504 R. E. HANDSCHUMACHER AND A. D. WELCH 



caused 50% inhibition of the activity of thymidylate synthetase, whereas 

 fluorodeoxyuridine and fluorouridylic acid were inactive even at 500 times 

 this concentration. The hydroxymethylation of deoxycy tidy lie acid was 

 insensitive to all fluoro derivatives tested, including 5-fluorodeoxycytidine- 

 5'-phosphate. 387a 



Another enzymic site at which fluorouracil derivatives act is uridine 

 phosphorylase. 388 The partially purified enzyme from Ehrlich ascites tumor 

 cells is inhibited by fluorouracil to the extent of 50%, when its activity 

 on the phosphorolysis of uridine is tested at a molar ratio of 1:1. These 

 enzyme preparations also catalyze the formation of fluorouridine from 

 ribose-1 -phosphate and fluorouracil and phosphorylate the ribonucleoside 

 in the presence of ATP and Mg ++ to form the 5'-phosphate. Furthermore, 

 in a cell line of this tumor selected for resistance to fluorouracil, 389 the pres- 

 ence of uridine phosphorylase activity could not be detected. This apparent 

 shut down of preformed pyrimidine utilization also involved deoxyuridine, 

 since its phosphorylase was not found in the fluorouracil-resistant subline, 

 although it was present in the sensitive line. The effects of fluorouridine 

 on uridine kinase have not been reported, but fluorouracil is inactive as an 

 inhibitor; however, fluorouracil-resistant cell lines of Ehrlich ascites tumor 

 and leukemia L-1210 possess much lower levels of this enzyme. In a differ- 

 ent study 389a with Ehrlich ascites tumor cells which have been selected for 

 resistance to fluorouracil, the analog failed to inhibit the incorporation of 

 formate into DNA thymine, but still partially blocked the incorporation of 

 uracil into RNA. These results have been interpreted to indicate unimpaired 

 formation of fluorouracil ribonucleotides and a change in the affinity of 

 thymidylate synthetase for the deoxyribonucleotide of fluorouracil. 



Since fluorouracil or fluorouridine, but not fluorodeoxyuridine, inhibits 

 the incorporation of orotic acid as well as uracil into RNA pyrimidines, an 

 enzymic site of action at the ribonucleotide level beyond uridylic acid is 

 suggested, but this has not yet been demonstrated. 390 However, it has been 

 shown through the use of a mutant of E. coli that fluorouracil did not in- 

 hibit accumulation of total proteins, although it completely blocked the 

 induction of /3-galactosidase activity, presumably by blocking synthesis of 

 new RNA. 391 In mammalian cells, however, this must not be a prominent 



387a S. S. Cohen, personal communication (1959). 



388 O. Skold, Biochim. el Biophys. Acta 29, 651 (1958). 



389 P. Reichard, (). Skold, and G. Klein, Mature 183, 939 (1959). 



389a C. Heidelberger, P. B. Dunneberg, G. Kaldor, and O. Ghobar, Federation Proc. 

 18, 244 (1959). 



390 I. Melnick, A. Cantarow, K. E. Paschkis, Arch. Biochem. Biophys. 74, 281 (1958). 



391 J. Horowitz, J. J. Saukkonen, and E. Chargaff, Biochim. el Biophys. Acta 29, 222 

 (1958). 



