8488 Clin Pathol 1996;49:848-852 Method for quantifying expression of functionally active topoisomerase II in patients with leukaemia J Clin Pathol: first published as 10.1136/jcp.49.10.848 on 1 October 1996. Downloaded from A R Cattan, D Levett, E A Douglas, P G Middleton, P R A Taylor Abstract phenotype in vivo of the eventual failure of Aims-To produce a method to measure response to treatment seen in patients with and quantify enzymatically active topo- leukaemia.6 isomerase II in normal and neoplastic The topoisomerases are a ubiquitous family human cells. ofenzymes seen in bacteria (DNA gyrases) and Methods-A crude cell lysate from density in eukaryote organisms where two major forms separated mononuclear cells from either exist: topoisomerase I which catalyse single peripherial blood or bone marrow was strand DNA breaks and ligations; and topo- prepared as a source of topoisomerases. isomerase II which catalyse the cleavage and Using the lysate, minicircles from the ligation of double strand DNA breaks. These Crithedia kinetoplast DNA complex were enzymes are believed to play important roles in decatenated before being separated by DNA replication, repair and in gene transcrip- agarose gel electrophoresis and visualised tion (for review see 7 8). In mammalian cells using ethidium bromide/ultraviolet fluor- there are two forms (a and [) oftopoisomerase escence. II.9 Little is still known about their explicit Results-Cell number, sample volume roles although there are reported associations and drug inhibition concentration re- with the cell cycle'0 and changes in expression quired to produce reliable and reproduc- of the two forms in cell lines under cytotoxic ible assay conditions were established. drug selection."1 Intra- and interassay standards were A variety of drugs used in the treatment of included which permitted the quantifica- leukaemia, including the anthracyclines (dau- tion of active topoisomerase II in matched norubicin, idarubicin), epipodophyllotoxins peripheral blood, bone marrow, presenta- (VP-16) and anthraquinone (mitoxantrone), tion, and relapse samples from patients are known to target topoisomerase II, and may with acute lymphoblastic leukaemia. Ac- be partly responsible for the antineoplastic tive topoisomerase II has been converted effects found. Attempts to measure activities of to a unit scale which has been used to topoisomerase II and changes in their proper- compare topoisomerase II activities in ties in mammalian cellular preparations have cells from patients with normal blood and been largely restricted to cell lines.'2-14 Topo- http://jcp.bmj.com/ bone marrow samples. isomerase II activities in clinical leukaemic Conclusions-There was no change in samples have been reported using slot blots to topoisomerase II activities between sam- detect specific mRNAs'5 16 and by western blot- ples taken at presentation and those taken ting to detect protein and determine protein during a recurrence. However, topoisom- concentrations.'6 17 As topoisomerase II expres- erase II activity in leukaemic blast popula- sion has been shown to be decreased in cell tions was increased compared with lines exposed to cytotoxic agents,'7 18 several on October 2, 2021 by guest. Protected copyright. topoisomerase II activity in normal cells. groups have attempted to show a similar corre- (7 Clin Pathol 1996;49:848-852) lation in clinical specimens with the hope of relating this to clinical outcome. Although it Keywords: drug resistance, topoisomerases, leukaemia. has been reported that topoisomerase II expression changes during treatment, most investigations have been unable to correlate At presentation, most acute leukaemias show this expression with remission status.'5-'` Ellis some response to treatment; however, with et all9 have described a filter based assay to time most adult patients succumb to their dis- quantify biologically active topoisomerase II in ease due to relapse. Following relapse, treat- a cell line with the intention of using this ment protocols become less effective and drug method in an attempt to predict the develop- concentrations are escalated with the addition ment of drug resistance to the topoisomerases. of other chemotherapeutic agents. This phe- Quantification of topoisomerase II by either Department of nomenon of failing response to therapy with measurement of mRNA or specific protein and Haematology, increased tumour load is often referred to col- the use of this to correlate topoisomerase II University of lectively as the development of drug resistance. activity with drug resistance phenotype does Newcastle upon Tyne, There are many in vitro and in vivo models not take into account that the protein may be Royal Victoria Infirmary, Newcastle used to describe the development of drug non-functional. It is known that phosphoryla- upon Tyne NEI 4LP resistance, including the multidrug resistance tion status affects topoisomerase II function phenotype or MDR,' drug detoxification,' and that this may