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.

Total RNA from normal human ovary tissue was purchased 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 manufacturer's protocol. Semi-quantitative RT-PCR was performed 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 β-actin, c-Fos and each HOX gene. Relative expression was calculated using the Livak comparative Ct method (2^-ΔΔCt) 10 .

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 detection kit (BD Pharmingen).

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 (120 μM). The cells were washed twice with PBS, visualised 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 incubated 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).

SK-OV3 cells were seeded at a concentration of 4 × 10⁴ cells/ml on 60 mm tissue culture dishes and were incubated 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 buffer containing protease and phosphatase inhibitor cocktails 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 transferred 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.

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 pressure 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 subsequent 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 treatment group to allow the amount of HXR9 peptide in tumours to be assessed. Tumours were excised from three mice at 3, 12 and 24 hours after iv administration of 100 mg/Kg HXR9. Total cellular protein was extracted and 10 μg were dot blotted on nitrocellulose membrane and probed with a rabbit polyclonal anti-HXR9 antibody. Quantification was achieved using a standard series of HXR9 dilutions.

Data are given as means ± SEM of at least three independent experiments. Significant effects were determined by 2-tailed Student's t tests (p < 0.05), or the Mann-Whitney test for mouse tumour modelling experiments. For the analysis of the QPCR for HOX gene expression the Bonferroni correction was applied to account for multiple comparisons. There are 39 HOX genes thus the limit for a significant result is p < 0.0013.

Previous studies have examined the expression of HOX genes in ovarian tumours but to date this had not been done in a comprehensive manner, looking at all 39 members of this family. Thus, we measured the relative expression of HOX genes in normal ovarian tissue and in the derived cell lines SK-OV3 and OV-90 using semi-quantitative PCR (Fig 1). This revealed that multiple HOX genes had a dysregulated pattern of expression in SK-OV3 cells, but generally not in OV-90 cells, in comparison to the normal ovary. In SK-OV3 the A family of the HOX genes showed a statistically significant up regulation of HOXA5, HOXA9, and HOXA13. The HOXB and HOXD families were mainly expressed only in SK-OV3 cells; HOXB5, HOXD9, and HOXD11 showed significantly increased expression.

In order to assess the efficacy of HXR9 in vivo we established a xenograft model of SK-OV3 in nude mice by injecting cells into the flank. When the average tumour volume had reached 100 mm³ mice were given an initial dose of HXR9 of 100 mg/Kg iv, followed by twice weekly iv treatments at 10 mg/Kg. Tumour growth in HXR9 treated mice was retarded by 46% at 32 days compared to the control group (Fig 7a). RNA was extracted from tumours at the end of 32 days in order to measure cFos expression by QPCR; cFos was found to be expressed at an 18-fold higher level in tumours from HXR9 treated mice than in tumours from untreated mice (Fig 6).

The uptake of HXR9 by tumours after the initial dose was measured by excising tumours at 4, 12 and 24 hours and assessing the amount of HXR9 using an anti-HXR9 antibody. This indicated that there is a rapid accumulation of peptide in the first four hours and that the amount of peptide remains stable beyond 24 hours (Fig 7b).