Deoxyuridine Monophosphate Research Articles

Comparison of the Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine and 3′-Fluoro-3′-Deoxythymidine in Lymphocytes in the Presence of 5-Fluoro-2′-Deoxyuridine

The mechanism of action of 2′, 3′-dideoxythymidine analogues such as azidothymidine (AZT) and fluorothymidine (FLT) is thought to be chain termination and inhibition of reverse transcriptase by the triphosphate. However, little triphosphate is formed intracellularly relative to monophosphate and diphosphate. Azidothymidine and fluorothymidine are phosphorylated intracellularly by the thymidine salvage pathway and therefore must compete with thymine nucleotides for phosphorylation, which may limit the rate of phosphorylation. We have investigated the degree to which competition with thymidine limits the phosphorylation of azidothymidine and fluorothymidine. In this paper we show that inhibition of thymidylate synthase by 5-fluoro-2′-deoxyuridine monophosphate increases the phosphorylation of azidothymidine and fluorothymidine by reducing the pools of thymine nucleotides and therefore removing the competition. 5-fluoro-2′-deoxyuridine abolished the pools of TTP and dGTP and caused the appearance of dUTP in treated lymphocytes. Three times more triphosphate was formed in cells treated with 5-fluoro-2′-deoxyuridine. Ten times more fluorothymidine triphosphate than azidothymidine triphosphate was formed. The results suggest that while competition with intracellular thymine nucleotides does hinder the phosphorylation of AZT and FLT, the major limiting factor is their ability to act as substrates for the phosphorylating enzymes.

Cell cycle- and differentiation stage-dependent variation of dUTPase activity in higher plant cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a key enzyme in pyrimidine nucleotide metabolism, specifically hydrolyzes deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate and inorganic pyrophosphate. This enzyme activity has been studied in cellular extracts from Allium cepa root meristem cells with two specific aims: (i) to determine how the properties of the plant enzyme compare with those of dUTPase purified from other sources, and (ii) to analyze the relationship between dUTPase activity and cell proliferation and cell differentiation. Plant dUTPase is highly specific for dUTP, with an apparent Km of 6 microM, is highly sensitive to EDTA and it is probably a metalloenzyme. Our results demonstrate the presence of high levels of dUTPase in both resting and proliferating root meristem cells. The enzyme activity appears to be tightly regulated during the cell cycle. dUTPase activity increases at the G1/S boundary, remains high throughout S phase, and shows almost undetectable levels during G1 and G2. We have also found that dUTPase activity in differentiated cells, located in the mature portion of the root, is barely detectable. Altogether our results indicate that dUTPase activity is modulated by the proliferation rate and that this activity progressively decreases as cells initiate their differentiation program.

The Cyclic Dimer of 5-Fluoro-2′-Deoxyuridylic Acid: A Potent Anticancer Agent

Abstract As part of a synthetic program on cyclic oligomers of DNA, the cyclic dimer of 5-fluoro-2′-deoxyuridylic acid (FdUMP) was synthesized. The fully protected dimer 5 was obtained following Catlin and Cramer′s phosphotri-ester strategy. Autocondensation and deprotection then afforded the title compound 9 [cyclo(5FdUp5FdUp)] in excellent yield. In vitro, 9 proved slightly less active than FdUrd in inhibiting the proliferation of various murine and human tumor cells, but, in vivo, 9 was equally effective, and less toxic than 5-FdUrd in inhibiting adenocarcinoma tumor growth in mice.

Interaction of deoxyuridine with fluorouracil and dipyridamole in a human colon cancer cell line

