BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 dGFaiegsneuerarenadt1io∆nNoβf-sctaatbelneinHiCn 1a1tecterllacliyncelsineexdperpesesnindgenβt-gmaalancnteorsi- Generation of stable HC11 cell lines expressing β-galactosi- dase and ∆ Nβ-catenin in a tetracycline dependent manner. (a) HC11-lacZ cells – β-galactosidase activity of cell lysates following incubation for three days at the indicated tetracy- cline concentrations. β-galactosidase expression is effectively repressed at 20 ng/ml tetracycline. The assay was carried out in triplicate and results are presented as the mean β-galactos- idase activity (normalised to the protein concentration of the samples) ± standard error. (b) Western blot of total cell lysates from HC11-∆Nβ-catenin cells cultured ± tetracycline. The N-terminal deletion mutant of β-catenin is detected by both its myc-epitope tag and the anti-β-catenin antibody. tTA was detected only in the absence of tetracycline, confirming the autoregulatory nature of the induction system. gFCeiognneufsrireimn2arteiospnoonfseditffoer∆eNntβia-lceatxepnrienssoivoenreoxfppruetsastioven target Confirmation of differential expression of putative target genes in response to ∆Nβ-catenin overexpression. The expression level of each gene in the HC11-lacZ (L) and HC11-∆Nβ-catenin (∆N) cell lines was assessed by Northern blotting. The fold-induction was quantitated using a phos- phorimager under non-saturating conditions, normalised to the expression of GAPDH mRNA in the samples and is expressed as fold change relative to the expression level of that gene in the HC11-lacZ cell line. Validation of targets Five genes were selected for further study – Extracellular Matrix Protein 1 (Ecm1), Autotaxin, Receptor Activity Modify- ing Protein 3 (Ramp3), Cd14 and Hypoxia Inducible Gene 2 (Hig2). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ∆Nβ-catenin (Figure 2). RNA samples used for Northern blotting were from independent induction experiments to those used for microarray analysis, thus demonstrating repeatedly by two distinct methods that the transcript levels of these genes are altered in cells overexpressing ∆Nβ-catenin. The expression level of each of the transcripts was quantitated using a phosphorimager and normalised to the expression of Gapdh mRNA in the samples. The data in Figure 2 rep- resent film exposure times ranging between 24 and 72 hours. Quantitations were performed using short (one hour or less) exposures to a phosphorimager screen, such that the signal intensity was not saturating. Molecular cloning of mouse Hig2 Hypoxia Inducible Gene 2 encodes a 63 amino acid polypeptide and was one of several genes identified in a screen for genes regulated by hypoxia in a human cervical epithelial cell line [31]. HIG2 shares no sequence similar- ity with other known proteins. In order to facilitate the functional analysis of this gene, ESTs were identified which encoded mouse and rat Hig2, and the sequences of chimpanzee and baboon were inferred from genomic sequence data. A multiple alignment of the inferred amino acid sequences shows that these polypeptides are highly similar (Fig 3A). Analysis of these sequences using a Kyte-Doolittle hydrophobicity plot showed that the N- termini of these proteins contain a series of hydrophobic amino acids (Fig 3B). This region of hydrophobicity was reminiscent of a signal peptide and sequence analysis using the signal peptide prediction program, SignalP [32,33] supported this possibility. Hig2 has an N-terminal signal peptide and is secreted To investigate the subcellular localisation of Hig2, a Hig2- EGFP fusion gene was constructed and expressed in both HC11 and Madin-Darby Canine Kidney (MDCK) cells by transient transfection (Figure 4a and 4c). In both cell lines, Hig2-EGFP is localised to large round vesicle-like structures in the cytoplasm. Similar observations were made in HEK-293 cells (data not shown). The fluores- cence was detected predominantly around the periphery of these structures suggesting that they do not consist of solid masses of aggregated protein. When two aspartate residues were introduced to the putative signal peptide by site-directed mutagenesis, Hig2(Y8V9/D8D9)EGFP, this distinctive subcellular localization was abolished (Fig 4B and 4D). These large structures did not colocalize with either markers of mitochondria (pDsRed2-mito, Clon- tech), nor lipid droplets (Nile Red, Molecular Probes) nor with markers of endosomes or lysosomes (pulse-chase analysis with TRITC-dextran); data not shown. However, in live HC11 cells transfected with Hig2-EGFP, observa- tions at high magnification revealed that the cytoplasm of Page 4 of 9 (page number not for citation purposes)