Metabolism in Tissue Culture Cells Derived from Rat Hepatomas

II. 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 , 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) 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 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 , 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 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 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 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 , 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 , (Gdr) io-' M. and (Adr) block cell growth in cultures of Fractionation of cells.—Except where noted otherwise, appendix cells (48), and various derivatives of 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 will be discussed in which contained the intracellular acid-soluble compounds, the third paper of this series (i5). was decanted and saved for assay of radioactivity. C. To the pellet 0.5 ml of H20 was added; it then was MATERIALS AND METHODS resuspended as described above (B). One milliliter of Novikoff hepatoma-derived suspension cultures were 0.2 N NaOH was added, and the contents were mixed with cultivated, and cell concentrations were determined by the vortex mixer, heated in a water bath at 80°Cfor using haemocytometer as previously described (30). Sev 20 mlii, and chified to 0°C. Two volumes of 10 % TCA

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@ (3.0 ml) were added, and the contents were mixed and •-NTL4j@Ta' co@c. 14MEDIUM N @ 6- WIIT1AL Tdr CONC. IN MEDIUM N @ centrifuged as described in B. Preliminary experiments 0- IN@AL Tdr CONC@INNEDIUN N £- INITIAL @[email protected]@i@ ii MEDIUM 0-3 N revealed that the supernatant, while containing essentially U-CONTROI@SNO Ta' @ioMIEThOFTERIN 6 all of the digestion products of the ribonucleic acid (RNA) .J5 (13), did not contain a significant amount of radioactivity, or of DNA, as measured by the diphenylamine reaction i@-@ M (10) ; it therefore was discarded. E2 D. DNA was extracted essentially according to the UI method of Schneider (42), as follows. The pellet was resuspended as described in B, in 0.75—2.0 ml of 1120, depending on the pellet volume (usually less than 0.1 ml, @j6 10-i NJ in which case the minimum volume was used). An equal volume of 10 % TCA was added, and the contents were mixed with a stirring rod. Stirring continued intermit In tently during the extraction (20 mm at 90°C),after which 0 the contents were cooled and centrifuged as in B. This procedure extracted more than 90 % of the radioactivity 0 24 40 56 in the pellet and represented the optimal time and tem HOURSAFTER 9JBCULTURE perature for extraction of DNA as measured by the di CHART 1.—Relationship of thymidine (Tdr) concentration in phenylamine reaction. the medium to growth of amethopterin-blocked N1-S4 cells. Amethopterin, i0@ M; adenosine, 5 X i0' M; serine, i0@ M; and Estimation of radioactivity.—For liquid scintillation glycine, i0-@M; all final concentrations, added at time of sub counting, 0.i-ml aliquots of samples were added to glass culture. Control flasks received adenosine, serine, and glycine. vials, 10 ml of scintillator solution (naphthalene, 80 gm; All values are averages of duplicate flasks. Experiment 1. 2 ,5-diphenyloxazole, 5 gm ; a-naphthylphenyloxazole, 50 mg; dissolved in 1 liter of a mixture of 5 parts xylene, 5 parts dioxane, and 3 parts ethanol) (21) were added and the sample radioactivity was measured as described (i6), using a two-channel liquid scintillation spectrometer (automatic Tri-Carb liquid scintillation spectrometer, Packard Instrument Co., La Grange, Illinois). Prelimi nary experiments determined that the quenching factors for water and 1 : 10 dilution of medium No. 7i in water were the same, and that the supernatants containing the acid-soluble compounds, RNA hydrolysate, and extracted DNA (all basically 5 % TCA), all quenched to the same degree but more than water or the i :10 dilution of me Tdr I0@M IO@M IO@M (O@M dium No. 71 in water. Therefore, in correcting the data INITiALCONCENTRATION for quenching, the appropriate predetermined factor was CHART 2.—Distribution of thymidine (Tdr)-2-14C in amethop term-blocked Ni-S4 cells. Amethopterin, adenosine, serine, and used. glycine added as described in Chart 1, at time of subculture. All Diphertykimine reaction.—The Burton modification (7) values are averages of duplicate flasks. Experiment 1. of the Dische reaction (10) was employed, with Adr used as a standard. the endogenous production of dTMP via thymidylate Analysis of intracellular acid-soluble compounds.—Ali synthetase is blocked, but other cell activities are not quote of cell suspensions were chilled quickly to 0°Cand hindered except as they depend on dTMP and/or DNA washed twice in ice-cold 5-77. The pellets were resus synthesis. Thymidine-2-1@C was added to the flasks, and pended in ice-cold 0.4 N perchioric acid and centrifuged. the final concentrations at time of subculture ranged from The supernatants were decanted, neutralized with KOH 10-6 to 10—sM. The results (Chart i) show that 0-6M to PH 7.0-8.0 and chromatographed by the system of Tdr was not sufficient to support normal logarithmic Ivesetat.(i9). growth, and that i0@ M Tdr was inhibitory. On the Auloradiography.—Cells were washed twice in 5-77 and other hand, iO—@andi0@ M Tdr permitted normal cell smeared on glass slides. After being air-dried, the smears multiplication as measured both by diphenylamine and by were washed twice in ice-cold 5 % TCA and twice in ice cell counts. An examination of the curve for iO_6M cold H2O. Stripping film was applied to the slides which Tdr reveals that the cells apparently began to exhaust the then were exposed, developed, and stained as described supply of Tdr by 24 hr, and by 40 hr could multiply no (24). longer. This is verified in Chart 2, in which the medium curves reflect the concentration of Tdr in the medium, RESULTS 100 % being equivalent to the initial concentration in each Experiments were carried out to determine the effects of case. The radioactivity in the acid-soluble fractions, Tdr deficiency and Tdr excess. Cells were subcultured though significant, represented a small proportion of the into medium No. 71 containing amethopterin, adenosine, total count in the sample and for this reason is not included serine, and glycine as described (30). In this situation in Chart 2.

