RNA synthesis inhibitors alter the subnuclear distribution of DNA topoisomerase I.
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-beta-D-ribofuranosylbenzimidazole (DRB) at concentrations that inhibited [3H]uridine incorporation into RNA by > or = 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 microM topotecan or 1.25 microM 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.
Buckwalter
C A
CA
Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905, USA.
Lin
A H
AH
Tanizawa
A
A
Pommier
Y G
YG
Cheng
Y C
YC
Kaufmann
S H
SH
D000818:Animals; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D006224:Cricetinae; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D012313:RNA / Q000096:biosynthesis*; D019772:Topotecan; D014407:Tumor Cells, Cultured
D004004:Dichlororibofuranosylbenzimidazole; D012313:RNA; D019772:Topotecan; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin