Morgan et al. BMC Cancer 2010, 10:89 http://www.biomedcentral.com/1471-2407/10/89 the PBX-binding peptide HXR9 [6] triggers apoptosis in SK-OV3 cells both in vitro and in vivo. Methods Maintenance of SK-OV3 and OV-90 in culture The human ovarian adenocarcinoma-derived cell lines SK-OV3 and OV-90 were obtained from the American Type Culture Collection (LGC Promochem, Teddington, UK). The cells were cultured in McCoy’s 5A modified medium (Sigma, Poole, UK) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (Invitrogen Ltd, Paisley, UK) and 1% penicillin (10,000 U/ml)/ streptomycin (10 mg/ml) (Sigma). Cell cultures were maintained at 37°C in a humidified, 5% CO2 incubator. Semi quantitative PCR Total RNA from normal human ovary tissue was pur- chased from Applied Biosystems (Warrington, UK). RNA was isolated from SK-OV3 and OV-90 cells using the RNeasy® Plus Mini Kit (Qiagen Ltd, Crawley, UK) according to the manufacturer’s instructions. cDNA was synthesised from RNA using the Cloned AMV First Strand Synthesis Kit (Invitrogen) following the manufac- turer’s protocol. Semi-quantitative RT-PCR was per- formed using the Stratagene MX3005P Real Time PCR machine (Agilent Technologies UK Ltd, Stockport, UK) and SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma). Oligonucleotide primers were designed to facilitate the unique amplification of b-actin, c-Fos and each HOX gene. Relative expression was calculated using the Livak comparative Ct method (2-ΔΔCt) [10]. Analysis of cell death and apoptosis Cell viability was measured via the MTS assay (Promega, Southampton, UK) according to the manufacturer’s instructions. Briefly, cells were plated in a 96-well plate at a concentration of 1 × 105 cells/ml and incubated for 24 hours. Cells were treated overnight with the active peptide HXR9 or the control peptide CXR9 at a range of dilutions. The IC50 of the cells was determined by plotting a dose-response curve. To detect morphological changes consistent with apoptosis, cells were harvested by incubating in trypsin-EDTA (Sigma) at 37°C until detached and dissociated. Apoptotic cells were identified using a Beckman Coulter Epics XL flow cytometer (argon laser, excitation wavelength 488 nm, FL-2 and FL-4 detectors) and the Annexin V-PE apoptosis detec- tion kit (BD Pharmingen). Imaging of the cells For phase contrast micrographs, cells were plated into 60 mm tissue culture dishes at a concentration of 1 × 105 cells/ml and incubated at 37°C for 24 hours. Cells were treated overnight with HXR9 (120 μM) or CXR9 Page 2 of 9 (120 μM). The cells were washed twice with PBS, visua- lised using a Nikon Eclipse TE100 inverted microscope and images recorded using a Nikon DS-L camera and capture software (Nikon Instruments Europe B.V., Amstelveen, The Netherlands). For fluorescent images of HXR9 localisation, 35 mm culture dishes were seeded with 2 × 105 cells and incu- bated as previously described. The cultures were treated with HXR9-FITC conjugate (30 μM) for 1 hour. The cells were fixed with 10% formalin in neutral-buffered saline (Sigma). Cells were visualised using a Nikon Eclipse TE 2000-S fluorescent microscope and images recorded using a DMX 1200F camera and NIS Elements capture software (Nikon Instruments). Detection of PARP cleavage by western blotting SK-OV3 cells were seeded at a concentration of 4 × 104 cells/ml on 60 mm tissue culture dishes and were incu- bated at 37°C. When cells approached 75% confluence, cultures were treated overnight with HXR9 (120 μM) or CXR9 (120 μM). Lysates were prepared with RIPA buf- fer containing protease and phosphatase inhibitor cock- tails and EDTA (Perbio Science UK Ltd, Cramlington, UK). The protein concentration was quantified using the bicinchoninic acid assay (Perbio) according to the manufacturer’s instructions. Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and trans- ferred to polyvinylidene fluoride membrane. Anti-PARP antibody (Biomol International, Exeter, UK) was used to detect both cleaved and full-length PARP. To visualise proteins, the ECL Western blotting detection system (Amersham Biosciences, Little Chalfont, UK) was used. Mice and in vivo trial All animal experiments were conducted in accordance with the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the Welfare of Animals in Experimental Neoplasia [12] and approved by the St. George’s Hospital Medical School Ethical Review Committee. The mice were kept in positive pres- sure isolators in 12 hour light/dark cycles and food and water were available ad libitum. Athymic nude mice were inoculated subcutaneously with a suspension of 2.5 × 106 SK-OV3 cells in culture media (100 μl). Once tumours reached volumes of approximately 100 mm3, mice received an initial dose of 100 mg/Kg CXR9 or HXR9 intravenously, with subse- quent dosing of 10 mg/Kg twice weekly. Each treatment group contained 10 mice. The mice were monitored carefully for signs of distress, including behavioural changes and weight loss. An additional 10 mice were included in each treat- ment group to allow the amount of HXR9 peptide in tumours to be assessed. Tumours were excised from