Inhibition Of Ribonucleoside Diphosphate Reductase Research Articles

Selective inhibition of ribonucleotide reductase by the monofunctional alkylating agent 5(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954)

The monofunctional alkylating agent 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) is a potent and selective inhibitor of the growth of the Walker carcinoma ( ld 50 0.002 μg ml ). Growth inhibition by CB 1954 is accompanied by a rapid inhibition of DNA synthesis with little effect on RNA and protein biosynthesis. Interference with the biosynthesis of DNA by CB 1954 has been shown to be due to inhibition of ribonucleoside diphosphate reductase. The order of effectiveness of inhibition of the enzyme by CB 1954 in four cell lines parallels the tumour growth inhibitory activity. Furthermore, analogues of CB 1954 with methyl substitution on the amide group were less effective inhibitors of the growth of the Walker carcinoma and also less potent inhibitors of ribonucleotide reductase. Treatment of Walker cells with CB 1954 caused a rapid inhibition of enzyme activity and a corresponding fall in the levels of deoxyribonucleoside triphosphates which corresponded with the effect of the drug on DNA synthesis. A human bladder carcinoma has been shown to be particularly susceptible to growth inhibition by CB 1954. Measurement of the effect of CB 1954 on ribonucleotide reductase from biopsy specimens of human tumours could provide a basis for patient selection for therapy by this agent.

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells.

Ornithine decarboxylase activity increases at least 4-5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G(1) and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G(1)-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.

Mutagenicity of thymidine to cultured Chinese hamster cells.

THYMIDINE starvation is known to be both toxic and mutagenic to various prokaryotes, including Escherichia coli1,2, Bacillus subtilis3 and bacteriophage T4 (refs 4–6). Base excess, as well as base starvation, can also be mutagenic. For instance, Bernstein et al.5 showed that a superabundance of exogenous thymidine (4 mM) produced an increased reversion rate for some T4 amber mutants. In mammalian cells a superabundance of exogenous thymidine is toxic, perhaps by inhibiting DNA synthesis7–9. The mechanism for these effects may be related to the observations that dTTP inhibits the enzymatic reduction of CDP to dCDP by ribonucleoside diphosphate reductase10 and also that thymidine reduces the dCTP pools of Chinese hamster cells to 90% of normal11. We report here that a superabundance of exogenous thymidine is both toxic and highly mutagenic to V-79 Chinese hamster cells but that hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, is toxic but not mutagenic.

Studies on the selective toxicity of guanazole in mice

Guanazole ( 3,5-diamino- 1,2,4-triazole (GZ), an antileukemic agent effective against acute myelocytic leukemia, seems to exert its action through inhibition of ribonucleoside diphosphate reductase. Using recently developed technique for drug infusion in unrestricted mice, 5–30 mg/ml guanazole was infused at a rate of 0.2–0.3 ml/hr in normal mice and mice bearing leukemia L 1210 or a subline resistant to guanazole (L1210/GZ). Incorporation of 14C -uridine (UR) into DNA was considerably reduced in spleen, thymus and bone marrow; incorporation of 3H -thymidine was reduced to a much greater extent in these tissues. The inhibition of precursor incorporation was dependent on the concentration of guanazole used and was somewhat correlated to the intracellular drug concentration in different tissues. This inhibition was in the following order: L 1210 spleen > normal spleen > L1210/GZ spleen. Analysis of free intracellular ribonucleotide pools indicated the presence of a high IMP and a low ATP pool in normal spleen, and a moderate IMP and a high ATP pool in spleen infiltrated by cells from either leukemia; this profile resembled that observed in intraperitoneally growing L 1210 cells. Guanazole infusion resulted in the reversion of pools profile from that characteristic of L 1210 spleen to that of normal spleens. The morphological form and microscopic characteristics of L 1210 spleen from mice treated with GZ also resemble those of spleen from non-leukemic mice. Study of in vitro uptake of guanazole in leukemia L 1210, indicated that the transport occurred by a passive diffusion process. In the subline, L1210/GZ, the initial rate of uptake was 1/3rd to 1/4th of that in the parent line. This suggested that a generalized change in membrane architecture is likely to have accurred in L1210/GZ. The results of this study are consistent with the idea that in different tissues in mice, the mechanism of action of guanazole is dependent upon inhibition of ribonucleotide reductase, whereas its selectivity of action is dependent upon differences in uptake.

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.

Adaptation of the cells of Escherichia coli to the presence of hydroxyurea increases the level of inorganic pyrophosphatase activity

Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, completely arrested the net synthesis of DNA for 3–4 h, when it was added in 30 mM concentration to growing cultures of Escherichia coli K12. Thereafter the net synthesis of DNA started again, although slowly, and simultaneously with it the formation of inorganic pyrophosphatase activity was stimulated leading to a 2-fold increase in the specific activity of the enzyme in 2–3 h. Subsequently cell division began again. In this way a new steady state, stable in the presence of hydroxyurea, was reached. This new state was characterized by the high specific activity of inorganic pyrophosphatase, a small but constant amount of DNA/cell mass (1/4 of the normal value), and large elongated cells. All these changes were slowly reversed during 5–6 h, when the cells were transferred into a drug-free medium. The activity of isoleucyl-tRNA synthetase, assayed as a control, did not change significantly in the presence of hydroxyurea. Hydroxyurea had no effect on the activity of inorganic pyrophosphatase in vitro.

Potential antitumor agents. 6. Possible irreversible inhibitors of ribonucleoside diphosphate reductase.

Potential antitumor agents. 6. Possible irreversible inhibitors of ribonucleoside diphosphate reductase.

Mechanism of inhibition of ribonucleoside diphosphate reductase by ga-( n)-heterocyclic aldehyde thiosemicarbazones

Methyl and benzo derivatives of 2-formylpyridine thiosemicarbazone were shown to be potent inhibitors of the ribonucleoside diphosphate reductase of the Novikoff hepatoma. Blockade of enzyme activity correlated with the ability of the various agents to retard the incorporation of 3H-thymidine into DNA of Sarcoma 180 ascites cells in vitro. Structure-activity relationships suggested that position six of 2-formylpyridine thiosemicarbazone and position three of 1-formylisoquinoline thiosemicarbazone are comparable with respect to orientation at the inhibitor binding site and that the enzyme has little bulk tolerance at this locus.

Inhibition of ribonucleoside diphosphate reductase by 1-formylisoquinoline thiosemicarbazone and related compounds.

Inhibition of ribonucleoside diphosphate reductase by 1-formylisoquinoline thiosemicarbazone and related compounds.