Studies on Fluorinated Pyrimidines IV. Effects on Nucleic Acid Metabolism in Vivo*

PETERB. DANNEBERG,!BETTYJo MONTAG,ANDCHARLESHEIDELBERGER

(McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison 6, Wis.)

In conjunction with the studies on the metabo tion of labeled ureidosuccinic acid into nucleic acid lism and incorporation of labeled 5-fluorouracil and pyrimidines and that 5-bromodeoxyuridine inhib 5-fluoroorotic acid into RNA (2), investigations its the incorporation of thymidine into nucleic acid have been carried out to determine the effects of thymine (7, 8). various fluorinated pyrimidine derivatives as in Prusoff et al. (20) have shown that 6-azathy- hibitors of nucleic acid biosynthesis. Because the midine inhibits the in vitro incorporation of for- fluorine atom in the pyrimidine molecule is at mate-C14 into DNA thymine in bone marrow and tached to the same carbon that bears the methyl Ehrlich ascites carcinoma cell suspensions. group of thymine, it was early surmised that these Stone and Potter have measured the effect of compounds would block the methylation reactions a large number of pyrimidine analogs, including leading to the metabolic formation of DNA thy 5-fluorouracil and 5-fluoroorotic acid, on the con mine. Preliminary evidence that this is so was version of orotic acid-C14 into acid-soluble uridine presented in a report from this laboratory (12). nucleotides in a soluble preparation from rat liver Although there has been abundant work on the (23, 24). effects of antifolics, alkylating agents, azaserine, In the present paper we report on the in vivo N-methylformamide, etc., on the processes of nu effects of 5-fluorouracil, 5-fluoroorotic acid (5), cleic acid biosynthesis, cf. Skipper (22), only 5-fluorouridine, and 5-fluoro-2'-deoxyuridine (6) scanty effort has been devoted to studios of the on the incorporation of formate-C14 into nucleic effects of purine or pyrimidine analogs on the acid purines and pyrimidines, uracil-2-C14, orotic incorporation of normal precursors into nucleic acid-6-C14, and thymidine-6-H3 into nucleic acid acids, such as those carried out here. In the bio pyrimidines; and of P32into DNA and RNA. chemical screening of a large series of compounds, Davidson and Freeman have demonstrated the MATERIALS AND METHODS inhibition of P32 incorporation into DNA of the In all experiments, Swiss albino mice obtained from Taconic 755 tumor by 6-mercaptopurine and 8-azaguanine Farms, Germantown, N.Y., bearing 6-day-old transplants of (44); LePage and Greenlees have found that in the Ehrlich ascites carcinoma were used. Unless otherwise indicated, the inhibitors were injected 30 minutes before the in vitro Ehrlich ascites cells the incorporation of labeled precursors, and the mice were sacrificed 12 hours after glycine-2-C14 into nucleic acid purines was inhib administration of the precursor. All injections were intra- ited by 6-mercaptopurine, but not significantly by peritoneal. Each experimental group consisted of three mice, 8-azaguanine (17); Heidelberger and Keller re whose livers, spleens, and ascites cells were pooled. Duplicate ported that, in slices of Flexner-Jobling carcinoma, isolations were carried out on each pooled tissue. The doses of inhibitors ordinarily used were 150 mg/kg of 5-fluorouracil and the incorporation of formate into nucleic acid pu 105 mg/kg of 5-fluoroorotic acid. These doses produced sig rines was inhibited by 6-mercaptopurine, 8-aza nificant tumor inhibition with a single injection (14). The guanine, and 2,6-diaminopurine (15). Eidinoff has proteins were precipitated and defatted in the usual way (25). found that 5-aminouridine inhibits the incorpora- The tissue precipitates thus obtained were made up to 8 ml. with 10 per cent NaCl, phenolphthalein was added, and the pH * This work was supported in part by a grant-in-aid of the was adjusted with l N NaOH until the indicator was red; then Wisconsin Division of the American Cancer Society; a grant, 0.5 ml. of saturated NaHCOj solution was added to maintain C-2832, from the National Cancer Institute, National Insti- a slight alkalinity. The samples were extracted for 30 minutes tures of Health, U.S. Public Health Service; and by a grant-in- with frequent stirring at room temperature and, following aid from Hoffmann-LaRoche, Inc. Preliminary reports of part centrifugation, were extracted with the same volume of 10 per of this work appeared in Fed. Proc., 16, 194, 1957, and Abstr. cent NaCl for 1 hour at 95Â°C.The hot extraction was repeated Am. Chem. Soc. Meeting, Sept. 8-13, p. 20C, 1957. for 30 minutes with 5 ml. of NaCl. The combined hot extracts t Lederle Post-Doctorate Fellow in Oncology. and the cold extract were separately filtered through glass wool into 5 volumes of 95 per cent ethanol to precipitate the sodium Received for publication October 25, 1957. nucleates. This amount of ethanol prevents the co-precipita- 329

