Morgan et al. BMC Cancer 2010, 10:89 http://www.biomedcentral.com/1471-2407/10/89 only in SK-OV3 cells; HOXB5, HOXD9, and HOXD11 showed significantly increased expression. Targeting HOX/PBX dimers in SK-OV3 and OV-90 Results from previous studies [9,11] together with our data show that many HOX genes are upregulated in ovarian cancer. We therefore decided to study the role of HOX genes in SK-OV3 and OV-90 cells by interfer- ing with HOX function. Previous work has shown that disrupting the interaction between HOX proteins and their PBX co-factor can trigger apoptosis in melanoma and renal cancer cells [6,7]. PBX binding to HOX modi- fies the selection of DNA binding sites and hence the identity of HOX target genes. A short peptide, HXR9, was previously shown to disrupt the formation of HOX/ PBX dimers, alter transcriptional regulation by HOX proteins and thereby induce apoptosis [6,7]. HXR9 con- tains nine repeated arginine residues to facilitate cellular uptake, probably through endocytosis [13]. In order to show that HXR9 was able to enter cells, SK-OV3 cul- tures were incubated with a fluorescently labelled ver- sion of the peptide. This indicated that HXR9 was indeed taken up by SK-OV3 cells and tended to accu- mulate in the nucleus (Fig 2). Furthermore, HXR9 killed SK-OV3 cells with an IC50 of approximately 70 μM, although it did not kill OV-90 cells (Fig 3a). Cell death in SK-OV3 cells was accompanied by characteristic changes in cellular morphology (Fig 3b). HXR9 treatment causes apoptosis Flow cytometric analysis was used to distinguish between apoptotic and necrotic cell death. SK-OV3 cells Page 4 of 9 were stained with Annexin-V-PE to detect the externali- sation of phosphatidylserine, which occurs during the early stages of apoptosis. Cells were counterstained with the vital dye 7AAD. When compared with controls, a significantly higher proportion of SK-OV3 cells treated with HXR9 were shown to be in late apoptosis (P < 0.001) but few cells were found to be in the early apop- totic or necrotic state (Fig 4). Further, immunoblotting indicated that there was a reduction in full-length PARP in HXR9-treated SK-OV3 cells (Fig 5); PARP cleavage is another characteristic event of apoptosis [14]. Previous studies have shown that upregulation of cFos is a key event in HXR9-mediated apoptosis in both mel- anoma and renal cancer cells [6,7]. Semi-quantitative PCR revealed that cFos expression is on average 50 times greater in SK-OV3 cells treated with HXR9 (Fig 6) than in either untreated or CXR9-treated cells, whilst HXR9 treatment of OV-90 cells did not cause a signifi- cant increase in cFos expression (Fig 6). HXR9 retards tumour growth in vivo In order to assess the efficacy of HXR9 in vivo we estab- lished 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 Figure 2 Uptake of fluorescently labelled HXR9 by SK-OV3 cells. Cells were incubated for one hour with an HXR9-FITC conjugate (30 μM) (green). HXR9 is confined to the nucleus in the majority of cells.