Effect of certain uracil analogs on incorporation of uracil into RNA

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THE

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281

2. VISCHER, E., MEYSTRE, C., AND WETTSTEIN, A., Helv. Chim. Acta 38,835 (1955). 3. NOBILE, A., CHARNEY, W., PEARLMAN, P. L., HERZOQ, H. L., PAYNE, J. C., TULLY, M. E., JEVNIK, M. A., AND HERSHBERG, E. B., J. Am. Chem. Sot. 77, 4134 (1955). 4. STOUDT,T. H., MCALEER, W. J., CHEMERDA, J. M., HIRSCHMANN, R. F., MARLATT, VIRGINIA, AND MILLER, R., Arch. Biochem. and Biophye. 69, 304 (1955). 5. VISCHER, E., AND WETTSTEIN, A., Ezperientia 9, 371 (1953). Research Laboratories, Chemical Division, Merck & Co., Inc. Rahway, New Jersey Received December 10, 1967

Effect of Certain Uracil Analogs on Incorporation

T. H. STOUDT W. J. MCALEER M. A. KOZLOWSKI VIRGINIA MARLATT

of Uracil into RNA’, 2

Evidence that uracil may be utilized preferentially in the biosynthesis of nucleic acids in tumors (1,2) has stimulated interest in the possible tumor-inhibitory activity of uracil analogs. We have found 2-thiouracil to be without influence on the growth of transplanted tumors in the rat (hepatoma, lymphosarcoma, Walker), although it effectively inhibits the induction of hepatomas by 2-acetaminofluorene (3). 6-Azauracil (4), and particularly 5-fluorouracil (5), have been found to possess marked tumor-inhibitory activity. Heidelberger et al. (5), reported significant depression of incorporation of formate-Cl4 into nucleic acid thymine in mouse liver, spleen, and ascites cells following administration of 5-fluorouracil. Their suggestion that one of the important actions of this compound involves reactions concerned with the conversion of formate to the methyl group of thymine is supported by the recent observations of Eidinoff et al. (6), who found that 5-fluorouracil depressed incorporation of erotic acid-6-Ci4, but not of thymidine-2-Ci4, into the DNA thymine of human tumor transplant slices. Another site of action is suggested by the results obtained by incubating rat hepatoma slices with uracil-2-C” in the presence of 2-thiouracil, 6-azauracil, and 5-fluorouracil (Table I). These three analogs depressed incorporation of uracil into hepatoma RNA in molar ratios of 1: 1.5-Fluorouracil was the most active and 2-thiouracil the least active, the former exerting a marked effect in a molar ratio of 1: 10. These findings suggest an action on the reactions involved in the conversion of uracil to uridylic and cytidylic acids, into which the major portion of uracil-2-Cl4 is incorporated in vivo (1) and in vitro (7). 1 Aided by a grant (C-1307 (C5)) f rom the National Cancer Institute, National Institutes of Health, U. S. Public Health Service. * Uracil-2-Cl4 was obtained from the Volk Radiochemical Co., Chicago, Ill., on allocation by the U. S. Atomic Energy Commission. 5-Fluorouracil was obtained from Hoffmann-LaRoche, Inc., Nutley, N. J., and 6-azauracil from Dr. Arnold D. Welch, Yale University, and Dr. George Hitchings, Wellcome Research Laboratories, through the Cancer Chemotherapy National Service Center, National Institutes of Health.

282

LETTERS

TO THE

TABLE

EDITORS

I

Effect of Uracil Analogs on Incorporation RNA of Transplanted Hepatoma

Cts./min./mg. RNA

Uracil 5omM

5omM 89 mM 89 mM 5omM

5omM 5omM 5omM

5omM 5omM

of Uracil4Y Slices

-

2-Thiouracil5 mM 2-Thiouracil89 mM -

6-Azauracil 5 mM 6-Aaauracil 50 mM -

5-Fluorouracil 5-Fluorouracil

5 mM 50 mM

in

% inhibition

415 348 461 290

16 37

413 353 116

15

413 149 44

64 90

72

Experimental Procedure. The tissue used was a transplanted hepatoma originally induced in A x C rats by 2-acetylaminofluorene. Slices (0.7-1-O g.) were incubated at 37°C. with 0.05 mM of uracil-2-V (56 ~c./mM) in an oxygen atmosphere, with shaking, for 2 hr. in 10 ml. of the following medium: 100 parts 0.154 M NaCl; 4 parts 0.154 M KCl; 1 part 0.154 M KCl; 1 part 0.154 M KHZPO, ; 1 part 0.154 M MgS01.7Hz0; 12 parts 0.1 M PO1 buffer, pH 7.4; 5 parts 0.308 M glucose. The uracil and analogs were added from the following stock solutions in the buffer mixture: uracil-2-C:” 10 PM/ml.; 2.thiouracil, 25&I/ml.; 6-asauracil and B-fluorouracil, 15 &/ml. RNA, DNA, and acid-soluble nucleotides were isolated and analyzed (8,9) by conventional procedures. The samples were plated by evaporation and counted in a gas-flow counter. REFERENCES