tion of sodium chloride, which sometimes accompanies the specific activities of two to three separate experi sodium nucleates when small volumes of ethanol are used for ments, with duplicate analyses on two pools of the precipitation. The sodium nucleates were hydrolyzed with 2 ml. of 70 per cent perchloric acid at 95Â°C.for 1| hours (19), tissues. The reproducibility of the experiments was the solution was diluted 1:10 with water and passed through fairly good. However, there were a few widely 8 X 0.5 cm. columns of Darco, G-60, charcoal. The inorganic aberrant values. These were included in the aver salts were removed by washing with 50 ml. of water, and the ages quoted when the radioactivity in the samples purine and pyrimidine bases were eluted with ethanol:cone. counted was statistically significant and the spec- NH4OH:water, 73:3:24 (v/v). The mixture was dried and separated by one-dimensional paper chromatography with trophotometric purity of the bases was adequate. isopropanol :cone. HC1: water, 170:41:39 (v/v) according to It has previously been reported from this labo Wyatt (26). This method gave good separation of all the bases ratory (12) that the incorporation of labeled for if the hot and cold extracted nucleates were run separately. The positions of the spots were located with an ultraviolet lamp, mate into the methyl group of DNA thymine is and they were cut out with a paper punch (li-in, diameter), strongly inhibited by 5-fluorouracil and 5-fluoroo- assayed for radioactivity in flow counters, and corrected for rotir acid. This is further confirmed by the experi self-absorption. The paper discs were eluted with 0.1 N HC1 at 87Â°C.for 12 hours, and the absorption at 260, 280 m^, and ments given in Table 1, where it may be seen that at the absorption maximum of the individual bases was deter both drugs produced essentially complete inhibi mined. The identity and purity of the bases were established by tion of this process in spleen and ascites cells and TABLE1 EFFECTSOFS-FLUOROURACILANDS-FLUOROOROTICACIDONNUCLEICACIDBIOSYNTHESIS INMICEBEARINGTHEEHRLICHASCITESCARCINOMA Drug given 30 minutes before labeled precursor. Mice sacrificed 12 hours after precursor.

CONTROLS5-FluorouracilCENT SPECIFIC ACTIVITY OP ACTIVITYOP S-Fluoroorotic acid AMÃ“LES/PRECURSORLABELED ACIDBASEDNA L*SCONTROLSAcounts/min/^mole4134958141410115164045576086,00036,60093401201130551272039119,90014,20021418401410196026,000768034909636281770437017507540663051424101620486026,90016,7009060108026304470PERmg/kg)L321097196238720113622243325168S4716159560225SO582199052A612260985826232219Â¿91294938l28788469406555102320191404543Ã(150 mgAg) (10Â« 46260672267399306118129A883841255893192424421317141 UOUBEFormate-C14 IOUuItUracil-2-C14 thymineMixed cytosine" adenine" guanineDNA 10uuOrotic-6-C14acid thymineRNA uracilMixed cytosineDNA 10Â«uuThymidine-6-H3 thymineRNA uracilRNA cytosineDNA cytosineDNA 5Phosphate-P32 thymineRNADNASPECIFIC <0.1"No.EXP.32113222211121NUCLEIC

* L = liver; S = spleen; A = ascites cells.

