Biochemical Screening of Pyrimidine Antimetabolites I

Biochemical Screening of Pyrimidine Antimetabolites I. Systems with Oxidative Energy Source*

JOSEPHE. STONEANDVANR. POTTER

(McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis.}

Orotic acid (uracil-4-carboxylic acid) has been the livers were quickly excised and placed in a chilled bath of isotonic saline solution. A 20 per cent homogenate in chilled shown under both in vivo and in vitro conditions, 0.25 M sucrose was made with the use of an all-glass Potter- in both microorganisms and mammals, to be a pre Elvehjem homogenizer, and Ihis homogenate was centrifuged cursor of the mono-, di-, and triphosphate pyrim- at approximately 600 g for 10 minutes to remove nuclei and idine nucleotides, the pyrimidine coenzymes, and whole cells. also of the pyrimidine moieties in both ribo- and Portions of 0.8 ml. of the cytoplasmic liver fraction were deoxyribonucleic acid (5, 6, 11, 12, 13, 15-17). placed in 25-ml. Erlenmeyer flasks, each of which contained 2.20 ml. of a reaction mixture which had the following com This study was prompted by the current position: progress in the study of nucleic acid metabolism Potassium glutamate 15.0 AmÃ³les and its relationship to tumor growth and metabo Potassium fumarate 6.0 /Â«moles lism. The purposes of this research are the Potassium pyruvate 15.0 /iiiiole-s selection of agents as possible components of KHjPO, 15.0 /Â¿moles sequential (9) or concurrent blocks (4), the clari MgCl2 9.0 /uÃ±Ã³les fication of biochemical pathways, and the develop Ribose-5-phosphate (RSP) 6.0 /Â¿moles Uridine-5-monophosphate (UMP-5') 1.0 /imoles ment of concepts and technics in a biochemical Orotic acid-6-C" 0.3 /tmoles approach to pharmacological research. Adenosine triphosphate (ATP) 3.0 /uÃ±Ã³les The data presented here represent the results of A sufficient quantity of sucrose to make an isotonicity equiva a screening program with a biochemical system lent to 0.25 Msucrose. which converts orotic acid to the uridine nucleo A sufficient quantity of water to make a volume of 2.20 ml. tides. Normal rat liver cytoplasm furnished the The pH was adjusted to 7.2-7.4 with 0.2 N KOH. All flasks enzyme source, and oxidative substrates furnished were in duplicate. Drugs were added to the system in a drug: orotic acid ratio of 10:1 (3.0 /IMof the drug to 0.3 /IMof the the source of energy for the reactions. A primary orotic acid).2 Whenever possible, the drugs were added in objective of the study was to ascertain which aqueous solution; however, when necessary, acetate solutions drugs could be shown to act upon one or more or propylene glycol solutions were utilized. In these cases, ap steps in the conversion of orotic acid to the uridine propriate control flasks were added. The reaction mixtures were incubated with constant agita nucleotides. tion for 20 minutes at 30Â°C.The reactions were terminated by the addition of 1.50 ml. of 1.5 N perchloric acid to each flask, MATERIALS AND METHODS which resulted in a final perchloric acid concentration of 0.5 N. It has been demonstrated that under in vitro The resulting mixture was centrifuged at 600 g for 10 minutes conditions orotic acid is converted to uridine and the pellet discarded. The acid-soluble supernatant liquid was neutralized with 2.0 NKOH with phenol red as an internal nucleotides by preparations of normal rat liver indicator, and the neutral solution was then allowed to stand (6, 10). The latter observation is the basis of the for 10 minutes at â€”¿10Â°C.for the precipitation of residual test system used in this study. The drugs were potassium perchlorate. After recentrifugation, the supernatant added to a standardized system, and the con fluid was placed upon 5-cni. Dowex l (X10) aniÃ³nexchange resin columns (14). version of orotic acid to the uridine nucleotides The Chromatographie columns were submitted to the elu- was measured and compared with the conversion tion scheme which is summarized in Table 1. in appropriate controls. Fractions 1, 2, and 3 were not examined and were discarded White rats of either sex,1weighing 150-450 gm., were used (see below). Fractions 4, 5, 6, and 7 were read at 260 and 275 in this study. The animals were sacrificed by decapitation, and 1Obtained from the Holtzman-Rolfsmeyer Rat Company, * This work was supported in part by a grant (No. C2409) Madison, Wis. from the National Cancer Institute, National Institutes of 2The orotic acid used in this study was labeled in carbon 6. Health, United States Public Health Service. Its specific activity was 4.1 X 10* counts/min/mg (5.75 /imoles). Approximately 213,000 counts/min were added in Received for publication July 10, 1956. 0.3 /imoles added to each flask. 1033

