SUBCELLULAR REDISTRIBUTION OF DNA TOPOISOMERASE-I IN ANAPLASTIC ASTROCYTOMA-CELLS TREATED WITH TOPOTECAN

DNA topoisomerase I is the cytotoxic target for chemotherapeutic agent s of the camptothecin class, The cytotoxicity of these drugs is though t to be mediated by a dose-dependent increase in topoisomerase I molec ules 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 mu M 9-dimethylaminomethyl-10-hydroxycam ptothecin (topotecan; TPT) or 5 mu M 7-ethyl-10-hydroxycamptothecin (S N-38), We postulated that the decline in number of complexes seen afte r 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 t o DNA, To investigate these possibilities, we incubated SJ-G5 cells fo r 20-60 min with 0.25-5 mu M TPT and analyzed the cells for amount and localization of topoisomerase I by indirect immunofluorescence staini ng and fluorescence digital imaging microscopy, We verified the result s obtained with fluorescence digital imaging microscopy by rapid fract ionation of nuclear and cytoplasmic proteins, separation of these prot eins 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 mu M TPT, A small (25% ) but significant (P < 0.05) decrease in the amount of nuclear topoiso merase I was also observed during this time course, Simultaneously, th e cytoplasmic content of the M(r) 67,000 form of topoisomerase I incre ased 50-100%, Preincubation of cells with 10 mu M cycloheximide for 10 min prevented the increase of topoisomerase I in the cytoplasm, indic ating that the increase was due, at least in part, to de novo protein synthesis, Interestingly, chemotherapeutic agents other than camptothe cins were also found to dissociate topoisomerase I from nucleoli. Thes e agents included m-AMSA (4'-(9-acridinylamino)methanesulfon-m-anisidi de), mitoxantrone, actinomycin D, and daunorubicin. Drugs such as 1-be ta-D-arabinofuranosylcytosine or hydroxyurea, which have no effect on RNA synthesis, did not induce the translocation, The biological signif icance of the ability of camptothecins to redistribute their own drug target is under investigation.