ACCUMULATION OF 5-METHYLTETRAHYDROFOLIC ACID AND FOLYLPOLYGLUTAMATE SYNTHETASE EXPRESSION BY MITOGEN-STIMULATED HUMAN-LYMPHOCYTES

The accumulation of 5-methyl[H-3]tetrahydrofolic acid (5CH3[H-3]FH4) b y phytohaemagglutinin stimulated lymphocytes (PHA-L) cultured in folat e free media was investigated to determine the mechanism of uptake of 5CH3FH4 and the requirement of the cells for this vitamin as assessed by monitoring de novo thymidine synthesis. When grown in 20 nm 5CH3[H- 3]FH4 PHA-L accumulate radiolabel at a rate of 0.04 pmol/h/10(6) cells . This doubles the endogenous folate pool of unstimulated cells (0.6+/ -0.16 pmol/10(6) cells) in about 15 h. Uptake proceeded via a saturabl e process, independent of a high affinity folate receptor as assessed by ligand binding and by Northern and Western blot analysis. However, transport was blocked by probenecid, which is consistent with an anion carrier mechanism. Unstimulated cells lacked folylpolyglutamate synth etase (FPGS) activity and did not express significant amounts of FPGS mRNA. After 48 h of mitogen stimulation there was a 4-10-fold increase in FPGS mRNA and folylpolyglutamate formation (Glu(greater-than-or-eq ual-to 5)) was essentially simultaneous with 5CH3[H-3]FH4 transport. I ncreasing extracellular folate to 2 muM only increased intracellular f olate 8-fold, but the length of the folylpolyglutamates decreased. The increased folate did not increase de novo thymidine synthesis compare d to cells grown in physiological folate. We conclude that mitogen sti mulation activates the process(es) for folate accumulation, especially FPGS, and that physiological uptake (0.04 pmol/h/10(6) CellS) is adeq uate for meeting the cells' need for the vitamin.