the ratio of the absorbance at 280/260 DIM,and the jumÃ³les/disc a lesser effect in liver. Formate was also found to were determined at the point of maximum absorption. The be incorporated into mixed nucleic acid cytosine, specific activities of the bases were then computed and ex pressed as counts/min/iimole. The tritium-containing thymine possibly by carbon dioxide fixation, but this proc samples were counted directly in flow counters on the paper ess was not significantly inhibited by the fluorin discs. No attempt was made to correct for self-absorption, since ated pyrimidines. It is known from the excellent the discs had identical thicknesses. The quantities of DNA and work of Friedkin (9) and Cohen (3) that the RNA were determined by the diphenylamine and orcinol reac tions, respectively (21). The P3*radioactivity was determined methylation reaction leading to thymidylic acid in dipping counters. is catalyzed by a tetrahydrofolic acid coen/yme. The sodium formate-C14 and orotic acid-6-C14were obtained The same type of coenzyme has been shown by from Tracerlab, Inc., the uracil-2-C14 from Volk Radio- Goldthwait et al. (10) to be involved in the con chemical Corp., the thymidine-6-H3 from Schwarz Labora version of formate into the 2- and 8-carbons of tories, Inc., and the P32from the Oak Ridge National Labora the purines. That 5-fluorouracil and 5-fluoroorotic tory, all on allocation from the U.S. Atomic Energy Commis sion. All fluorinated pyrimidines were obtained from Dr. acid act specifically to inhibit the reaction leading Robert Duschinsky, of Hoffmann-LaRoche Inc. (5, 6). to the formation of the methyl group of thymine, and not as folie acid antagonists, is shown by the RESULTS lack of effect of the compounds on the in vivo The design of the experiments has been de conversion of formate into the mixed nucleic acid scribed under "Materials and Methods." The data adenine and guanine (Table 1). reported in Table 1 usually represent the mean The use of uracil-2-C14 as a precursor (16)

afforded the opportunity of studying the effects into RNA in liver and ascites cells, but not in of 5-fluorouracil and 5-fluoroorotic acid on its spleen. Considerable inhibition of the incorpora conversion into RNA uracil, DNA thymine, and tion of P32 into DNA was observed in the drug- mixed nucleic acid cytosine. The results (Table treated mice, particularly in the ascites cells. The 1) show that the conversion of uracil into DNA lack of complete inhibition of this process is in thymine was inhibited to about the same extent accord with the results of Davidson and Freeman as the incorporation of formate into DNA thymine (4), who never obtained an inhibition of more than under the conditions of these experiments, except 50 per cent of the incorporation of P32 into the that there was less inhibition by 5-fluoroorotic acid DNA of Adenocarcinoma 755 with a series of in the spleen in this case. The conversion of uracil compounds. Although our inhibitions were some into RNA uracil was inhibited by the fluorinated times greater than 50 per cent, no account was pyrimidines to a somewhat lesser extent than its taken in this work of the specific activity of the conversion into DNA thymine, particularly in the inorganic phosphate in the tissues, which David ascites tumor cells. This was also in general true son and Freeman (4) have shown to be affected for the conversion of uracil into nucleic acid by the drug treatment. cytosine. In the experiments with orotic acid as the pre cursor, a greater incorporation into the RNA X FU TUMOR uracil was observed in the liver than in the tumor, 100 <2>FULIVER in agreement with the results of Hurlbert and Ã•FU INTESTINE Potter (11) and of Heidelberger et al. (16). 5- DFO TUMOR 75' Fluorouracil and 5-fluoroorotic acid almost com CJFO LIVER pletely inhibited the conversion of the precursor into DNA thymine, a result comparable to that 50' obtained when uracil was the precursor. In both cases, the inhibition was greater in ascites cells than in liver. There was considerable, but not 25' complete, inhibition by both drugs of the conver sion of orotic acid into RNA uracil, the quantities, of which were again similar to those found with 0.15 0.5 5 10 25 100 uracil as the precursor. In the experiment with MG./KG. labeled orotic acid as the precursor, cytosine was CHART1.â€”Effectof fluorinated pyrimidines on the conver isolated from DNA and RNA separately, but there sion of formate-C14 into the methyl group of DNA thymine was no appreciable difference between the slight in tumor cells, intestine, and liver. Per cent of control thymine inhibitory effect of the drugs on the metabolic specific activity against dose. conversion of orotic acid into either RNA or DNA cytosine. In general there was a similar inhibition Since it was evident from the preceding work pattern found with orotic acid and uracil as labeled work and the in vitro experiments (1) that the precursors. fluorinated pyrimidines profoundly inhibit the When thymidine-H3 was used as a precursor, metabolic conversion of formate into the methyl 5-fluorouracil and 5-fluoroorotic acid produced a group of DNA thymine, it became of interest to stimulation of incorporation into DNA thymine establish an in vivo biochemical dose-response in all tissues relative to the controls. This observa curve for this reaction. Accordingly, the experiment tion is consistent with the hypothesis that these indicated in Chart 1 was carried out. Groups of drugs block the metabolic reaction leading to the three mice, bearing Ehrlich ascites carcinomas, de novo formation of the methyl group of DNA were injected intraperitoneally with various doses thymine, since the utilization of preformed thy- of 5-fluorouracil and 5-fluoroorotic acid, followed midine for DNA thymine biosynthesis would not 30 minutes later by formate-C14. Two hours later be blocked. The stimulation observed would be the animals were sacrificed, and the DNA thymine expected from an enhanced utilization and less was isolated from liver, ascites cells, and, in two ened dilution of the preformed thymidine owing to cases, the upper part of the small intestine. These the inhibition of the de novo pathway leading to specific activities were determined and compared DNA thymine synthesis. with those obtained from untreated controls. In When tracer amounts of phosphate-P32 were Chart 1 the logarithm of the dose is plotted against used as the nucleic acid precursor, 5-fluorouracil the per cent specific activity, as compared with and 5-fluoroorotic acid inhibited the incorporation that of the controls. If one compares, for example,

