Chemical synthesis and reagents
Quinoline aminopurine 1 (QAP 1), biotinylated purine analogue 2, purine analogues 3 and 4 (detailed synthesis will be described elsewhere), 17-AAG and ICRF-193 (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) were dissolved in DMSO to yield 10 mM final concentration and stored at -20°C. Doxorubicin (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) was dissolved in distilled water to yield 10 mM final concentration and stored at -20°C. All other chemical reagents were analytical grade quality. All cell culture reagents were purchased from AMIMED (Allschwil, Switzerland).
Full length tagged human topoisomerase II alpha protein production
Human topoisomerase II α cDNA was cloned in three successive steps. First the 5' region of the topoisomerase II α gene upstream of the EcoR1 site was amplified by PCR using an ATCC clone (#59748) as template and primers adding a Bgl2 site at its 5' end. The purified PCR product was digested with Bgl2 and EcoR1 and inserted into pFastBac1 (Invitrogen, Carlsbad, USA) previously digested with BamH1 and EcoR1. Then, an EcoR1/Pst1 fragment of the topoisomerase II α ATCC clone was inserted into the EcoR1/Pst1 sites of the plasmid previously obtained. Finally, the 3' end of topoisomerase II α downstream of Pst1 was amplified by a two-step PCR using the ATCC clone as template and primers adding a PreScission-His6-Strep tag and a Sph1 site at the 3' end. This product was subsequently digested with Pst1 and Sph1 and inserted into the corresponding sites of the plasmid previously obtained. The sequence of the inserted gene and flanking regions was verified by DNA sequencing. Topoisomerase II α protein was expressed in Sf21 cells (Invitrogen, Carlsbad, USA) using the Baculovirus system (multiplicity of infection 2.0, 72 h infection). The cell pellet was resuspended in 15 volumes ice cold lysis buffer (50 mM Na2HPO4 pH 7.7, 500 mM NaCl, 10 mM β-mercaptoethanol, 5 mM imidazol, 100 μg/ml PMSF, 2 mM MgCl2 and EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH)) and homogenized using a Heidolph Diax 600 homogenizer. Following centrifugation for 60 minutes at 48000 g and filtration through a 3 μm Millipore filter the lysate was loaded on a 5 ml HisTrap HP affinity column (GE Healthcare, Sweden), pre-equilibrated in buffer A (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM β-mercaptoethanol and 5 mM imidazol). The column was washed with buffer A containing 20 mM imidazole and the protein was eluted with a gradient of buffer B (250 mM imidazole in buffer A). Pooled fractions containing topoisomerase II protein were loaded on a SPX200 size exclusion column (16/20) equilibrated with 50 mM Tris HCl pH 7.7, 150 mM NaCl and 2 mM DTT, followed by loading on a 10 ml StrepTactin column (IBA, Germany) equilibrated with 100 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM β-mercaptoethanol, 1 mM EDTA. After a brief washing step, the enzyme was eluted with and stored in 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM β-mercaptoethanol, 1 mM EDTA and 2.5 mM desthiobiotin. Single-use aliquots of enzyme were flash-frozen in liquid nitrogen and stored at -80°C until use.