We have reported previously that dipyridamole increases the toxicity of 5-fluorouracil and alters fluorouracil metabolism in HCT 116 cells, producing a selective increase in fluorodeoxyuridine monophosphate (FdUMP) levels by blocking the efflux of fluorodeoxyuridine. Dipyridamole also blocks deoxyuridine efflux and prolongs the intracellular half-life of deoxyuridine monophosphate (dUMP). The significance of the effect of dipyridamole on FdUMP and dUMP levels was explored further. In cell growth experiments, 1–50 μM deoxyuridine enhanced the cytotoxicity of 5 μM fluorouracil in a dose-dependent manner, and ≥10 μM deoxyuridine increased the augmentation of fluorouracil toxicity produced by 0.5 μM dipyridamole. The effect of deoxyuridine on [6- 3H]fluorouracil metabolism was studied. After 4 hr, 25 μM deoxyuridine increased the amount of [ 3H]FdUMP formed 2- to 4-fold relative to that of fluorouracil ± dipyridamole alone. The mechanism by which deoxyuridine increased FdUMP was examined by measuring the distribution of [2′- 3H]deoxyuridine metabolites following exposure of 25 μM deoxyuridine ±5 μM fluorouracil. Tritium appeared in the FdUMP peak at 4 and 24 hr in cells exposed to fluorouracil and deoxyuridine, indicating that [ 3H]deoxyribose was transferred to fluorouracil. A large buildup of [ 3H]dUMP was seen in cells exposed to fluorouracil plus deoxyuridine for 4 and 24 hr compared to exposure to [ 3H]deoxyuridine alone, suggesting that dUMP may also inhibit catabolism of FdUMP. Since the increased FdUMP levels produced by dipyridamole did not appear to correlate with further depletion of thymidine triphosphate pools, the incorporation of [ 3H]fluorouracil metabolites into nucleic acids was monitored by cesium sulfate density centrifugation. Fluorouracil-RNA increased as a function of time (1, 2 and 13 pmol 10 6 cells after 4, 8 and 24 hr), but fluorouracil-DNA was detected only after 24 hr (0.5 pmoI 10 6 cells). Dipyridamole however, did not appear to alter the pattern of incorporation of fluorouracil into either RNA or DNA. Perturbations of endogenous dUMP levels by fluorouracil and dipyridamole were then studied. In cells exposed to fluorouracil alone, dUMP pools were unchanged from control at 2 hr, but they had increased 9-fold by 4hr (3362 pmol 10 6 cells). Simultaneous exposure to fluorouracil and dipyridamole resulted in a 1.5-fold (566 pmol 10 6 cells) and 13.6-fold (5049 pmol 10 6 cells) increase over control dUMP levels after 2 and 4hr respectively. The dUMP pools continued to enlarge through 24 hr. The effect of fluorouracil on DNA fragility was examined. In cells prelabeled with [ 14C]thymidine, there was no evidence of single-strand breaks in high molecular weight DNA after 4 or 24 hr of exposure to fluorouracil alone or with dipyridamole as measured by alkaline elution. In contrast, fluorouracil produced alkaline labile sites in newly synthesized DNA. Alkaline labile sites were also produced by exposure to dipyridamole. Concomitant exposure to FUra with dipyridamole and/or deoxyuridine resulted in a striking increase in the alkaline labile sites in DNA. These results suggest that effects on deoxyuridine metabolism may be important components of the interaction between fluorouracil and dipyridamole.

Comparison of Enzymes of Nucleotide Metabolism in Two Members of the Mycoplasmataceae Family

The presence or absence of some enzymes of nucleotide metabolism has been suggested by other workers as being of possible value as criteria for some genera within the Mollicutes. We assayed subcellular fractions of Mycoplasma mycoides subsp. mycoides strain Y and Ureaplasma urealyticum 960T (T = type strain) for these activities. U. urealyticum 960T had characteristics similar to those noted previously (Williams and Pollack, Int. J. Syst. Bacteriol. 35:227-230, 1985) for some other species in the Mycoplasmataceae in that it (i) does not show a deoxyuridine triphosphate nucleotidohydrolase, (ii) does possess deoxycytidine deaminase, (iii) does not show a specific deoxyuridine monophosphate phosphatase, and (iv) does possess deoxycytidine monophosphate deaminase. M. mycoides differs from these other members of the Mycoplasmataceae in that it (i) does possess a deoxyuridine triphosphate nucleotidohydrolase and (ii) does not show deoxycytidine or cytidine deaminase activity, but it is similar in that it does not show a specific deoxyuridine monophosphate phosphatase and does possess deoxycytidine monophosphate deaminase. Extracts of U. urealyticum were fractionated and assayed for activities of a range of enzymes of nucleotide metabolism. Our results indicate a capacity for salvage pathways of nucleotide synthesis with similarities to those already proposed for M. mycoides and Acholeplasma laidlawii B-PG9.

