Effect of 5-Bromodeoxyuridine on Deoxyribonucleic Acid- Thymine Synthesis and Cell Metabolism of Lymphatic Tissues and Tumors*

SAULKIT, CHARLESBECK,ODETTEL. GRAHAM,ANDARTHURGROSS

(Department of Biochemistry, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston 25, Texas)

5-Bromodeoxyuridine (BrUDr) is an effective under the same conditions. Although the incor inhibitor of the growth of E. coli and Lactobacitti, poration of isotopically labeled one-carbon com and this growth inhibition can be reversed by pounds into DNA-thymine was inhibited, neither thymidine (1-3). There is evidence that this thy- the labeling of acid-soluble thymine compounds, midine analog can replace the thymine of both serine, nor proteins was inhibited, nor was cell E. coli and bacteriophage deoxyribonucleic acid glycolysis or respiration significantly reduced. It (DNA) on an equimolar basis (7, 11, 12). In would therefore appear that BrUDr represents E. coli, nearly 50 per cent of the thymine can a relatively specific antagonist of the terminal be replaced. Whereas the physical properties and steps in the utilization of thymidine for DNA appearance of bacteriophage particles containing synthesis. incorporated 5-bromouracil appeared normal, a proportion were found to be nonviable. Litman MATERIALS AND METHODS and Pardee have observed that T2r2 stocks grown Cell suspensions of the following tissues were used in this in the presence of 5-bromouracil and sulfanilamide study: rat thymus, mouse spleen, lymphatic leukemia LL5147 under conditions in which the 5-bromouracil is (Ak mice) and E9514A (C3H mice), and lymphosarcoma 6C3HED. The care and handling of the tumor-bearing animals incorporated into the DNA may contain a high and preparation of the cell suspensions have been previously proportion of mutant types (11). The mutagenic described (9, 10). effect of 5-bromouracil in the presence of sulfanil Cell suspensions (approximately 20 mg. dry weight of tis sue) were incubated at 88Â°C.in Warburg vessels containing a amide must certainly be connected with a dis total volume of 2.45 cc. of fluid. The flasks also contained 2.5 turbance of DNA metabolism or structure. Anoth /'â€¢inÃ®lesofglutaminc, 30 /Â¿molesofglucose, 1 /Â¿moleofdeoxy- er instance of mutagenesis resulting from an inter cytidine, in some instances 1 Minole of thymidine, varying ference with thymine metabolism has been pre concentrations of BrUDr or other antimetabolites, and the labeled precursor. The incubation tune was generally 3 hours. sented by Coughlin and Adelberg (6), who ob Formaldehyde-C'4 (1 /imole [2 juc]per flask) was obtained served that specific thymine starvation is muta from Isotope Specialties, Inc. 5-Bromodeoxyuridine, 5-hy- genic in an E. coli strain which has a double re droxydeoxyuridine, and 5-bromouridine, bromouracil, and quirement for thymine and histidine. A hundred 6-mercaptopurine were purchased from the California Founda fold proportional increase of histidine-independent tion for Biochemical Research and Nutritional Biochemicals, mutants appeared among the survivors of 185 Inc. The authors are indebted to Dr. James F. Holland of the Roswell Park Memorial Institute, Buffalo, New York, for a gift minutes of thymine starvation. of 2-methylmercapto-4-amino-5-hydroxymethyIpryimidine. These facts prompted a study of the potential Acid-soluble thymine, thymidine, and thymidylate and role of BrUDr as an inhibitor of thymine bio DNA-thymine were extracted from the tissues, subjected to synthesis by tumors. The effect of the analog purification from radioactive contaminants, and assayed for on the incorporation of formaldehyde-C14 into radioactivity as previously described (10). Respiration and glycolysis were measured by conventional manometric proce thymine compounds under in vivo or in vitro dures. The methods for determining the specific activity of free conditions was investigated. Inhibition was ob serine and protein were those previously used in this laboratory served in the labeling of DNA-thymine, but the (9). Details concerning the in vivoexperiments are given in the labeling of DNA-adenine or guanine was reduced results section of this paper. only at high concentrations of BrUDr. Other RESULTS analogs of thymidine had no significant effect Effects of bromodeoxyuridine on the in vivo in * Aided in part by grants from the American Cancer Society corporation of formaldehyde-Câ„¢intoDNA-thymine. and the Leukemia Society, Inc. â€”C3H mice weighing 20-25 gm. were given in Received for publication January 9, 1958. oculations intraperitoneally of 0.2 cc. of un- 598