ATPase assay and kinetic analysis
DNA-dependent ATP hydrolysis of human topoisomerase II α was monitored by measuring the production of inorganic phosphate using acidic molybdate and malachite green . Specific ATPase activity of every batch was tested in a time-course to determine optimal incubation times and enzyme concentrations within the linear range of the assay. Reactions thus contained 10 – 40 nM topoisomerase II (dimer concentration) in reaction buffer (10 mM Tris HCl pH 7.5, 175 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 2 mM DTT, 2.5% glycerol and 1.7 nM supercoiled pUC18). The specific activity of the human topoisomerase II α enzyme measured in the ATPase assay was 0.24 μmol PO4·min-1·mg-1. Compounds were initially screened at 10 μM concentration and IC50 values were determined if inhibition was greater than 70%. Compounds at the indicated concentrations were added as serial dilutions prepared in DMSO and preincubated for 10 minutes at 37°C on an orbital shaker. Reactions were started by the addition of 400 μM ATP (Sigma, catalog number A2383) and allowed to proceed for 30 minutes. Reactions were terminated by the addition of 200 μl malachite green reagent and the OD was read immediately at 630 nm on a SpectraMax Plus microtiter plate reader using SoftMax Pro software (Molecular Devices). For kinetic analysis, reactions were carried out in duplicate as described above either in the presence of vehicle (DMSO) or QAP 1. Following a preincubation for 5 minutes, ATP was added at the indicated concentrations to start the reactions, which were allowed to proceed for 17 minutes before stopping and reading as above. From these values the OD values obtained at each respective ATP concentration without enzyme were subtracted for background correction. The low signal to noise ratio of the malachite green assay did not permit the study of a broad range of inhibitor concentrations in competition experiments. Low inhibitor concentrations did not result in enough inhibition to clearly demonstrate the effect of the inhibitor and higher inhibitor concentrations gave very weak signals. A compromise was found by carrying out the competition experiments with a single concentration of the inhibitor that gave a good inhibition of enzyme activity while keeping a measurable signal. The equations used to fit these experiments are:
Each competition experiment was fitted (Grafit version 5.04 – Erithacus Software) with these 3 equations and the models that gave unrealistic inhibition constants (above 10 mM) and/or large standard errors (similar to the value of the fitted parameter) were rejected. Then, the F-test was carried out to identify the best fitting model. An alternative method was used to determine the mode of action of the inhibitor. Dose-response experiments were carried out at different ATP concentrations and the corresponding IC50
s determined. According to the Cheng-Prusoff equation for linear competitive inhibition [25
The IC50 of a competitive inhibitive inhibitor should increase in a linear fashion with the concentration of ATP. The measured IC50s were then plotted in function of the ATP concentrations and linear regression analyses were carried out. For calculation of Michaelis-Menten kinetic parameters  data were plotted in XLfit 4 (XLfit 4 curve fitting software for Microsoft Excel, ID Business Solutions Ltd, Guildford, Surrey, United Kingdom) and linearized according to Eadie-Hofstee. In the latter plot data points obtained at the lowest ATP concentration were omitted as these are the most error prone. Mean Km and Vmax values were calculated from two independent experiments.
DNA decatenation assay
Reagents for the assay were purchased from TopoGEN (catalog number 1001–1, TopoGEN, Inc., Port Orange, FL). Topoisomerase II was extracted according to the manufacturer's instructions from nuclei of HL-60 cells (ATCC; American Type Culture Collection, Manassas, Virginia, USA) and contained both isoforms as assessed by Western blotting. Calculation of specific topoisomerase II decatenation activity was based on complete decatenation of a given amount of catenated input DNA (100 ng kDNA) by a defined amount of nuclear extract containing the alpha and beta isoforms or of a defined amount of purified enzyme in a particular amount of time. The topoisomerase II activity from nuclei decatenated 0.07 ng catenated kDNA · min-1 · ng-1 extract. Purified topoisomerase II alpha (see above) decatenated 0.13 ng catenated kDNA · min-1 · ng-1. Vehicle (DMSO) or drug substance at concentrations to yield the desired end concentrations were added and samples were preincubated for 5 minutes at 37°C and 400 rpm in an Eppendorf thermomixer. Reactions were started by adding ATP (to 450 μM final concentration) to each sample and incubation was continued for 20 minutes. Reactions were terminated by placing the samples on ice and adding 4 μl stop/gel loading buffer. 20 μl of each sample were separated by 1% agarose gel electrophoresis. Gels were analyzed under a UV transilluminator and decatenated kDNA products were quantified using AlphaEaseFC (FluorChem 8900) image analysis software version 3.2.3 (Alpha Innotech, San Leandro, CA).
