The Wnt/Wg signal transduction pathway is an evolutionarily conserved pathway that plays an important role in the developmental program of many organisms (for review see [1–4]). Genetic epistasis tests in Drosophila, in combination with biochemical experiments performed in Xenopus and in tissue culture cell lines, have contributed much information to the molecular mechanisms underlying Wnt signaling. In recent years, the discovery of the genes and their protein products that are involved in Wnt signaling has led to the development of a complex signaling network that includes various scaffolding proteins, kinases, and transcription factors. For a detailed description of the proteins involved, please see The Wnt Genes Webpage . With respect to the canonical Wnt/β-catenin pathway, in the absence of Wnt/Wg signaling, cells maintain low cytoplasmic levels of the oncoprotein β-catenin or Armadillo (Arm), its Drosophila homolog. β-catenin is a multifunctional molecule that acts at the plasma membrane in adherens junctions, can be found in the cytoplasm, and is also detected in the nucleus as part of a transcription factor complex. Upon phosphorylations at its amino terminus by an isoform of casein kinase 1 (CK1) and by glycogen synthase kinase-3β (GSK-3β), β-catenin is targeted for ubiquitin-mediated degradation by the "destruction complex" consisting of GSK-3β, the tumor suppressor protein adenomatous polyposis coli (APC), axin, and a member of the SCF ubiquitin ligase complex, β-TrCP/Slimb [6, 7]. In order to initiate signaling, Wnt must bind to its receptor Frizzled and its co-receptor LRP, leading to β-catenin stabilization. β-catenin levels now rise in the cytoplasm, reaching a critical amount that enables β-catenin to translocate to the nucleus where it interacts with Lef/TCF [8, 9] and activates target gene expression.
Besides playing a role in differentiation and development, the Wnt/Wg signaling pathway has also been implicated in tumorigenesis . The Wnt-1 oncogene was originally discovered as int-1, a gene whose activation upon insertion of the mouse mammary tumor virus results in the formation of mouse mammary tumors [10, 11]. Although Wnt-1 is not expressed in the normal mouse mammary gland, expression of Wnt-1 in transgenic mice results in the formation of mammary tumors. In addition, overexpression of Wnt-1 in mouse mammary epithelial cell lines such as C57MG or RAC311 results in their transformation . Furthermore, in Rat1 fibroblasts, Wnt-1 overexpression induces serum-independent cellular growth in a manner that correlates with an increase in cytoplasmic β-catenin . These results suggest that perhaps Wnt-1-mediated transformation occurs through an increase in cytoplasmic β-catenin levels because transformation can take place in the absence of Wnt-1 signaling upon overexpression of a stable form of β-catenin. Other cell lines, such as NIH3T3 cells, have also been employed to study the response of endogenous β-catenin to Wnt-1 signaling and have arrived at similar conclusions: Wnt-1 signaling allows for accumulation of β-catenin to levels that enable it to form a complex with Lef/TCF proteins to activate transcription .
Expression of some Wnts has been correlated with the development of human cancer. Recently, increased expression of Wnt-1 mRNA was observed in cell lines derived from a human gastric cancer (OKAJIMA), pancreatic cancer (BxPC-3), and in 50% of primary gastric cancers . The Wnt 2 gene is up-regulated at the mRNA level in gastric and esophageal carcinomas as well as in colorectal tumors at various stages . Wnt-5a mRNA is expressed at very low levels in breast cell lines and normal breast tissue, but benign proliferations and invasive cancers exhibit a ten-fold and four-fold higher level of Wnt-5a mRNA, respectively . Wnt-5a is also up-regulated at the RNA level in lung, breast, prostate carcinomas and melanomas . Wnt10B mRNA is elevated in primary breast carcinomas and in several noncancerous and cancerous breast cell lines . Finally, Wnt-13 mRNA is expressed in several adult tissues and in three human cancer cell lines: HeLa cells (cervical cancer) and in MKN28 and MKN74 (gastric cancer), potentially implicating this gene in tumorigenesis . Thus, the up-regulation of Wnts is correlated with the development of multiple tumor types, providing additional evidence that Wnt proteins and the pathway(s) they regulate play an important role in the control of cell growth and differentiation.
