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. 2004 May 12:4:6.
doi: 10.1186/1471-213X-4-6.

Identification of Wnt responsive genes using a murine mammary epithelial cell line model system

Lisa Taneyhill  1 , Diane Pennica
Affiliations

Identification of Wnt responsive genes using a murine mammary epithelial cell line model system

Lisa Taneyhill et al. BMC Dev Biol. .

Abstract

Background: The Wnt/Wg pathway plays an important role in the developmental program of many cells and tissues in a variety of organisms. In addition, many Wnts and components of their downstream signaling pathways, such as beta-catenin and APC, have been implicated in tumorigenesis. Over the past years, several genes have been identified as Wnt responsive, including c-myc, siamois, and cyclin D1.

Results: In order to identify additional genes responsive to Wnt signaling that contribute to the transformed phenotype, we performed a cDNA subtractive hybridization screen between a mouse mammary epithelial cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG). The screen identified a total of 67 genes to be up-regulated in response to Wnt signaling. Of these 67 genes, the up-regulation of 62 was subsequently confirmed by Northern and dot blot analyses (and, for a subset, semi-quantitative PCR) of RNA isolated from C57MG cells subjected to (1) an independent Wnt-1 retroviral infection, and (2) co-culture with Wnt-1 expressing cells. Among the confirmed Wnt-1 responsive genes, we further characterized a mouse homolog of the human transcription factor Basic Transcription Element Binding protein 2 (BTEB2), Wnt-1 Responsive Cdc42 homolog (Wrch-1), and Wnt-1 Induced Secreted Protein (WISP-1).

Conclusion: Several novel genes were identified in this screen, as well as others that have been shown previously to be regulated by Wnt signaling, such as connexin43. The results indicate that cDNA subtractive hybridization is a useful method for identifying genes involved in the process of Wnt-1-induced transformation.

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Figures

Figure 1
Figure 1
Northern blot analysis of putative Wnt-1 responsive genes after retroviral infection of C57MG cells. C57MG cells were independently infected with either a Wnt-1 or empty vector retrovirus. Forty-eight hours post-infection, cells were split into media containing 2.5 μg/ml puromycin. Infected cells were passaged in the presence of puromycin for three weeks after which time total RNA was isolated from each cell line when cells were approximately 95% confluent. Northern blot analysis was performed using five μg of total RNA, and probes were prepared from the sequences obtained. Blots were subsequently probed for GAPDH. Signals were quantitated using the PhosphorImager.
Figure 2
Figure 2
Semi-quantitative RT-PCR (QRT-PCR) confirmation of putative Wnt-1 responsive genes after retroviral infection of C57MG cells. C57MG cells were infected with either a Wnt-1 or empty vector retrovirus. Forty-eight hours post-infection, cells were split into media containing 2.5 μg/ml puromycin. Infected cells were passaged in the presence of puromycin for three weeks after which time total RNA was isolated from each cell line. Reverse transcription was performed on the indicated amounts of total RNA, followed by PCR using primers designed for each sequence of interest and TaqMan probes (Applied Biosystems). triose phosphate isomerase (tpi) was used as a control in each QRT-PCR experiment.
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