Topoisomerase II α and β were affinity precipitated with biotinylated inhibitor as follows. Exponentially growing HL-60 cells were collected on ice and incubated with hypotonic buffer (10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 1× protease inhibitor cocktail (Complete Mini, Roche Diagnostics GmbH)) for 15 minutes, then 0.6% v/v NP-40 was added and cells were disrupted using a Dounce homogenizer. Lysates were transferred to 1.5 ml Protein LoBind Eppendorf tubes and nuclei were collected after centrifugation at 8000 rpm for 10 minutes and washed twice with hypotonic buffer. To extract and release chromatin associated topoisomerase II nuclei were incubated for 30 minutes in DNAse buffer (20 mM Tris HCl pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 25 mM NaCl, 0.5 mM EDTA, 1% NP-40, 1 mM DTT, 1 × protease inhibitor cocktail, 0.1 mM PMSF) containing 40 U DNAse I (Roche Diagnostics GmbH) followed by addition of twice concentrated nuclear extraction buffer (20 mM HEPES pH 7.5, 3 mM MgCl2, 20 mM KCl, 50 mM β-glycerophosphate, 840 mM NaCl, 1 mM EDTA, 50 mM PNPP, 1 mM DTT, 1 mM PMSF) and rotation for 1 h in the coldroom. The soluble fraction was obtained following centrifugation at 13000 rpm for 15 minutes. 25 μg nuclear extract were used for the affinity precipitation. The salt concentration was adjusted to 150 mM with nuclear extraction buffer (without NaCl) and the sample volume was toped off to 200 μl with precipitation buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 25 mM β-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 15 mM PPI, 1 mM PMSF). Compounds were preincubated for 30 minutes prior addition of biotinylated purine analogue 2 for 2 h. Streptavidin agarose beads (Novagen, 69203) were washed twice with PBS, blocked for 30 minutes with PBS containing 1% BSA, washed twice with PBS and finally with precipitation buffer. 40 μl equilibrated streptavidin agarose beads were added to the sample and incubation was continued for 1 h. Beads were pelleted by centrifugation at 7000 rpm for 5 minutes and washed thrice with precipitation buffer. During the last wash the beads were transferred to a new tube and the bound fraction was released by boiling for 5 minutes in Laemmli sample buffer. Topoisomerase II α and β were detected by Western blot as described below.
Topoisomerase II α and β siRNA
0.3 × 106 HeLa T-Rex cells (Invitrogen, Paisley, United Kingdom UK) per 6-cm plate were seeded in 2.6 ml of medium without antibiotics. The day after seeding, cells were transfected for 72 hours with stealth RNA oligos (Invitrogen, Paisley, United Kingdom UK): stealth control oligo 1: 5'ACUUCCGAUUCGUGUAACCGAC UUU3'; and stealth control oligo 2: 5'GGGCUA GUUAUAGUCGACACUGGUA3') or with stealth RNA oligos directed against topoisomerase II α and β transcripts (α1: 5'ACUCAGCCUCUUAUGUGCCAAGUUU3'; α2: GGACAACAUUUG AUCCAAGAUCUUA3' and β1: 5'GGGUGAUCUUGAUACUGCAGCAGUA3'). Lipofectamine™ 2000 was used to transfect oligos according to the manufacturer's instructions. Lipofectamine containing medium was replaced 6 hours after transfection by 3 ml of fresh culture medium. Cells were extracted in 200 μl lysis buffer (50 mM Tris-HCl, pH 7.5; 5 mM EDTA; 420 mM NaCl; 0.5% Nonidet P-40 (NP-40); 1 mM Benzamidine; 20 mM NaF and 1.5 mM MgCl2) freshly supplemented with 1 mM PMSF (from 100 mM stock solution in ethanol), 1 mM DTT (from 1 M stock solution) and 1× protease inhibitor cocktail. Lysates were centrifuged at maximum speed in an Eppendorf centrifuge for 10 minutes at 4°C. The nuclear pellet was resuspended in 22 μl of Laemmli sample buffer and boiled at 95°C for 5 minutes. 15 μl of nuclear extracts were subjected to 6% denaturing SDS-PAGE, transferred to PVDF membranes and probed with the anti-topoisomerase II β antibody (sc-13059, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:1000 in PBS 0.1% Tween) using appropriate secondary antibody and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech Inc., Buckinghamshire, United Kingdom). After stripping the membrane it was re-probed with a goat anti-topoisomerase II α antibody (sc-5348, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:2000 in PBS 0.1% Tween). Immunoreactive bands were revealed by HRP-conjugated protein G (Zymed, CA; diluted 1:3000 in PBS 0.1% Tween) using ECL reagents.
