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 $\text{±,}\text{l}\text{-5,10-}\text{methylenetetrahydrofolate}$ 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.     

Repair replication induced by 3H incorporated into HeLa DNA

On the average, one disintegration of ³H within HeLa DNA induces the same amount of repair replication as does 0.34 rad of externally administered 300 kV X-radiation.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.     

DNA lesions,chromosomal aberrations and G2 delay in CHO cells cultured in medium containing bromo- or chloro-deoxyuridine

Experiments have been performed to investigate whether BrdUrd- and CldUrd-substituted DNA contains lesions causing a delay in cell-cycle progression and induction of chromosomal aberrations. The presence of lesions has been determined directly by alkaline sucrose gradient and nucleoid sedimentation analysis and indirectly by screening for induced chromosomal aberrations. The influence of inhibitors of DNA repair (caffeine and 3-aminobenzamide) or DNA synthesis (hydroxyurea) on the frequencies of such aberrations has been estimated. It is found that BrdUrd and CldUrd are cytotoxic when present in DNA. No randomly located DNA breaks could be detected under neutral conditions, but BrdUrd-substituted DNA was found to contain numerous alkali-labile sites. CldUrd at high concentrations causes G₂ delay, similar to the action of known DNA-damaging agents. The extent of delay depends on the pattern of incorporation of the analogue, i.e., incorporation for two cell cycles causes the longest delay, growth for 12 h in CldUrd followed by 12 h in dThd-containing medium causes a lesser delay and the delay is not significant when the cells are incubated in the analogue for only 12 h prior to fixation. Numerous chromatid type aberrations are present in cells incubated at the highest CldUrd concentration, and their induction follows the pattern of induction of G₂ delay, indicating the sharing of a common lesion. Caffeine, 3-aminobenzamide and hydroxyurea increase the number of chromosomal aberrations when added 2 h before fixation. The significance of these results is discussed.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

Inhibition of DNA synthesis and cell death

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytotoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.     

Evidence for the involvement of cytochrome P-450-dependent monooxygenase(s) in the formation of genotoxic metabolites from N-hydroxyurea

Hydroxyurea induces DNA repair replication in the cytochrome P-450-containing C2Rev7 rat hepatoma cell line. Repair is severalfold increased by pretreatment of the cells with dexamethasone, which induces cytochrome P-450-dependent monooxygenase activities in these cells. In the dedifferentiated hepatoma line H5, which strongly expresses cytochrome P-448 but no cytochrome P-450-dependent enzyme activities, hydroxyurea is not genotoxic. The results support the notion that the formation of genotoxic metabolites from hydroxyurea is mediated by a cytochrome P-450-dependent enzyme.