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 5CH₃ [³H₃]H₄ folate at 60 min, showing that within this period most of the [³H₃]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 HC0 [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 LIZIOR 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.