The Mechanism of Deoxyribonucleic Acid-Thymine Biosynthesis by Lymphatic Tissues and Tumors*

SAULKIT

(University of Texas, 31. D. Anderson Hospital and Tumor Institute, Department of Biochemistry, Houston, Texas)

The utilization of labeled carbon from formate- that free orotic acid is utilized for DNA-T syn C14(3, 11, 12, 33), serine-3-C14 (2, 3), glycine-2-C14 thesis in vivoin the rat and mouse (11,14,17) and, (2, 3), or methionine-methyl-C14 (12) for the bio in vitro, in cat spleen, rat liver (35), or Ehrlich synthesis of the methyl group of deoxyribonucleic tumor cells (.17).Free thymine, uracil, or cytosine, acid-thymine (DNA-T) has been demonstrated in however, are poorly utilized for DNA-T synthesis the internal organs of the rat and chick, and in by the normal rat (13, 24), but free uracil is uti tissue slices of rat liver and cat spleen (19), rabbit lized to a somewhat greater extent by the mouse bone marrow (23, 31, 32), and Ehrlich ascites tu (17) and by rat hepatoma tissue (28). CyUdine mor cells (23). Folie acid derivatives function as and, to a lesser extent, uridine are readily utilized cofactors in the transfer of "one-carbon groups" to for the synthesis of both ribo- and deoxyribonu the methyl group of DNA-T. The incorporation of cleic acid pyrimidines, as is deoxycytidine for formate into the DNA-T of rabbit bone marrow DNA pyrimidines (9, 10, 24-27). Thymidine-2-C14 (32) or Ehrlich ascites cells (23) is inhibited by is incorporated into DNA-T but not into other aminopterin, and the inhibition is partially re nucleic acid pyrimidines (7, 8, 24). The utilization versed by leukovorin. Aminopterin inhibited and of thymidine for DNA-T synthesis has been dem citrovorum factor partially reversed the inhibition onstrated in bacteria (1), plant tissues, and proto of the incorporation of uracil-2-C14 deoxyriboside zoa, as well as in animal tissues (7, 8). (UDr) into DNA-T (5, 6). Incorporation of radio In experiments in vivo,Reichard (24) has shown active thymidine (TDr) into DNA-T was not in that at high dose levels 5-methyluridine-2-C14 is hibited by aminopterin. In the synthesis of the incorporated into the DNA-T of the regenerating methyl group of DNA-thymine from H2C14OONa liver of the rat, but not into the DNA-T of rat or L-serine-3-C14-H2, 0.9 and 1.5 atoms of deu intestine. Inasmuch as the pattern of utilization terium, respectively, accompanied the labeled car resembled that of free thymine, it was suggested bon (2, 3). On the other hand, during the conver that the 5-methyluridine was utilized only after sion of the beta carbon of serine and of H2C14OONa conversion to free thymine. The utilization of to carbon atoms 2 and 8 of purines, an extensive deoxyuridine for the formation of DNA-T, but labilization of deuterium took place. These differ not of DNA-cytosine, took place rapidly, under ences are probably attributable to the fact that a conditions where free uracil was not utilized. How hydroxymethyl-Ã®olicacid derivative is required for ever, the utilization pattern resembled that of thymine biosynthesis and a/or/WT/Z-folicacidderiv thymidine rather than uridine (6, 24). Deoxyuri- ative for purine biosynthesis. dine-2-C14 was also utilized for free thymidine and Although the precursors of the methyl group of for DNA-T synthesis, but not for DNA-cytosine DNA-T have been elucidated, the nature of the or ribonucleic acid pyrimidine synthesis, by chick acceptor compounds for the "one-carbon com or rabbit bone marrow cells (5, 6). Nonlabeled pound" has until recently remained obscure. It is thymidine significantly reduced the incorporation conceivable that orotic acid, uracil, cytosine, ribo- of labeled uracil deoxyriboside into DNA-T by or deoxyribonucleosides or -nucleotides of the bone marrow cells (5, 6). pyrimidine bases, or dihydro derivatives of the Experiments on the incorporation of labeled above compounds could function as acceptors for formate into DNA-T of rabbit bone marrow and the "one-carbon compound." It has been shown Ehrlich tumor cells were recently reported by * Aided in part by grants from the Leukemia Society, Inc., Prasoff and coworkers (22, 23). The specific ac and the American Cancer Society. tivity of DNA-T was increased by the addition Received for publication August 25, 1956. of uridine (UR), cytidine (CR), or deoxycytidine ifi

