DNA topoisomerase I is the cytotoxic target for chemotherapeutic agents of the camptothecin class. The cytotoxicity of these drugs is thought to be mediated by a dose-dependent increase in topoisomerase I molecules bound to DNA, resulting in DNA damage and cell death. We observed that in SJ-G5 human anaplastic astrocytoma cells growing in culture, the maximum number of topoisomerase I-DNA complexes occurred 5–15 min after the addition of 0.25–25 µm 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan; TPT) or 5 µm 7-ethyl-10-hydroxycamptothecin (SN-38). We postulated that the decline in number of complexes seen after 15 min might be due to decreases in the amount of topoisomerase I or the redistribution of this enzyme such that it could no longer bind to DNA. To investigate these possibilities, we incubated SJ-G5 cells for 20–60 min with 0.25–5 µm TPT and analyzed the cells for amount and localization of topoisomerase I by indirect immunofluorescence staining and fluorescence digital imaging microscopy. We verified the results obtained with fluorescence digital imaging microscopy by rapid fractionation of nuclear and cytoplasmic proteins, separation of these proteins by polyacrylamide gel electrophoresis, and densitometric scanning of immunoblots. Results showed that topoisomerase I dissociated from nucleoli within 60 min after treatment with 1–5 µm TPT. A small (25%) but significant (P < 0.05) decrease in the amount of nuclear topoisomerase I was also observed during this time course. Simultaneously, the cytoplasmic content of the Mr 67,000 form of topoisomerase I increased 50–100%. Preincubation of cells with 10 µm cycloheximide for 10 min prevented the increase of topoisomerase I in the cytoplasm, indicating that the increase was due, at least in part, to de novo protein synthesis. Interestingly, chemotherapeutic agents other than camptothecins were also found to dissociate topoisomerase I from nucleoli. These agents included m-AMSA (4′-(9-acridinylamino)methanesulfon-m-anisidide), mitoxantrone, actinomycin D, and daunorubicin. Drugs such as 1-β-d-arabinofuranosylcytosine or hydroxyurea, which have no effect on RNA synthesis, did not induce the translocation. The biological significance of the ability of camptothecins to redistribute their own drug target is under investigation.
This work was supported by NIH Grants CA63516, CA23099, and CA21765 (CORE) and by the American Lebanese and Syrian Associated Charities.