Thymidylate synthesis in a folate deprived cell line

Abstract

Abnormalities of the de novo and salvage pathways of thymidylate synthesis have been investigated in a folate deprived lymphoblastoid cell line. Impaired DNA synthesis was observed, with an increased percentage of cells in S and G2 phase, whereas the mitotic index was decreased. Thymidylate synthesis along the salvage pathway was markedly increased, with a higher activity of thymidine kinase and higher uptake of 3H-thymidine (3H-TdR). The same abnormalities were observed when cells were treated with 5-fluorodeoxyuridine or methotrexate. The de novo pathway was slightly modified with a nearly normal incorporation of 3H-6 deoxyuridine (3H-UdR) and a moderate decrease of thymidylate synthetase activity; in contrast, the uptake of 3H-dU was markedly inhibited in drug-treated cells. Preincubation with cold deoxyuridine (10(-4) M) did not suppress the uptake of 3H-TdR as efficiently as in control cells; with increasing concentration of dU to 10(-2) M, this suppressive effect became almost complete. This high concentration of cold dU exerted a competitive inhibition on thymidine kinase. The deoxythymidine triphosphate (dTTP) pool was increased in deficient cells and it was only slightly increased by addition of cold dU (10(-4) M) in the culture medium whereas a substantial expansion of this pool was observed in control cells treated under the same conditions. These data do not necessarily exclude a defect of thymidylate synthesis along the de novo pathway in the folate deficient cells. the normal incorporation of 3H-dU could be explained by a decreased isotope dilution due to a reduced deoxyuridine monophosphate (dUMP) pool. This pool could be decreased by feedback inhibition by dTTP on some enzymatic activities, mainly deoxycytidylate deaminase. The enlarged dTTP pool which probably derives mainly from the salvage pathway could be poorly functional for DNA replication according to the model of compartmentation of DNA precursors.