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1957 American Association for Cancer Research. KITâ€”DNA-Thymine Biosynthesis by Lymphatic Tissues and Tumors 57 with nonlabeled L-serine so that the total L-sm'ne added per (CDr), and was markedly depressed by thymidine, aminopterin, or azathymidine, but not by free flask was 2.4 AmÃ³les.) At the end of the incubation period, the contents of azathymine. the Warburg vessels were decanted into centrifuge tubes In the experiments described in the present pa containing an equal volume of 40 per cent ethanol. The per, the mechanism of DNA-T formation was vessels were rinsed with 1 cc. of 20 per cent ethanol, and the studied in vitro in the Ehrlich ascites tumor, in rinse was added to the centrifuge tubes. As the concentration of free thymine, thymidine, and thymidylic acid is extremely three lymphatic tumors of mice, and in mouse low in tissues, nonlabeled thymine, thymidine, and thymidy spleen, rat thymus, and rabbit appendix cells. late were added to facilitate the isolation of the thymine deriva Either serine-3-C14 or formate-C14 was used as the tives. The contents of the centrifuge tubes were heated at about precursor of the methyl group of thymine. In addi 75" C. for 5 minutes to coagulate the proteins. In the experi tion to the labeling of DNA-T, the labeling of free ments with labeled formate, 0.1 cc. of concentrated nonradio- active formic acid was added. The tubes were centrifuged, thymine, thymidine, and thymidylate was meas and the supernatant was collected. The precipitate was washed ured. It was therefore necessary to establish con with 3 cc. of 20 per cent ethanol, and the ethanol wash was com ditions for the separation of the labeled thymine bined with the previous supernatant. derivatives from other radioactive substances. Supernatant fraction.â€”The supernatant fraction was de salted by passing the extract through a 3 cm. X 9 mm. Dowex- This was effected by carrying out a partial group 50-H cation exchange resin (15). The resin was washed with an separation and desalting of tissue extracts by ion additional 8 cc. of water, and the eluate and washings were exchange resin chromatography, and by subjecting combined. In addition to the inorganic cations, serine, and the fraction which contained the thymine com other free amino acids, adenine or cytosine nucleosides were re pounds to two-dimensional paper chromatog tained on the resin column. raphy. Details are described in the "Materials and The eluate, which contained the thymine derivatives, was concentrated under reduced pressure. Aliquots were then taken Methods" section of this paper. for two-dimensional paper chromatography (Whatman 3 MM In agreement with the experiments of Prusoff filter paper). The solvents were isopropanol:ammonia:water and associates (22, 23), Reichard (24), and Fried- (140:5:55) and Â¡sopropanol:cone. HCl:water (130:18:52). The ultraviolet-absorbing areas were detected with the aid of kin and Roberts (5, 6), the present studies show a mineralight lamp, and the thymine, thymidine, and thymidy that uracil deoxyriboside and cytosine deoxyribo- late spots were cut from the paper and eluted with water into side are potential acceptors for formate-C14 or for stainless steel planchÃ©is.Samples were dried and assayed for the jScarbon of serine. That the thymidine which radioactivity by means of a Tracerlab gas flow counter and is thereby formed is in equilibrium with the DNA- superscaler. Radioautograms were usually prepared from paper chromatograms. It was possible to verify in this way T precursors in the tumor tissues, as in various that the ultraviolet-absorbing areas due to the thymine deriva normal tissues, is also suggested by the fact that tives coincided with the darkened areas of the no-screen x-ray nonlabeled thymidine inhibited the incorporation film and that they were well separated from other radioactive of serine-3-C14 and formate-C14 into DNA-T. areas of the paper. Nucleic acidfraction.