RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I

Christopher A. Buckwalter, Amy H. Lin, Akihiko Tanizawa, Yves G. Pommier, Yung Chi Cheng, Scott H. Kaufmann

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

The acute effect of RNA and DNA synthesis inhibitors on DNA topoisomerase (topo) I localization within cells was examined. Indirect immunofluorescence revealed that topo I was distributed throughout the nuclei but was concentrated in nucleoli of untreated K562 leukemia cells and A549 non-small cell lung cancer cells. Treatment with the DNA polymerase inhibitor aphidicolin did not alter this distribution. In contrast, 30-60 min after addition of the RNA synthesis inhibitor 5,6-dichloro-1-β-D- ribofuranosylbenzimidazole (DRB) at concentrations that inhibited [3H]uridine incorporation into RNA by ≥50%, topo I was visible throughout the nuclei without nucleolar accentuation. Western blotting and activity assays confirmed that the amount of topo I polypeptide and topo I activity were unaltered by the brief DRB 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 translocation 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 topo I to nonnucleolar regions of the nucleus in a variety of cell types. In DC3F hamster lung fibroblasts, 2.5 μM topotecan or 1.25 μM camptothecin 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 accumulation of topo I in the nucleolus is related to ongoing RNA synthesis but also raise the possibility of screening for some types of camptothecin resistance at the single-cell level using a rapid immunofluorescence-based assay.

Original languageEnglish (US)
Pages (from-to)1674-1681
Number of pages8
JournalCancer research
Volume56
Issue number7
StatePublished - Apr 1 1996

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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    Buckwalter, C. A., Lin, A. H., Tanizawa, A., Pommier, Y. G., Cheng, Y. C., & Kaufmann, S. H. (1996). RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I. Cancer research, 56(7), 1674-1681.