DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.     

Alterations in deoxynucleoside triphosphate metabolism in DNA damaged cells: identification and consequences of poly(ADP-ribose) polymerase dependent and independent processes

Treatment of L1210 cells with increasing concentrations of MNNG produces heterogeneous perturbations of cellular deoxynucleoside triphosphate pools, with the magnitude and direction of the shift depending on the deoxynucleotide and on the concentration and time of exposure of the DNA damaging agent. 5 microM MNNG stimulated an increase in dATP, dCTP and dTTP but dGTP pools remained constant. These increases were not affected by 3-aminobenzamide, indicating that the pool size increases were produced by poly(ADP-ribose) polymerase independent reactions. 30 microM MNNG caused a time dependent decrease in dATP, dGTP, dTTP and dCTP. The dGTP pool was most drastically affected, becoming totally depleted within 3 hours. The fall in all 4 dNTP pools was substantially prevented by 3-aminobenzamide, suggesting that the decrease in dNTPs following DNA damage is mediated by a poly(ADP-ribose) polymerase dependent reaction. Severe depression of dGTP pools consequent to NAD and ATP depletion may provide a metabolic pathway for rapidly stopping DNA synthesis as a consequence of DNA damage and the activation of poly(ADP-ribose) polymerase.     

Rapid changes in deoxynucleoside triphosphate pools in mammalian cells treated with mutagens

In this communication we describe the rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes.     

Unbalanced Deoxyribonucleotide Synthesis Caused by Methotrexate

The effect of methotrexate on the free intracellular pools of thymidylate triphosphate (dTTP) and deoxyadenosine triphosphate (dATP) in normal human phytohaemagglutinin-transformed lymphocytes has been studied. Methotrexate caused a fall in the dTTP pool ranging from 38% to 88% and a rise in the dATP pool ranging from 24% to 185%.A rise in the free intracellular pool of dATP is thought to inhibit both rubonucleotide reduction and polynucleotide ligase, an enzyme concerned in DNA synthesis and repair. The hypothesis is suggested here that folate deficiency per se, as well as a functional folate deficiency induced by methotrexate may cause reduced DNA synthesis, megaloblastic changes, and chromosome abnormalities by producing a rise in the free intracellular pool of dATP as well as by causing a fall in free intracellular dTTP.     

Deoxyribonucleoside triphosphate pools in regenerating rat liver: effect of hydroxyurea and exogenous deoxypyrimidines

Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals.     

Deoxyribonucleoside triphosphate pools of herpes simplex virus infected cells: the influence of selective antiherpes agents and the role of the deaminase pathway.

The effect of 5-methoxymethyl-2'-deoxycytidine (MMdCyd), in combination with tetrahydrodeoxyuridine (H4dUrd) and 5-methoxymethyl-2'-deoxyuridine (MMdUrd) on deoxyribonucleoside triphosphate pools was assessed. The dNTP pool content was almost 5 times as high in herpes simplex virus (HSV) infected VERO cells compared with mock-infected cells. Significant differences in dNTP pool sizes were observed with the different treatments. Treatment of HSV-infected cells with MMdCyd and MMdUrd resulted in a massive expansion of the dTTP pool, whereas pools of dCTP and dGTP were not affected substantially. MMdUrd and MMdCyd produced dATP pools that were 4 and 2.5 times that of the controls, respectively. Treatment with H4dUrd resulted in the dCTP pool increasing 12 times and barely detectable levels of dTTP. MMdCyd in combination with H4dUrd resulted in a marked reduction of the total deoxyribonucleoside triphosphate level. These results indicate that during viral replication the bulk of the thymidine nucleotides are derived from the dCyd/dCMP deaminase de novo pathway.     

