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

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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

Fingerprint

Nucleic Acid Synthesis Inhibitors
Type I DNA Topoisomerase
Camptothecin
Topotecan
RNA
Aphidicolin
Protein Synthesis Inhibitors
K562 Cells
Poisons
Uridine
Cycloheximide
Indirect Fluorescent Antibody Technique
Non-Small Cell Lung Carcinoma
Cricetinae
Fluorescent Antibody Technique

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

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.

RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I. / Buckwalter, Christopher A.; Lin, Amy H.; Tanizawa, Akihiko; Pommier, Yves G.; Cheng, Yung Chi; Kaufmann, Scott H.

In: Cancer Research, Vol. 56, No. 7, 01.04.1996, p. 1674-1681.

Research output: Contribution to journalArticle

Buckwalter, CA, Lin, AH, Tanizawa, A, Pommier, YG, Cheng, YC & Kaufmann, SH 1996, 'RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I', Cancer Research, vol. 56, no. 7, pp. 1674-1681.
Buckwalter CA, Lin AH, Tanizawa A, Pommier YG, Cheng YC, Kaufmann SH. RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I. Cancer Research. 1996 Apr 1;56(7):1674-1681.
Buckwalter, Christopher A. ; Lin, Amy H. ; Tanizawa, Akihiko ; Pommier, Yves G. ; Cheng, Yung Chi ; Kaufmann, Scott H. / RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I. In: Cancer Research. 1996 ; Vol. 56, No. 7. pp. 1674-1681.
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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.",
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