The essentiality of folate for the maintenance of deoxynucleotide precursor pools, DNA synthesis, and cell cycle progression in PHA-stimulated lymphocytes.

The fidelity and progression of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in DNA replication and repair. Because folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of this study was to determine the effect of folate deprivation on deoxynucleotide pool levels and cell cycle progression. Primary cultures of phytohemagglutin (PHA)-stimulated splenocytes were used as the cellular model. T-cells and macrophages were purified from spleen cell suspensions obtained from F344 rats and recombined in culture. The cells were harvested after a 66-hr incubation with PHA and analyzed for nucleotide levels by reverse-phase HPLC with diode array detection. The proportion of cells in the different phases of the cell cycle was determined by bivariate flow cytometric measurement of bromodeoxyuridine (BrdU) incorporation and DNA content (propidium iodide staining). PHA-stimulated T-cells cultured in medium lacking folate and methionine manifested significant decreases in the deoxynucleotides dCTP, dTMP, dGTP, and dATP relative to cells cultured in complete medium. The reduction in dNTP pools was associated with a decrease in the corresponding ribonucleotide pools. Flow cytometric analysis revealed a 2-fold increase in S and G2/mitosis (G2/M) DNA content in PHA-stimulated cells cultured in the medium lacking folate and methionine, which suggests a delay in cell cycle progression. These alterations in DNA content were accompanied by a 5-fold decrease in BrdU incorporation relative to PHA-stimulated cells cultured in complete medium.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
The synthesis and turnover of deoxynucleoside triphosphate (dNTP) pools are tightly coupled to DNA synthesis (1). Because folate-derived one-carbon groups are essential for the de novo synthesis of purines and pyrimidines, it was of interest to determine the effect of folate deprivation on nucleotide pools, DNA synthesis, and cell cycle progression. In addition to folate, methionine was omitted from the medium to increase the intracellular folate requirement and further stress folate availability for dNTP biosynthesis (2,3). The irreversible diversion of 5,10-methylene tetrahydrofolate to 5-methyl-tetrahydrofolate for the regeneration of methionine further compromises folate availability for de novo nucleotide synthesis (Fig. 1). Since dNTPs are the immediate precursors for the polymerases involved in DNA replication and repair, the fidelity of DNA synthesis is critically dependent on the correct balance and availability of deoxynucleotides (4,5). Several studies have shown that dNTP imbalance in vitro induced by antifolate drugs will promote certain genetic (and cancer-associated) lesions including folate-sensitive fragile site expression (6,7), DNA strand breakage (8), errorprone DNA repair (9), and mutagenesis (10) tions of intracellular folate and methionine deficiency by omitting these essential nutrients in the medium of cultured splenocytes. Lymphocytes cultured in vitro can be stimulated to proliferate via mitogen exposure, and they provide a convenient model for the study of aberrant DNA metabolism and cell cycle progression under conditions of folate deprivation. The results of this study indicated that folate/methionine deprivation in phytohemagglutinin (PHA)-stimulated T-cells is associated with a decrease in intracellular nucleotide pools, an arrest in DNA synthesis, and a delay in cell cycle progression. These alterations did not occur when folate and methionine were added to the deficient medium.

Spleen Cell Suspensions
Spleens were aseptically removed and single-cell suspensions prepared by gentle teasing with forceps and aspirations through a 21-gauge needle in Medium 199 (Gibco, Grand Island, NY). The suspension was processed for purification of macrophage and T-cell populations. Macrophages were isolated by adherence to petri dishes for 60 min. at 37°C in a humidified CO2 incubator. The nonadherent cells were decanted and further enriched for T-cells by elution through nylonwool columns as described by Julius et al. (11). The plastic adherent macrophages were removed by vigorous washing with Ca2+-Mg2+-free Hank's balanced salt solution. The viability of both the macrophage and Tcell populations was >95% as determined by Trypan blue exclusion. Purifled populations of macrophages and T-cells were recombined in a 1:10 proportion (5 x 106 macrophages: 5 x 107 T-cells) in duplicate 25-mL flasks and cultured in a total volume of 10 mL Medium 199 (M199) plus 5% fetal bovine serum (Hyclone Laboratories, Logan, UT). Aliquots of cells from the same cell suspension were cultured in duplicate in one of three different media: complete M199, deficient M199 lacking in folate and methionine, or deficient M199 supplemented with 1.0 mg/L folic acid and 15 mg/L methionine. Phytohemagglutinin (Sigma Chemicals, St. Louis, MO) was added at 4 ,g/mL as a proliferative stimulus. Cells cultured in complete M199 without PHA served as control. After 66 hr, cells were harvested for HPLC and flow cytometric analysis.

Cell Extraction for HPLC Analysis
Cells were transferred to 15-mL tubes and centrifuged at 200 g for 5 min. at 4°C. The supernatant 174 BeR -DEOXYNUCLEOTIDE POOL IMBALANCE AND CELL CYCLE PROGRESSION was removed, and the cell pellet was suspended in 0.5 mL ice-cold 0.6 M trichloroacetic acid (TCA), vortexed vigorously, and kept on ice for 20 min. After centrifugation, the acidic supernatant was transferred to a microcentrifuge tube containing 0.55 mL ice-cold freon-trioctylamine as previously described (12). The mixture was vortexed for 15 sec, and after centrifugation at 4°C, the lower phase was carefully removed by aspiration, leaving the aqueous solution of nucleotides. Samples were lyophylized and subsequently stored at -70°C. Just before analysis, cell extracts were resuspended in 100 gL of 0.2 M ammonium phosphate, pH 5.35, and filtered through a 0.4-,um microfuge filter unit. The injection volume was 20 ,uL.

