thymidine-5--triphosphate and Neoplasms--Experimental

Potential sources of errors in 3H-thymidine use in cell proliferation studies are reviewed. Many factors affect the uptake of this agent into newly synthesized DNA: predominance of denovo and salvage pathways of thymidilate biosynthesis, activity of certain enzymes, size of thymidilate and TTP endogenous pools, differences in 3H-thymidine utilization rate during S-period, its catabolism in cell and tissue culture systems, degradation by bacteria, its reutilization in vivo and in vitro, effects of the isotope activity, of the exposition time of autoradiographs and of the beta-particles absorbance in the specimen. Practical recommendations are given to avoid these errors.

Topics: Animals; Autoradiography; Cell Cycle; Cells, Cultured; DNA; DNA, Neoplasm; Humans; Interphase; Neoplasms; Neoplasms, Experimental; Thymidine; Thymidine Kinase; Thymine Nucleotides; Tritium

We examined whether an increase in the salvage and/or the de novo synthesis of thymidine (TdR) can explain the elevated DNA synthesis rate found up to 15-20 h after combined treatment with cisplatin and 5-fluorouracil (5-FU), compared with single-drug regimen. The salvage and the de novo pathways of TdR in Bp8 mouse ascites tumor cells were reduced equally after the combined treatment and the single-drug treatments. The inhibition of the de novo pathway of TdR was confirmed by a reduced thymidylate synthase activity, as measured in cell extract. A marked imbalance of the deoxyribonucleotide triphosphates were found, in particular between the deoxypyrimidines. These imbalances were similar between the 5-FU single-drug treatment and combined treatment. We conclude that neither the extracellular TdR salvage nor the de novo synthesis of TdR explain the relatively elevated DNA synthesis rate after combined treatment. We suggest that the supra-additive effect of the combined treatment is due to an interaction between the elevated DNA synthesis, the imbalanced deoxyribonucleotides and the cisplatin-induced DNA cross-links, and possibly also due to a higher concentration of 5-FU incorporated into DNA.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Chromatography, High Pressure Liquid; Cisplatin; Cross-Linking Reagents; DNA, Neoplasm; Fluorouracil; Male; Mice; Neoplasms, Experimental; Thymidine; Thymidylate Synthase; Thymine Nucleotides; Tumor Cells, Cultured

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.

Topics: Affinity Labels; Allosteric Regulation; Allosteric Site; Animals; Cell Line; Kinetics; Lymphoma; Mice; Mutation; Neoplasms, Experimental; Nucleosides; Nucleotides; Ribonucleotide Reductases; Thymine Nucleotides

The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.

Topics: Affinity Labels; Allosteric Site; Animals; Binding Sites; Binding, Competitive; Cell Line; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Lymphoma, Non-Hodgkin; Mice; Mutation; Neoplasms, Experimental; Ribonucleotide Reductases; Thymine Nucleotides; Ultraviolet Rays

Topics: Adenine Nucleotides; Animals; Ascites; Carbon Isotopes; Carcinoma, Hepatocellular; Chromatography; Cytosine Nucleotides; Deoxyribonucleosides; DNA; DNA, Neoplasm; Guanine Nucleotides; Liver; Liver Neoplasms; Metabolism; Mice; Neoplasms, Experimental; Nucleotidases; Nucleotides; Phosphorus Isotopes; Polyphosphates; Research; Thymine Nucleotides

Topics: Ascites; Chromatography; Metabolism; Neoplasms; Neoplasms, Experimental; Nucleotides; Phosphates; Phosphorus Isotopes; Research; Spectrophotometry; Thymidine; Thymine Nucleotides; Tritium