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Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland.

Barker HE, Smyth GK, Wettenhall J, Ward TA, Bath ML, Lindeman GJ, Visvader JE - BMC Dev. Biol. (2008)

Bottom Line: In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered.Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia. barker@wehi.edu.au

ABSTRACT

Background: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene.

Results: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.

Conclusion: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells.

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Overexpression of DEAF-1 in MCF10A cells leads to enhanced cellular proliferation within acini. A) MCF10A-EcoR cells transduced with either a Deaf-1-expressing or empty pBabe-puro retrovirus were analyzed by western blotting of whole cell lysates using anti-DEAF-1 polyclonal antisera. Blotting for tubulin provided a loading control. B) Control or Deaf-1 expressing MCF10-EcoR cells were plated at 4,000 cells/well in eight-well glass chamber slides. Wells were pre-coated with Matrigel and the final culture medium contained 20 ng/ml EGF and 2% Matrigel. Acini were fixed in 2% paraformaldehyde after 8 days in culture, immunostained with anti-DEAF-1 (green), counterstained with DAPI (blue) and acini visualized by confocal microscopy (DAPI = uv; and DEAF-1 = 488 nm). Scale bar represents 47.6 μm. C) Transduced acini were grown as in B) from control MCF10A-EcoR cells, DEAF-1-transduced-MCF10A cells, and malignant MCF10CA1h cells. Cells were fixed in 2% PFA after 6, 8 and 10 days in culture. Acini were immunostained with anti-Ki67 (green) and counterstained with DAPI (blue) following 6, 8 and 10 days in culture. At least three independent experiments were performed. Acini were visualized with a confocal microscope (DAPI = uv; and Ki67 = 488 nm). Scale bar represents 47.6 μm.
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Figure 1: Overexpression of DEAF-1 in MCF10A cells leads to enhanced cellular proliferation within acini. A) MCF10A-EcoR cells transduced with either a Deaf-1-expressing or empty pBabe-puro retrovirus were analyzed by western blotting of whole cell lysates using anti-DEAF-1 polyclonal antisera. Blotting for tubulin provided a loading control. B) Control or Deaf-1 expressing MCF10-EcoR cells were plated at 4,000 cells/well in eight-well glass chamber slides. Wells were pre-coated with Matrigel and the final culture medium contained 20 ng/ml EGF and 2% Matrigel. Acini were fixed in 2% paraformaldehyde after 8 days in culture, immunostained with anti-DEAF-1 (green), counterstained with DAPI (blue) and acini visualized by confocal microscopy (DAPI = uv; and DEAF-1 = 488 nm). Scale bar represents 47.6 μm. C) Transduced acini were grown as in B) from control MCF10A-EcoR cells, DEAF-1-transduced-MCF10A cells, and malignant MCF10CA1h cells. Cells were fixed in 2% PFA after 6, 8 and 10 days in culture. Acini were immunostained with anti-Ki67 (green) and counterstained with DAPI (blue) following 6, 8 and 10 days in culture. At least three independent experiments were performed. Acini were visualized with a confocal microscope (DAPI = uv; and Ki67 = 488 nm). Scale bar represents 47.6 μm.

Mentions: To generate human breast epithelial cells stably overexpressing human DEAF-1 protein, MCF10A cells were initially transfected with a vector containing the mouse Ecotropic Receptor (EcoR) gene to generate MCF10A-EcoR cells. These cells were subsequently infected with an ecotropic pBabe-puro retrovirus encoding DEAF-1. Western blotting of whole cell lysates confirmed that DEAF-1 was overexpressed in DEAF-1-transduced MCF10A-EcoR cells relative to control cells transduced with an empty vector (Fig. 1A).


Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland.

Barker HE, Smyth GK, Wettenhall J, Ward TA, Bath ML, Lindeman GJ, Visvader JE - BMC Dev. Biol. (2008)

Overexpression of DEAF-1 in MCF10A cells leads to enhanced cellular proliferation within acini. A) MCF10A-EcoR cells transduced with either a Deaf-1-expressing or empty pBabe-puro retrovirus were analyzed by western blotting of whole cell lysates using anti-DEAF-1 polyclonal antisera. Blotting for tubulin provided a loading control. B) Control or Deaf-1 expressing MCF10-EcoR cells were plated at 4,000 cells/well in eight-well glass chamber slides. Wells were pre-coated with Matrigel and the final culture medium contained 20 ng/ml EGF and 2% Matrigel. Acini were fixed in 2% paraformaldehyde after 8 days in culture, immunostained with anti-DEAF-1 (green), counterstained with DAPI (blue) and acini visualized by confocal microscopy (DAPI = uv; and DEAF-1 = 488 nm). Scale bar represents 47.6 μm. C) Transduced acini were grown as in B) from control MCF10A-EcoR cells, DEAF-1-transduced-MCF10A cells, and malignant MCF10CA1h cells. Cells were fixed in 2% PFA after 6, 8 and 10 days in culture. Acini were immunostained with anti-Ki67 (green) and counterstained with DAPI (blue) following 6, 8 and 10 days in culture. At least three independent experiments were performed. Acini were visualized with a confocal microscope (DAPI = uv; and Ki67 = 488 nm). Scale bar represents 47.6 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Overexpression of DEAF-1 in MCF10A cells leads to enhanced cellular proliferation within acini. A) MCF10A-EcoR cells transduced with either a Deaf-1-expressing or empty pBabe-puro retrovirus were analyzed by western blotting of whole cell lysates using anti-DEAF-1 polyclonal antisera. Blotting for tubulin provided a loading control. B) Control or Deaf-1 expressing MCF10-EcoR cells were plated at 4,000 cells/well in eight-well glass chamber slides. Wells were pre-coated with Matrigel and the final culture medium contained 20 ng/ml EGF and 2% Matrigel. Acini were fixed in 2% paraformaldehyde after 8 days in culture, immunostained with anti-DEAF-1 (green), counterstained with DAPI (blue) and acini visualized by confocal microscopy (DAPI = uv; and DEAF-1 = 488 nm). Scale bar represents 47.6 μm. C) Transduced acini were grown as in B) from control MCF10A-EcoR cells, DEAF-1-transduced-MCF10A cells, and malignant MCF10CA1h cells. Cells were fixed in 2% PFA after 6, 8 and 10 days in culture. Acini were immunostained with anti-Ki67 (green) and counterstained with DAPI (blue) following 6, 8 and 10 days in culture. At least three independent experiments were performed. Acini were visualized with a confocal microscope (DAPI = uv; and Ki67 = 488 nm). Scale bar represents 47.6 μm.
Mentions: To generate human breast epithelial cells stably overexpressing human DEAF-1 protein, MCF10A cells were initially transfected with a vector containing the mouse Ecotropic Receptor (EcoR) gene to generate MCF10A-EcoR cells. These cells were subsequently infected with an ecotropic pBabe-puro retrovirus encoding DEAF-1. Western blotting of whole cell lysates confirmed that DEAF-1 was overexpressed in DEAF-1-transduced MCF10A-EcoR cells relative to control cells transduced with an empty vector (Fig. 1A).

Bottom Line: In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered.Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia. barker@wehi.edu.au

ABSTRACT

Background: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene.

Results: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target.

Conclusion: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells.

Show MeSH
Related in: MedlinePlus