alter the antineoplastic effect Correspondence to: changes in both the level and structure of the of the drugs in question.8 In an attempt to Dr A R Cattan. drug target,3 and changes in DNA repair address this problem, we have developed an Accepted for publication mechanisms.4 All of these mechanisms may assay to measure biologically active topo- 16 July 1996 work alone or in concert to produce the isomerase II in leukaemic samples. We report Topoisomerase II in leukaemia 849 Table 1 Patient details PREPARATION OF CRITHEDIA DNA Crithedia fasciculata (a gift of Dr I Hickson, Patient Age Sex Diagnosis Presenting Remission status Anti-leukaemic chemotherapy given Oxford) was grown in brain heart infusion number phenotype WBC x achieved prior to sample J Clin Pathol: first published as 10.1136/jcp.49.10.848 on 1 October 1996. Downloaded from 109/l medium (Difco) containing 10 ig/ml hemin, Control 73 M B-CLL Chlorambucil (832 mg), 100 gg/ml penicillin, 0.125 ,ug/ml streptomycin prednisolone (520 mg) at 280C to 1-5 x 107/ml. DNA was prepared ALL1 23 F Calla+ 190 Complete Induction therapy + intensive from the harvested protozoa (2 x 10") using ALL remission therapy + consolidation + allograft ALL2 48 M T-ALL 357 Complete Induction therapy + intensive standard lysis and chloroform/alcohol extrac- remission therapy + consolidation + allograft tion methods. The mixture of genomic and ALL3 39 M preB-ALL 63.3 Complete Induction therapy + intensive kinetoplast DNA was treated with RNase A remission therapy + consolidation + allograft ALL4 16 M Calla+ 44.2 No remission Induction therapy + intensive (bovine pancreas; Sigma, Poole, Dorset, UK) ALL therapy for 60 minutes at 37°C (50 jg/ml final concen- ALL5 62 F B-ALL 19.4 No remission None tration) and then mixed with molecular weight ALL6 16 M Null 44.8 No remission None ALL marker (5:3 ratio Hind III digested bacterio- ALL7 21 M Calla+ 13.7 No remission None phage k markers; Integra Biosciences) to form ALL the target DNA for the topoisomerase II assay. ALL8 47 F Null 43.4 Complete None ALL remission ALL9 * 42 F preB-ALL 63.7 Complete None ASSAY OF TOPOISOMERASE II ACTIVITY remission The assay is an adaption of the assay of Davies *Leukaemia secondary to treatment for Hodgkin's disease; WBC = white blood cell. et al.22 Briefly, 5 gl reaction buffer (50 mM Tris (pH 7.9), 120 mM KCI, 10 mM MgCl2, 0.5 the characterisation of this assay and the mM EDTA, 30 gg/ml bovine serum albumin, 5 expression of functional topoisomerase II in mM DTT, 5 mM ATP), 5 ,ul adriamycin (380 leukaemic blasts taken from patients with acute ng/ml in water), 24.5 gl water, 7.5 pl cell lysate, lymphoblastic leukaemia (ALL). and 8 gl Crithedialk DNA phage were mixed in a microcentrifuge tube, incubated at 30°C for Methods 30 minutes and the reaction stopped by the PATIENTS addition of 12.5 jtl stop solution (5% w/v SDS, The diagnosis of leukaemia was made by a 30% glycerol, 2.5 jtg bromophenol blue). consultant haematologist using Romanovsky stained blood and marrow smears and was PROTEIN ESTIMATION confirmed by appropriate cytochemical stain- Protein content in lysates was measured ing and immunophenotyping of the leukaemic according to the method of Bradford,23 blasts. Details of the patients at presentation adapted for use on a 96 well plate reader (Mul- are shown in table 1. All cases were of de novo tiskan MCC 340). leukaemia except for patient ALL9 where the leukaemia was considered secondary to previ- GEL ELECTROPHORESIS AND DENSITOMETRIC ous chemotherapy for Hodgkin's disease. All MEASUREMENTS patients received initial chemotherapy accord- Assay samples were run for 16 hours in 0.8% http://jcp.bmj.com/ ing to the NEALL III Protocol.20 Remission (w/v) agarose using standard DNA electro- induction therapy was with vincristine, pred- phoresis conditions and bands visualised with nisolone, adriamycin, and cytosine. If complete ethidium bromide. Following electrophoresis remission (defined as <5% blasts in an the resulting separations of DNA molecules adequately cellular marrow) was not achieved were visualised by ultraviolet illumination of by seven weeks, then further intensive treat- the DNA-ethidium complexes (302 nm trans- ment with high dose cytosine and mito- illumination) and the image stored electroni- on October 2, 2021 by guest. Protected copyright. xantrone was given. Four patients did not cally for further analysis (UVP 2000, Ultaviolet achieve complete remission. Two patients had Products Ltd). The units of topoisomerase II recognised adverse factors (ALL6, Ph chromo- were calculated by adjusting the ratio of the some positive; ALL7, B-ALL) and a third total area of topoisomerase II to k marker (ALL8) evolved to acute myeloid leukaemia bands to those produced by an aliquoted con- after five weeks on treatment. Consolidation trol lysate in that particular test and corrected treatment was with vincristine, steroids, cyto- to the relevant point on a standard curve (fig sine, asparaginase, methotrexate, and 6-thio- 1).