Effects of 5-mercapto-2'-deoxyuridine on the incorporation of nucleosides into RNA and DNA in a primary lymphocyte culture system.
The effects of 5-mercapto-2'-deoxyuridine (MUdr) on DNA synthesis in a primary murine spleen lymphocyte culture system stimulated by phytohemagglutinin (PHA) were studied. Inhibition of thymidine incorporation into acid insoluble nucleic acid material was 50% at 0.5 mM MUdR concentration, while inhibition of deoxyuridine incorporation into acid-insoluble nucleic acids was 50% at 0.01 mM MUdR. Time course studies, at 0.5 and 0.05 mM MUdR, showed that the magnitude of inhibition of incorporation for thymidine and deoxyuridine, respectively, increased from a time point after PHA stimulation when increased synthesis of thymidine kinase and thymidylate synthetase had leveled off. At 1 mM MUdR, total cellular DNA in cultures was decreased 43% at 42 hr after PHA stimulation. Neither the total number of cells nor the percentage of PHA-transformed cells was decreased in comparison to that of controls. MUdR therefore blocks the increase in DNA content of lymphocytes that is initiated during the S phase of the cell cycle. Millimolar levels of MUdR inhibited incorporation or uridine, adenosine, and cytidine into acid-insoluble material in pha-stimulated primary murine lymphocyte cultures. Total cellular RNA synthesis was inhibited at these levels of MUdR, with no differential effects on 4, 18, or 28 S RNA species observed. Uptake of these nucleosides into the total cellular acid-soluble material was not blocked. Uptake of different labeled nucleosides into cellular, acid-soluble pools occurs at different rates. Thus, choice of a suitable minimum pulse time to achieve saturation for different labeled nucleosides must relate to this consideration. Thymidine kinase from whole-cell sonic extracts of PHA-stimulated lymphocytes was inhibited 65% by 1 mM MUdR at 24 and 48 hr after stimulation. Uridine kinase extracted from the PHA-stimulated cells was also significantly inhibited by 1mM MUdR at 24 hr (56%). Exogenous guanosine incorporation into lympohcyte acid-insoluble material is increased by MUdR. This increased utilization of exogenous nuceloside is apparently the result of MUdR inhibition of conversion of adenosine to guanine nucleotides within the lymphocytes and a consequent diminution of the total intracellular guanine nucleotide pool size. The active inhibitory compound is the deoxyribonucleoside or deoxyribonucleotide. Comparison with the riboside analog 5-mercaptouridine showed that MUdR was a more efficient inhibitor of nucleoside incorporation.
D000818:Animals; D004247:DNA / Q000096:biosynthesis*; D003857:Deoxyuridine / Q000494:pharmacology; D006151:Guanosine / Q000378:metabolism; D037102:Lectins; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009705:Nucleosides / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis*; D013154:Spleen / Q000166:cytology; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors; D013997:Time Factors; D014541:Uridine Kinase / Q000037:antagonists & inhibitors
D037102:Lectins; D009705:Nucleosides; D009711:Nucleotides; D011506:Proteins; D006151:Guanosine; D013891:Thiouridine; D012313:RNA; D004247:DNA; D013937:Thymidine Kinase; D014541:Uridine Kinase; D003857:Deoxyuridine