The basis for the proliferation-dependent cytotoxicity of
methotrexate has been investigated in mice bearing the L5178Y
ascites leukemia.
Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing
ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase
tumor cells accumulated 8-fold higher levels of
methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing
tumor cells were observed in the cellular levels of unmetabolized
methotrexate. Intestinal mucosa and bone marrow from non-
tumor-bearing mice resembled slowly growing
tumor cells and had markedly lower levels of
methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of
methotrexate polyglutamates in the logarithmically growing
tumor cells was consistent with an increased synthesis of
methotrexate polyglutamates in these cells. The enhanced
methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular
methotrexate transport, transmembrane efflux of
methotrexate, or hydrolysis of
methotrexate polyglutamates.
Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells.
Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased
methotrexate polyglutamate accumulation and
thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of
methotrexate for
tumors with a high growth fraction is a consequence of the rapid rates of both cellular
methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by
thymidylate synthase.