Relation of Folic Acid Reductase to Amethopterin Resistance in Cultured Mammalian Cells

Abstract

SUMMARY Two lines of cultured Sarcoma 180 cells resistant to amethop- terin have been studied. Resistance developed gradually in a medium containing hypoxanthine (AH cells; 67-fold resistant), and in a medium containing thymidine (AT cells; 174-fold resistant). The rate of decrease of resistance in the absence of the drug was independent of the type of medium used; after one month of cultivation, the cells were about half as resistant. The requirement for folic or folinic acids for growth was not altered during the development of resistance. Folinic acid prevented competitively the inhibition of growth caused by amethopterin; 9 times more amethopterin than folinic acid was necessary for inhibition of the growth of sensitive Sarcoma 180 cells, whereas 600 times more was necessary for inhibition of AH cells and 1500 times more for inhibition of AT cells. The relationship between folinic acid and amethopterin is discussed and a suggestion is made concerning the possible site of action involved. The sensitivity to the growth-inhibitory effects of 6-mercaptopurine, 6-diazo-5-oxo-n-norleucine, and 5-fluoro- deoxyuridine remained unchanged despite the development of amethopterin resistance. The resistant AH cells were found to contain 65 times more folic acid reductase than the sensitive Sarcoma 180 cells, and the AT cells contained 155 times more. Only the quantity of this enzyme had increased (or the number of active sites per enzyme molecule) whereas the kinetic characteristics, Michaelis constant, K,, value for folic acid (1.1 to 1.25 X 1O-5