PMID: 8603418Apr 1, 1996Paper

Subcellular redistribution of DNA topoisomerase I in anaplastic astrocytoma cells treated with topotecan

Cancer Research
M K DanksD O Whipple

Abstract

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 microM 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan; TPT) or 5 microM 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 microM 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 den...Continue Reading

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