THE EFFECT OF FOLIC-ACID AND OR METHIONINE DEFICIENCY ON DEOXYRIBONUCLEOTIDE POOLS AND CELL-CYCLE DISTRIBUTION IN MITOGEN-STIMULATED RAT LYMPHOCYTES/

Folate deficiency will induce abnormal deoxynucleoside triphosphate (d NTP) metabolism because folate-derived one-carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Und er conditions of methionine deprivation, a functional folate deficienc y for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous met hionine resynthesis from homocysteine. The purpose of the present stud y was to examine the effect of nutritional folate and/or methionine de privation in vitro on intracellular dNTP pools as related to DNA synth esis activity and cell cycle progression. Primary cultures of mitogen- stimulated rat splenic T-cells were incubated in complete RPMI 1640 me dium or in custom-prepared RPMI 1640 medium lacking in folic acid and/ or methionine. Parallel cultures, initiated from the same cell suspens ion, were analysed for deoxyribonucleotide pool levels and for cell pr oliferation. The distribution of cells within the cell cycle was quant ified by dual parameter flow cytometric bromodeoxyuridine/propidium io dide DNA analysis which allows more accurate definition of DNA synthes izing S-phase cells than the traditional DNA-specific staining with pr opidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool a nd an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels we re significantly reduced with single or combined deficiencies of folat e and methionine. These nucleotide pool alterations were associated wi th a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G(2) +M phase of the cell cycle. Folate depri vation in the presence of adequate methionine produced a moderate decr ease in DNA synthesizing cells over the 68 h incubation. However, meth ionine deprivation, in the presence or absence of folate, severely com promised DNA synthesis activity. These results are consistent with the established 'methyl trap' diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide s ynthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the av ailability of folate one-carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression a nd DNA synthesis.