deoxyguanosine-triphosphate and Carcinoma--Ehrlich-Tumor

The analysis of deoxyribonucleoside 5'-triphosphates (dNTPs) in cell extracts by high-pressure liquid chromatography [C. Garrett , and D.V. Santi (1979) Anal. Biochem. 99, 268-273] requires the prior, selective degradation of ribonucleoside 5'-triphosphates ( rNTPs ) that are present in the extracts in large quantities. When this method was used for quantifying the dNTPs in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and dATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. It was found that the material which gave this peak was formed during the periodate oxidation of rNTPs in the presence of methylamine, and that it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was found to be effective in preventing the partial loss of dGTP in the oxidation procedure. By using this improved method, the dNTP contents of the extracts of Ehrlich ascites tumor cells have been measured in an accurate and reproducible manner. The analysis requires about 10(6) cells, and all four dNTPs can be quantified in 2.5 h, starting from the harvest of the cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Extracts; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Male; Methylamines; Mice; Oxidation-Reduction; Periodic Acid; Ribonucleotides; Thymine Nucleotides; Tissue Extracts

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Deoxyguanine Nucleotides; Macromolecular Substances; Mice; Molecular Weight; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases

The nature of the inhibition of Ehrlich tumor cell ribonucleotide reductase by combinations of agents directed at the non-heme iron-containing component and the effector-binding component was studied with the use of isobolograms. From these studies, it was determined that the combinations of pyrazoloimidazole (IMPY) and dialdehyde of inosine, IMPY and deoxyguanosine triphosphate (dGTP), IMPY and deoxyadenosine triphosphate (dATP), and IMPY and deoxythymidine triphosphate (dTTP) gave synergistic inhibition of cytidine diphosphate reductase. The combination of dATP and dGTP also gave synergistic inhibition. The combinations of hydroxyurea and IMPY, 4-methyl-5-aminoisoquinoline thiosemicarbazone (MAIQ) and IMPY, and dialdehyde of inosine and dialdehyde derivative of 5'-deoxyinosine gave antagonistic inhibition. Other combinations utilizing MAIQ and dATP, MAIQ and dGTP, MAIQ and dTTP, hydroxyurea and dGTP, and hydroxyurea and dTTP gave inhibition which was additive.

Topics: Aldehydes; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Drug Interactions; Drug Synergism; Edetic Acid; Hydroxyurea; Imidazoles; Inosine; Isoquinolines; Mice; Pyrazoles; Ribonucleotide Reductases; Thiosemicarbazones; Thymine Nucleotides