â€”Theinsoluble residue from the initial extract was washed with ethanol, ethanol-ether, and petroleum MATERIALS AND METHODS ether-ether-acetone (4). The dry nucleoprotein was suspended in 0.1 cc. of 70 per cent perchloric acid (20) and heated for 1 Cell suspensions were prepared from Lymphosarcoma hour in a steam bath. To the hydrolysate 0.5 cc. of water was 6C3HED (Gardner), spleens from an animal bearing lymphatic added, and any insoluble material was sedimented by centrif- leukemia LL-5147, and from normal mouse spleen, rat thymus, ugation. Aliquots of the supernatant were taken for two- or rabbit appendix, as previously described (4, 15). Ascites dimensional paper chromatography. The solvents were: tumor cells were obtained from mice previously given inocula isopropanol: cone. HC1:water (130:33.2:36.8) (35) and tions intraperitoneally of 0.25 cc. of the Ehrlich ascites tumor butanol :ammonia :water (172:8:20). Chart 1, which shows or ascites leukemia E-9514A. The ascites cell suspensions the location of various compounds in these solvent systems, were centrifuged in a clinical centrifuge, and the tumor cells demonstrates that thymine, uracil, cytosine, 5-methyl cyto were resuspended in 2 volumes of modified Krebs-Ringer solu sine, guanine, and adenine were well separated. Xucleotides tion containing phosphate (0.01 M) and bicarbonate (0.028 M) did not migrate appreciably in the butanol-ammonia-water buffer (4). Tumors LL-5147 and E-9514A were obtained from solvent system, but these substances did move from the origin Jackson Memorial Laboratory, Bar Harbor, Maine. in the isopropanol-ammonia-water solvent system. Cell suspensions were incubated for 3 hours at 38Â°C.in Thymine and in some cases the other nucleic acid bases Warburg vessels which also contained glucose, labeled sub were cut from the paper, and the radioactivity was determined strate, buffer solution, and, in some experiments, either as described above. Quadruplicate chromatograms were pre pyrimidine derivations or free amino acids, or both. The total pared for each experimental sample. Two papers were used fluid volume per flask was 2 cc. The concentration of the pyrimi for determining the radioactivity. The thymine was extracted dine derivatives was 0.5-1.0 mM in the early experiments and from the third and fourth papers with 5 cc. of 0.1 NHC1,and the 5 mMin later experiments. DL-Serine-3-C14waspurchased from amount of thymine present was determined spectrophotomet- Isotope Specialties, Inc.; formate-C14 from Tracerlab, Inc., rically with a Beckman DK-2 spectrophotometer. The refer or the Nuclear Chemical Corporation. Labeled substrates ence cells contained 0.1 N HC1 extracts of paper blanks pre were added to each flask as follows: (a) 3.3 nc. (0.7 /imole) of pared from areas of the paper chromatograms which showed formate-C14 or (6) 2 ite. (2.4 /Â¿moles)of DL-serine-3-C14.(In no ultraviolet absorption under the mineralight lamp. The some experiments, however, the labeled DL-serine was diluted optical density of the samples was recorded between 300 m/u

