Nucleotide Pool Imbalance Research Articles

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.

Imbalance of total cellular nucleotide pools and mechanism of the colchicine-induced cell activation.

Treatment with colchicine or vinblastine, both inhibitors of microtubule assembly, renders quiescent 3T3 cells in an "activated state" as evidenced by induction of DNA synthesis and other criteria. Microtubule disassembly caused by colchicine or vinblastine brings about a dramatic expansion of total cellular UTP pools with a concomitant diminution in total cellular ATP pools, thus resulting in a marked imbalance in total cellular nucleotide pools. Colchicine and vinblastine also stimulate total cellular RNA synthesis without enhancing uridine phosphorylation, suggesting that these drugs affect the G1 phase of the cell cycle at a point beyond the enhancement of uridine phosphorylation that usually accompanies mitogenic stimulation of quiescent mammalian cells. The markedly expanded cellular UTP pools appear to be necessary for initiation of the colchicine-stimulated DNA synthesis because decreasing cellular UTP pools by addition of D-glucosamine results in a selective inhibition of DNA synthesis in the colchicine-stimulated, but not control, cells. Furthermore, D-glucosamine exerts its inhibitory effect only when it is present in the cultures within the first 14 hr after colchicine treatment. When added at 21 hr, D-glucosamine still decreases cellular UTP pools, but it is no longer inhibitory for DNA synthesis, which commences 14-16 hr after colchicine stimulation. Taxol, an antitumor drug, prevents microtubule disassembly and also blocks such events as expansion of total cellular UTP pools and stimulation of RNA and DNA synthesis, indicating that microtubule depolymerization acts as a primary event initiating the process of cell activation induced by colchicine.

Orotic Acid, a New Promoter for Experimental Liver Carcinogenesis

Male Fischer 344 rats initiated with 1,2-dimethylhydrazine 2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to a diet containing 1% orotic acid for 13 months developed a 100% incidence of hepatocellular carcinoma. The creation of nucleotide pool imbalances by dietary orotic acid, for e.g., an increase in uridine nucleotides and a decrease in adenine nucleotides, was considered as a possible mechanism for the promotional effect of orotic acid on liver carcinogenesis. The significance of this hypothesis is that altered nucleotide pools affect both genomic as well as membrane organization. Consistent with this hypothesis is our finding that feeding rats with a diet containing 1% orotic acid for 10 weeks resulted in a liver DNA damage as monitored by its slower sedimentation in alkaline sucrose gradients compared to the corresponding controls. To assess the general applicability of this hypothesis, nucleotide pool imbalances were created by using methods other than feeding orotic acid and their effect on the incidence of gamma-glutamyltransferase positive foci in carcinogen initiated rats was determined. The results obtained indicated that rats initiated with 1,2-dimethylhydrazine.2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to diet deficient in arginine, a regimen that causes an increased synthesis and excretion of orotic acid, or were fed diets containing 1% thymidine or 1% thymine developed greater number of gamma-glutamyltransferase positive foci compared to the corresponding controls fed the basal diets. These results were interpreted to indicate that orotic acid exerts its promotional effect probably by creating an imbalance in nucleotide pools. One of the mechanisms by which an imbalance of nucleotide pools influences the pathogenesis of the carcinogenic process may be by inducing perturbations in the DNA.

Variation of mutation and recombination frequencies over a range of thymidylate concentrations in a diploid thymidylate auxotroph

A diploid yeast thymidylate auxotroph was grown under conditions of thymidylate stress ranging from depletion to excess levels of the nucleotide. High concentrations of thymidylate were mutagenic and recombinagenic whereas starvation for thymine nucleotides was recombinagenic and only slightly mutagenic. These results are discussed in relation to possible mutagenic and recombinagenic mechanisms of nucleotide pool imbalances.

Association of mutator activity with UV sensitivity in an aphidicolin-resistant mutant of Chinese hamster V79 cells.

The spontaneous mutation rates of an ultraviolet light (UV)-sensitive aphidicolin-resistant mutant (aphr-4-2) and its revertants have been determined by 2 techniques. By using the fluctuation analysis, the mutant and its thymidine (TdR)-prototrophic 'revertant' were found to exhibit elevated spontaneous mutation rates at the 6-thioguanine- and diphtheria-toxin-resistant loci. In contrast, the TdR-auxotrophic 'revertant' did not show this property. Similar results were obtained by the multiple replating technique. From these comparative studies and other previous characterizations, it appears that a single gene mutation is responsible for the following pleiotropic phenotype: slow growth. UV sensitivity, high UV-induced mutability, high frequency of site-specific bromodeoxyuridine (BrdU)-dependent chromosome breaks and enhanced spontaneous mutation rate. Recent studies indicate that the mutation may be on the gene for DNA polymerase alpha. The results further indicate that thymidine auxotrophy or imbalance in nucleotide pools is not necessarily associated with the mutator activity in mammalian cells.

Hypermutability of a UV-sensitive aphidicolin-resistant mutant of Chinese hamster fibroblasts

An ultraviolet light (UV)-sensitive thymidine auxotroph of Chinese hamster V79 cells that exhibits pleiotropic effects such as a high level of deoxycytidine triphosphate, slow growth, sensitivity to cytidine, and high frequencies of site-specific bromodeoxyuridine-dependent chromosomal aberrations was selected by its resistance to aphidicolin. The UV-induced mutability of this mutant and one of its revertants, which retains some of the phenotypes listed above, was studied in 3 mutation assay systems. The results showed that the mutant was hypermutable for ouabain and diphtheria-toxin-resistant mutations compared to wild-type V79 cells at the same UV dose or the same survival level. The mutant exhibits a delayed expression of maximal frequency of induced 6-thioguanine-resistant mutants. When maximal frequencies are compared at the same UV dose, the mutant also has higher mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase locus. The revertant was similar to the wild-type in UV sensitivity and mutability. Our results showed that UV sensitivity and hypermutability are correlated and controlled by a single gene. Thymidine auxotrophy or nucleotide pool imbalance apparently did not contribute to the UV sensitivity and mutability of the mutant.