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Vol. 54, Issue 1, 78-85, July 1998
and II
in Leukemic
Cells: Isoform-Specific Cleavable Complexes Visualized and Quantified
In Situ by a Novel Immunofluorescence Technique
Department of Biochemistry and Genetics (E.W., K.P., C.A.A.),
LRF
Unit (A.J.F.), and
Cancer Research Unit (M.J.T.), The Medical School,
The University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH,
United Kingdom
We have shown that both DNA topoisomerase (topo) II
and 
are in
vivo targets for etoposide using a new assay which directly measures
topo II and cleavable complexes in individual cells after
treatment with topo II targeting drugs. CCRF-CEM human leukemic cells
were exposed to etoposide for 2 hr, then embedded in agarose on
microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from
drug-stabilized cleavable complexes remained associated with the DNA.
The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the or isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled
slow scan charged coupled device camera was used to capture images. A
dose-dependent increase in green immunofluorescence was observed when
using antisera to either the or isoforms of topo II, indicating
that both isoforms are targets for etoposide. We have called this the
TARDIS method, for trapped in agarose DNA immunostaining. Two key
advantages of the TARDIS method are that it is isoform-specific and
that it requires small numbers of cells, making it suitable for
analysis of samples from patients being treated with topo II-targeting
drugs. The isoform specificity will enable us to extend our
understanding of the mechanism of interaction between topo II-targeting
agents and their target, the two human isoforms.
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