and 240 m/x. The shape of the curves corresponded to that of and thymine are also shown in Table 1. Attention thymine standards. The thymine concentration was calculated should be directed to the sum of the radioactivity from the optical density values at the maxima of the curves. The specific activity of the DNA-T (counts/min//imole) was of these two compounds, since the separation on then determined. the paper chromatograms was not in all cases com plete. Also, it is probable that some TDr may have RESULTS been converted to thymine as a result of nucleosi- The effect of uracil deoxyriboside (UDr) on thy dase or phosphorylase activity and that some mine and DNA-T synthesis.â€”Table 1 shows that cleavage may have occurred during the handling the addition of low concentrations of UDr to the of the tissue extracts. incubation medium stimulated the incorporation The incorporation of formate-C14 into free of formate-C14 into DNA-T, whereas thymidine thymidine plus thymine (T) was greatly stimulated inhibited the incorporation markedly. Similar re by the addition of UDr (Table 1), but, in contrast sults were obtained by Prusoff and coworkers (23). to the labeling of the DNA-T, the labeling of free The total counts per minute in free thymidine thymidine plus thymine was increased by adding

.9 TDr TDrP ..8 /T v UDr 7 CDr U

.-.6 ala asp ser ClJiPglyc

.5 HMC ARP CR o /, es ADr A :â€¢â€¢â€¢- u R -1â€”3

0Â£ oes.

.7 .6 .5 .4 .3 NH3-Butanol-H2O <-

CHART1.â€”Two-dimensional chromatogram showing sepa HMC, hydroxyinethylcytosine; C'RP, cytidylate; G, guanine; ration of nucleic acid derivatives and some amino acids. The O, orotic acid; T, thymine; TDr, thymidine; TDrP, thymidyl- following abbreviations were used: A, adenine; AR, adenosine; ate; U, uracil; UR, uridine; UDr, deoxyuridine; URP, uri- ADr, adenine deoxyriboside; ARP, adenylate; C, cytosine; dylate; asp, aspartate; ala, alanine; glyc, glycine; glut-NHj, CR, cytidine; CDr, deoxycytidine; MeC, 5-methylcytosine; glutainine; and ser, serine.

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1957 American Association for Cancer Research. KITâ€”DNA-Thymine Biosynthesis by Lymphatic Tissues and Tumors nonlabeled thymidine to the experimental flasks. E-9514A, rat thymus, and mouse spleen cells This was probably attributable to the fact that (Table 3). In every case, the addition of UDr to more radioactivity was thereby "trapped" in the the medium greatly increased the radioactivity of thymidine "pool." TDr plus T, usually by a factor of 5 or more. When UDr and TDr were both added to the In the presence of UDr, most of the radio incubation vessels, the specific activity of DNA-T activity was found in TDr rather than in thymine was greatly reduced, as in the case of the addition in all tissues except the Ehrlich ascites tumor of TDr alone, while the radioactivity of the TDr plus T was greatly increased, as in the case of the TABLE 2 EFFECTOFTHYMIDINEONDNA-T TABLE 1 SYNTHESISFROMSERINE-S-C'Â« EFFECT OF DEOXYURIDINEORTHYMIDINE ON UTILI (TUMOR6C3HED) ZATIONOFFORMATE-C14BYEHRLICH CELLS DNA DNA-T(counts/min/ Thymine Adenine ADDITION (counts/min/Vniole} + T Addition /imole) (counts/min) Control 760 1,000 TDr 360 850 Inactivated at zero time 1806,70010,8001,1001,100TDr2501005204,1001,1005,1002907,0009004,800 Control (Tables 1 and 3). It is possible that the higher lev UDr els of free thymine radioactivity of the Ehrlich TDr tumor reflect differences in the pyrimidine nucleo- UDr+TDr sidase or phosphorylase activity of that tissue or T = Umiliile. differences in the endogenous thymine and thymi UDr = urteil deoxyriboside; TDr = thymidine. Pyrimidine nucleosides present at 1 mM final concentra dine pools. Table 4 shows that the conversion of tions. serine-3-C14 to TDr plus T by the Gardner tumor increased as the concentration of UDr was in addition of UDr alone. If the cells were killed by creased. The specific activity of the DNA-T of the adding acid at the start of the incubation, very Ehrlich, E-9514A, and LL-5147 tumors was also little radioactivity was found in the thymine increased by about 30 per cent by adding UDr to compounds. the incubation medium (Table 3). On the other Table 2 shows that the addition of TDr to the hand, the specific activity of DNA-T of the Gard incubation medium also greatly reduced the in ner tumor or rat thymus cells was not increased corporation of serine-3-C14 into Gardner tumor by the addition of UDr (0.5 or 5.0 mM/flask), DNA-T but that the specific activity of the ade- nor was it appreciably altered when the incuba nine of the DNA was affected but little. tion medium was supplemented with glutamine, Similar experiments with both serine-3-C14 and the essential amino acids, and UDr. Thus, a gross formate-C14 were carried out with tumors LL-5147, stimulation of the conversion of formate-C14 to TDr

