39. ANTIMETABOLITES AND NUCLEIC ACID METABOLISM 501 



t eristic of early death. Although similar studies with a fluorouracil-resistant 

 subline of this tumor revealed little change in the DXA content of cells 

 exposed to the drug, an unexplained doubling of the amount of RNA and 

 protein in the resistant cells was recorded. A similar depression of the syn- 

 thesis of DNA with continued production of protein was noted in cultures 

 of E. coli exposed to fluorouracil. 373 



The specific sites of action of fluorouracil, though suggested by the above- 

 described studies carried out in vivo, are best examined by consideration of 

 the metabolism of this analog and the effects of these metabolites on en- 

 zymic reactions in viiro. In general, it may be stated that fluorouracil under- 

 goes all the anabolic reactions of uracil, with the obvious exception of 

 methylation at position 5 of the derivative corresponding to 2'-deoxyuridy- 

 lic acid. Following intraperitoneal injection of fluorouracil-2-C 14 into mice 

 bearing Ehrlich ascites cells and other tumors, the corresponding ribonu- 

 cleoside, and its mono-, di-, and triphosphates have been isolated by ion- 

 exchange chromatography. 366 Small amounts of 5-fluorocytidine and its 

 5'-phosphate have been isolated from the acid-soluble fraction of Ehrlich 

 ascites cells incubated with 5'-fluorouridine 374 (see below); the presence of 

 higher phosphates of these cytidine analogs has yet to be demonstrated. 



Minor amounts of diphosphate derivatives of fluorouridine, probably 

 analogous to the uridine-containing coenzymes involved in carbohydrate 

 metabolism, were also detected. Following ion-exchange separation of the 

 ribonucleotides in the acid-soluble extract of Ehrlich ascites cells incubated 

 in vitro with fluorouracil, 5-fluoro-2'-deoxyuridylic acid was found to com- 

 prise 11 % of the fluorouridylic peak. 374 The deoxy derivative was identified 

 by enzymic dephosphorylation and rechromatography with a borate buffer 

 to separate the resultant 5-fluorodeoxyuridine from the corresponding ribo- 

 nucleoside. The presence of this deoxyribonucleotide in the acid-soluble 

 portion of the cell is of considerable importance in explaining the biochem- 

 ical activities of fluorouracil. Di- or triphosphates of fluorodeoxyuridylic 

 acid were not detected, 374, 375 an observation compatible with the finding 

 that deoxyuridylic acid, unlike thymidylic acid, is not converted to higher 

 phosphates. 376 Incorporation of fluorouracil into the RNA of many mouse 

 and human tissues also occurs and the mixed 2'- and 3'-monophosphates of 

 fluorouridine have been isolated following hydrolysis of the RNA with 

 alkali. 366 The extent of replacement of normal pyrimidines under the con- 



373 J. Horowitz, J. J. Saukkonen, and E. Chargaff, Biochim. et Biophys. Acta 29, 223 

 (1958). 



374 E. Harbers, N. K. Chaudhuri, and C. Heidelberger, J. Biol. Chem. 234, 1255 

 (1959). 



375 L. Bosch, E. Harbers, and C. Heidelberger, Cancer Research 18, 335 (1958). 



376 M. J. Bessman, I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms, and A. 

 Kt)rnberg, Proc. Natl. Acad. Set. U. S. 44, 633 (1958). 



