STUDIES ON DRUG RESISTANCE IN MAMMALIAN CELLS I. AMETHOPTERIN RESISTANCE IN MOUSE FIBROBLASTS

¹ Department of Pharmacology, Stanford University School of Medicine, Stanford, California

The growth of the mouse fibroblast ("L" cells, clone 929) is inhibited when cultured in vitro in the defined medium described by Eagle (1955) at a concentration of amethopterin of 10^-7 M. Variants of this cell line have been obtained which are resistant to amethopterin at concentrations of 10^-7, 10^-6, and 10^-5 M.

Resistance persists for many months in cells cultured in the absence of drug, although there is an apparent decrease in the degree of resistance with time.

The 10- and 100-fold resistant mutants require the same amounts of folic acid (10^-6 M) or N5-formyl-THFA (10^-8 M) as the normal cell line for optimal growth, and demonstrate toxic effects of vitamin deprivation sooner than the normal cell line.

The same concentration of amethopterin, 3 x 10^-8 M, was required for 50% inhibition of growth of normal cells in the presence of 10^-6 M folic acid or 10^-8 M N₅-formyl-THFA.

Folic acid serves as a weak competitive antagonist to amethopterin. N5-formyl-THFA is strictly competitive with amethopterin, the competitive ratio (inhibitor to metabolite) for 50% inhibition of growth being 3, 30 and 300 for the normal cell, 10- and 100-fold resistant mutants, respectively.

It is concluded that the site of action of amethopterin in these cells is most likely concerned with some step in tetrahydrofolic acid utilization rather than the reduction of folic acid.

Resistance in these cells cannot be explained by better utilization of folio acid, nor by any metabolic alteration leading to independence of folic acid-like compounds. Other possible explanations of the mechanism of resistance are discussed.

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