BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 Background The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organo- genesis [1]. Wnt-1 is the prototypic member of this large family of secreted glycoproteins and was originally identi- fied as a gene insertionally activated by mouse mammary tumour virus [2]. Wnt-1 is one of a number of Wnt family members which act to control the cellular level of β-cat- enin. Wnt proteins bind seven-pass transmembrane receptors of the Frizzled family, and a signal is transduced via Dishevelled to a complex which contains the Adeno- matous Polyposis Coli (APC), Axin and Glycogen Syn- thase Kinase-3β (GSK-3β) proteins [3,4]. This signal antagonizes the phosphorylation of β-catenin by GSK-3β. There are four phosphorylation sites in the N-terminus of β-catenin which, in the absence of Wnt signal, are phos- phorylated by Casein Kinase I alpha and GSK-3β [5,6]. This phosphorylation leads to the ubiquitination and sub- sequent proteasomal degradation of β-catenin [7]. Inhibi- tion of β-catenin phosphorylation by Wnt signalling leads to the accumulation of β-catenin which forms a bipartite complex with members of the TCF/LEF transcription fac- tor family and activates the transcription of target genes, a process which is regulated by multiple interacting factors [8]. Overexpression of Wnt-1 in the mammary glands of trans- genic mice leads to extensive hyperplasia and tumorigen- esis [9]. APC was identified as the tumour suppressor gene mutated in the hereditary colorectal cancer syndrome, Familial Adenomatous Polyposis [10,11]. Mutations in Axin and β-catenin have also been detected in tumours of the colon and other tissues [12]. Deregulation of this pathway appears to be play a contributory role in a signif- icant proportion of human tumours of epithelial origin and hence, the identification of effector genes of this path- way is an important step towards the elucidation of the mechanisms involved. Many of the Wnt targets thus far identified are cell-cycle regulators [13,14] and transcription factors [15-20], and function in a cell-autonomous manner, providing insight into the mechanisms by which tumour cells deregulate proliferation and inhibit apoptosis. Tumours are complex organs composed of tumour cells, stromal fibroblasts, endothelial cells and cells of the immune system; and reciprocal interactions between these cell types in the tumour microenvironment are necessary for tumour growth [21,22]. Here we postulate that proteins secreted by Wnt/β-catenin tumour cells and receptors expressed by these cells may play roles in mediating interactions between neighbouring tumour cells or between tumour cells and their microenvironment. Consequently, in this study we have focussed our attention on identifying novel genes encoding receptors and secreted proteins. Methods Cell culture All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cul- tured in 5% CO2 at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 µg/ml insulin, 5 ng/ml epidermal growth factor and 50 µg/ml gentamycin [23]. HC11-lacZ and HC11-∆Nβ-cat- enin cells were routinely cultured in 2 µg/ml tetracycline to repress transcription of the tetracycline-regulated trans- gene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum. The HC11-lacZ and HC11-∆Nβ-catenin cell lines were generated by infecting the cells with an ecotropic retrovi- rus (TRE-tTA) in which the tTA cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own tran- scription in an autoregulatory manner [24]. HC11 cells expressing tTA were subsequently infected with ecotropic retroviruses derived from RevTRE (Clontech) which directed the expression of either β-galactosidase or ∆Nβ- catenin in a tetracycline dependent manner. Bosc23 cells were used to produce ecotropic retroviruses [25]. Cells were transiently transfected with the appropri- ate retroviral construct and the supernatant was collected 48 hours post-transfection. Polybrene was added to a final concentration of 5 µg/ml and the supernatant was added to HC11 cells for 24 hours. HC11 cells were then sub- jected to antibiotic selection using either 250 µg/ml G418 or 200 µg/ml hygromycin B as appropriate. RNA isolation Cell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitro- gen). Total RNA was isolated according to the manufac- turer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufac- turer's instructions. Northern blotting 10 µg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech) in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) forma- mide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 µg/ml denatured herring sperm DNA. Radiola- belled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Page 2 of 9 (page number not for citation purposes)