The Turnover of Folate Coenzymes in Murine Lymphoma Cells

• Published in: The Journal of biological chemistry Vol. 248; no. 17; pp. 5932 - 5936
• Main Authors: Nixon, Peter F., Slutsky, Gerald, Nahas, Aly, Bertino, Joseph R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-09-1973; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Biological Assay; Carbon Isotopes; Cell Line; Chromatography, Ion Exchange; Coenzymes - metabolism; Culture Media; Drug Stability; Folic Acid - analysis; Folic Acid - metabolism; Glutamates - metabolism; Lactobacillus casei; Lymphoma - metabolism; Methionine - metabolism; Methotrexate - pharmacology; Mice; Neoplasms, Experimental - metabolism; Pancreas - enzymology; Peptides - metabolism; Protein Binding; Tetrahydrofolate Dehydrogenase - metabolism; Tetrahydrofolates - metabolism; Thymine Nucleotides - biosynthesis; Thymine Nucleotides - metabolism; Tritium
• DOI: 10.1016/S0021-9258(19)43490-X
• EISSN: 1083-351X
• ISSN: 0021-9258

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.