nan in a Beckman model DU spectrophotometer. The radio 4) UMP-5' + ATP ^ UDP + ADP (3). activity of the samples was determined by plating a 0.2-ml. 4a) UDP + ATP ^Â±UTP + ADP (3). aliquot from each tube upon weighed aluminum planchets 4b) UTP-> UDPX (UDP conjugates) and which were inscribed with a circle 3.8 sq. cm. in area. Approxi mately 0.05 ml. of a 0.1 per cent solution of gelatin was used to UTPXS (3). spread the solution upon the inscribed area.8 The samples were 5) The ATP renewal is accomplished by oxida- then dried under a heat lamp, and the determination of radio tive phosphorylation with the Krebs cycle activity was done in gas-flow proportional counters.4 It should be emphasized that to insure activity of the system fortified at three points. all glassware should be washed in sulfurie-nitric acid cleaning solution and glass-distilled water. All reagents should also be In testing drugs against the standard system, prepared with glass-distilled water. the following points were taken as criteria of the activity of the system: Under control conditions, the standard sys 1. The total radioactivity in the UTP-UTPX tem5 converts an average of 7 per cent (range, 3-12 complex is the most reliable index to the effect of per cent among all preparations used) of the orotic the drug upon the conversion of orotic acid to the acid to the uridine nucleotides. The UTP-UTPX6 uridine nucleotides. In this study it has been taken complex accounts for 80-90 per cent of the orotic as the major criterion. acid converted. 2. The quantity of nucleotide in the UTP- TABLE1* SCHEDULEOFELUENTSANDFRACTIONSCOLLECTED Fraction Ml/total No. samples/ no. Eluent fraction fraction Compounds eluted 1 HjO 30 Nucleosides, bases 2 3.0 N formic acid t 150 Adenosine monophosphate (AMP), UMP-5', orotic acid 3 0.3 N ammonium formate! 30 Orotic acid and ADP 4 0.4 N ammonium formate 30 Mixed uridine coenzymes (UDPX's) 5 0.7 N ammonium formate 30 Uridine diphosphate (UDP) 6 l. 0 N ammonium formate 30 Uridine triphosphate (UTP) and UTPX 7 1.25 N ammonium formate 30 ATP * AHsubstances eluted in thÃ©Chromatographiescheme presented in Table 1 were characterized by either chromatography of known samples or reference to previous research done in this laboratory (3). t All formic acid solutions were at pH 2.O. ÃŽAllammonium formate solutions were at pH 5.O.