Histone extracts of HL-60 or HeLa T-Rex cells for Western blot analysis were prepared as follows: Following treatments, medium was removed and cells were collected/washed with PBS. Cells were centrifuged for 5 minutes at 1500 rpm and 4°C and the cell pellet was resuspended in 300 μl of ice-cold hypotonic buffer (see above). Cells were transferred in a 1.5 ml Eppendorf tube and incubated for 15 minutes on ice. Thereafter, NP-40 was added to the cells to a final concentration of 0.6% (v/v) and cells were disrupted by pumping 10–15 times through a 1 ml syringe connected to a 23-gauge needle. The extract was centrifuged for 10 minutes at 8000 rpm and 4°C. The supernatant (cytoplasmic fraction) was removed and the nuclear pellet was washed once with 500 μl of ice-cold hypotonic buffer for 4 minutes at 8000 rpm. Nuclear proteins were extracted by addition of 22 μl sample buffer and boiling for 3 minutes. Samples were resolved by 15% SDS-PAGE following transfer to PVDF membrane. After blocking, membranes were incubated either with anti-γH2AX (clone JBW301, 05–636, Upstate, Charlottesville, VA, diluted 1:1000 in PBS containing 0.1% Tween 20 and 5% milk) or histone H3 (catalog number 9715, Cell Signaling Technologies Inc., Beverly, MA, diluted 1:1000 in TBS containing 0.1% Tween 20 and 5% milk) antibodies overnight at 4°C. The corresponding horseradish peroxidase conjugated secondary antibodies were diluted 1:3000 and incubated for 1 hour at room temperature and immunoreactive bands were revealed as described above.
In-Cell Western analysis was carried out as follows. HeLa T-Rex cells were seeded at the cell density of 15 × 103 cells per well in black 96-well plates (Greiner Bio-one Vacuette, St Gallen, Switzerland). The day post-seeding, cells were pretreated with increasing concentrations of either ICRF-193, QAP 1 or vehicle (DMSO) for 30 minutes followed by a 2 hours treatment with 1 μM of doxorubicin, or were only treated with the same concentrations of ICRF-193, QAP 1 or vehicle (DMSO) for 2 hours. Treatments were done in triplicate. Cells were directly fixed by adding 100 μl of 3.7% formaldehyde (diluted 1:10 in PBS from a 37% formaldehyde solution; Sigma, Buchs, Switzerland) per well for 10 minutes at room temperature. Cell membranes were permeabilized by 4 washes with 100 μl per well of PBS containing 0.25% Tween-20 for 4 minutes. Cells were blocked with 50 μl per well of Odyssey Blocking Buffer (OBB; LI-COR Biosciences GmbH, Bad Homburg, Germany) for 2 hours at room temperature with gentle agitation. 25 μl per well of γH2AX antibody (clone JBW301, catalogue number 05–636, Upstate, NY) diluted 1:500 in OBB were incubated overnight at room temperature with gentle agitation. Cells were washed 5 times with 50 μl per well of PBS containing 0.1% Tween-20 for 3 minutes and stained with 25 μl per well of goat anti-mouse InfraRedDye 800 antibody (Rocklands Immunochemicals, Inc., Gilbertsville, PA) diluted 1:800 in OBB containing 0.2% Tween-20 for 1 hour at room temperature in the dark. An additional wash with 50 μl per well of PBS containing 0.1% Tween-20 was performed before DNA staining with 50 μl per well of TO-PRO-3 iodide (642/661) (Invitrogen AG, Basel, Switzerland) diluted 1:5000 in OBB for 1 hour at room temperature in the dark. Finally, cells were washed 4 times with 50 μl per well of PBS containing 0.1% Tween-20 for 3 minutes, liquid was removed and plates were kept at 4°C until reading. γH2AX signals were read using the LI-COR Odyssey Infrared Imager with the Odyssey v1.1 software using the 800 nm channel. Blank values correspond to non-specific γH2AX signal detected in wells without primary antibody staining. The means of these values were subtracted from the γH2AX signals to correct for non-specific background. The resulting values for each sample were normalized to the DNA quantity by measuring the corresponding TO-PRO-3 iodide signals. The TO-PRO-3 iodine signals were detected by using the 700 nm channel. The percentage of γH2AX signal was determined for each concentration of ICRF193 and QAP 1 with or without doxorubicin treatment by setting the value for the treatment with vehicle and 1 μM doxorubicin to 100%. To display relative suppression of γH2AX induction and catalytic topoisomerase II inhibition in cells the values measured in the presence of catalytic inhibitor were subtracted from the corresponding value measured at the same concentration of catalytic inhibitor but in the presence of 1 μM doxorubicin. These data were plotted in XLfit 4 (XLfit 4 curve fitting software for Microsoft Excel, ID Business Solutions Ltd, Guildford, Surrey, United Kingdom) to determine the IC50 values of the catalytic topoisomerase II inhibitors by using four parameter logistic calculation.