the in vivo inhibition of this reaction at a dose of effective dose most commonly given daily in 1.25 mg/kg, essentially complete inhibition of the tumor-screening experiments (14). In Chart 2 the reaction in the ascites cells of fluorouracil-treated per cent specific activities of DNA thymine as mice is found; no inhibition was observed in the compared with that of the untreated controls is liver or intestines of the same animals. 5-Fluor- plotted against time. The drug was administered oorotic acid had no significant activity in liver or intraperitoneally at zero time; at the number of ascites cells at this dose. Thus, this experiment hours afterward shown on the chart, formate-C14 shows that there was a greater in vivo biochemical was injected, and the animals were sacrificed 2 effect produced by 5-fluorouracil in tumors than hours later. It will be observed that the metabolic in liver and intestines. At a dose of 6.25 mg/kg conversion of formate into the methyl group of of 5-fluoroorotic acid, there was considerable inhi DNA thymine in the tumor cells was essentially bition of this reaction in tumor, but not in liver. completely inhibited for 12 hours and rose to only In general, both 5-fluorouracil and 5-fluoroorotic 22 per cent of the controls at 24 hours. On the acid inhibited the conversion of formate into the other hand, in liver the inhibition was never com plete and rose to 42 per cent of the controls at 12 hours and 112 per cent at 24 hours. The initial dip in the liver curve probably reflected a delay in absorption. Thus, it is evident that, at least at this dose, 5-fluorouracil exerted an effect of longer duration on this biosynthetic reaction in ascites cells than in liver. This represents a still further example of the selective action of fluorouracil against tumor cells. This finding is similar to that of LePage et al. (18), who found a longer duration of azaserine inhibition of de novo purine biosynthesis in tumor cells than in spleen.

OH OH

CHART2.â€”Duration of effect of 5-fluorouracil at 25 mg/kg on the conversion of formate-C" into DNA thymine in liver CH2OH and ascites cells in vivo. Per cent of control thymine specific activity against time between injections of the drug and the H H formate. OH OH 5-FUUOROURlDlNE 5-FLUORO-2'-OEOXYURIDINE methyl group of DNA thymine to a greater extent FUR FUDR RO 5-0338 RO 5-0360/2 and at a lower dosage in ascites cells than in liver and spleen. CHART3.â€”Structure, abbreviation, and code numbers of Although it might be argued that, since the the nucleosides of 5-fluorouracil. drugs and precursors were injected intraperitoneally and thus came in direct contact with the During the course of these investigations the tumor cells, it is not justifiable to consider these riboside and deoxyriboside of 5-fluorouracil, 5- results as in vivo evidence for selective action on fluorouridine, and 5-fluoro-2'-deoxyuridine were tumor cells, it may be pointed out that with synthesized by Duschinsky et al. (6). These com 5-fluoroorotic acid no such wide differential was pounds have been screened against various tumors, found between tumor cells and liver as that ob and the results will be reported elsewhere (13). In served with 5-fluorouracil. This mode of adminis mice bearing the Ehrlich ascites tumor, both ri- tration was selected, because these drugs do not bosides, whose structures are shown in Chart 3, prolong the life of mice bearing the Ehrlich ascites are more effective than 5-fluorouracil in increasing tumor when injected at other sites. the life span of the animals. It next became desirable to extend these studies In view of the demonstration that 5-fluorouracil to a third dimension and determine the duration is converted into acid-soluble nucleotides of vari of effect in various tissues. To make the experiment ous levels of phosphorylation (2) and that deoxy- practical, it was limited to a consideration of liver fluorouridylic acid is also produced (1), it seemed and ascites cells at a single dose of 5-fluorouracil. likely that these nucleosides should also inhibit The dose selected was 25 mg/kg, which is the the metabolic conversion of formate into the