Kinetics of UV-induced changes in deoxynucleoside triphosphate pools in Chinese hamster ovary cells and their effect on measurements of DNA synthesis

Measurements of dNTP pools following exposure of Chinese hamster ovary cells to ultraviolet radiation reveals a rapid accumulation of cellular dTTP and a rapid loss of cellular dCTP. Exposure to 3-, 10- or 20 Jm-2 results in a 3-, 4- or 5.4-fold increase in cellular dTTP, respectively, within the first 10 min after exposure. dTTP levels then decrease noticeably, approaching the control value 3 to 5 hr later. In contrast, dCTP levels decrease rapidly within 10 min after exposure, ultimately to 1/10 that observed in the unirradiated control population. Recovery to normal dCTP levels is slow, taking in excess of 12 hr. No change in dATP is observed for 1-2 hr; subsequently, a moderate decrease in dATP levels occurs which is then followed by recovery, beginning 8 hr after irradiation. These results contrast with changes in dNTP pools observed in Chinese hamster V-79 cells exposed to mutagens. Measurements of rates of DNA synthesis by pulse-labeling cells with [3H]thymidine are also apparently affected by UV-induced transient deviations in the endogenous radiospecific activity of the labeled precursor.     

Diverse deoxyribonucleotide profiles in cultured human cells with differential sensitivity to thymidine

Intracellular deoxyribonucleotide pools were examined before and after thymidine treatment in highly sensitive T-lymphoid cells, relatively resistant B-lymphoid cells and moderately sensitive melanoma cells. Among the 4 cell lines studied, proportions of the 4 deoxyribonucleotide pools varied appreciably while ribonucleotide profiles were similar. The ratio of dGTP to dCTP increased with sensitivity to thymidine. Increase in dTTP levels with increasing thymidine concentration was dependent on sensitivity of cells to thymidine and was accompanied by reduction in the dCTP pool. dGTP levels increased as did dTTP levels in all cells, while dATP pool expansion correlated with thymidine sensitivity. The results indicate an additional aspect of purine deoxyribonucleotide involvement in the growth inhibitory effects of thymidine.     

The pool size of deoxyguanosine 5′-triphosphate and deoxycytidine 5′-triphosphate in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 − C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/10⁶ cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied.     

A Review Comparing Deoxyribonucleoside Triphosphate (dNTP) Concentrations in the Mitochondrial and Cytoplasmic Compartments of Normal and Transformed Cells

The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10^?7) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm.     

Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.     

Elevation and hardening of the fertilization membrane in sea urchin eggs : Role of the soluble fertilization product

Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 ? C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/10⁶ cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of adenosine deaminase deficiency

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented ("reversed") to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. "Reversal" restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. "Reversibility" of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.     

Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.     

Deoxyribonucleoside triphosphate and DNA synthesis in synchronized cultures of Chlamydomonas

Pool sizes of dATP, dTTP, dGTP and dCTP were determined during the life cycle of Chlamydomonas using light-dark synchronized cultures. The pools of all four nucleotides were small until the start of the DNA synthesis, when they all increased in close time relationship with the increase in rate of DNA synthesis. The dTTP and dATP pools increased more than 200-fold while the pools of dCTP and dGTP expanded approx. 10 times.     

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.     

Deoxynucleotide pools and DNA synthesis in resting and PHA-stimulated human lymphocytes treated with mutagens.

In resting and PHA-stimulated PBL treated with uv light or MMS we measured the sizes of the dTTP and dATP pools and the variation of ATP content taken as an indicator of cytotoxicity. The effects on DNA synthesis were examined by measuring DNA repair (unscheduled DNA synthesis) in resting PBL and the inhibition of DNA replication in stimulated PBL. While both treatments affected DNA synthesis, only MMS perturbed dNTP pools and decreased the intracellular concentration of ATP. All the effects were more evident in cycling than in resting lymphocytes.     

Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism

Purine and pyrimidine deoxyribonucleoside metabolism was studied in G1 and S phase human thymocytes and compared with that of the more mature T lymphocytes from peripheral blood. Both thymocyte populations have much higher intracellular deoxyribonucleoside triphosphate (dNTP) pools than peripheral blood T lymphocytes. The smallest dNTP pool in S phase thymocytes is dCTP (5.7 pmol/10(6) cells) and the largest is dTTP (48 pmol/10(6) cells), whereas in G1 thymocytes, dATP and dGTP comprise the smallest pools. While both G1 and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. We have studied ribonucleotide reduction and deoxyribonucleoside salvage in S phase thymocytes in the presence of extracellular deoxyribonucleosides. Based on these studies, we propose a model for the interaction of deoxyribonucleoside salvage and ribonucleotide reduction in S phase thymocytes. According to this model, extracellular deoxycytidine at micromolar concentrations is efficiently salvaged by deoxycytidine kinase. However, due to feedback inhibition of deoxycytidine kinase by dCTP, the maximal level of dCTP which can be achieved is limited. The salvage of both deoxyadenosine and deoxyguanosine (up to 10(-4) M) is completely inhibited in the presence of micromolar concentrations of deoxycytidine, whereas the salvage of thymidine is unregulated resulting in large increases in dTTP levels. Moreover, significant amounts of the salvaged deoxycytidine is used for dTTP synthesis resulting in further increase of dTTP pools. The accumulated dTTP inhibits the reduction of UDP and CDP while stimulating GDP reduction and subsequently also ADP reduction. The end result of the proposed model is that S phase thymocytes in the presence of a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthesized primarily by ribonucleotide reduction. Using the proposed model, it is possible to predict the relative intracellular dNTP pools found in fresh S phase thymocytes.     

Deoxyribonucleotide pools as targets for mutagenesis by N-methyl-N-nitrosourea

This paper describes a biological test of the hypothesis that one or more components of the intracellular nucleotide pool represent a significant target for the mutagenic effects of alkylating agents. In other words, we ask whether mutagenesis can occur either through alkylation of susceptible nucleotide residues in DNA, or through alkylation of a free nucleotide, followed by its incorporation into DNA. Our approach is based upon the premise that if a nucleotide pool is a mutagenic target, then transient expansion of that pool should increase the target size and enhance mutagenesis following subsequent treatment with an alkylating target.

Working either with V79 hamster lung fibroblasts or Chinese hamster embryo fibroblasts (CHEF/18), we treated cells for 30 min, under conditions that expanded one or more pools of deoxyribonucleoside triphosphates. This was followed immediately by a 30-min treatment with 0.5 mM N-methyl-N-nitrosourea. After 8 days of additional culture for recovery of cells and expression of mutations, we plated in selective media to determine the abundance of 6-thioguanine-resistant mutants in each culture.

We found that conditions which expand pools of either dATP or dTTP and dGTP stimulate mutagenesis by MNU, with the degree of stimulation varying in different experiments from 2-to 6-fold. Although alternate interpretations can be entertained, the data are consistent with the hypothesis that nucleotide pools represent alkylation targets. A biochemical test of the hypothesus us warranted.

During our studies we made several other noteworthy observations: (1) treatment of V79 cells with mutagen alone does not significantly affect dNTP pools; (2) deoxynucleotide pool perturbations are quite short-lived following transfer of cells to normal medium; (3) deoxyuridine is significantly more effective than thymidine in expanding dTTP pools; (4) deoxyuridine by itself is significantly mutagenic, particularly to CHEF/18 cells.     

Lack of specific correlation of the deoxycytidine triphosphate pool level with rate of DNA synthesis.

The levels of the four deoxyribonucleoside triphosphate pools and the distribution of cells in the various phases of the cell cycle have been examined in Chinese hamster cells as thymidine, present as a regular constituent in the growth medium, was removed in stages. The results indicate that: 1. Duration of the DNA synthetic phase was lengthened when thymidine was removed from the growth medium. 2.Temporally correlated with lengthening of the DNA synthetic phase upon thymidine removal was a 7-fold increase in level of the dCTP pool, reduction in the dGTP pools, and little or no change in dATP pool. 3.Radioactive labeling procedures indicated that expansion of the dCTP pool could be completely accounted for by increased ribonucleotide reductase activity and that the dTTP pool switched from a largely exogenous thymidine source to endogenous dTTP synthesis as the extracellular thymidine concentration was reduced. 4.Deoxyuridine and thymidine were apparently transported by the same system in Chinese hamster cells, while deoxycytidine was transported by a different system. Although deoxycytidine transport was unaffected by thymidine, phosphorylation of intracellular deoxycytidine compounds to the triphosphate level was stimulated by thymidine. Cytidine transport was not significantly affected by thymidine.