The known reactions included in the test sys UTPX fraction is an indication of the trans- tem may be summarized as follows: phosphorylase ability of the system in the presence 1) R5P + ATP -> PRPP (phosphoribosylpyro- phosphate) + AMP (7). specific activity of 1200 counts/min//*mole. However, this sys 2) Orotic acid + PRPP â€”¿Â»Orotidylicacid (orotic tem was unsatisfactory as a test object because of the extreme ly low percentage of orotic acid converted to the uridine acid ribotide) + PP (pyrophosphate) (8). nucleotides and because of excessive variation between dupli 3) Orotidylic acid -> UMP-5' + C02 (8). cate flasks (20-40 per cent). It was found that, with this and all subsequent systems investigated, the major uridine derivative 3The gelatin solution facilitates spreading of the plated produced was uridine triphosphate (UTP). This fraction con solutions; it also equalizes the crystallization of the ammonium tains small amounts of a uridine nucleotide (Dr. L. Hecht, formate present (M. J. Johnson, personal communication). unpublished data) whose specific activity is about 60 per cent 4After being counted, the plates were again weighed, and of that of the UTP. This compound has not been completely correction was made for self-absorption. The assistance of Dr. characterized and is designated as UTPX because it follows UTP on the chromatogram. The radioactivity of the UTP- Charles Heidelberger and his staff in radioactivity determina UTPX fraction has been adopted as the major criterion of tions is gratefully acknowledged. activity. 6The first system investigated consisted essentially of the The second system was further modified by the deletion of same oxÂ¡dativesubstrates employed in the standard system, the fructose and the reduction of the UMP-5' pool to 1.0 including 3.0 jumÃ³lesof ATP, a relatively large amount of /Â¿mole.Thissmall amount of UMP-5' serves as a marking pool, UMP-5' (6 /Â¿moles),and a pool of 3.0 /Â¿molesoforotic acid and its phosphorylation to UTP-UTPX serves as a measure which contained 0.3 /Â¿molesofthe labeled compound. These ment of the transphosphorylative ability of the preparation. substances were incubated with 0.8 ml. of 20 per cent rat liver Amounts of UMP-5' greater than 1 /Â¿moledepress the con homogenate for 20 minutes at 30Â°C.Under these conditions, version of orotic acid into the uridine nucleotides by actual the conversion of orotic acid to the uridine nucleotides could inhibition, aside from the effects due to competition for high not be demonstrated; however, under these conditions the energy phosphate and the dilution of the radioactivity. This UMP-5' pool was almost entirely converted to uridine. aspect will be treated fully in a later paper. Ribose-5-phosphate The addition of 6.0 /Â¿moleseachof fructose and ribose-5- was shown to be present at optimal amounts when used at a phosphate to the system outlined above and the substitution of level of 2.0 /xmoles/ml. Fructose may be used in place of ribose- the cytoplasmic fraction of rat liver in the place of the whole 5-phosphate; however, its inclusion adds a number of addi homogenate resulted in the production of UTP which had a tional reactions to an already complex system.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. STONEANDPOTTERâ€”ScreeningofPyrimidine Antimetabolites 1035 of the drug. Theoretically, the specific activity of UDP, and UTP-UTPX are good indices of a pri the UTP-UTPX (counts/min/Mmole) can be taken mary effect upon oxidative phosphorylation rather as an indication of the effect of the drug upon than upon orotic acid metabolism per se. oxidative phosphorylation. However, it must be The total radioactivity found in UDP, UDPX's, noted that the large ATP pool used in the standard UMP-5', or in orotic acid itself can be used as an system is sufficient for the transphosphorylation additional criterion of the conversion of orotic of the UMP-5' pool to UTP.6 Under these condi acid. However, these fractions (with the exception tions, the UMP-5' pool is converted to UTP re of the orotic acid fraction) contain relatively little gardless of the effect a drug may have on the radioactivity and are not so reliable as the UTP TABLE2* DRUGSWHICHDEMONSTRATEDNoSIGNIFICANTACTIONAGAINST THESTANDARDSYSTEM Compound no. Name 1. 2-Hydroxy-4-amino-S-carbethoxypyrimidine 2. 2-Ammo-4-hydroxy-5-carbethoxypyrimidine 8. 2-Mercapto-4-amino-S-carbethoxypyrimidine 4. 2-Ethoxy-4-hydroxy-5-carbethoxypyrimidine 5. 2-Ethylmercapto-4-chloro-5-carbethoxy pyrimidine 6. l-(D-glucopyranosyl)-carbamylcytosine 7. 2-EthyImercapto-4-ammo-5-4^1orophenoxymethylpyrimidine 8. 2-Ethylsulfonyl-4-amino-S-(2,4-dichlorophenoxymethyl)-pyrimidine 9. 2-Hydroxy-4-amino-5-aminomethylpyrimidine 10. 2,4-Diethoxy-5-carbethoxypyrimidine 11. Na-carbethoxy-L-asparagine 12. Na-carbethoxy-D-asparagine 13. 4-Amino-5-hydroxymethylpyrimidine 14. 5-Methyl-orotic acid 15. 5-Iodo-orotic acid 16. 4-Methyl-uraciI 17. 4-Methyl-2-sulfo-uracil 18. 4-Trifluoromethyl-2-sulfo-uracil 19. 2-Mercapto-orotic acid 20. 5-Amino-orotic acid 21. Thiothymine 22. Bromouracil 23. 4-Oxy-5-(o,p-dichlorophenoxy)-pyrimidine 24. Thiocytosine 25. Thiouracil 26. Azathymine 27. Cytosamine * Drugs nos. 1â€”15,28-35,40, and 46-51 were supplied by Sharpe and Dohme, Inc. Nos. 19, 20, 36-38, and 41 were supplied by Lederle Laboratories. Nos. 21-26, 44, and 45 were supplied by Welcome Research Laboratories. Nos. 27 and 52 were supplied by Eli Lilly and Co. Nos. 16-18, 39, 42, and 43 were supplied by Dr. Charles Heidelberger of this department. energy source of the system. However, in cases complex. The UMP-5' fraction is extremely of an extreme inhibitory effect of an agent upon small and is separated only with difficulty from the energy source, the absolute amounts of ATP, the orotic acid. The orotic acid fraction itself is 6During the course of a later investigation, an experiment extremely radioactive, and its net conversion is was performed in which the following changes were made in low. For these reasons, the UMP-5' peak and the the test system employed in this study: The ATP pool was orotic acid peak are eluted from the column with reduced from 3.0 Â¿imolesto 0.5 /linole, and the nuclei were washed with chilled 0.25 Msucrose solution and the washings 3.0 N formic acid and discarded. added to the cytoplasmic fraction. The time of incubation was increased to 45 minutes. These modifications resulted in the RESULTS AND DISCUSSION conversion of 47 per cent (45.6-50.0 per cent) of the orotic The drugs which have been tested in this study acid. Moreover, under these conditions the specific activity of the UTP complex is a valid measurement of the oxidative have been divided into three groups on the basis capacity of the system. It should be noted that this modifica of their effects upon the test system. Those com tion allows for a finer differentiation between drug action upon pounds which depressed the conversion of orotic oxidative phosphorylation and upon orotic acid metabolism acid 0-20 per cent were regarded as demonstrating per se. Such a system, because of its ability to convert a high proportion of orotic acid at low molari! ics to uridine nucleo- no significant effect upon orotic acid metabolism tides, could be used for production of the latter in radioactive (Table 2). form. Those agents which gave an inhibition of 20-40

