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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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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.
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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.

Mentions: In order to confirm the results of the screen, C57MG cells were infected with a Wnt-1 or empty vector retrovirus and passaged in tissue culture for three weeks to mimic the conditions under which the original screen was performed. After this time period and when cells were approximately 95% confluent, RNA was isolated from the Wnt-1-infected cells (C57MG/Wnt-1) and empty vector-infected cells (C57MG/Vector), and Northern blot analysis was done using probes prepared from the sequences. Results from several of these Northern blots are shown in Figure 1. The Northern blots show that the screen yielded many genes that are up-regulated to varying levels. Connexin43 showed greater than twenty-fold induction in response to Wnt-1 signaling, and the remaining clones were induced less than ten-fold. Thymosinβ4, cyclin G, caldesmon, BTEB2, protein phosphatase V, and histone H3.3 are found at higher levels in the C57MG/Wnt-1 cell lines compared to the C57MG/Vector control. FKBP12 showed very little change in mRNA expression levels between the two cell lines. This result suggests that SSH can identify subtle differences in transcript levels between two cells lines. Therefore, we have identified a group of genes which are up-regulated in the presence of Wnt-1 signaling three weeks after Wnt-1 retroviral infection. These genes may or may not be direct responsive genes of Wnt-1 signaling, as many of these genes may be activated as a secondary cascade of gene expression in response to Wnt-1.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: In order to confirm the results of the screen, C57MG cells were infected with a Wnt-1 or empty vector retrovirus and passaged in tissue culture for three weeks to mimic the conditions under which the original screen was performed. After this time period and when cells were approximately 95% confluent, RNA was isolated from the Wnt-1-infected cells (C57MG/Wnt-1) and empty vector-infected cells (C57MG/Vector), and Northern blot analysis was done using probes prepared from the sequences. Results from several of these Northern blots are shown in Figure 1. The Northern blots show that the screen yielded many genes that are up-regulated to varying levels. Connexin43 showed greater than twenty-fold induction in response to Wnt-1 signaling, and the remaining clones were induced less than ten-fold. Thymosinβ4, cyclin G, caldesmon, BTEB2, protein phosphatase V, and histone H3.3 are found at higher levels in the C57MG/Wnt-1 cell lines compared to the C57MG/Vector control. FKBP12 showed very little change in mRNA expression levels between the two cell lines. This result suggests that SSH can identify subtle differences in transcript levels between two cells lines. Therefore, we have identified a group of genes which are up-regulated in the presence of Wnt-1 signaling three weeks after Wnt-1 retroviral infection. These genes may or may not be direct responsive genes of Wnt-1 signaling, as many of these genes may be activated as a secondary cascade of gene expression in response to Wnt-1.

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