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1958 American Association for Cancer Research. KIT et al.â€”5-Bromodeoxyuridine and Cell Metabolism 599 diluted ascites fluid containing E9514A tumor cells. to DNA. BrUDr at levels ranging from 0.32 After 7 days of tumor growth, the mice were to 3.2 milJimoles was effective in reducing the divided into two groups of three. One group labeling of DNA-thymine. In the experiments was given injections intraperitoneally of 5.4 /Â¿moles described above, 1 /Â¿moleof deoxycytidine was of BrUDr, the other of physiological saline. Five present in the incubation medium. As reported minutes later, 4 //.c.(2 /Â¿moles)offormaldehyde-CH previously, the presence of deoxycytidine greatly was administered by the same route, and 2 hours increases the conversion of the one-carbon pre afterwards the animals were sacrificed. BrUDr cursor to thymine compounds (10). Inhibition markedly inhibited the incorporation of the pre of DNA-thymine labeling was, however, obtained cursor into the DNA-thymine of the tumor cells whether or not deoxycytidine was included in or the spleen cells from the tumor-bearing animal the medium (Table 3). Several experiments were but did not significantly affect the labeling of also performed in which nonradioactive thymidine DNA-adenine or guanine (Table 1). was added to the incubation flasks. Thymidine TABLEl EFFECTOFS-BROMODEOXYURIDINEONINCORPORATION OF FORMALDEHYDE-C14 INTO DNA In Vivo

Thymine Adenine Guanine TISSUE Control BrUDr* Control BrUDr* Control BrUDr* E9514A 1250 450 3500 3270 2090 2260 Spleen 1300 580 4710 3940 1210 1490 E9514A 1370 270 2580 2040 * Bromodeoxyuridine (5.4 /Â¿moles)injected 5 minutes prior to a test dose of 4 /ic. of HjC"O (2 AmÃ³les), and animals sacrificed 2 hours later.

TABLE 2 INHIBITIONBYS-BROMODEOXYURIDINEOFINCORPORATION OF FORMALDEHYDE-C14 INTO DNA-THYMINE in Vitro Cose, DNA ACID-SOLUBLE (milli- TAG T TDr TDrP TISSUE ADDITION moleÂ«) (countÂ«/min//imole) (total counts/min) Gardner Control 4930 2800 1970 3710 10500 470 BrUDr 1.6 1390 3040 1860 3100 8670 360 LL5147 Control 1985 1150 3210 650 BrUDr 0.8 496 1240 4230 1570 Thymus (rat) Control 11920 3410 580 5010 2225 BrUDr 0.8 6180 3190 565 6745 2220 BrUDr 1.6 4480 1840 910 8705 2680 BrUDr 2.4 3610 1595 1010 5440 1845 Abbreviations: T = thymine; A = adenine; G = guanine; TDr thymidine; TDrP = thymidylate; BrUDr = 5-bromodeoxyuridine.