per cent were placed in an intermediate group respiratory enzymes. The agents found active (Table 3). against this system were then tested against a The drugs which depressed orotic acid con- more specific system with a glycolytic energy version 40-100 per cent were selected for further source. The differential screening systems show study (Table 4). clearly which agents are active against orotic acid In general, oxidative phosphorylation is per- metabolism per se and which are active against haps not the energy source of choice in a bio- oxidative phosphorylation alone, chemical-pharmacological screening system. This Differential screening with multiple systems source of energy has certain disadvantages, can also give information on agents with multiple Among these are the rigorous standards of cleanli- points of action and could be an aid in the correla- ness which must be observed and the instability of tion of biochemical and pharmacological phe- the enzyme system. However, the most serious nomena. Programs of this type should also give disadvantage lies in the fact that the energy rise to a dictionary of drugs whose point or points source may be the most sensitive component of the of action are more or less known. It would seem system. Agents which act upon one or more steps that such a body of knowledge would be invalu- in the oxidative cycle could appear to be inhibitors able in integrative biochemistry, of reactions in which they actually have no effect. The thirteen agents which demonstrated some However, in this study oxidative phosphoryla- inhibitory capacity can be divided into four tion was deliberately chosen as an energy source chemical and to some extent functional groups, because of its extreme sensitivity. Under these Group 1 consists of the 5-substituted analogs conditions, it allows for the screening not only of of orotic acid and 5-diazo-uracil. The chloro, orotic acid antimetabolites but also of agents bromo, and diazo analogs of orotic acid demon- which may have an effect upon some of the strated moderate inhibition (average, 45 per cent

TABLE 8 DRUGSWHICHDEPRESSEDTHECONVERSIONOF OHOTICACIDBY20-40 PER CENT Compound no. Name 28. 2-Ethylmercapto-4-hydroxy-5-carbethoxypyrimidine 29. 2-Ethylrnercapto-4-amino-5-aminomethylpyrimidine 30. 2,4-Diamino-5-carbethoxypyrimidine 31. 2-Mercapto-4-amino-5-hydroxymethylpyrimidine 32. 2-Ethylmercapto-4-amino-5-hydroxymethylpyrimidine 33. D-dihydro-orotic acid 34. 5-Hydroxymethylcytosine 35. Fumarylurea 36. 5-Carboxy-orptic acid 37. 2-Amjno-orotic acid 38. 6-Acetic-erotic acid 39. 4-Trifluoromethyl-uracil