HPLC Chromatographic Conditions
HPLC analyses were performed on a Beckman System Gold HPLC system consisting of a programmable solvent module (Model 126) and a diode array detector (Model 168) with an Econosphere C-18 reverse-phase column (5 ,um, 300 x 4.6 mm, Alltech Associates). Absorbance was monitored at 260 nm using diode array detection. Two buffers were used: buffer A consisted of 0.2 M (NH4)H2PO4 in 1.0 M KC1, pH 5.35, and buffer B was made of 0.2 M (NH4)H2PO4 with 1.25 M KCl and 10% methanol, pH 5.0. Buffers were prepared fresh daily in double-distilled water, filtered (0.2-,um filter, Alltech Associates) and degassed by helium purging before use. Samples were eluted isocratically at a flow rate of 0.8 mL/min with 100% buffer A for 8 min. At t = 8 min, a 15-min linear gradient to 80% buffer B was initiated. At t = 35 min, the system returned to 100% buffer A (over 0.2 min) to begin equilibrating for the next sample. A 15-min reequilibration period was required between samples. The identity of dNTP peaks in cell extracts was verified by comparing retention times with dNTP standards and by examining the increase in peak area after quantitative dNTP addition. Peak purity was confirmed by diode array detection. Quantitation of pool sizes was accomplished using individual calibration curves for each nucleotide and Beckman System Gold Software.

Bivariate Flow Cytometric Analysis of BrdU Incorporation and DNA Content
Cultured cells were pulse labeled with 10 ,uM bromodeoxyuridine (BrdU) for 60 min at 37°C. Those cells actively synthesizing DNA during this interval will incorporate BrdU into their DNA. After BrdU exposure, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU monoclonal antibody and propidium iodide exactly as described by the vendor (Becton-Dickinson, San Jose, CA). Control samples were similarly processed but without prior exposure to BrdU. Flow cytometric analysis of individual nuclei (10,000/sample) was conducted using a FACScan flow cytometer (Becton-Dickinson) equipped with an argon laser tuned to 488 nm. List mode data were converted to a bivariate display of FL1 versus FL2 Fluorescence (BrdU-FITC versus propidium iodide). The percentage of cells in GI, S, and G2/M phases of the cell cycle (propidium iodide) and those actively incorporating BrdU were calculated using the Lysys II program from Becton-Dickinson. The macrophage population was excluded from analysis by gating on the DNA histogram.

Results
Effect of Folate Availability on Deoxynucleotide Pools in PHA-Stimulated T-Cells Figure 2 shows the HPLC profiles of nucleotide pools obtained from cells cultured in a) complete M199 without PHA, b) complete M199 plus PHA, c) deficient M199 (lacking in folate and methionine) plus PHA, and d) deficient M199 (supplemented with folate and methionine) plus PHA. Quantitative analysis of pool sizes expressed as pmole/106 cells is presented in Table   1. Relative to PHA-stimulated cells cultured in complete M199, the ribonucleotides CTP, UTP, GTP, and ATP and the pteridine ribonucleotide NAD were consistently reduced in the cells cultured in deficient M199 lacking in folate/methionine. Similarly, the deoxyribonucleotides dCTP, dUTP, dGTP, dTMP, and dATP were found to be reduced in cells cultured in the deficient medium. In contrast, nucleotide levels of cells cultured in the deficient M199 supplemented with folate and methionine were found to exceed those observed  Table   2. In the cells cultured in complete M199, PHA stimulation resulted in a marked increase in BrdU incorporation as expected (Fig. 3 A,B). However, BrdU incorporation in PHA-stimulated cells cultured in the deficient medium was reduced 5-fold to that equivalent to cells cultured without PHA in the complete medium ( Fig. 3A,C). The proportion of PHA-stimulated cells in S and G2/M in the deficient medium was increased 2fold compared to PHA-stimulated cells cultured in the complete medium and may reflect a delay in cell cycle progression (Fig. 3B,C). Supplementation of the deficient medium to control levels of folate and methionine allowed the cells to progress normally through the cell cycle with kinetics comparable to cells cultured in complete M199 (Fig. 3B,D).

Discussion
Agents or conditions that alter the balance of intracellular deoxynucleotides have been previously shown to alter the rate of DNA synthesis and cell-cycle progression. For example, lymphocytes exposed to antifo-late drugs such as 5-fluorouracil or methotrexate exhibit deoxynucleotide pool imbalance and arrest of DNA synthesis (8,13). Hydroxyurea suppresses replicative DNA synthesis by inhibiting ribonucleotide reductase and depleting dNTP pools (14,15). DNA replication fork movement has been shown to be   reduced in human megaloblastic lymphocytes (16) that also exhibit dNTP pool imbalance. In other studies, alterations in dNTP pools have been associated with DNA strand breakage (8), mutagenesis (10,17), and error-prone DNA repair (18,19). In the present study, the effect of folate and methionine deprivation on nucleotide and deoxynucleotide pools and cell-cycle kinetics was evaluated in primary cultures of PHAstimulated T-cells. The observed decrease in T-cell nucleotide and deoxynucleotide derivatives of adenine, guanine, uridine, and cytidine under conditions of folate deficiency would suggest a decrease in folatedependent de novo nucleotide synthesis and/or an increase in nucleotide degradation.
The decrease in nucleotide pool levels in T-cells cultured under folate-deficient conditions was associated with a delay in cell-cycle progression and an apparent arrest in DNA synthesis. Taken together, the data presented support the hypothesis that alterations in dNTP pool size may directly or indirectly affect the rate of DNA synthesis and cell-cycle progression.