Camptothecin-stabilised topoisomerase I-DNA complexes in leukaemia cells visualised and quantified in situ by the TARDIS assay (trapped in agarose DNA immunostaining).
We have shown that the TARDIS assay (trapped in agarose DNA immunostaining) can be used to detect DNA-topoisomerase I (topo I) cleavable complexes in situ in individual cells following treatment with topo I-targeting drugs. This assay is a modification of the assay for DNA-topoisomerase II (topo II) cleavable complexes (Willmore et al., Mol Pharmacol 53: 78-85, 1998). Drug-stabilised topo I-DNA complexes were detected in situ by topo I-specific primary antibodies and then visualised using fluorescein isothiocyanate conjugated second antibodies. Immunofluorescence was then quantified using a cooled slow-scan coupled device camera and image analysis procedures. Camptothecin (CPT) 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 between 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 immunofluorescence detected by the TARDIS assay was specific for topo I-targeting agents. Hence, the TARDIS assay provides a powerful tool to determine the levels of drug-stabilised cleavable complexes in whole cells and thereby aid in the understanding of the mechanism of interaction between topo I-targeting drugs and their target.
Padget
K
K
School of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK.
Carr
R
R
Pearson
A D
AD
Tilby
M J
MJ
Austin
C A
CA
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology*; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin