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



Studies in vitro with leucocytes obtained from human subjects have dem- 

 onstrated remarkable differences between the cells characteristic of acute 

 leukemia, chronic granulocytic leukemia, and chronic lymphocytic leu- 

 kemia. 62 Thus, the two former types of cells utilized formate-C 14 rapidly 

 and steadily for at least 6 hours in vitro, incorporations which were stimu- 

 lated by folinic, but not by folic acid, and markedly inhibited by high con- 

 centrations of amethopterin (an inhibition prevented by folinic acid). On 

 the other hand, cells from normal individuals or from patients with chronic 

 lymphocytic leukemia took up but little formate and were essentially un- 

 affected by amethopterin. Since amethopterin, even at very high levels, as 

 with mouse leukemia cells, did not suppress completely the incorporation 

 of formate by cells of patients with chronic granulocytic leukemia, the exist- 

 ence of an amethopterin-resistant biochemical pathway for formate utiliza- 

 tion was considered. However, it should be emphasized here that the effect 

 of amethopterin appears to be exerted primarily on the formation of FH 4 

 and probably affects but little, if at all, the activity of preformed coenzyme 

 stored within the leukemic leucocytes. That the coenzyme content of such 

 cells may not be optimal, however, is indicated by the finding that the 

 addition of folinic acid in vitro stimulated the incorporation of formate, 

 while the finding that folic acid was without such activity may indicate 

 that the folic acid reductase activity is extremely low in these cells, or that 

 DPNH or TPNH may be limiting. It would be of much interest to examine 

 FH 2 -reductase activity of the cells, since, as previously discussed, the syn- 

 thesis of thymidylic acid (and presumably of other compounds, e.g., methio- 

 nine-methyl) appears to involve the formation from / 5 " 10 FH 4 of FH 2 and 

 its amethopterin-sensitive reconversion to FH 4 . 



In view of these results (and those described in the first paragraph of this 

 section), it will perhaps appear surprising that amethopterin is so rarely 

 used in the treatment of chronic granulocytic leukemia. However, the host- 

 toxicity observed with amethopterin is usually much greater in adults than 

 in children and other agents are employed if possible. 



Recent clinical studies with halogenated derivatives of amethopterin in 

 which the p-aminobenzoic acid portion of the molecule is substituted with 

 a single (3'-) or two (3', 5'-) chlorine atoms, or with bromine (3'-) and chlo- 

 rine (5'-) atoms, although based on very encouraging findings in the treat- 

 ment of mouse leukemia, 63 and markedly lower toxicity in rats and dogs 



62 R. J. Winzler, A. D. Williams, and W. R. Best, Cancer Research 17, 108 (1957); 

 R. J. Winzler, in "The Leukemias: Etiology, Pathophysiology, and Treatment" 

 (J.W. Rebuck, F. H. Bethell, and R. W. Monto, eds.), p. 567. Academic Press, New 

 York, 1957. 



63 A. Goldin, J. Natl. Cancer Inst. 22, 811 (1959) ; A. Uoldin and R. Humphreys, Proc. 

 Am. Assoc. Cancer Research 3, 22 (1959). 



