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Method for quantifying expression of functionally active topoisomerase II in patients with leukaemia
A R Cattan1, D Levett, E A Douglas
1Department of Haematology, University of Newcastle upon Tyne, Royal Victoria, Infirmary.
Aims:
To produce a method to measure and quantify enzymatically active topoisomerase II in normal and neoplastic human cells.
Methods:
A crude cell lysate from density separated mononuclear cells from either peripherial blood or bone marrow was prepared as a source of topoisomerases. Using the lysate, minicircles from the Crithedia kinetoplast DNA complex were decatenated before being separated by agarose gel electrophoresis and visualised using ethidium bromide/ultraviolet fluorescence.
Results:
Cell number, sample volume and drug inhibition concentration required to produce reliable and reproducible assay conditions were established. Intra- and interassay standards were included which permitted the quantification of active topoisomerase II in matched peripheral blood, bone marrow, presentation, and relapse samples from patients with acute lymphoblastic leukaemia. Active topoisomerase II has been converted to a unit scale which has been used to compare topoisomerase II activities in cells from patients with normal blood and bone marrow samples.
Conclusions:
There was no change in topoisomerase II activities between samples taken at presentation and those taken during a recurrence. However, topoisomerase II activity in leukaemic blast populations was increased compared with topoisomerase II activity in normal cells.