Four folate analogues, methotrexate, aminopterin, 10-deazaaminopterin, and 10-ethyl-10-deazaaminopterin were assessed for their ability to be metabolized to poly-γ-glutamyl derivatives in three tumor lines which vary in their sensitivity to these agents. Cytotoxicity of the four analogues against the murine L1210 leukemia and the human Manca B cell leukemia, as determined by a 3-h clonogenic assay, showed aminopterin and the two 10-deazaaminopterin compounds to be approximately equivalent for each cell type and were 3- to 10- (L1210) and 7- to 14-fold (Manca) more potent than methotrexate. In murine Sarcoma 180 cells, 10-ethyl-10-deazaaminopterin and aminopterin were similarly potent but were 5- to 10-fold more potent than 10-deazaaminopterin and 40- to 80-fold more potent than methotrexate. These results could be explained in part by the differences in transport properties and substrate activities for polyglutamylation for each analogue in these cell types.

Initial rates of polyglutamate accumulation of the four analogues, which were determined under conditions of comparable rates of drug entry into the three tumor cell lines, were 7- to 18-fold less than drug entry rates. In L1210 and Sarcoma 180 cells, the relative rates of polyglutamylation were in the order aminopterin > 10-ethyl-10-deazaaminopterin > methotrexate > 10-deazaaminopterin. In contrast, the relative rates of polyglutamylation in Manca cells were in the order 10-ethyl-10-deazaaminopterin ≅ aminopterin > 10-deazaaminopterin > methotrexate, suggesting that folylpolyglutamyl synthetase may have varying substrate preferences in different cell types. The maximum relative extents of total polyglutamate accumulation in L1210 cells were 85 to 95% of the total drug at 24 h. In Manca cells, the maximum polyglutamate accumulation was also 85 to 95%, but this was obtained by 6 h. However, in Sarcoma 180 cells, only aminopterin polyglutamates reached a similar maximum percentage of accumulation, while lower relative polyglutamate levels were achieved with the other analogues. Accumulation of individual polyglutamates in each cell line was similar for all analogues except aminopterin. For methotrexate and the two 10-deazaaminopterins, accumulation occurred mainly as the tetraglutamate or as higher polyglutamates. Aminopterin was accumulated mainly as the diglutamate, particularly in Manca cells where 70% of total drug was in the diglutamate form within the first 3 h and remained the predominant form for 24 h. Total intracellular drug continued to accumulate in all cell types during the 24-h period of drug exposure. This appeared to be accounted for by retention of longer-chain length polyglutamates. In L1210 cells incubated for 3 h with drug, efflux of methotrexate and aminopterin polyglutamates was found to be slower than the parent drug and decreased with the addition of polyglutamyl residues. In contrast, the efflux of methotrexate and aminopterin diglutamates from cells preloaded with these derivatives and the parent compounds was the same, and efflux overall was markedly more rapid.


This research was supported in part by Grants CA 08748, CA 18856, and CA 22346 from the National Cancer Institute. Presented in part at the 74th Annual Meeting of the American Association for Cancer Research, 1983 (26).

This content is only available via PDF.