Mutations in several components of the Wnt signaling pathway, such as APC and β-catenin, have also been linked to tumorigenesis. APC is a tumor suppressor gene that is mutated in up to 80% of human colon carcinomas . Mutations of APC result in the development of a form of inherited colon carcinoma called Familial Adenomatous Polyposis (FAP). Individuals with FAP develop multiple colonic polyps throughout their life, predisposing them to colon cancer. Elevated levels of β-catenin can also contribute to tumorigenesis. High levels of β-catenin are associated with several human cancers, including colon carcinomas, melanomas, pilomatricomas and hepatocellular carcinomas, either due to a nonfunctioning APC protein or to mutations that eliminate the phosphorylation sites within β-catenin [6, 22–25].
The diverse roles of Wnts in both development and tumorigenesis have fostered the search for Wnt responsive genes in these processes. Wg signaling in Drosophila is known to transcriptionally activate the expression of engrailed and Ultrabithorax through the Armadillo/dTCF complex [26, 27]. Besides these Drosophila homeobox genes, Wnt signaling through β-catenin in Xenopus results in the transcriptional induction of two additional homeobox genes, siamois  and twin . Other responsive genes of Wnt/Wg that are transcriptionally activated by β-catenin/Lef-TCF include the oncogenes cyclin D1 [30, 31], c-myc , and WISP-1 [33, 34]; c-jun and fra-1 , as well as the Xenopus fibronectin gene , connexin43 , matrilysin , BTEB2 , Wrch-1 , and membrane-type matrix metalloproteinase-1 (MT1-MMP) .
The canonical Wnt signaling pathway relies upon the activation of responsive gene expression through the β-catenin/Lef-TCF complex in response to the binding of Wnt to its receptor Frizzled. Evidence exists, however, to suggest that Wnts can signal through a β-catenin/Lef-TCF-independent mechanism to activate downstream gene expression. These "non-canonical" pathways rely on either (1) the phosphatidylinositol (PI) pathway to activate protein kinase C (PKC) and raise levels of intracellular calcium (Ca2+) in order to regulate responsive gene expression (the Wnt/Ca2+ pathway)  or (2) Wnt signaling through Dsh and the JNK (the Wnt/PCP pathway) [43, 44]. For example, Xenopus Wnt-5a, a Wnt that does not induce ectopic axis formation nor stabilizes β-catenin, employs the Wnt/Ca2+ pathway to exert its effects . Thus, it is apparent that not all Wnts initiate the transcriptional activation of their responsive genes through the β-catenin/Lef-TCF complex, suggesting that β-catenin stabilization is not the sole end result of all Wnt signaling and that other pathways can be stimulated upon binding of Wnt to the Frizzled receptor.
The current list of Wnt/Wg responsive genes contains primarily those genes whose expression is activated through the canonical pathway. In an attempt to identify additional downstream responsive genes of the Wnt signaling pathway that are relevant to transformation and potentially tumorigenesis, we performed a suppression subtractive hybridization (SSH) screen between a Wnt-1-expressing mouse mammary epithelial cell line (C57MG/Wnt-1) and the parental cell line (C57MG). SSH offers many advantages when compared to conventional methods, such as differential display (DD) and representational difference analysis (RDA), employed to identify mRNA and/or cDNA differences between two populations. Differential display is more suitable to different cell types rather than one cell type subjected to two different conditions, as differences in PCR amplification can make control mRNAs appear differentially expressed between two populations, and results are often not reproducible upon Northern blot analysis . Representational difference analysis  does not rely on suppression PCR to prevent the amplification of undesirable sequences. SSH, on the other hand, permits the rapid isolation of both rare and abundant messages from the tester population in two hybridization steps followed by two PCR reactions. It has been widely used over the past five years to identify syntenin and other TNF-inducible genes in endothelial cells , to contrast gene expression profiles between non-tumorigenic rat embryo fibroblasts and H-ras transformed cells , to identify differences between the bacterial genomes Escherichia coli and Salmonella typhimuriu , and to generate a testis-specific cDNA library, where rare sequences were enriched over 1000-fold in one round of subtractive hybridization .
Using the technique of SSH, we previously reported the identification and characterization of a mouse homolog of BTEB2 , Wrch-1 , and WISP-1 [33, 34]. This report details the remaining results of the subtractive hybridization screen; in particular, the identification of 59 additional genes confirmed to be up-regulated in response to Wnt signaling. These results reveal the gene expression changes that occur in a tissue culture model system in response to Wnt signaling and may help explain the resulting transformed phenotype.