The Turnover of Folate Coenzymes in Murine Lymphoma Cells

To estimate the turnover of 5-CH3-H4-folate in murine lymphoma cells L1210, L1210R (a methotrexate-resistant subline), and L5178Y, suspensions of whole cells were allowed to concentrate 5-[14C]CH3-[9,3',5'-3H]H4-folate; analysis of cell extracts showed that, for each cell line, 81 to 85% of the total cell [14C]CH3 groups were transferred to nonfolate compounds within 5 min and 82 to 91% at time intervals up to 60 min. The initial transfer of 14C appeared to be into [14C]methionine, but insoluble cell materials were also progressively 14C labeled. Of the total cell 3H, more than 87% remained identified as 5-CH3-[3H3]H4-folate at 60 min, showing that within this period most of the [3H2]H4-folate derived from 5-CH3-[3H3]H4-folate returned to maintain the labeling of the pool of 5-CH3-[3H3]H4-folate. To estimate the flux of folates through the pathway of thymidylate biosynthesis, L1210 and L1210R cells were allowed to concentrate either 5-CH3-[9,3',5'-3H]H4-folate in the presence of methotrexate or 5-HCO-[6-3H]H4-folate. Of total 3H taken up as 5-HCO-[6-3H]H4-folate, 28% appeared to be transferred to thymidylate in 60 min by L1210 cells and 52% by L1210R cells. In methotrexate-treated L1210 cells, 23% of the total 3H taken up as 5-CH3-[3H3]H4-folate was accumulated in 60 min as [3H3]H2-folate, a product of thymidylate biosynthesis. However, in cells of the methotrexate-resistant L1210R line, no [3H3]H2-folate was accumulated by the use of 2 mM methotrexate despite the demonstrated high flux of folates through the pathway of thymidylate biosynthesis. These data show the significance, for methotrexate resistance, of the 11-fold increase of dihydrofolate reductase in L1210R cells.