Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase.

In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system.

Biosynthesis of cytidine diphosphate diglyceride by human platelets.

Homogenates of human platelets contain an enzyme which catalyzes the formation of cytidine diphosphate diglyceride from cytidine triphosphate and phosphatidic acid. The enzymatic activity could not be dissociated from platelet particles and the greatest specific activity was found in the membrane fraction. The K(m) for cytidine triphosphate was 0.16 mmole/liter and the apparent K(m) for phosphatidic acid was 6.2 mmoles/liter. The pH optimum was 7.0 and the most effective buffers were triethanolamine-HCl and Tris-HCl. The reaction was dependent on the presence of divalent cations, magnesium being the most effective of those investigated. Monovalent cations did not alter the reaction rate. Evidence is presented that the cytidine diphosphate diglyceride produced can serve as a precursor for the synthesis of phosphatidylinositol. No difference was found in the enzymatic activity in platelets from normal subjects and from patients with diseases known to interfere with platelet thromboplastic function.

Consequences of methotrexate inhibition of purine biosynthesis in L5178Y cells.

Addition of 1 muM methotrexate to cultures of L5178Y cells results in an initial inhibition of thymidine, uridine, and leucine incorporation into acid-insoluble material followed, after about 10 hr, by a partial recovery in the extent of incorporation of these precursors. Acid-soluble adenosine triphosphate and guanosine triphosphate concentrations are greatly reduced initially, but guanosine triphosphate concentrations appear to recover partially by 10 hr. Acid-soluble uridine triphosphate and cytidine triphosphate concentrations initially increase after methotrexate treatment but then, with time, they too decline. Hypoxanthine and guanine are more effective than is adenine in overcoming the methotrexate-induced inhibition of thymidine incorporation. These results suggest that, in the presence of methotrexate, guanine nucleotides become limiting for nucleic acid synthesis before adenine nucleotides do. The block of purine de novo synthesis in L5178Y cells by methotrexate is almost complete and is not reversed with time. This suggests that the additional purine nucleotides that are available for nucleic acid synthesis 8 to 10 hr after addition of methotrexate are being derived from nucleic acid breakdown. Consistent with this is the observed reduction in the number of polyribosomes and hence, presumably in messenger RNA levels.

The effect of chemical mutagens on purine and pyrimidine nucleotide biosynthesis.

Nucleotide biosynthesis in Novikoff hepatoma cells is markedly altered by a variety of chemical mutagens, whether the mechanism of mutagenesis is by base substitution, covalent binding (adduct formation), intercalation, or cross-linking of DNA. The compounds investigated (N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, 9-aminoacridine, and mitomycin C), at concentrations that cause some inhibition of RNA and DNA synthesis, bring about a large increase in the pool levels of all four nucleoside triphosphates. At the same time, reactions leading to the synthesis of CTP from exogenous uridine and GTP and ATP from exogenous hypoxanthine are severely inhibited. The formation of UTP from uridine and ATP from adenosine, by more direct phosphorylation reactions, appears relatively unaffected. The increase in nucleotide pool size cannot be accounted for by a corresponding increase in de novo purine and pyrimidine nucleotide synthesis, as experiments with labeled formate and aspartate show similar inhibitions by the mutagens. With the salvage precursors, [3H]uridine and [3H]hypoxanthine, the mutagens can produce a widely divergent reduction in the labeling of RNA-CMP versus RNA-UMP and of RNA-GMP versus RNA-AMP, mostly a result of these agents causing large differences in the specific activities of the respective triphosphate precursors. These observations suggest that, in addition to the reactions with DNA, nucleotide biosynthesis could be another important biochemical target of chemical mutagens.

Cyclopentenyl cytidine analogue. An inhibitor of cytidine triphosphate synthesis in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of cytidine, cCyd, was investigated in human colon carcinoma cell line HT-29. Upon exposure of cells to 10(-6)M cCyd, cell viability was reduced to 20% of control, whereas cytocidal activity was not present after 2 hr of drug exposure. Cell lethality was partially reversible by Urd, Cyd or dCyd at 10(-6)M cCyd, and fully reversible by these nucleosides at 2.5 X 10(-7)M cCyd. The incorporation of [14C]dThd and [3H]Urd into DNA and RNA was inhibited by 50% by exposure for 2 hr to 2.5 X 10(-7) and 1.5 X 10(-6)M cCyd respectively. Upon 24 hr of drug exposure, the IC50 for RNA synthesis was reduced 2.5-fold, whereas DNA synthesis was almost totally inhibited. cCyd produced a rapid and preferential reduction of CTP synthesis with a half-life of 1 hr at 10(-6)M drug. The IC50 of cCyd for reducing CTP concentrations after 2 hr of drug exposure was 4 X 10(-7)M. Concomitant with the reduction of CTP levels was the inhibition of transcription of rRNA and, to a lesser extent, tRNA, without changes in the processing nucleolar RNA. No changes in the size of DNA were produced following treatment with cCyd. These results indicate that cCyd is a potent and rapid inhibitor of CTP synthesis and that this effect correlates with its cytocidal activity.