In vitro effect of 5-bromodeoxyuridine.â€”The TABLE 3 presence of BrUDr also resulted in a marked In Vitro EFFECTOFS-BROMODEOXYURIDINEONTHE LA in vitro inhibition of the incorporation of formalde- BELINGOFTHYMINECOMPOUNDSOFLYMPHOSARCOMA hyde-C14 into the DNA-thymine of rat thymus, 6C3HED WITHORWITHOUTDEOXYCYTIDINEADDED lymphatic leukemias LL5147 and E9514A, and DNA ACID-SOLUBLE lymphosarcoma 6C3HED (Table 2). The specific ADDI-ATioNS T T TDr TDrP activity of DNA-adenine or guanine was not af (counts/min/pmole) (total counts/min) Control3710CDr* 2480 1010 660 130 fected at low concentrations of BrUDr but was 5850BrUDr* progressively reduced at higher concentrations. 1460 7200 270 440CDr*38705260 There was no apparent reduction of the labeling 1010 1000 80 of acid-soluble thymine, thymidine, or thymid- 1970 5070 1640 7480 290 ylate. Instead, the total radioactivity of the acid- BrUDr soluble thymine compounds was increased as DNA * One /umole of deoxycytidine and 2.17 /amÃ³lesof bromode- biosynthesis decreased. It is therefore unlikely oxyuridine per flask. Each flask also contained 30 /Â¿molesglu that the BrUDr was inhibiting the methylation cose, and 2.5 jumÃ³lesglutamine, and 1 /umole (2 /Â¿c.)of formaldehyde-C14. Total volume, 2.45 cc. of the thymidine precursor. Rather, the effect Abbreviations: CDr = deoxycytidine; BrUDr = bromode- was apparently on the conversion of the thymidine oxyuridine.

markedly depressed the labeling of DNA-thymine, Effect of compounds related to 5-bromodeoxyuri- but not of DNA-adenine. Because the size of dine.â€”The effect of various compounds related to the free thymidine pool was increased, the total BrUDr on the labeling of thymine compounds was radioactivity of free thymine compounds was in investigated. At comparable concentrations, bro- some cases increased. The labeling of acid-soluble mouracil, bromouridine, 5-hydroxydeoxyuridine, thymine compounds was investigated in the pres and^-methyhnercapto-e-amino-S-hydroxymethyl- ence of BrUDr and thymidine but in the absence pyrimidine were essentially ineffective as inhibi of deoxycytidine. Under these conditions also, tors. The presence of 6-mercaptopurine also pro BrUDr did not inhibit the labeling of acid-soluble duced no marked inhibitions (Table 5). thymine compounds (Table 4). 5-Bromodeoxyuridine and respiration and glycolysis.â€”Concentrations of BrUDr which inhibited TABLE 4 the labeling of DNA-thymine did not significantly S-BROMODEOXYUHIDINE AND THE INCORPORATION OF affect cell respiration or glycolysis (Table 6). FORMALDEHYDE-C" INTO ACID-SOLUBLE THYMINE Incorporation of formaldehyde into protein and COMPOUNDSOFTUMORLLS147INTHEPRESENCEOF free serine.â€”No inhibition by BrUDr on the in THYMIDINE* corporation of formaldehyde-C14 into protein or T TDr TDrP free serine was noted. Rather, there was a small (total counts/mm) increase in labeling (Table 7). These experiments Control 1900 1320 430 further suggest that BrUDr does not intervene BrTJDr 1940 1700 480 in cell metabolism at the level of one-carbon (0.99 millimoles) transfer reactions. * Each flask contained 5 AmÃ³lesthymidine, 2.5 /Â¿molesglu- Effect of 5-bromodeoxyuridine on the growth of tamine, and 30 AmÃ³lesglucose. Total volume: 2.45 cc. There was no deoxycytidine present. tumor E9514A.â€”Sixteen male C3H mice weigh-

TABLE 5 THYMIDINEANALOGSANDTHEINCORPORATIONOFFORMALDEHYDE-C" INTOTHYMINECOMPOUNDS CONC. DNA ACID-SOLUBLE (milli- TAG TDr TDrP Tisera6C3HEDE9514ARat (counts/mÃŒD//imole) (total counts/min) 4930 2800 1970 3710225023003290329517901700174014401750580920950510820105007520943011200109002640177013907680763050101030015000474036704705354805006705805104102230252022303710652026802330 7130 3480 2530 4330 3830 2370 5170 4140 3090 5920 4120 2680 40004720367050304000119008480782013000112002240021700233002870221034102640235028502760

thymusRat

thymusADDITIONControlBrU*BrUBrUBrUControlHS-purine*MAHMP*ControlHS-purine*ControlMAHMP*MAHMPHOUDr*BrUR*mo!11.23222242.2.

* Abbreviations: BrU = 5-bromouracil; HS-purine = 6-mercaptopurine; MAHMP = 2-methylmercapto-4- amino-5-hydroxymethylpyrimidine; HOUDr = 5-hydroxydeoxyuridine; BrUR = 5-bromouridine; T = thymine; TDr = thymidine; TDrP = thymidylate; A = adenine; G = guanine.

