Hormonal alteration of methotrexate and folate polyglutamate formation in cultured hepatoma cells

Glutamylation of the antifolate methotrexate in H35 hepatoma cells was stimulated by physiologic concentrations of insulin and dexamethasone. At saturating concentrations of the hormone a 2.7-fold stimulation could be obtained with insulin (65 nM, 16-h exposure) and a 1.8-fold stimulation with dexamethasone (100 nM, 16-h exposure). The increases in glutamylation caused by the hormones were not additive, and both were inhibited by actinomycin D and cycloheximide. N6,O2'-dibutyryl cAMP and theophylline caused a modest reduction of glutamylation in control and dexamethasone-treated cultures, but repressed the stimulation caused by insulin by approximately one-third. Enhancement of synthesis by dexamethasone and insulin was associated with increases in the tri-, tetra-, and pentaglutamate derivatives of methotrexate, with little change in intracellular methotrexate and methotrexate diglutamate. When the conversion of folinic acid into the folylpolyglutamate pool was examined in folate-depleted H35 cells, insulin and dexamethasone had similar effects. The results suggest that these hormones play a role in the glutamylation of the folate coenzymes in a liver-derived transformed cell line in culture and that these effects are also reflected in the interaction of the cells with antifolates such as methotrexate.