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U

244056 T1MEINHC@.@ @@ I0@ IO@ INITIALMOLARITYOFTdr INMEDIUM CHART 4.—Effect on cell growth of variation in the intracellular CHART 3.—Relationship of extracellular thyinidine (Tdr) con concentration of thymidine (Tdr) compounds. All values by centration to intracellular concentration of Tdr compounds in calculation from the data in Charts 1 and 3, and the cell volume, amethopterin-blocked N1-S4 cells. Amethopterin, adenosine, 2.4 X 10—'ml (see text) . The values on the abscissa represent serine, and glycine added as described in Chart 1, at time of sub the quotient : nanomoles of intracellular acid-soluble Tdr com culture. All values are averages of duplicate flasks. Experiment pounds per 106 cells (Chart 3) divided by the volume of 10' 1. nM = nanomoles = 10' moles. cells (2.4 X 10—'ml). The values on the ordinate are the % increase in total cell deoxyribonucleic acid (DNA)/ml of sus Intracellular acid-soluble thymidine compounds.—The pension during the subsequent 16-hr interval (calculated from the technics employed permitted estimates of the size of the data of Chart 1). The initial extracellular Tdr concentrations intracellular pool of acid-soluble Tdr compounds including (in the media at time of subculture) were, from left to right, io-.6 M, i0@ M, 10@ M, and 10' M for the points in the lower curve, Tdr and the various Tdr , all of which are and the same for the points in the upper curve. Experiment 1. precursors for DNA-thymine. From Chart 3 it is ob Sample calculation: Initial Tdr concentration, 106 M; interval, vious that when the Tdr concentration in the medium is 24-40 hr. increased, the intracellular Tdr compounds also are in Calculation of ordinate: creased. The values were obtained by calculation from Cell DNA/ml at 40 hr the total intracellular acid-soluble radioactivity, the total — Cell DNA/ml at 24 hr x 100=ordinatevalue number of cells in each sample, and the specific activity of Cell DNA/ml at 24 hr of the added Tdr. Because the endogenous production More specifically, from Chart 1: of dTi\IP was blocked as described above, it was assumed —@@@ that by the time the cells had doubled (first 24 hr), using 1.95 @igDNA/ml 1.35 DNA/mt = 50% the Tdr of the niedium, the intracellular Tdr compounds 1.35 j@gDNA/mi would be totally derived from the Tdr of the medium and Calculation of abscissa: would have the same specific activity. This would obtain (a) if during a generation-time all the cells doubled their nmoles of intracellular acid—soluble Tdr compounds X 1 DNA and (b) if the intracellular pools of Tdr compounds 106cells X cell volume were small relative to the total amount of thymine in the = abscissa value DNA. The first condition was demonstrated by auto radiography and will be described in a subsequent para More specifically, from Chart 3 and the text: graph. That the second condition applies may be inferred 0.041 nmoles < 1 from the following calculations. Based on the data in 10$cells 2.4 X 10—'mi/lO' cells Chart 1, the average DNA content of 1O@cells is approxi @ mately 7.6 Using the thymine content of rat DNA, — 0.041 nmoles 1.69 X 10@ moles roughly 28 moles % (47), a value of 2.1 @igor 7 nano — 2.4 X 10—' ml liter moles (nmoles) of DNA-thymine/106 cells is obtained. Controls: Ordinate values only were plotted for the controls, This is more than twice the pool size found in the highest because knowledge of the intracellular pools in the controls is external concentration of Tdr (i0@) M, Chart 3). In lacking. Indeed it is intended to infer such knowledge from the addition, if the cells ordinarily had a large reserve pool of experimental curves, using the intracellular Tdr concentrations which permitted a growth rate most closely approaching that of Tdr compounds, the addition of amethopterin would not the control suspensions. Thus, for the 24—40-hrinterval, an shut off DNA synthesis abruptly, whereas preliminary intracellular concentration of 4 X 10' M is inferred for the control experiments indicated that in less than an hour after addi because this level in the experimental cells allowed them to grow tion of amethopterin all DNA synthetic activity had at almost exactly the same rate as the control cells. For the 40-56-hr cells, this value would be about 1.7 X 10@ M, which in stopped (unpublished data). this interval permitted cell growth in the experimental flasks at It was desirable to obtain an estimate of the intracellular the same rate as the control cells.

