Two different species of dihydrofolate reductase in mammalian cells differentially resistant to amethopterin and methasquin.

The disparate cross-resistance behavior of certain amethopterin-resistant Chinese hamster cell lines to the quinazoline antifolate, methasquin, prompted an investigation of the possibility of the existence of more than one species of dihydrofolate reductase in these cell populations. The dihydrofolate reductase of four amethopterin-resistant Chinese hamster sublines (DC-3F8/A50, 460-fold resistant; DC-3F8/A55, 4,455-fold resistant; DC-3F8/A75, 21,400-fold resistant; and DC-3F/A3, 108,400-fold resistant) and of the parental cell lines (DC-3F and DC-3F8) was partially purified and characterized. Kinetic analyses of the reduction of folate and dihydrofolate as well as inhibition analyses revealed that DC-3F8/A50 and DC-3F8/A75 synthesize the parental type of enzyme, whereas DC-3F/A3 and DC-3F8/A55 synthesize a structurally dissimilar enzyme, designated the A3 reductase. The parental enzyme interacts stoichiometrically with amethopterin and with the quinazoline as well. Inhibition of the A3 reductase is stoichiometric only with methasquin. The reversible inhibition of the A3 enzyme by amethopterin reflects a weak interaction of enzyme and this agent. The decreased sensitivity of the latter enzyme to amethopterin explains, in part, the disparate response to the quinazoline of the two most amethopterin-resistant sublines, DC-3F8/A75 (5,727-fold cross-resistant to methasquin) and DC-3F/A3 (208-fold cross-resistant). This observation in amethopterin-resistant mammalian cell populations of a structurally altered dihydrofolate reductase with decreased affinity for amethopterin, but with parental-type sensitivity to methasquin, strengthens the possibility that amethopterin may act as a mutagenic agent and suggests that methasquin may have clinical value in treating amethopterin-resistant neoplastic disease.