BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 FHiig2u-rEeG5FP fusion protein is secreted from transfected cells Hig2-EGFP fusion protein is secreted from transfected cells. Western blot analysis of total cell lysates and conditioned medium from (1) untransfected HEK293 cells and cells trans- fected with plasmids encoding (2) EGFP, (3) Hig2-EGFP, (4) Hig2(Y8V9/D8D9)EGFP. Oligomers of HIG2-EGFP formed by intermolecular disulphide bridges are indicated by arrowheads. secreted protein is not glycosylated (data not shown). Pre- vious studies using GFP fused to a signal peptide directing entry into the ER demonstrated that, in this redox environment, the cysteine residues of GFP form intermolecular disulphide bridges which result in oligomerization of GFP molecules [35]. Oligomers of Hig2-EGFP were detected (Fig 5, black arrowheads) but no oligomerization of EGFP was observed. Hig2 itself does not contain cysteine residues, thus the oligomeriza- tion is mediated by the EGFP domains. These data are consistent with HIG2-EGFP entry into the classical secre- tory pathway. Collectively, these data demonstrate that Hig2 contains a functional N-terminal signal peptide and is likely a secreted protein. Expression of HIG2 in human tumours To investigate the relevance of HIG2 in human tumours, the expression level of this gene was examined in 68 tumour cDNA samples compared to normal adjacent tis- sue from the same patients using a Matched Tumour/Nor- mal cDNA blot (Clontech) (Figure 6A). The levels of HIG2 were approximately similar in most of the tumour types examined but were strongly and consistently downregu- (a6Far8i)rgapAuyarntceaieolyn6nsttisasinoifnHgImG2atcmhReNdAtumexopurresasniodnnornmaahlusammapnlecsDfNroAm (a) Analysis of HIG2 mRNA expression on a human cDNA array containing matched tumour and normal samples from 68 patients. For each pair, the tumour sample is on top. The bottom row of the array contains cDNA from cell lines (left to right: HeLa, Daudi, K562, HL-60, G361, A549, MOLT-4, SW480 and Raji). (b) Quantitative analysis by phosphorim- ager. Vertical dashed lines indicate ± 2-fold difference. HIG2 expression is broadly similar in most pairs, but is strongly and consistently downregulated in most of the kidney and stom- ach tumours analysed. lated in most of the cases of kidney and stomach tumours analysed. These data suggest that the downregulation of HIG2 observed upon deregulated β-catenin signalling in vitro may be of clinical relevance in human tumours. Discussion cDNA microarray analysis of the transcriptional changes resulting from overexpression of a constitutively active β- catenin revealed a panel of putative target genes of the Wnt/β-catenin pathway in mouse mammary epithelial cells. This differential expression was confirmed by North- ern blotting in five cases. Autotaxin was originally identified as a secreted enzyme with potent motility stimulating activity [36] and has Page 6 of 9 (page number not for citation purposes)