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1958 American Association for Cancer Research. DANNEBERG et al.â€”Studies on Fluorinated Pyrimidines. IV 333 methyl group of DNA thymine. Accordingly, dose- greater tumor-inhibitory activity in animals main response experiments were carried out in vivounder tained on a diet free of nucleic acids and their conditions comparable to those described in Chart precursors. 1. The results are shown in Chart 4. It has been previously demonstrated (2) that At the very small doses employed in this experi 5-fluorouracil and 5-fluoroorotic acid are incor ment, both nucleosides completely inhibited the porated into ribonucleic acid. Now it has been methylation reaction in the tumor cells. In the shown, in addition, that these drugs inhibit various case of 5-fluorouridine there was no inhibition of processes of ribonucleic acid biosynthesis. With thymine biosynthesis in the livers at these dose the data now at hand it is not possible to de levels. However, in the ascites cells there was termine which of these biochemical effects is complete inhibition at 5 mg/kg and very little in responsible for the tumor-inhibitory properties of hibition at 0.05 mg/kg of 5-fluorouridine. When the this series of compounds, or whether both are inhibitory effects of 5-fluorouracil and 5-fluorouri required. The in vivo results reported here are in dine are compared, it is found, from Charts 1 and complete agreement with the findings obtained in 4, that this metabolic reaction occurred in the vitro in glycolyzing suspensions of Ehrlich ascites tumor cells to an extent of 30 per cent of the con trols at a dose of 0.5 mg/kg, whereas, with an % 100- equimolar dose of fluorouracil (0.25 mg/kg), the DFUDR TUMOR specific activity of the DNA thymine was 65 per cent of the controls. Therefore, the riboside of â€¢FUDR LIVER 5-fluorouracil was somewhat more effective than 80- A FUR TUMOR the free base in inhibiting this reaction in vivo. With 5-fluoro-2'-deoxyuridine, there was a 50 per A FUR LIVER 60- cent inhibition in liver of the conversion of formate into DNA thymine at all three doses, and com plete inhibition in the tumor cells, even at 0.05 40- mg/kg. Thus, with the two nucleosides there was some selective action on the tumor, at least as compared with liver, and the 5-fluoro-2'-deoxy- 20- uridine has been demonstrated to be an extraor dinarily potent inhibitor of DNA thymine for mation ire vivo. It is also the most powerful inhib .05 0.5 5.0 MG./KG. itor of the series in vitro (1). CHART4.â€”Effectof fluorouracil nucleosides on the conver sion of formate-C" into DNA thymine. Per cent of control thy DISCUSSION mine specific activity against dose. It is evident from the foregoing that 5-fluor ouracil and 5-fluoroorotic acid are powerful inhib cells reported in an accompanying paper (1) and itors of certain processes of both UNA and DNA serve to strengthen the conclusions drawn from pyrimidine nucleic acid biosynthesis and exert a that work. Since interpretation of the results and somewhat selective effect on tumor cells. More the localization of the metabolic block (s) produced over, these compounds, as well as 5-fluorouridine by the fluorinated pyrimidines must take into and 5-fluoro-2'-deoxyuridine, completely inhibit account both in vivo and in vitro experiments, the conversion of formate into the methyl group detailed discussion of this subject will be given of DNA thymine in Ehrlich ascites tumor cells in the next paper (1). in vivo. As indicated from the P32experiments, this block is sufficient to inhibit appreciably the process SUMMARY of DNA biosynthesis in vivo. However, the experi 1. The effects of 5-fluorouracil and 5-fluoroorot- ments with labeled thymidine clearly show that ic acid on nucleic acid biosynthesis in livers, DNA biosynthesis can occur in the presence of spleens, and Ehrlich ascites carcinoma cells have fluorinated pyrimidines, provided that exogenous been studied in vivo in mice preformed thymidine is supplied. This is further 2. These drugs inhibited the conversion of ura- evidence for the location of the block at the cil-2-C14and orotic-6-C14 acid into DNA thymine, reaction leading to the formation of the methyl RNA uracil, and, to a lesser extent, into nucleic group of DNA thymine. Because the utilization acid cytosine. The inhibition was usually greater of exogenous thymidine is not blocked by these in spleen and ascites tumor cells than in liver. drugs, one might predict that they would show 3. The conversion of formate-C14 into the meth-

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Peter B. Danneberg, Betty Jo Montag and Charles Heidelberger

Cancer Res 1958;18:329-334.

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