TABLE 3 EFFECTOFDEOXYURIDINEONTHYMIDINESYNTHESISBYLYMPHATICTISSUESANDTUMORS DNA-T + TDr Tissue Substrates (counts/min//jmolc) (counts/mm) Gardner Serine Control 460 520 UDr 1,900 3,720 Gardner Formate Control 2,100 280 1,100 UDr* 1,900 1,500 14,700 LL-5147 Formate Control 2,100 630 UDr 2,700 830 5,000 Ehrlich Serine Control 2,400 190 670 UDr 3,200 2,000 600 E-9514A Formate Control 1,500 1,250 3,400 UDr* 2,200 1,240 19,000 Thymus Serine Control 1,400 UDr 4,900 Thymus Formate Control 1,400 540 4,100 UDr* 1,500 2,500 29,000 Spleen Serine Control 600 400 UDr 300 5,400 * UDr concentrations: 5.0 mM. In all other experiments, the concentration of UDr was 0.5 mM.

plus T was observed under conditions in which no of the formate and the serine j3-carbon, or it was effect on the specific activity of DNA-T was readily deaminated to deoxyuridine. In the former found. Although UDr is a suitable acceptor for instance, 5-methyl cytosine deoxyriboside (or 5- formate-C14 or the hydroxymethyl group of serine, hydroxymethyl cytosine deoxyriboside) would be other factors appear to limit the synthesis of the product which would be deaminated to thy- DNA-T by the Gardner tumor or thymus cells. midine (or 5-hydroxymethylthymidine). Effect of cytidine (CR) and cytosine deoxyribo- Cytidine was much less effective than CDr or side (CDr).â€”The effect of the addition of cytidine UDr in increasing the radioactivity of TDr plus T (CR) or of CDr on the incorporation of formate- (Table 5) but nevertheless increased the specific C14 or serine-3-C14 into thymine derivatives is activity of DNA-T in the experiments with each of the tissues. A stimulating effect of CDr and of CR on DNA-T biosynthesis by Ehrlich tumor cells TABLE 4 was also reported by Prusoff and associates (22, EFFECTOF DEOXYUKIDINECONCEN 23). Presumably, CR was active in increasing the TRATIONONTHYMIDINESYNTHE incorporation of formate-C14 to TDr and T be SISFROMSERINE-S-C14BYGARDNER TUMORCELLS cause of a rapid conversion to CDr (27, 28). The effect of CR addition on the metabolism of the UDr concentration T+TDr (mM) (counts/mm) Gardner tumor and thymus cells is to be con 0 1,100 trasted with that of UDr. In the latter instance, 0.25 2,300 the labeling of TDr plus T was markedly stimu 0.50 2,900 lated, but the specific activity of DNA-T was but 0.75 3,400 1.0 3,500 little affected. This suggests that cytosine deriva-

