Role for cytosolic folate-binding proteins in the compartmentation of endogenous tetrahydrofolates and the 5-formyl tetrahydrofolate-mediated enhancement of 5-fluoro-2'-deoxyuridine antitumor activity in vitro.
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.
Matherly
L H
LH
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
Czajkowski
C A
CA
Muench
S P
SP
Psiakis
J T
JT
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D005467:Floxuridine / Q000494:pharmacology*; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D002955:Leucovorin / Q000378:metabolism; D007939:Leukemia L1210 / Q000378:metabolism* / Q000473:pathology; D013763:Tetrahydrofolates / Q000378:metabolism*
D002352:Carrier Proteins; D013763:Tetrahydrofolates; D005467:Floxuridine; D005492:Folic Acid; D002955:Leucovorin