IV POISONING OF THE SPINDLE 943 



In summary, it would appear that antimetabolites capable of interfering in the 

 synthesis of protein, of ribonucleic acid, or of high-energy phosphate bonds might 

 interfere with proper spindle assemblage or in its function, which seems to be in 

 part subject to chromosomal control. 



(a) Antifolics 



The tendency of antifolics to arrest mitosis in metaphase has already been 

 mentioned. That this is probably related to an influence on nucleic acid or 

 nucleotide metabolism is evident from what is known concerning the role in i- 

 carbon transfer played by some derivative of folic acid, such as the citrovorum 

 factor, or N-5-formyl-5,6,7,8-tetrahydropteroylglutamic acid {cf. Schlenk, 1955), 

 or the possibly dinucleotide coenzyme formed from the citrovorum factor and 

 ATP (Nichol et al., 1955). A relevant finding is that of Skipper, Morgan and 

 Bennett (1952), who showed that a-methopterin decreases the incorporation of 

 labeled formate and glycine into adenine of adenosine triphosphate in mice with 

 about the same order of activity as it inhibits their incorporation into nucleic 

 acid adenine. Actions of folic acid analogues, of folic acid itself and of analogues 

 of the precursors of folic acid or folinic acid may be considered here. 



Metaphase arrest was reported in onion roots as an effect of sulfanilamide 

 (Berger, 1944). This effect as well as the preprophasic inhibition caused by o.i or 

 0.5% sulfanilamide can be reversed with /?-aminobenzoic acid, which is a pre- 

 cursor of folic acid (Hindmarsh, 1949). The analogue, aminopterin, causes 

 mitotic aberrations in plants (Himes and Leuchtenberger, 1950; Jakowska, Ni- 

 grelli and Goldsmith, 1950). 



Folic acid itself injected in a dose of about 200 mg/kg into mice causes meta- 

 phases to accumulate in Ehrlich ascites tumor cells, with chromosomal clumping 

 (Lettre and Landschiitz, 1947). Folic acid in such a massive dose might well act 

 as an antagonist of its functional derivative. Folic acid has also been described as 

 being more strongly inhibitory to growth in onion root tips than 5-aminouracil 

 (Duncan and Woods, 1953). 



Antifolic acids have been studied in tissue culture by several investigators. 

 Hughes (1950) found that 0.5 mM aminopterin completely arrests mitosis in 

 chick cells in metaphase, while 5.5 mA/ a-methopterin causes only partial meta- 

 phase arrest. On rat fibroblasts, the optimum concentration of aminopterin for 

 metaphase blocking is 0.2 mAf (Benitez, Murray and Chargaff, 1954). Metaphase 

 arrest has been said to set in within a few min. (Hughes, 1952b), 15 min. (Jacobson, 

 1954a), or 20 min. (Benitez, Murray and Chargaff, 1954) but to disappear some 

 hours after antifolics are added to the medium. 



.Metaphase arrest was seen even after 24 h. in cultures of mouse embryonic skin 

 and sarcoma 180 exposed to 4-aminopteroylglutamic acid and its analogues con- 

 taining threonine or alanine in place of the glutamic acid moiety, but not in cul- 

 tures exposed to 4-aminopteroyltryptophan (Biesele, 1954a). These analogues are 

 also antifolics (Oleson, 1950). 



Antagonism studies showed that folic acid applied even in ten times the con- 

 centration of aminopterin does not block mitotic arrest by the latter in cultures of 

 chick osteoblasts and fibroblasts (Jacobson, 1954b). Citrovorum factor does block 



Lileralure p. g4y 



