Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells.
The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.
Baram
J
J
Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892.
Chabner
B A
BA
Drake
J C
JC
Fitzhugh
A L
AL
Sholar
P W
PW
Allegra
C J
CJ
D000217:Acyltransferases / Q000378:metabolism*; D001943:Breast Neoplasms; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D013056:Spectrophotometry, Ultraviolet
C055914:10-formyldihydrofolate; D005492:Folic Acid; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D008727:Methotrexate