4 Calculated from Chart 1 as follows: molar concentration of the Tdr compounds and to compare DNA/mi at time of subculture or 0.68 pg DNA/mI the effects on cell growth of alterations in this level Cells/ml at time of subculture 0.895 X 10' cells/mi (Chart 4). The % increases in cell DNA/ml were ob

= 7.6 pg DNA/10' cells tamed from the data in Chart i, and the intracellular con

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The nature of the intracellular Tdr compounds after the administration of radioactive (@Hor ‘4C)Tdrto the suspensions was determined. The experiments were car ned out as described in “Materials and Methods―. It was found, over a wide range of concentrations (i0'—10@ M) of added Tdr, that whatever the amount, the Tdr taken into the cells was converted quite rapidly into dTTP (80—90%) and dTDP (10—20%). It was not possible to detect significant intracellular amounts of dTMP or Tdr in any of these experiments. An indication of the rapidity of attainment of a stable intracellular concentration of Tdr compounds, after addition of radioactive Tdr to the suspensions, can be obtained from an examination of Chart 5. These results are similar to those reported for Ehrlich ascites cells (20) and for a strain of Lackbacillus acidophilus (32) ; in both cases a stable equilibrium was ADDITiON obtained in less than 10 mm. In addition, preliminary OF Tè-2-14C experiments (Chart 6) revealed that even in cells incu CHART 5.—Uptake of thymidine (Tdr) into intracellular acid bated at 4°CTdr was taken into the acid-soluble fraction, soluble fraction. N1-S4 cells, no amethopterin, adenosine, serine, or glycine. Initial cell concentration approximately 700 X and that by 1 hr the intracellular level of Tdr compounds 10'/nil. All values are averages of duplicate flasks. Experiment was already almost one-half that of cells incubated at 2. nM = nanomoles = 10' moles. 37°C. Apparently DNA synthesis was shut off, inasmuch as no isotope was incorporated during this time. While amethopterin was not included in these experiments (Charts 5 and 6), a comparison of the data with those of Chart 3 will reveal similar levels of intracellular acid-sol uble Tdr compounds from 10 mm to 56 hr for a given concentration of Tdr in the medium. By the latter time, however, the Tdr concentration in the medium had dropped significantly in the i0@ and i0@ M flasks (Chart 2), and the intracellular levels therefore were somewhat I lower. The relationship of the negative feedback of dTTP on Tdr kinase (19) to the rapid achievement of a ADDITION stable intracellular level of Tdr nucleotides, even in the OF Tdr-2-14C presence of large concentrations of Tdr in the medium, is CHART 6.—Effect of temperature (°) on uptake of thymidine not clear. Because (ATP) re (Tdr) into acid-soluble fraction. Tdr concentration in medium; verses this feedback inhibition (i9) knowledge of the 10—0M. 37°Ccurve from Experiment 2 (Chart 5). Cell concen tration approximately 181 X 10'/ml. All values are averages of intracellular levels of ATP will be required before any duplicate flasks. Experiment 3. pM = picomoles = 1012 moles. conclusions can be made. Autoradiography.—Autoradiography was carried out centrations of Tdr compounds (abscissa) were estimated with 3HTdr, as described, to obtain an estimate of the by dividing the values for nanomoles of Tdr/iO' cells homogeneity of the cell population with respect to DNA (Chart 3) by the volume of 1O@cells,2.4 X 10@ ml.' It synthesis during logarithmic growth. As anticipated, is recognized that these calculations require the assump only some of the cells (approximately 50 %) were syn tion that the cell is a sac of freely diffusible fluid, and thesizing DNA (as evidenced by labeling) during a 1-hr that in reality it probably is not. Relative to this point, exposure to the isotope. When the exposure was in Behki and Schneider (2) have reported that most of creased to a large fraction of the generation time (iO hr), the deoxynucleotides of regenerating rat liver or of Novi most of the cells were labeled and, after a 25-hr exposure, koff hepatoma are found in the cell nuclei, when these are essentially all were labeled heavily. These findings sup isolated in nonaqueous solvents. Because such localiza port the assumption that during logarithmic growth all tion of Tdr compounds in the nucleus or in any other part the cells undergo synthesis of DNA during one generation of the cell would increase the local concentration it will time. be implicit that “intracellularconcentration― actually Dependence of amethopterin-blocked cells on exogenously represents the minimum intracellular concentration. A supplied thymidine.—That the cells blocked by amethop further assumption is necessary: that the intracellular Tdr term were dependent on Tdr supplied in the medium for a is predominantly in one form (as dTTP, for example), and thymine source was demonstrated by the failure of such it will be shown subsequently that this is justified. cells to grow if Tdr was not added (30). Furthermore, if the quantity of added Tdr was low (i0' M), the cells were S Obtained by measuring the volume (1.10 ml) of a pellet formed in centrifuging 920 ml of a suspension of 5 X 10' cells/mi; able to grow until the Tdr was exhausted (Charts 1 and 2) the cell concentration was determined by using a hemocytometer after which growth stopped, indicating that growth in the (30). presence of amethopterin depended on a continuous sup

