intracellular nucleotides of deoxycytidine and the final concentration of Tdr was calculated from the (Cdr), since this compound added to the culture medium concentration of the stock solutions of Tdr, which had been will restore normal cell multiplication (29, 30, 48). It is assayed by ultraviolet absorption. Although the stocks possible also to produce a specific thymine deficiency by had peen prepared in 5-77 which contained phenol red, adding amethopterin to the culture medium, provided a 1/1000 dilution of S-77 in 102 N HC1 had an absorbance that suitable levels of adenosine, serine, and glycine are at 267 ms of less than 0.01, thus permitting an accurate added to relieve other deficiencies induced by the amethop assay on stock solutions of initial Tdr concentration of term (18, 30, 40). Among purines, deoxyguanosine (Gdr) io-' M. and deoxyadenosine (Adr) block cell growth in cultures of Fractionation of cells.—Except where noted otherwise, appendix cells (48), and various derivatives of adenine the sample material was maintained at a temperature of (1, 22, 23, 33, 48) including Adr-N-oxide, a metabolic approximately 5°C. product of Adr, inhibit the synthesis of DNA in Ehrlich A. Ten milliliters of cell suspension containing 10@—i0@ ascites cells in vitro (13). While deoxyadenosine tniphos cells/mi were placed in a 12-mi centrifuge tube and cen phate (dATP), deoxyguanosine triphosphate (dGTP), and trifuged at approximately 1000 X g for 5—10mm. An thymidine triphosphate (dTTP) all exert negative aliquot of the supernatant medium was diluted 1: 10 in feedback on the production of Cdr nucleotides from cyti H20 for subsequent assay of radioactivity. The pellet of dine nucleotides (37, 38), it is not clear if Adr and Gdr cells was washed twice in 5 ml of 5-77 by resuspension operate in the same manner as Tdr to inhibit cell growth. with a vortex mixer and centnifugation as described above. The purpose of the present study was to measure certain Preliminary experiments showed that these washes did not parameters of the metabolism of Tdr added to suspension remove any significant quantities of radioactivity, and the cultures derived originally from the Novikoff rat hepa supernata@its therefore were discarded. toma (30). These cells have the desirable characteristics, B. After the 2nd wash, i ml of H20 was added to the for a study of this type, of lacking Tdr nucleoside deoxy pellet, which then was resuspended with the vortex mixer ribosyl transferase (49), and also the ability to catabolize and homogenized at 5°Cusing an ultrasonic disintegrator thymine and uradil (30). Among the questions studied (Disontegrator, system forty; Ultrasonic Industries, Inc., are the intracellular form of the Tdr added to the cultures, Albertson, L.I., N. Y.) operating at a frequency of 90 kc. and the extracellular concentration versus the intracellular To the homogenate was added an equal volume (1 ml) of concentration of Tdr compounds and the resulting influ io%tnichioraceticacid(TCA),andthecontentswere ence on the growth rate. The relationship of the intra mixed with the vortex mixer. After centnifugation at cellular concentration of Tdr compounds to the metab approximately 1000 X g for i0—i5 mm, the supernatant, olism of other pyrimidine nucleosides will be discussed in which contained the intracellular acid-soluble compounds, the third paper of this series (i5).
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