RNA-SYNTHESIS INHIBITORS ALTER THE SUBNUCLEAR DISTRIBUTION OF DNA TOPOISOMERASE-I

The acute effect of RNA and DNA synthesis inhibitors on DNA topoisomer ase (topo)I localization within cells was examined. Indirect immunoflu orescence revealed that topo I was distributed throughout the nuclei b ut was concentrated in nucleoli of untreated K562 leukemia cells and A 549 non-small cell lung cancer cells, Treatment with the DNA polymeras e inhibitor aphidicolin did not alter this distribution. In contrast, 30-60 min after addition of the RNA synthesis inhibitor 5,6-dichloro-1 -beta-D-ribofuranosylbenzimidazole (DRB) at concentrations that inhibi ted [H-3]uridine incorporation into RNA by greater than or equal to 50 %, topo I was visible throughout the nuclei without nucleolar accentua tion, Western blotting and activity assays confirmed that the amount o f topo I polypeptide and topo I activity were unaltered by the brief D RB treatment, Within 30 min of DRB removal, topo I relocalized to the nucleoli in the absence or presence of the protein synthesis inhibitor cycloheximide. Collectively, these results suggest a reversible trans location of topo I out of the nucleoli when RNA synthesis is inhibited , Treatment with the topo I poisons topotecan or camptothecin, agents that also inhibit RNA synthesis, likewise caused redistribution of top o I to nonnucleolar regions of the nucleus in a variety of cell types, In DC3F hamster lung fibroblasts, 2.5 mu M topotecan or 1.25 mu M cam ptothecin was sufficient to cause this topo I redistribution, In DC3F/ C-10 cells that contain a mutant camptothecin-resistant topo I, topo I relocalization required 50-fold higher concentrations of topotecan or camptothecin but not DRB, These observations not only suggest that ac cumulation of topo I in the nucleolus is related to ongoing RNA synthe sis but also raise the possibility of screening for some types of camp tothecin resistance at the single-cell level using a rapid immunofluor escence-based assay.