TABLE 5 EFFECTOFCYTIDINEORDEOXYCYTIDINEONTHYMINESYNTHESIS BYLYMPHATICTISSUESANDTUMORS + TDr TiuueGardnerEhrlichE-9514ASpleenThymusRabbit (counts/mm) Formate Control 2,100 280 1,100 CDr* 5,600 820 19,000 CR*Serine 3,5002,400 1703,000190 Control CDr 3,000 940 2,600 CRFormate 3,1001,500 9001,300 6503,400 Control CDr* 2,109 1,600 11,100 CR*Serine 2,7002,200 3,300600 4,400400 Control CDrFormate 1,700540 1,1004,100 Control CDr* 15,700 1,600 10,200 CR*Formate 9,7007,800 500550 4,8001,800 ap Control pendixSubstrates CDr*DNA-T(counts/min/pmole)16,000T 1,660670 13,000 CR = cytidine. CDr = deoxycytidine. * Concentration of CDr and CR, 5 mM; in all other experiments, 0.5 mM.

shown in Table 5. The addition of CDr greatly tives were limiting in the conversion of serine-3- stimulated the incorporation of label into both C14or formate-C14 to DNA-T by the latter tissues. the DNA-T and the TDr plus T of three tumors Effect of thymine, cytosine, uracil, and uridine.â€” and of the thymus, appendix, or spleen cells. CDr Thymine-2-C14 (0.9 mM) was not significantly in was particularly effective in increasing the specific corporated into the DNA-T of the three lympho- activity of DNA-T of the Gardner tumor and the mas or of mouse spleen cells. Furthermore, the thymus cells, the tissues which were not stimulated addition of nonlabeled thymine, uracil, cytosine, by the addition of UDr. The radioactivity of T or of uridine to the incubation medium had very plus TDr was also greatly increased by the addi little effect on the conversion of formate-C14 of tion of CDr. Either the CDr was itself an acceptor serine-3-C14 to TDr plus T (Table 6) of the Gard-

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1957 American Association for Cancer Research. KITâ€”DNA-Thymine Biosynthesis by Lymphatic Tissues and Tumors 61 ner tumor, tumor LL-5147, or rat thymus cells; ing of thymidylate was small compared with the nor did the presence of U, C, or UR affect the effect on the labeling of TDr plus T. Total thymid specific activity of the DNA-T of these tissues. ylate radioactivity was not increased by the addi Prusoff and associates (22) have observed, how tion of uridine or cytidine. ever, that the addition of uridine increased the Incorporation of formate-C1* into methylcytosine specific activity of the DNA-T of the Ehrlich of DXA.â€”The concentration of 5-methylcytosine tumor. The negative results reported here are in the DNA of various animal species varies noteworthy in view of the markedly positive re from 0.008 to 0.075 moles/4 moles of nucleotide sults with CDr or UDr. (36). Thus, the amount of DNA-5-methylcyto- The labeling of thymidylate.