TABLE 4* DRUGSWHICHINHIBITEDTHECONVERSIONOFOROTICACIDBY40-100PER CENT UTP-UTPXUTP-UTPX ATP radioactivity /Â¿moles pmoles Compound aÂ«per cent as per cent ns per cent no. Name of control of control of control 40. 5-Chloro-orotic acid 55 (2) 139 138 41. 5-Bromo-orotic acid 33 (3) 129 131 42. 5-Diazo-orotic acid 47(3) 95 83 43. 5-Diazo-uracil 27(3) 64 57 44. 2,4-Diammo-5-(3',4'-dichlorophenyl)-6-methylpyrimidine 25 (2) 38 27 45. Daraprim 9(2) 14 17 46. 2-Benzylmercapto-4-amino-5-carbethoxypyrimidine 29 (2) 90 93t 47. 2-Ethylmercapto-4-amino-5-chlorornethylpyrimidine 35 (2) 118 84 48. 2-Benzylmercapto-4-araino-5-hydroxymethylpyrimidine 9 (2) 48 60 49. 2-Ethoxy-4-amino-5-carbethoxypyrimidine 17 (2) 73 63 50. 2-Ethylmercapto-4-amino-5-carbethoxypyrimidine 11 (2) 47 43 51. N"-carbethoxy-DL-asparagine 50 (l) 91 52. Amicetin 41 (2) 63 50 * Figures in parentheses give number of experiments. t In one experiment a duplicate flask was extremely aberrant and demonstrated a total count which was 180 per cent of the control levels. This drug is extremely insoluble.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. STONEANDPOTTERâ€”ScreeningofPyrimidine Antimetabolites 1037 of control). This inhibition is accompanied by an in a semispecific system which essentially tested actual increase in the amount of UTP-UTPX and orotic acid conversion only. These data will be in ATP. From these data it would appear that presented in another publication. these agents have no inhibitory effect upon oxidative phosphorylation and that the inhibition oc curs in the steps between orotic acid and UMP-5'. SUMMARY Fifty-two pyrimidine derivatives and related The occurrence of abnormal metabolites or the "piling up" of other metabolites could not be compounds have been tested for orotic acid anti- metabolite activity against a biochemical system demonstrated because of the low net conversion of which converts orotic acid to the uridine nucleo orotic acid. The inhibition produced by 5-diazo- tides. Thirteen of these drugs have been demon uracil would seem to be of a different type be strated to be active against one or more of the cause of the depression of the amounts of triphos- components of the system. phate which accompany the inhibition. It should be noted that the 5-carboxy, amino, methyl, and REFERENCES iodo analogs of orotic acid were ineffective in this 1. FALCO,E. A.; HITCBINGS,G. H.; RÃœSSEL,P. B.; and system. The 2-amino and 2-mercapto analogs VANDEBWEBÃ•T,H. Antimalarials as Antagonists of were likewise ineffective. Purines and Pteroylglutamic Acid. Nature, 164:107-8, Group 2 includes Daraprim (2,4-diamino-5-par- 1949. achlorophenyl-6-ethylpyrimidine) and its analog 2. FLTNN,E. H.; HINMAN,J. W.; CARÃ“N,E.L.; and WOOLF, 2,4-diamino-5- (3',4'-dichlorophenyl) -6-methylpy- D. O. The Chemistry of Amicetin, a New Antibiotic. J. Am. Chem. Soc., 76:5867-71,1953. rimidine. In this system these drugs are marked 3. HERBERT, E.; POTTER, V. R.; and TAKAGI, Y. The inhibitors of orotic acid conversion to the uridine Phosphorylation of 5'-Uridine Nucleotides by Cell Frac nucleotides (average, 17 per cent of control). tions from Rat Liver. J. Biol. Chem., 213:923-40, 1955. 4. HITCHINGS,G. H. Purine and Pyrimidine Antagonists. However, this phenomenon is accompanied by Am. J. Clin. Nutrition, 3:321-27,1955. marked depression of the amounts of triphosphate. 5. HUHLBEHT,R.B., and POTTER,V. R. Nucleotide Metabo Thus, it would seem that these drugs have a lism. I. The Conversion of Orotic Acid-6-C14to Uridine marked effect upon the energy source of the sys Nucleotides. J. Biol. Chem., 209:1-21, 1954. 