Camptothecin-stabilised topoisomerase I-DNA complexes in leukaemia cells visualised and quantified in situ by the TARDIS assay ((t)under-barrapped in(a)under-barga(r)under-barose (D)under-barNA (i)under-barmmuno(s)under-bartaining)

We have shown that the TARDIS assay ((t) under bar rapped in (a) under bar garose (D) under bar NA (i) under bar mmuno (s) under bar taining) can be u sed to detect DNA-topoisomerase I (topo I) cleavable complexes in situ in i ndividual cells following treatment with topo I-targeting drugs. This assay is a modification of the assay for DNA-topoisomerase II (topo II) cleavabl e complexes (Willmore et al., Mol Pharmacol 53: 78-85, 1998). Drug-stabilis ed topo I-DNA complexes were detected in situ by topo I-specific primary an tibodies and then visualised using fluorescein isothiocyanate conjugated se cond antibodies. Immunofluorescence was then quantified using a cooled slow -scan coupled device camera and image analysis procedures. Camptothecin (CP T) was shown to stabilise topo I-DNA cleavable complexes in whole cells in a dose-dependent manner in both CCRF-CEM and K562 cells and in lymphoblasts from an adult with newly diagnosed acute myeloid leukaemia treated ex vivo with CPT. In K562 cells, cleavable complexes were found to be maximal betw een 30 and 90 minutes continuous exposure of CPT, and approximately 78% of cleavable complexes formed in these cells were found to be reversed within 5 minutes of drug removal. It has also been shown that the immunofluorescen ce detected by the TARDIS assay was specific for topo I-targeting agents. H ence, the TARDIS assay provides a powerful tool to determine the levels of drug-stabilised cleavable complexes in whole cells and thereby aid in the u nderstanding of the mechanism of interaction between topo I-targeting drugs and their target. (C) 2000 Elsevier Science Inc.