The characteristics and consequences of folate depletion in hepatoma cells in vitro by inhibition of dihydrofolate reductase

Abstract Growth of rat hepatoma cells in subtoxic concentrations of the DHFR inhibitor metoprine caused a marked time and concentration dependent reduction in cellular folates. As much as 75% total cellular folates can be lost without impairing growth. Increasing the concentration of metoprine into a range that causes inhibition of growth results in no further reduction in cellular folates. This effect is presumably mediated through inhibition of DHFR and several mechanisms are discussed which may account for these results. Cells grown in medium in which the concentration of folate is changed from 4 μ m to 20 n m had intracellular folate levels that were reduced 85%. This is nearly the same reduction caused by treating cells grown in normal medium (4 μ m folate) with continuous, subtoxic levels of metoprine. The reduction in cellular folates caused by growth in n m folic acid caused enhanced growth inhibitory activity of several antifolates. On a concentration basis metoprine was 12-fold more active under these conditions, PDDF was 37-fold more active and DDATHF was 44-fold more active. The reason for the enhanced sensitivity to PDDF and DDATHF may also be analogous to the reason for their synergism with the low concentration of metoprine and trimetrexate (12).