Biosynthesis of rat brain phosphatidylethanolamines from intracerebrally injected ethanolamine.

[2-3H]Ethanolamine was injected intracerebrally into male rats and the brains of the animals immediately removed by particular procedures at regular intervals over the first 1200 sec. The incorporation of radioactivity into brain phosphorylethanolamine, cytidine-5'-diphosphate (CDP) ethanolamine and phosphatidylethanolamines was examined and quantitated. The nature of phosphatidylethanolamine molecular subspecies, which became labelled, was also investigated after isotope administration. Phosphorylethanolamine, CDP-ethanolamine and phosphatidylethanolamines were all labelled already 5 sec after the administration of labelled ethanolamine. The specific radioactivities of different phosphatidylethanolamine molecular subspecies varied according to the time elapsed from the injection to the sacrifice of the animals. This last result, together with the data on time course of labelling of ethanolamine phosphoglycerides and their precursors, provides indications that this base may be incorporated into lipids not only by net synthesis pathway, but also by base-exchange reaction.

Decreased utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides in rat liver after administration of alpha-hexachlorocyclohexane.

Administration of alpha-1,2,3,4,5,6-hexachlorocyclohexane (alpha-HCH) to rats decreased the utilization of [2-14C]orotic acid for the synthesis of liver cytidine nucleotides. The specific radioactivities of uridine components of the acid-soluble pool and rRNA increased during the first hours of treatment with the drug. Later on the specific radioactivities of uridine nucleotides remained unchanged, while those of cytidine components decreased gradually. Administration of hydrocortisone increased the incorporation of labelled orotic acid into rRNA cytidylic acid.

Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine.

Following the administration of D-galactosamine the utilization of [2-14C] orotic acid for the synthesis of the cytidine components of the acid-soluble extract and liver RNA cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application of low doses of the drug which do not interfere with the specific activity of the uridine components of the acid-soluble extract or of liver RNA uracil. Simultaneously the administration of [U-14C]cytidine paralleled by its enhanced liver uptake. The total amount of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied by the increased level of microsomal cytochrome P-450.

Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.

Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria.

Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.

[Sister chromatid exchanges under bromine incorporation into DNA cytosine nucleotides. II. Utilization of 3H-deoxycytidine as a DNA cytosine precursor]. / Sestrinskie khromatidnye obmeny pri vkliuchenii broma v tsitozinovye nukleotidy DNK. II. Ispol'zovanie 3H-dezoksitsitidina v kachestve predshestvennika tsitozina DNK.

The incorporation of 3H-deoxycytidine (3H-Cdr) in the presence of thymidine (Tdr) into cultured human blood lymphocytes has been studied. The analysis of the label in interphase nuclei as well as in chromosomes at metaphase was carried out. The labeling was much higher when 3H-Cdr (0.5 to 1.0 C/ml, 2--4 x 10(-5) mM) was added to the cultures simultaneously with Tdr (4 x 10(-1) mM). This observation is considered as an indication that in the presence of high doses of Tdr exogeneous Cdr is utilized to synthesize cytosine of DNA rather than thymidine. During the first hours after its addition, the bulk of 3H-Cdr is eliminated from the culture medium. At 12 hrs of the incubation, the medium seems to be free of the nucleoside as shown particularly from the single chromatid localization of the label in chromosomes of the second mitosis. The incorporation into lymphocytes of 3H-Tdr administered in the same dose under the same conditions was registered for the whole period of observation (24 hrs). The data obtained are discussed in relation to lymphocyte catabolism of exogeneous nucleosides.

[Effect of acetylcholine on incorporation of 14C-precursors into uridylic and cytidylic nucleotides of the RNA of digestive glands]. / Vkliuchenie 14C-predshestvennikov v uridilovye i tsitidilovye nukleotidy RNK pishchevaritel'nykh zhelez pod vliianiem atsetilkholina.

The incorporation rate of [2-14C]orotic acid and [2-14C]uridine into the cytidylic RNA nucleotides is significantly lower than into the uridylic ones. In the liver it was twice as low as in the stomach mucosa or in pancreas of albino rats. The administration of acetylcholine in combination with proserine has no influence on the RNA content and its nucleotide composition in the tissues. The administered drugs however caused changes in the relation of the incorporation rates of both labels into uridylic and cytidylic RNA nucleotides, which evidences for the uridylic nucleotide synthesis. In the liver such changes are not detected, but utilization of the labeled uridine is shown to be more intensive for the cytidylic RNA nucleotides synthesis.