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



in what appears to be "thymineless" death. 493 However, under these condi- 

 tions, little or no azathymidine was found in the DNA. Once initiated, the 

 death process usually could not be interrupted by the addition to the cul- 

 ture of massive amounts of thymidine. When added to cultures in concen- 

 trations insufficient to abolish reproduction, azathymidine was incorporated 

 into the DNA of S. faecalis in an amount equivalent to 18 % of the normal 

 thymine content. Clearly, the extent of inhibition and the degree of incor- 

 poration into DNA are essentially unrelated, since maximal incorporation 

 occurs under conditions which do not result in lethal effects on the cells. 



Evidence concerning the mechanism of action of azathymine and its de- 

 oxyribonucleoside has been obtained primarily from studies with rabbit 

 bone marrow and with Ehrlich ascites cells in vitro. i9i ~ in The incorporation 

 of formate into DNA thymine in these systems was blocked by azathymi- 

 dine (but not by azathymine), whereas incorporation of thymidine in ascites 

 cells and bone marrow was relatively insensitive to azathymidine; how- 

 ever, direct comparison of these results is difficult, because in the former 

 case the unnatural deoxyribonucleoside is competing with what is believed 

 to be very small pools of thymine nucleotides. From the urine of mice re- 

 ceiving radioactive azathymine there have been isolated the ribonucleoside 

 of the analog, as well as a catabolic derivative, and also relatively large 

 amounts of uracil. 497 Formation of the ribonucleoside (the structure of 

 which has been proven by chemical synthesis 498 ), and of the deoxyribonu- 

 cleoside by a soluble fraction from liver has been demonstrated 499 ; however, 

 these compounds did not influence the formation of the corresponding de- 

 rivatives of thymine in this system. 499 



A chemical synthesis of the 3'- and 5'-mono- and 3',5'-diphosphates of 

 azathymidine has been reported, 500 but the biological activity of these de- 

 rivatives has been tested only in whole-cell systems in which they are less 

 active than the corresponding deoxyribonucleoside. An investigation of the 

 effects of these derivatives on the enzymic steps responsible for the synthe- 

 sis of thymidylic acid and its subsequent incorporation into DNA would be 

 desirable. 



493 W. H. Prusoff, J. Biol. Chem. 226, 901 (1956). 



494 A. D. Welch, W. H. Prusoff, and L. G. Lajtha, Trans. Assoc. Am. Physicians 68, 

 112 (1955). 



496 W. H. Prusoff, L. G. Lajtha, and A. D. Welch, Biochim. et Biophys. Acta 20, 209 

 (1956). 



496 W. H. Prusoff, Biochem. Pharmacol. 2, 221 (1959). 



497 W. H. Prusoff and R. A. Gaito, Abstr. 131st Meeting Am. Chem. Soc, Miami, Flor- 

 ida, p. 2C (1957). 



498 R. H. Hall, J. Am. Chem. Soc. 80, 1145 (1958). 



499 W. H. Prusoff and R. A. Gaito, Federation Proc. 17, 292 (1958). 

 600 R. H. Hall and R. Hazelkorn, /. Am. Chem. Soc. 80, 1138 (1958). 



