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

Taneyhill L, Pennica D - BMC Dev. Biol. (2004)

Bottom Line: 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).The results indicate that cDNA subtractive hybridization is a useful method for identifying genes involved in the process of Wnt-1-induced transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA, USA. lziemer@caltech.edu

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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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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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.

Mentions: Several of the clones confirmed by Northern blot analysis were also found to be up-regulated in the C57MG/Wnt-1 cells by semi-quantitative RT-PCR (QRT-PCR) analysis performed on the same RNA prepared for use in the previously described Northern blot experiments. A list of these genes is found in Table 1, and Figure 2 shows typical QRT-PCR results for four genes: an unknown gene (483), Wrch-1, BTEB2, and IGF-BP5. In this experiment, different amounts of RNA were subjected to QRT-PCR using the TaqMan method (Applied Biosystems). Each gene analyzed shows a higher expression level in the C57MG/Wnt-1 cell line than in the parental C57MG cell line. Wrch-1 and IGF-BP5 show striking patterns of expression in that they are barely detectable in the C57MG cell line. BTEB2 and the unknown gene 483 are also expressed at higher levels in the C57MG/Wnt-1 cell line, although expression of each gene can be detected to some extent in the parental cell line. In general, the QRT-PCR data are in good agreement with the results obtained by Northern blot analysis, and overall the QRT-PCR technique offers a more sensitive and quantitative approach to assess changes in gene expression levels. In some cases good probes for Northern blot analysis could not be prepared and/or little change was observed in transcript levels between the cell lines; however, the ability to perform QRT-PCR for these candidates enabled us to determine if these genes were responsive to Wnt signaling (for example, thrombospondin and FKBP12, respectively).


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

Taneyhill L, Pennica D - BMC Dev. Biol. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC425575&req=5

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.
Mentions: Several of the clones confirmed by Northern blot analysis were also found to be up-regulated in the C57MG/Wnt-1 cells by semi-quantitative RT-PCR (QRT-PCR) analysis performed on the same RNA prepared for use in the previously described Northern blot experiments. A list of these genes is found in Table 1, and Figure 2 shows typical QRT-PCR results for four genes: an unknown gene (483), Wrch-1, BTEB2, and IGF-BP5. In this experiment, different amounts of RNA were subjected to QRT-PCR using the TaqMan method (Applied Biosystems). Each gene analyzed shows a higher expression level in the C57MG/Wnt-1 cell line than in the parental C57MG cell line. Wrch-1 and IGF-BP5 show striking patterns of expression in that they are barely detectable in the C57MG cell line. BTEB2 and the unknown gene 483 are also expressed at higher levels in the C57MG/Wnt-1 cell line, although expression of each gene can be detected to some extent in the parental cell line. In general, the QRT-PCR data are in good agreement with the results obtained by Northern blot analysis, and overall the QRT-PCR technique offers a more sensitive and quantitative approach to assess changes in gene expression levels. In some cases good probes for Northern blot analysis could not be prepared and/or little change was observed in transcript levels between the cell lines; however, the ability to perform QRT-PCR for these candidates enabled us to determine if these genes were responsive to Wnt signaling (for example, thrombospondin and FKBP12, respectively).

Bottom Line: 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).The results indicate that cDNA subtractive hybridization is a useful method for identifying genes involved in the process of Wnt-1-induced transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA, USA. lziemer@caltech.edu

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.

Show MeSH
Related in: MedlinePlus