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Accumulation of 5‐methyltetrahydrofolic acid and folylpolyglutamate synthetase expression by mitogen stimulated human lymphocytes
Author(s) -
Fort Daniel W.,
Lark Richard H.,
Smith Angela K.,
MarlingCason Margaret,
Weitman Steven D.,
Shane Barry,
Kamen Barton A.
Publication year - 1993
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1993.tb03133.x
Subject(s) - intracellular , phytohaemagglutinin , biology , biochemistry , stimulation , extracellular , thymidine , folate receptor , receptor , endogeny , microbiology and biotechnology , endocrinology , in vitro , cancer cell , genetics , cancer
Summary. The accumulation of 5‐methyl[ 3 H]tetrahydrofolic acid (5CH 3 [ 3 H]FH 4 ) by phytohaemagglutinin stimulated lymphocytes (PHA‐L) cultured in folate free media was investigated to determine the mechanism of uptake of 5CH 3 FH 4 and the requirement of the cells for this vitamin as assessed by monitoring de novo thymidine synthesis. When grown in 20 n m 5CH 3 [ 3 H]FH 4 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 saturable 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 synthetase (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 ≥5 ) was essentially simultaneous with 5CH 3 [ 3 H]FH 4 transport. Increasing extracellular folate to 2μ m only increased intracellular folate 8‐fold, but the length of the folylpolyglutamates decreased. The increased folate did not increase de novo thymidine synthesis compared to cells grown in physiological folate. We conclude that mitogen stimulation activates the process(es) for folate accumulation, especially FPGS, and that physiological uptake (0·04 pmol/h/10 6 cells) is adequate for meeting the cells' need for the vitamin.