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ently by starvation for deoxycytidylate (dCMP) (29, 30). Similar findings were reported by Eagle et at. (ii) for cer tamaminoacids(value,lysine,andthreonine)essential z to the growth of HeLa cells. These workers found that Q U) minimal intracellular concentrations of these compounds z w a- were required, ranging from 1 to 5 X 0@M, depending on U) the particular amino acid. It was found further that the cells concentrated the amino acids from the medium ; that 0 is, the intracellular concentrations were higher than the -j extracellular concentrations. It can be seen from an examination of Chart 4 that with respect to Tdr a minimal -J -J intracellular concentration of Tdr compounds also is re LiJ U quired for growth, and, surprisingly, that this concentra tion is in the same range as those reported by Eagle et al. i@-@Nce\io-4 N Ude (ii) for the essential amino acids. Although the mech anisms are not clear, another similarity was found : the Novikoff cells apparently could not exhaust the medium CHART 7.—Utilization of 5-methyldeoxycytidine (5MeCdr) as Tdr completely (Chart 2, 10' M), nor could the HeLa cells a thymine source by amethopterin-blocked Ni-St cells. Ametho utilize all the essential amino acids when the concentra pterin, 106 M; adenosine, 5 X 10@ M; serine, 10@ M; and glycine, tions in the medium were too low. The Novikoff cells 10—iM; all final concentrations, added at time of subculture. Control flasks received adenosine, serine, and glycine. All values also concentrated the Tdr from the medium (4- to 5-fold at are averages of duplicate flasks ; time is in hours. Experiment 5. i0' M in the medium) but not as much as HeLa cells (Udr, .) concentrated the amino acids. At the higher internal concentrations of Tdr compounds growth was depressed ply of exogenous Tdr. When the quantity of thymine (Chart 4) ; but in the midranges the cells grew as well as incorporated into DNA over a specific interval was calcu did the unblocked control cells. These and the other data lated from radioactivity data, however, the value usually already presented imply that the normal range of concen was somewhat lower (10—30% less) than that calculated tration of intracellular dTTP in logarithmically growing from measurements made on aliquots of the same samples Novikoff tissue culture cells is approximately 5—10 X with the diphenylamine reaction, assuming a thymine 10—sM,although from the shapes of the 2 curves in Chart 4 content of 28 moles % the value for rat DNA (47). this obviously is only a rough estimate. It is of interest, The reasons for this discrepancy are not clear; however, however, to compare these concentrations with those re because of the apparent completedependenceof the blocked ported by Reichard (37) to be inhibitory for the produc cells on exogenous Tdr, and because the discrepancy was tion of dCMP from cytidylate by extracts of chick em variable, we feel that it probably is an artifact. In any bryos. It was found that, at about 10@ M, dTTP was case, the data do show that the bulk of DNA-thymine 50 % inhibitory, with 90 % inhibition occurring at approxi (measured by diphenylamine and calculation) incorporated mately 6 X i0-@ M. These figures are remarkably similar by amethopterin-blocked cells was derived from the to the intracellular concentrations of dTTP which begin medium. to inhibit the growth of the Novikoff cells, probably by a 5-Methykleoxycytidine as a thymine source.—Because similar mechanism, although the estimation of these con amethopterin interferes with the methylation of deoxy centrations in the latter case is admittedly indirect and (Udr) monophosphate (31), 5-methyldeoxycytidine inferential. (5-MeCdr) was tried as a possible precursor of DNA Effect (iThcell multiplication of limiting thymidine triphos thymine ; this compound being chosen because the methyl phate production.—Because cell growth is apparently re group is already attached to position of the pyrimidine lated to the maintenance of the intracellular pools of ring. It was found that the amethopterin block could be dTTP at minimal levels, the possibility should be con relieved completely by the addition of this compound at sidered of depressing the rate of DNA synthesis by con 10—sM(Chart 7). It is of interest to note that i0@ M troffing the rate of production of dTTP. Because Tdr 5-MeCdr produced a lower growth rate that nevertheless added to the medium is concentrated so rapidly by the was logarithmic; at i0@ M some growth took place for cells (Charts 5 and 6), it would be difficult to maintain a 24 hr, but after that the cell population appeared to de stable but restricted level of this compound in the medium. chine. As anticipated, neither Udr nor Cdr at i0@ M When, however, 5-MeCdr is utffized to relieve the thy wduld support the growth of amethopterin blocked cells. mine deficiency (Chart 7), there apparently are some rate limiting steps in its conversion to dTTP, possibly at the DISCUSSION deamination step, although it is not known whether this Intracellular concentration of thymidine compounds.—It occurs at the nucleoside or level in this system. may be seen from the data in Chart 4 that large changes In any case, a much higher concentration in the medium in the intracellular quantities of Tdr compounds have an (10-i M) is required to restore normal growth than would effect on cell multiplication. If too little be present, the be necessary with Tdr (i0@—i0@M),and further, at an cells are starved for thymine and cannot synthesize DNA; intermediate level (i0@ M) growth does occur but at a in the presence of too much, growth is depressed, appar lower rate (Chart 7). Although the over-all rate of DNA

