930 ANTIMETABOLITES AS MITOTIC POISONS 13 



in cells of mouse sarcoma 1 80 than in cells of embryonic mouse skin by amino- 

 pterin, a-methopterin, 4-aminopteroylthreonine, and 4-aminopteroylalanine in 

 concentrations from 0.2-1.0 or 5.0 xnM. 4-Aminopteroyltryptophan is more in- 

 hibitor)' to cells of embryonic mouse skin. 



(b) Antiglutamines 



The amide group of glutamine furnishes the nitrogen for positions 3 and 9 of 

 the purine skeleton (cf. Reichard, 1955), and this is the basis for an additional 

 set of antimetabolites. Azaserine, or O-diazoacetyl-L-serine, inhibits certain 

 tumors (Stock, Reilly, Buckley, Clarke and Rhoads, 1954) and interferes with the 

 role of glutamine in purine biosynthesis, causing the accumulation of certain 

 acyclic ribotide precursors of inosinic acid (Hartman, Levenberg and Buchanan, 

 1956). Azaserine thus inhibits incorporation of formate (Skipper, Bennett and 

 Schabel, 1954) and of glycine (Greenlees and LePage, 1956) into nucleic acid. 



Azaserine strongly inhibits mitosis in mouse tissue cultures (Biesele, 1958 a). 

 Exposure for 24 h. to o.i mM azaserine depresses mitotic incidence in mouse 

 sarcoma 180 cells to about 10 percent of the control value but in mouse embryonic 

 skin cells to only about 70 percent of the control. A partial reversal of the mitotic 

 inhibition from azaserine can be achieved with L-histidine, but L-glutamine is 

 completely effective (Biesele, 1958 b). Similar effects are achieved with y-glutamyl 

 hydrazide. It is somewhat less toxic than azaserine but also suppresses mitosis 

 difTerentially in sarcoma 180 cells. The mitotic suppression can be reversed in 

 mouse tissue cultures and in cultures of certain human carcinoma cell strains with 

 glutamine (Biesele, 1958 b). 



Azaserine at 0.2 mg/kg decreases the mitotic index in 4 ascites tumors of the 

 mouse for 1 2 hours after injection ; at the same time, it inhibits incorporation of 

 glycine-2-^'*C into their acid-soluble and nucleic acid purines (Fernandes, LePage 

 and Lindner, 1956). 



(c) Purine and pyrimidine analogues 



Structural analogues of the physiological purines are often inhibitory anti- 

 metabolites. Many of them depress mitotic incidence, as is illustrated by Kihl- 

 mann's results (1952) with 8-ethoxycaffeine. Pea roots were suspended in 7.5 mM 

 8-ethoxycafTeine for 3 h. and then returned to tap water. Mitotic incidence, which 

 had been about 6%, fell to less than i percent in the first 1 2 hours but then returned 

 to the original level by 24 hours of the recovery period. 



Mouse embryonic skin cells in culture suffer a greater depression of mitosis 

 from exposure to 2.0 mM 2-ethoxycaffeine for 2 days than do mouse sarcoma 

 180 cells (Biesele, Berger, Clarke and Weiss, 1952). CafTeine and 8,8'-ethylene- 

 diaminoditheophylline behave in the reverse fashion. 



Studies of the reversal of the inhibitions caused by substituted purines and purine 

 analogues have indicated generally complex situations. Kidder and Dewey (1949) 

 concluded that the methylxanthines inhibit more than one enzyme system in the 

 ciliate, Tetrahymena geleii, because their inhibition can only be partly reversed 

 with guanine or other purines. Caffeine inhibition of conidial growth of the asco- 

 mycete, Ophiostoma multiannulatum, is likewise only partially reversed with 