1. RUTMAN, R. J., CANTAROW, A., AND PASCHKI~, K. E., Cancer Research 14, 119 (1954). 2. HEIDELBEROER, C., LEIBMAN, K. C., HARBERS, E., AND BHARGAVA, P. M., Cancer Research 17, 399 (1957). 3. PASCHIKIS, K. E., CANTAROW, A., AND STASNEY, J., Cancer Research 8, 257 (1948). 4. HAKALA, M. T., LAW, L. W., AND WELCH, A. D., Proc. Am. Assoc. Cancer Research 2(2), 113 (1956). 5. HEIDELBEROER, C., CHANDHURI, N. K., DANNEBERG, P., MOOREN, D., GRIESBACH, L., DUCHINSKY, R., SJXNITZER, R., PLEVIN, E., AND SCHEINER, J., Nature 179, 663 (1957). 6. EIDINOFF, M. L., KNOLL, J. E., AND KLEIN, D., Arch. Biochem. and Biophys. 71, 274 (1957). 7. CANTAROW, A., WILLIAMS, T. L., PASCHKIS, K. E., AND BERQGREN (submitted for publication).

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8. HERBERT, E., POTTER, V. R., AND HECHT, L. I., J. BioE. Chem. 226,659 9. SCHNEIDER, W. C., J. Biol. Chem. 161. 293 (1945).

Department of Biochemistry, and Division of Endocrine and Cancer Research, Jefferson Medical College,

(1957).

IRVINCJ MELNICK ABRAHAM CANTAROW KARL E. PAXHKIS

Philadelphia, Pennsylvania Received January 16, 1968

Synergistic

Effect of Gibberellin

and 6-(Substituted)-purines of Lettuce Seed

on Germination

Several 6-(substituted)aminoand thiopurines (l-3), as well as gibberellin (4)) have recently been shown to stimulate seed germination, under nonoptimal temperature conditions and in the absence of light. Since these germination effects were quite similar, it was decided to test for a possible synergistic relationship between these two structurally dissimilar compounds. Such a synergism was observed when lettuce seed were pre-soaked in various combinations of these derivatives, and allowed to germinate in the dark at 30”. Lettuce seed (Early Curled Simpson) were pre-soaked in solutions containing 0, 1, or 10 r/ml. of the purine analog in the presence of 0, 30, or 100 -y/ml. of gibberellin. Higher concentrations of the purine derivatives are toxic, and higher concentrations of Gibberellin tended to become toxic in this assay system. After pre-soaking in the mixed solution for 8 hr., the seed were drained, placed on wet filter paper in covered petri dishes, and allowed to germinate at 30” in the dark for 24 hr. All handling operations were carried out in the presence of blue light (fluorescent light filtered through four layers of Du Pont 300 MSC dark blue cellophane). The results of these experiments are presented in Table I. During the time interval used in these experiments, none of the seed pre-soaked in 6-(substituted)purine solutions at the concentrations studied produced greater than 15% germination, with the exception of 6-benzylaminopurine and kinetin at concentrations of 10 r/ml., which gave 23 and 29% germination, respectively. These values, within the experimental error of the technique, represent comparable activity. Gibberellin, at 30 and 100 r/ml., gave 11 and 15% germination respectively. A combination of these compounds yielded, in every case, a marked, stimulation of rate of lettuce seed germination. For example, a mixture of 10 y of 6-beneylaminopurine and 30 y of gibberellin gave 8570 germination. The thiopurine analogs were less effective; however, a stimulation was observed when the seed were pre-soaked in a mixture containing gibberellin over that of seed soaked in either of the compounds alone. The structural feature of the purine derivative initiating this activity does not appear to be solely the purine nucleus, since adenine, at similar concentrations, gave no stimulatory effects either alone or in the presence of gibberellin. The fact that both the thio- and amino- analogs affect the rate of germination of lettuce seed indicates that structural specificity is not very exacting. However, it appears that the purine structure itself may be necessary to produce an active analog since the corresponding 4-(substituted)-