â€”Radioactivity sine was too low to be detected by paper chro- from serine-3-C14 or fonnate-C14 was observed in matograms under conditions where the amount of adenine or cytosine on the chromatograms TABLE 6 was not excessive. Small amounts of nonlabeled EFFECTOFTHYMINE,CYTOSINE,URACIL,ORURIDINE 5-methylcytosine were therefore added to the ONTHYMINEPLUSTHYMIDINESYNTHESIS nucleic acid extracts to facilitate the isola +(counts/mÃ®n)4504141,5001,2007509507306309206001,4001,3001,700860600TDr520460tion of this substance. Chart 1 shows that 5- Tissue SubstratesSerineFormateFormateFormateSerineSerineControlTControlCControlUURControlUURControlUURControlCTmethylcytosine was adequately separated from Gardner the known purine and pyrimidine compounds of nucleic acids. The total radioactivity of the nu- Gardner TABLE 8 Gardner EFFECT OF DEOXYURIDINE ON THE INCORPORATIONOF URACiL-2-C" LL-5147 INTO DNA-T (EHRLICH) DNA-Thymine Added (counts/min/Â¿jmoJe) Thymus Control 2,700 UDr 1,500 UDr concentration, 1 mM, Ehrlich cleic acid 5-methylcytosine of rat thymus and tumor E-9514A was determined after incubating TABLE 7 the cells with formate-C14. In the former case, EFFECTOFDEOXYURIDINEORDEOXYCYTIDINEON 260 counts/min and in the latter case, 550 counts/ THEINCORPORATIONOFFORMATE-C"INTOTHY min were found. The total methylcytosine radio MIDYLATEOFTUMORSANDLYMPHATICTISSUES activity was increased, by the addition of CDr or TOTAL COUNTS/MIN CR to the medium, to 630 and 932 counts/min, TISSUE6C3HED6C3HED*E9514A*LL-5147EhrlichThymusCDr3,30012,00015,00015,0002,700*UDr1,100 plus respectively, in the case of the thymus cells, and 2,2008,100 1044 and 960 counts/min, respectively, in the case 10,00015,000 14,0001,600 of the tumor cells. On the other hand, the addition 2,1002,700 of UDr or UR to the incubation medium did not 3,60016,000 increase the total radioactivity recovered in nucleic (rat)*Appendix* 18,0002,600plus (rabbit)CONTBOL acid 5-methylcytosine. UDr and CDr concentrations, 5.0 mM; in all other experi Effect of uracil deoxyriboside on incorporation of ments, 0.5 mM. uracil-2-Cli into DNA-thymine.â€”An experiment was performed on the utilization of uracil-2-C14 by free thymidylic acid. The total radioactivity was the Ehrlich ascites cells. The addition of non- of the same order of magnitude in thymidylate as labeled UDr to the medium reduced the incorpora in thymine plus thymidine in those experiments tion of uracil-2-C14 into DNA-T by about 44 per in which UDr or CDr was added to the incubation cent (Table 8). This is consistent with the possi medium. In the absence of UDr or CDr, thymidy bility that UDr is in rapid equilibrium with late radioactivity greatly exceeded that of thy DNA-T precursors. mine plus thymidine. The addition of UDr or CDr to the medium in DISCUSSION creased the total radioactivity of thymidylate in Formate or the hydroxymethyl group of serine most but not all the experiments. This is illustrated is readily transferred to deoxyuridine in lymphatic in Table 7. The effect of UDr or CDr on the label tissues and tumors as in other tissues (5, 6, 22, 24).

