A major portion of the intracellular folates in L1210 cells grown in (6R,S)-5-formyltetrahydrofolate (
leucovorin) was bound to cytosolic
proteins when
cell extracts were fractionated by rapid gel filtration or adsorption with
activated charcoal. Only low levels of intracellular folates were associated with mitochondria (less than 5%).
Protein-bound folates comprised 37-100% of the cytosolic cofactors following growth in 2-600 nM
5-formyltetrahydrofolate. Total intracellular folates increased in proportion to the changes in media
folate concentration; however, binding was saturable. The maximum level of
protein-bound folates in L1210 cells was 66 pmol/mg
protein.
Protein-bound folates were also detected in HT29 human
colon adenocarcinoma cells grown in
5-formyltetrahydrofolate (maximum, 11 pmol/mg
protein). For both lines,
folate binding was specific for the
tetrahydrofolate and
5,10-methylenetetrahydrofolate pool, and, to a lesser extent,
5-methyltetrahydrofolate. Extremely low levels of
protein-bound 5-formyl-, 10-formyl-, and 5,10-methenyltetrahydrofolates were measured, even though considerable amounts were detected intracellularly. Pentaglutamyl folates were the predominant cofactor forms in L1210 cells; conversely, the tetraglutamates were the most abundant
protein-bound
folate derivatives. Increasing media concentrations of
5-formyltetrahydrofolate potentiated
5-fluoro-2'-deoxyuridine cytotoxicity. For L1210 cells, essentially all of the intracellular
tetrahydrofolate and
5,10-methylenetetrahydrofolate fraction was
protein bound over the concentration range of
5-formyltetrahydrofolate which maximally augmented fluoropyrimidine cytotoxicity. The relative changes in the 50% inhibitory concentrations for
fluorodeoxyuridine directly approximated the increases in the levels of
protein-bound
tetrahydrofolates in L1210 cells. There was no direct relationship between the levels of unbound folates and
fluorodeoxyuridine cytotoxicity. Similar results were obtained with HT29 cells. The major
folate-
binding protein in L1210 cells eluted during Sephacryl S-300 chromatography with a molecular weight of approximately 200,000; a small amount of a higher molecular weight
folate-
binding protein (Mr 450,000) was also detected. These findings support the concept of a compartmentation of endogenous folates involving specific binding to cytosolic
proteins. These associations may regulate reduced
folate availability for metabolic processes, and also mediate utilization of
5,10-methylenetetrahydrofolate for ternary complex formation with
thymidylate synthase in cells treated with fluoropyrimidines. In this fashion, the levels of
protein-bound
tetrahydrofolates could represent an additional, previously unrecognized, determinant of fluoropyrimidine pharmacological activity toward mammalian cells.