6. HURLBEBT,R. B., and REICHAHD,P. The Conversion of tem. The effect of these drugs upon orotic acid Orotic Acid to Uridine Nucleotides in Vitro. Acta Chem. metabolism cannot be ascertained until they are Scandinav., 9:251-Â«2,1955. tested against a system whose energy source is 7. KORNBEHG,A.;LIEBERMAN,I.;and SIMS,E. S. Enzymatic unaffected by them. Daraprim has been shown by Synthesis and Properties of 5-Phosphoribosyl Pyrophos- other workers to be a folie acid antagonist (1). The phate. J. Biol. Chem., 215:389-402, 1955. 8. LIEBERMAN,L;KORNBERG,A.;and SIMS,E. S. Enzymatic action shown here may be a corollary of this or Synthesis of Pyrimidine Nucleotides, Orotidine-5'-Phos- may be an additional point of action. phate and Uridine-5'-Phosphate. J. Biol. Chem., 216:403- Group 3 is composed of the 2,5-substituted 15, 1955. 4-amino pyrimidines. The inhibition was accom 9. POTTER,V.R. Sequential Blocking of Metabolic Pathways in Vivo. Proc. Soc. Exper. Biol. & Med., 76:41-46, 1951. panied by moderate depression of the amounts of 10. POTTER,V.R.; HECHT,L.; and HERBERT,E.Incorporation both uridine triphosphate and ATP. In this case, of Pyrimidine Precursors into Ribonucleic Acid in a Cell- the data derived from a system of this type cannot free Fraction of Rat Liver. Biochem. & Biophys. Acta, differentiate between action upon orotic acid 20:439-40, 1956. 11. REICHARD,P. On the Turnover of Purine and Pyrimidine metabolism and the energy source. This group of Polynucleotides in the Rat Determined with N16. Acta drugs demonstrates the variability between dupli Chem. Scandinav., 3:422-32, 1949. cate flasks and experiments. This phenomenon 12. REICHARD,P., and BERGSTROM,S. Synthesis of Poly may be due to variation in solubility with slight nucleotides in Slices from Regenerating Liver. Acta. Chem. variation in pH. These compounds all have an Scandinav., 6:190-91, 1951. 13. SCHMITZ,H.; HURLBERT,R. B.; and POTTER, V. R. amino group in position 4, and it would seem that Nucleotide Metabolism. III. Mono-, Di-, and Triphos- a large variety of groups can be used in positions phates of Cytidine, Guanosine, and Uridine. J. Biol. 2 and 5. It is difficult to draw conclusions from these Chem., 209:41-54, 1954. data, because a potentially active compound may 14. SIEKEVITZ,P., and POTTER,V. R. Radioactive Labeling of Intramitochondrial Nucleotides during Oxidative Phos be almost completely insoluble. phorylation. J. Biol. Chem., 216:237-55, 1955. The antibiotic Amicetin (2) produced an aver 15. WEED,L. L. The Incorporation of Radioactive Orotic Acid age inhibition of 41 per cent of the control levels; into the Nucleic Acid Pyrimidines of Animal and Human there was moderate diminution of the amounts of Tumors. Cancer Research, 11:470-73, 1951. triphosphate. 16. WEED,L. L., and WILSON,P. W. The Incorporation of C1Â« N" carbethoxy-DL-asparagine diminished orotic Orotic Acid into the Nucleic Acid Pyrimidines in Vitro. J. Biol. Chem., 189:435-41, 1951. acid conversion to 50 per cent of the controls. 17. â€¢¿â€”. Studies of Pyrimidine Nucleotides with Orotic The drugs discussed above were reinvestigated Acid-2-C" and PÂ».Ibid., 216:745-49, 1953.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. Biochemical Screening of Pyrimidine Antimetabolites: I. Systems with Oxidative Energy Source

Joseph E. Stone and Van R. Potter

Cancer Res 1956;16:1033-1037.

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