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. GENTRY et al.—Pyrimidine Metabolism. II 515 synthesis is thus lowered by the apparent restriction of the 14. Frenkel, E. P., Sugino, Y., Bishop, R. C., and Potter, R. L. production of dTTP, it is not known if the increased Effect of x-Radiation on DNA Metabolism in Various Tissues of the Rat. VI. Correlative Morphologic and Biochemical generation time results from a longer period for interphase Changes during the Regeneration of the Thymus. Radiation DNA synthesis, or from a lengthening of telophase + Res., 19: 701—16,1963. G-i, as suggested by Siskin and Kinosita (45). 15. Gentry, G. A., Morse, P. A., Jr., and Potter, V. R. Pyrimidine When the rate of production of dTTP is too slow, which Metabolism in Tissue Culture Cells Derived from Rat Hepa appears to be the case with i0@ M 5-MeCdr (Chart 7), cell tomas. III. Relationship of Thymidine to the Metabolism of Other Pyrimidine Nucleosides in Suspension Cultures Derived growth is retarded greatly and the cultures stop growing from the Novikoff Hepatoma. Cancer. Res., U: 517—25,1965. after a relatively short time. When the cells do not have 16. Gentry, G. A., Walker, B. M., and Randall, C. C. Correlated any source of thymine (Chart 7, Cdr and Udr) they main Autoradiographic and Biochemical Study of DNA Labeling tain for approximately a generation time and then begin in Equine Abortion Virus Hepatitis. Virology, 16: 460-65, 1962. to disintegrate. This suggests that, although variations 17. Greulich, R. C., Cameron, I. L., and Thrasher, J. D. Stimu in the intracellular levels of dTTP can influence the over lation of Mitosis in Adult Mice by Administration of Thymi all rate of DNA synthesis, once the onset of DNA synthesis dine. Proc. Natl. Acad. Sci. U.S., 47: 743—48,1961. has occurred, a sufficient supply of thymine must be main 18. Hakala, M. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. Pyrimidine Metabolism in Tissue Culture Cells Derived from Rat Hepatomas: II. Thymidine Uptake in Suspension Cultures Derived from the Novikoff Hepatoma

Glenn A. Gentry, Paul A. Morse, Jr., David H. Ives, et al.

Cancer Res 1965;25:509-516.

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