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1957 American Association for Cancer Research. 62 Cancer Research It is not known whether 5-hydroxymethyluracil when Ehrlich tumor cells were incubated with for- deoxyriboside is an intermediate in the formation mate-C14 and UDr (Tables 1 and 3). Uracil-2-C14 of thymidine from deoxyuridine. Deoxycytidine is an effective precursor of DNA-T in this tumor and, to a lesser extent, cytidine are either acceptors (Table 8). Further experiments are required to of "one-carbon compounds" or are proximal pre ascertain whether uracil, uridine, and deoxyuri cursors of the acceptor compounds. Deoxyuridine dine are in rapid equilibrium or whether the spec may be an intermediate in the formation of thymi ificity of the Ehrlich tumor enzymes for "one- dine from deoxycytidine. The deamination of cy- carbon acceptor compounds" is less restricted than tosine compounds has been demonstrated in micro that of other tissues. The possibility that phos- organisms and in animal tissues (34). It is not un phorylated compounds or dihydroderivatives of likely that deoxycytidine also reacts with the "for cytosine, uracil, or orotic acid are acceptors for the mate donor" prior to deamination. The presence "one-carbon compound" also requires further of deoxycytidine increased the specific activity of study. DNA-methylcytosine in experiments with labeled formate. Further studies are required to assess the SUMMARY possible role of hydroxymethylcytosine deoxyribo The conversion of formate-C14 or serine-3-C14 side or 5-methylcytosine deoxyriboside in thymi to DNA-thymine and free thymine, thymidine, dine synthesis. and thymidylate was studied in vitro with cell The conversion of cytidine to thymidine prob suspensions of the Ehrlich ascites tumor, with ably occurs via deoxycytidine. The addition of three lymphatic tumors, and with normal mouse cytosine does not increase the labeling of T and spleen or rat thymus cells. In the presence of de TDr (Table 6). The experiments of Roll et al. (26) oxycytidine, deoxyuridine, or to a lesser degree and of Rose and Schweigert (27) indicate that cytidine may be converted to DNA-deoxycytidine cytidine, the total radioactivity of free thymidine without the cleavage of the pyrimidine-sugar link plus thymine was increased. In the presence of age. Experiments with deoxycytidine-N15 (25) deoxycytidine, cytidine, and in some cases deoxy uridine, the specific activity of DNA-T was also suggest that the reverse process, the conversion elevated. Deoxycytidine stimulated the conver of deoxycytidine to cytidine, occurs slowly or not sion of formate-C14 to DNA-methylcytosine. Non- at all in the rat. labeled thymidine reduced the conversion of for Nonlabeled thymidine reduced the incorpora mate-C14 or serine-3-C14 to DNA-thymine but in tion of formate to DNA-T but increased the radio creased the labeling of free thymidine. A decreased activity of TDr. This suggests that TDr is in rapid specific activity of DNA-T was also observed in equilibrium with DXA-T precursors. This con experiments in which uracil-2-C14 was the sub clusion is supported by the fact that labeled thy midine is rapidly converted to DNA-T in a variety strate and deoxyuridine was added to the incuba of tissues (1, 7, 8, 24). Kornberg et al. (16) have tion flasks. demonstrated the presence in E. coli cells of en REFERENCES zymes which convert TDr to thymidylate, thymi 1. DOWNING,M.,and SCHWEIGERT,B.S. Role of Vitamin B12 dine triphosphate, and a deoxyribopolynucleotide. in Nucleic Acid Metabolism. IV. Metabolism of C"-labeled In this system, nonlabeled thymidylate reduced Thymidine by Lactobacillus leichmanni. 3. Biol. Chem., the incorporation of TDr-2-C14 to polynucleotide. 220:521-26, 1956. Free thymidine, deoxycytidine, and deoxyuridine 2. ELWYN,D., and SPRINSON,D.B. The Extensive Synthesis occur in normal and tumor tissues of mice and of the Methyl Group of Thymine in the Adult Rat. J. Am. Chem. Soc., 72:8317, 1950. rats (18, 29, 30) and thymidine and deoxycytidine 3. . The Synthesis of Thymine and Purines from phosphates have been detected in thymus tissue Serine and Glycine in the Rat. J. Biol. Chem., 207:467-76, (21). The present results and those of Reichard 1954. (24), Friedkin and Roberts (5, 6), and Prusoff and 4. FARBER,E.; KIT, S.; and GHEENBERG,D. M. Tracer co-workers (22, 23) suggest that DNA-T may be Studies on the Metabolism of the Gardner Lympho- sarcoma. I. The Uptake of Radioactive Glycine into formed in many tissues by a series of reactions in Tumor Protein. Cancer Research, 11:490-94, 1951. volving CR, CDr, UDr, TDr, and TDrP as inter 5. FRIEDKIM,M., and ROBERTS,D. The in ritro Formation mediates: Other pathways of DNA-T synthesis of Thymidine from Uracil Deoxyriboside, an Aminop- may also be significant in certain tissues. In the terin Sensitive Reaction. Fed. Proc., 14:215, 1955. 6. â€”¿.Conversionof Uracil Deoxyriboside to Thymidine case of the Ehrlich tumor, the addition of non- of Deoxyribonucleic Acid. J. Biol. Chem., 220:653-60, labeled uridine increased the conversion of for- 1956. mate-C14 to DNA-T (23). Relatively high levels 7. FRIEDKIX,M.; TILSON,D.; and ROBERTS,D. Studies of of radioactivity were observed in free thymine Deoxyribonucleic Acid Biosynthesis in Embryonic Tissues

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