Chromosome segregation assay
HL-60 cells were treated overnight for 16 hours with 2 μg/ml aphidicolin (Sigma-Aldrich corporation, St. Louis, MO) and released by washing twice PBS and resuspending into fresh medium. After 6 hours release the cells were treated with 1 mM caffeine (Lancaster Synthesis, Heysham, Lancashire, UK) and with catalytic topoisomerase II inhibitor or vehicle for 2 hours. Cells were washed once with PBS and incubated with 56 mM KCl hypotonic solution for 30 minutes. Cells were pelleted and carefully resuspended in Carnoy's fixative. Cells were pelleted as above, supernatant was removed and cells were resuspended again in 2 ml fixative. Chromosome spreads were prepared by dropping 50 μl cell suspension onto microscopy slides. The slides were allowed to dry and then stained for 10 seconds in Hoechst 33342 dye (Molecular Probes, Eugene, Oregon, prepared 1:1000 in PBS from 10 mg/ml stock). After drying, mounting media (Vectashield®, Vector Laboratories, Burlingame, CA) and a cover slip were added. Samples were observed using an immersion oil lens (100 ×, Nikon, Plan Apo VC) by fluorescence microscopy (Nikon Eclipse, E600) in the DAPI channel. Images were acquired using analySIS® software (Soft Imaging System GmbH, Münster, Germany). Representative images were converted to grayscale, inverted and contrast adjusted with Adobe Photoshop. Mitotic cells were categorized as described .
Clonogenic growth assay with HCC1937 cells
HCC1937 BRCA1 mutant and HCC1937 BRCA1 reconstituted breast ductal carcinoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/l glucose and 1% (v/v) penicillin/streptomycin. Status of BRCA1 was corroborated by immunoprecipitation using 50 μg nuclear extracts and 1 μg mouse monoclonal antibody MS110 (ab16780, Abcam Inc., Cambridge, MA), which is directed towards the amino-terminus of the protein. Blots were probed with BRCA1 carboxy-terminal specific mouse monoclonal antibody D9 (sc-6954, Santa Cruz Biotechnologies, Santa Cruz, CA) and, following stripping, re-probed with antibody MS110. For assays, 5000 cells in 3 ml medium were seeded per well of 6 well plates in duplicates. The day after, vehicle (DMSO) or compounds were added. After one week medium was exchanged and vehicle or compounds were re-added for an additional two days. To assess proliferation the medium in each well was replaced with 1 ml medium containing 100 μl WST-1 reagent and incubation for 20 minutes at 37°C. Of each sample 110 μl supernatant were transferred into a 96 well plate and the OD was read at 450 nm (filter at 630 nm) on a SpectraMax Plus microtiter plate reader using SoftMax Pro software (Molecular Devices). For background correction, the blank value consisted of 100 μl medium containing 10 μl WST-1. Thereafter, colonies were stained by removing medium, washing cells once with PBS and incubation with 1 ml crystal violet solution for 30 minutes at room temperature. Cells were washed twice with PBS and colonies were counted using an Artec Counter (Model 880, Artec, Dyntatech Laboratories Inc.) with diameter cut-off set to 0.2 mm. Data were expressed as growth or colony numbers relative to vehicle control treatments (100%).