Excision repair of uracil in higher plant cells: Uracil-DNA glycosylase and sister-chromatid exchanges

Uracil is not a normal constituent of DNA. Under natural conditions, it may appear either by deamination of cytosine residues or by incorporation of deoxyuridine monophosphate (dUMP). Visible light irradiation of BrdUrd-treated cells efficiently leads, under experimental situations, to the formation of dUMP residues in DNA. Plant cells, like other living organisms, can eliminate this potentially harmful base from DNA by an excision repair pathway, uracil-DNA glycosylase being the first enzyme acting during the incision process. Purified plant uracil-DNA glycosylase is a low molecular weight enzyme (27–29.5 kD) that specifically releases uracil present in DNA by splitting off the sugar-base bond. This enzyme is non-competitively inhibited by uracil and 6-aminouracil, but not by thymine, both in vitro and in vivo. However, other structurally related compounds do not show any inhibitory effect. This characteristic poses a number of unaswered questions regarding its mechanism of action. At the chromosome level, dUMP residues appear to be sister-chromatid exchange (SCE)-initiating events. This has been demonstrated for dUMP residue introduced either by visible light exposure of BrdUrd-treated cells or by dUMP mis-incorporation instead of dTMP in cells treated with inhibitors of thymidylate synthetase. The excision repair of uracil in plants appears to be finely regulated in different cell types depending on their proliferation rate and their development stage. Thus, high levels of uracil-DNA glycosylase do not seem to be necessarily associated with DNA replication, since non-proliferating cells, natural constituents of dormant meristems, contain enzyme levels comparable to those found in proliferating tissues, where it is modulated: the higher the cell cycle rate (and the DNA replication rate) the higher the uracil-DNA glycosylase activity. Finally, this excision repair enzyme seems to be turned off as cells enter their differentiated state.

Induction of SOS responses in Escherichia coli by 5-fluorouracil

The inducibility of SOS responses by 5-fluorouracil (5-FU), which has been used as an antitumor drug, was studied in Escherichia coli cells which have different DNA repair capacities for UV lesions. Expression of the umuC gene was apparently induced by 5-FU in the wild-type and uvrA strains but not in lexA and recA strains. The inducibility of the umuC gene by 5-FU, the metabolite of which inhibits thymidylate synthetase, was abolished in cultures containing deoxythymidine monophosphate which is converted from deoxyuridine monophosphate by thymidylate synthetase. These results suggest that 5-FU may exert its SOS inducibility by inhibiting thymidylate synthetase and then disturbing DNA metabolism but not by incorporating 5-FU residues into RNA. Further, 5-FU weakly induced mutations in E. coli.

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2′-deoxyuridine (FdUrd) in combination with 2′-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10 −9 − 5 × 10 −7 M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70–80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10 −4 M. However, at the highest FdUrd dose tested (10 −7 M), 10 −4 M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6- 3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6- 3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2′-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2′-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.

Erythrocyte arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N) as indices of lead exposure.

The activities of three erythrocyte (rbc) enzymes, arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N), were compared in 16 lead workers and 14 age matched controls as correlates of blood lead (PbB) and unextracted zinc protoporphyrin (EP) concentrations. Subjects with PbB of 0.9-2.5 microM (19-52 micrograms/dl) had 6.5 +/- 0.6 IU of P5N activity with uridine monophosphate (UMP) as substrate, significantly less (p less than 0.001) than the 12.0 +/- 0.7 IU activity of controls with PbB 0.3-0.6 microM (6-12 micrograms/dl). The mean activity of rbc dP5N with either deoxyuridine monophosphate (dUMP) or thymidine monophosphate as substrate, and of rbc arginase, did not differentiate the two groups. The correlation coefficients of ln PbB with the selected substrates for P5N and dP5N were: UMP, r = -0.75; dUMP, r = -0.61; TMP, r = -0.23. The correlation coefficient of ln PbB and arginase activity was -0.03. Rbc P5N (UMPase) is a significant correlate of PbB, equivalent to rbc protoporphyrin. HPLC assay of rbc UMPase activity is a sensitive and rapid assay that appears to meet criteria for a reliable clinical laboratory index of blood lead concentrations.

