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Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with susceptibility to mammary cancer.

Ding L, Zhao Y, Warren CL, Sullivan R, Eliceiri KW, Shull JD - BMC Cancer (2013)

Bottom Line: Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM.Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin Madison, 1400 University Avenue, Madison, WI 53706, USA. shull@oncology.wisc.edu.

ABSTRACT

Background: We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

Methods: Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

Results: The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

Conclusions: We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

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Rat strain-specific effects of 17β-estradiol on luminal ectasia and expression of milk proteins. Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1, 3 or 12 weeks (n = 3). Nuclei were identified by staining DNA with DAPI (blue), luminal epithelial cells by immunostaining for K8 (red), and milk proteins were identified by immunostaining using a polyclonal antibody generated against milk specific proteins (green). Note that the mammary glands from E2 treated BN rats exhibit prominent ectatic lumens containing immunoreactive milk protein(s). Scale bars, 50 μm.
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Figure 4: Rat strain-specific effects of 17β-estradiol on luminal ectasia and expression of milk proteins. Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1, 3 or 12 weeks (n = 3). Nuclei were identified by staining DNA with DAPI (blue), luminal epithelial cells by immunostaining for K8 (red), and milk proteins were identified by immunostaining using a polyclonal antibody generated against milk specific proteins (green). Note that the mammary glands from E2 treated BN rats exhibit prominent ectatic lumens containing immunoreactive milk protein(s). Scale bars, 50 μm.

Mentions: IHC was performed using an antibody to milk proteins to evaluate mammary gland differentiation and to define the nature of the luminal ectasia observed in E2 treated BN rats (Figure 4). Immunoreactive milk proteins were detected in the lumens of sham treated ACI and BN rats and the amount of immunostaining did not differ discernibly between these rat strains. Milk proteins were also detected in the lumens of ACI rats treated with E2 for 1, 3 and 12 weeks. The most prominent feature of the mammary glands of E2 treated BN rats was the markedly dilated lumens that contain immunoreactive milk proteins. These data, together with data presented above, suggest that the primary response of the ACI mammary gland to E2 is cell proliferation, which leads to dramatic epithelial hyperplasia. By contrast, the primary response of the BN mammary gland to E2 appears to be differentiation to an active secretory epithelium associated with luminal ectasia and modest epithelial hyperplasia.


Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with susceptibility to mammary cancer.

Ding L, Zhao Y, Warren CL, Sullivan R, Eliceiri KW, Shull JD - BMC Cancer (2013)

Rat strain-specific effects of 17β-estradiol on luminal ectasia and expression of milk proteins. Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1, 3 or 12 weeks (n = 3). Nuclei were identified by staining DNA with DAPI (blue), luminal epithelial cells by immunostaining for K8 (red), and milk proteins were identified by immunostaining using a polyclonal antibody generated against milk specific proteins (green). Note that the mammary glands from E2 treated BN rats exhibit prominent ectatic lumens containing immunoreactive milk protein(s). Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924185&req=5

Figure 4: Rat strain-specific effects of 17β-estradiol on luminal ectasia and expression of milk proteins. Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1, 3 or 12 weeks (n = 3). Nuclei were identified by staining DNA with DAPI (blue), luminal epithelial cells by immunostaining for K8 (red), and milk proteins were identified by immunostaining using a polyclonal antibody generated against milk specific proteins (green). Note that the mammary glands from E2 treated BN rats exhibit prominent ectatic lumens containing immunoreactive milk protein(s). Scale bars, 50 μm.
Mentions: IHC was performed using an antibody to milk proteins to evaluate mammary gland differentiation and to define the nature of the luminal ectasia observed in E2 treated BN rats (Figure 4). Immunoreactive milk proteins were detected in the lumens of sham treated ACI and BN rats and the amount of immunostaining did not differ discernibly between these rat strains. Milk proteins were also detected in the lumens of ACI rats treated with E2 for 1, 3 and 12 weeks. The most prominent feature of the mammary glands of E2 treated BN rats was the markedly dilated lumens that contain immunoreactive milk proteins. These data, together with data presented above, suggest that the primary response of the ACI mammary gland to E2 is cell proliferation, which leads to dramatic epithelial hyperplasia. By contrast, the primary response of the BN mammary gland to E2 appears to be differentiation to an active secretory epithelium associated with luminal ectasia and modest epithelial hyperplasia.

Bottom Line: Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM.Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin Madison, 1400 University Avenue, Madison, WI 53706, USA. shull@oncology.wisc.edu.

ABSTRACT

Background: We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

Methods: Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

Results: The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

Conclusions: We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

Show MeSH
Related in: MedlinePlus