TABLE 6 5-BROMODEOXYUHIDINEANDRESPIRATIONOR GLYCOLYSISOFLYMPHATICCELLS TABLE 7 ANDTUMORS 5-BROMODEOXYURIDINE AND INCORPORATION OF FORMALDEHYDE-C" INTO ACID-SOLUBLE SERINE Qo,Control6.15.86.5BrUDr*6.05.76.8Ã¶SControl14.18.29.3BrUDr13.88.87.7ControlBrUDr* TIBSÃœI ORCELLPROTEINBYTUMOR6C3HED 6C3HED Counts/min/mg protein Total counts/min serine Spleen (mouse) 1370 60 ,800 Thymus (rat) 1510 72,300 * 3.91 AmÃ³lesbromodeoxyuridine added. All other flasks * 8.91 jumÃ³lesbromodeoxyuridine added. All flasks con contained 2.5 /Â¿molesglutamine, 1 /

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1958 American Association for Cancer Research. KIT et al.â€”5-Bromodeoxyuridine and Cell Metabolism 601 ing 18-22 gm. were inoculated intraperitoneal- prevent de novo thymidine synthesis (1, 4, 11). ly with 1.5 X IO6E9514A tumor cells. Beginning In neoplastic tissues, antifolic compounds can 3 days after the injection of the tumor, ten of be used to prevent the methylation of deoxy the mice were given injections each day of phys uridine. It will be of interest to observe whether iological saline and six of the mice of BrUDr the combined use of aminopterin and BrUDr (41.6 mg/kg body weight). Three of the control will prove more effective in prolonging the survival mice died 14 days after receiving the tumor, three of tumor-bearing animals than either alone (5). at 15 days, one at 16 days, one at 17 days, one at 19 days, and the last animal at 20 days after SUMMARY receiving the tumor. Two of the treated mice The effect of 5-bromodeoxyuridine and related died at 16 days, one at 17 days, one at 18 days, compounds on the metabolism of lymphatic leu- one at 19 days, and the final animal at 22 days kemias LL5147 and E9514A, lymphosarcoma 6C3- after receiving the tumor. The mean ( + the stand HED, rat thymus, and mouse spleen cells was ard error of the mean) survival of the control studied both in vivo and in vitro. It was observed and treated animals was 16 + 0.67 and 18 Â±0.93 that 5-bromodeoxyuridine reduced the incorpo days, respectively. It may be seen that BrUDr, ration of formaldehyde-C14 or formate-C14 into when administered alone, produced a slight but DNA-thymine. The incorporation of the isotope questionable prolongation of the survival time into DNA-adenine or guanine was affected only of mice with lymphatic leukemia, E9514A. at high concentrations of 5-bromodeoxyuridine. Several compounds related to bromodeoxyuridine DISCUSSION had no significant effect. There was no inhibition The results reported in this paper suggest that of the labeling of free thymine compounds, of BrUDr represents a relatively specific antagonist of the terminal steps of DNA-thymine synthesis. the hydroxymethylation reaction leading to serine synthesis, of the incorporation of the isotopie The analog does not prevent the methylation of deoxyuridine to form thymidine, the hydroxy- precursor into cell protein, or of cellular respiration methylation reaction leading to serine synthesis, or glycolysis. A slight but statistically question the anabolic reactions involved in the incorpora able increase of the survival time of mice bearing tion of the isotopie precursor into protein, or ascites tumor E9514A was observed after the daily the energy-yielding processes of glycolysis and administration of 41.6 mg/kg body weight of respiration. The above interpretation is supported 5-bromodeoxyuridine. by the preliminary findings of Eidinoff and co- workers (8). 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1958 American Association for Cancer Research. Effect of 5-Bromodeoxyuridine on Deoxyribonucleic Acid-Thymine Synthesis and Cell Metabolism of Lymphatic Tissues and Tumors

Saul Kit, Charles Beck, Odette L. Graham, et al.

Cancer Res 1958;18:598-602.

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