Recombination and amplification of pyrimidine‐rich sequences may be responsible for initiation and progression of the Xq27 fragile site: An hypothesis

A pyrimidine-rich sequence (PRS) of DNA is present as a normal sequence in the q27 band of the human X chromosome. Under conditions of pyrimidine nucleotide triphosphate deprivation during S phase, deoxyuridine monophosphate is misincorporated and has to be excised during G2 by DNA repair mechanisms. When a simple PRS is present on both homologous X chromosomes during oogenesis, PRS may undergo amplification through non-homologous crossing-over to produce the initial lesion of the fragile (X). Carriers of such initial lesions will be unaffected transmitting females or males. When an X chromosome bearing such an initial lesion is itself paired with a homologous X carrying a simple PRS during oogenesis, a much higher rate of non-homologous crossing-over may occur resulting in progression to an even longer stretch of pyrimidine rich DNA in this region; the increased length of PRS through amplification makes the region too long to be repaired during G2 and allows it to be seen as a fragile site in metaphase chromosome preparations. Furthermore, this amplified lesion may interfere with transcription of one or more genes in this region and produce the phenotype of the Martin-Bell syndrome.

Antifolate‐induced misincorporation of deoxyuridine monophosphate into DNA by cells from patients with the fragile X syndrome

The fragile site at Xq27 is expressed in vitro under conditions that lead to decreased intracellular thymidine triphosphate concentration, a condition which has also been shown to promote the misincorporation into DNA of deoxyuridine monophosphate (dUMP) in place of thymidine. We tested for increased whole-cell misincorporation of dUMP as a possible molecular mechanism for the expression of the fragile X abnormality. Neither deoxyuridine triphosphatase nor uracil-DNA-glycosylase, the two enzymes that normally prevent the accumulation of dUMP in DNA, was deficient in fragile X syndrome cells. Misincorporation of dUMP occurred in comparably low levels in both normal and fragile X syndrome lymphoblasts. Although these results provide strong evidence against generalized misincorporation of dUMP in fragile X syndrome cells, a substantial real difference present at Xq27 might not be detected in these studies of whole cells containing the diploid chromosome complement.

Pyrimidine Deoxyribonucleotide Metabolism in Members of the Class Mollicutes

Cell extracts from six Acholeplasma species, six Mycoplasma species, and Spiroplasma floricola 23-6T (T = type strain) were examined for enzyme activities of pyrimidine deoxyribonucleotide metabolism. All of these organisms had thymidine kinase and thymidine phosphorylase activities, and all lacked deoxycytidine triphosphatase activity. The 13 members of the Mollicutes were separated into three groups by the presence or absence of the following four enzyme activities: (i) the adenosine triphosphate-insensitive deoxyuridine triphosphate-specific hydrolyzing deoxyuridine triphosphatase, (ii) a deoxyuridine monophosphate phosphatase, (iii) deoxycytidine deaminase, and (iv) deoxycytidine monophosphate deaminase. Five of the six Acholeplasma species (all Acholeplasma species except Acholeplasma florum L1T) had all four enzymatic activities. The six Mycoplasma species only had the deoxycytidine and deoxycytidine monophosphate deaminase activities. The only two plant isolates studied, A. florum L1T and S. floricola 23-6T, lacked all four enzymatic activities.

Vanoxonin, a new inhibitor of thymidylate synthetase. III. Inhibition of thymidylate synthetase by vanoxonin-vanadium complex.

Quinquevalent vanadium complex with two mol of vanoxonin ligated by the two catechols was shown to be the active structure for inhibition of thymidylate synthetase. The catechol group of vanoxonin as the essential moiety for the inhibition of enzyme was further confirmed by studies of structure-activity relationships using the enzyme obtained from Ehrlich ascites carcinoma cells of mice. Vanoxonin-vanadium complex showed competitive inhibition with respect to deoxyuridylic acid but uncompetitive to 5,10-methylenetetrahydrofolate.

Metabolic fate of (E)-5-(2-bromovinyl)-2'-deoxyuridine in herpes simplex virus- and mock-infected cells.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is a potent antiherpes compound with far better activity against herpes simplex virus type 1 than type 2. To understand the role of drug metabolism in this differential antiviral activity, we examined the metabolic fate of this drug in virus-infected and mock-infected Vero cells by high-pressure liquid chromatography. After 8 h of incubation in which cells were exposed to 10 micrograms of the drug per ml, 63 pmol/10(6) cells of the parent compound was detected in acid-soluble extracts of mock-infected cells. Herpes simplex virus-infected cells, however, incorporated or metabolized, or both, up to 11,310 pmol/10(6) cells. Type 1-infected cells metabolized the drug to the triphosphate where as many as 5,565 pmol/10(6) cells were detected. In contrast, three strains of type 2-infected cells metabolized the drug to the monophosphorylated nucleotide and no further. The amount of drug getting into the cells was virus strain and inoculum dependent. These studies indicate that poor substrate acceptance of (E)-5-(2-bromovinyl)-2'-deoxyuridine monophosphate by herpes simplex virus type 2-specified thymidylate kinase is an important factor in situ in infected cells, preventing anabolism of the parent compound to its active triphosphorylated form. This may account for its type specificity.

Spectroscopic studies of ternary complexes of thymidylate synthetase, deoxyribonucleotides, and folate analogs.

Conformational changes accompanying the formation of binary and tightly bound ternary complexes of thymidylate synthetase and all possible combinations of three folate analogs (N-10-ethyl-quinazoline, folic acid triglutamate, and folic acid) and three deoxyribonucleotides (5-fluoro-2'-deoxyuridylic acid (FdUMP), 2'-deoxyuridylic acid (dUMP), and thymidylic acid (dTMP] were studied by means of ultraviolet difference spectroscopy. The amplitudes of the spectral changes upon ternary complex formation were 2-3-fold greater than those generated by formation of binary enzyme-nucleotide and enzyme-folate analog complexes. Difference spectra of the ternary complexes all showed a major increase in absorbance in the region of 320-340 nm, presumably due to perturbations of the folate analog chromophores, whereas decreases in absorbance occurred over a range of 260-310 nm. N-10-ethyl-quinazoline tended to form the complex with the greatest filtration efficiency on nitrocellulose filters, followed by folic acid triglutamate and folic acid, whereas among the nucleotides, the most stable complexes were formed with FdUMP, followed by dUMP and dTMP. A correlation was observed between the apparent stability of the ternary complex and the magnitude of the absorbance change in its difference spectrum. The formation of the various ternary complexes showed three different categories of rate behavior: 1) very rapid formation of the complex; 2) biphasic formation with a rapid phase and a slow phase requiring up to 90 min for completion; and 3) in the case of the ternary complex formed with enzyme, FdUMP, and folic acid, only a slow phase of binding. The slow formation of the latter complex was accompanied by concomitantly slow changes in the difference spectrum. However, in those cases of biphasic formation of the complexes, almost all of the spectral change occurred rapidly, and very little of it corresponded to the slow phase of complex formation. To accommodate these observations, a model is proposed involving a sequential interaction of the two subunits of thymidylate synthetase.

A homopolymer of the potent antitumor drug 2′-deoxy-5-fluorouridylate

Poly(5-fluoro-2′-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20°C lower than poly(dA) · poly(dT). A triple-stranded helix, poly(dA)·2 poly(dF 5U) was formed only in high salt (2.0 M NaCl).

Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells.

The double-stranded RNAs (I)n X (C)n and (A)n X (dUfl)n (dUfl is 2'-fluoro-2'-deoxyuridylic acid) have been compared as inhibitors of translation in cell-free systems from interferon-treated mouse L cells and from rabbit reticulocytes. In the interferon-treated mouse L-cell system, both double-stranded RNAs stimulated kinase activity, leading to phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha (eIF-2 alpha), but only (1)n X (C)n activated the (2'-5')-oligoadenylate synthetase. Moreover, in this system, (I)n X (C)n, but not (A)n X (dUfl)n, inhibited translation. Both (A)n X (dUfl)n and (I)n X (C)n also activated the rabbit reticulocyte kinase to phosphorylate protein P1 and eIF-2 alpha, but, in contrast to mouse L-cell systems, both (A)n X (dUfl)n and (I)n X (C)n were potent inhibitors of translation in reticulocyte lysates. These results indicate that protein P1 and eIF-2 alpha phosphorylation are not sufficient to cause inhibition of protein synthesis in interferon-treated mouse L-cell extracts. They further suggest that protein synthesis inhibition by (I)n X (C)n in extracts of interferon-treated L cells correlates better with activation of (2'-5')-oligoadenylate synthetase than with activation of the protein P1 and eIF-2 alpha kinase.

Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5- 3H]uridine monophosphate ([ 3H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [ 3H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [ 3H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [ 3H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of ±, l -5,10- methylenetetrahydrofolate is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.

The Effect of Folate Analogues and Vitamin B12 on Provision of Thymine Nucleotides for DNA Synthesis in Megaloblastic Anemia

AbstractThe role of vitamin B12 in the folate dependent biosynthesis of thymine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)–thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, NB-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl–FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl–FH4.

The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.