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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 mammary epithelial cell proliferation. A, Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1 week (n = 3). Column 1, nuclei identified by staining DNA with 4′,6-diamidino-2-phenylindole (DAPI, blue). Column 2, basal epithelial cells were identified by immunostaining for cytokeratin 5 (K5, green). Column 3, luminal epithelial cells were identified by immunostaining for cytokeratin 8 (K8, red). Colum 4, cells transiting S phase were identified by immunostaining for BrdU (yellow). Column 5, merged images from columns 1 through 4. Scale bars, 50 μm. B, The number of luminal epithelial cells (K8 positive) in S phase (BrdU positive) was quantified using a VectraTM multispectral fluorescence imaging system and illustrated as the percentage of total luminal epithelial cells. C, The number of luminal epithelial cells per field was quantified as an indicator of epithelial density. Each data bar in Panels B and C represents the mean ± standard error of the mean (SEM, n = 3). 1, p < 0.05 for comparison of E2 treated vs. sham treated rats of same strain. 2, p < 0.05 for comparison of E2 treated BN vs. treated ACI rats.
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Figure 2: Rat strain-specific effects of 17β-estradiol on mammary epithelial cell proliferation. A, Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1 week (n = 3). Column 1, nuclei identified by staining DNA with 4′,6-diamidino-2-phenylindole (DAPI, blue). Column 2, basal epithelial cells were identified by immunostaining for cytokeratin 5 (K5, green). Column 3, luminal epithelial cells were identified by immunostaining for cytokeratin 8 (K8, red). Colum 4, cells transiting S phase were identified by immunostaining for BrdU (yellow). Column 5, merged images from columns 1 through 4. Scale bars, 50 μm. B, The number of luminal epithelial cells (K8 positive) in S phase (BrdU positive) was quantified using a VectraTM multispectral fluorescence imaging system and illustrated as the percentage of total luminal epithelial cells. C, The number of luminal epithelial cells per field was quantified as an indicator of epithelial density. Each data bar in Panels B and C represents the mean ± standard error of the mean (SEM, n = 3). 1, p < 0.05 for comparison of E2 treated vs. sham treated rats of same strain. 2, p < 0.05 for comparison of E2 treated BN vs. treated ACI rats.

Mentions: Proliferation in defined mammary cell populations was quantified by IHC using antibodies to K5, a marker of basal epithelium, K8, a marker of luminal epithelium, and BrdU, a marker for cells that transited the S phase of the cell cycle within the 4 hours preceding euthanasia. Representative images from ACI and BN rats treated for 1 week with E2 and age matched, sham treated, control rats are illustrated in Figure 2A. Images generated at the 3 week and 12 week time points are appended as Additional file 2: Figure S1A and S1B, respectively. The mammary epithelia of both control and E2 treated ACI and BN rats were comprised of an outer layer of basal cells surrounding the inner luminal cells. Quantification by Vectra system demonstrated that the fraction of BrdU positive cells in the luminal epithelium of sham treated ACI and BN rats was below 1.0% at each of the time points and did not differ between strains (Figure 2A and 2B). Treatment with E2 dramatically induced proliferation within the luminal epithelium of ACI rats. The fraction of luminal cells staining positive for BrdU was increased to 10.6%, 8.2% and 5.8% in ACI rats treated with E2 for 1, 3 and 12 weeks, respectively. By contrast, E2 treatment increased the fraction of luminal cells staining positive for BrdU in BN rats to only 3.2% following 1 week and 1.8% following 3 weeks of treatment, and no significant increase was observed in BN rats treated with E2 for 12 weeks (Figure 2A and 2B). The fraction of S phase cells in the luminal epithelium of E2 treated ACI rats was significantly greater than in treated BN rats at each of the three time points. The difference in induction of luminal epithelial cell proliferation in these two rat strains was clearly reflected in the morphological and histological differences described above (Figure 1B, 1C and 2A), as well as in differences in epithelial density measured by quantifying the number of luminal epithelial cells per microscopic field. This indicator of epithelial density did not differ between sham treated ACI and BN rats at any of the time points examined (Figure 2C). The number of luminal epithelial cells per field was increased more than 6-fold in ACI rats treated with E2 for 1, 3 or 12 weeks, relative to age-matched control ACI rats. By contrast, the number of luminal epithelial cells per field was increased 1.7-, 2.4- and 3.2-fold in BN rats treated for 1, 3 and 12 weeks, respectively, relative to control BN rats. Together, these data demonstrate that the proliferative response of the luminal epithelium of ACI rats to E2 is markedly greater than that of BN rats. Proliferation in the basal epithelium was not quantified because the basal cells in E2 treated rats assumed an elongated morphology that made it difficult to assign a specific nucleus to the cells staining positive for K5.


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 mammary epithelial cell proliferation. A, Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1 week (n = 3). Column 1, nuclei identified by staining DNA with 4′,6-diamidino-2-phenylindole (DAPI, blue). Column 2, basal epithelial cells were identified by immunostaining for cytokeratin 5 (K5, green). Column 3, luminal epithelial cells were identified by immunostaining for cytokeratin 8 (K8, red). Colum 4, cells transiting S phase were identified by immunostaining for BrdU (yellow). Column 5, merged images from columns 1 through 4. Scale bars, 50 μm. B, The number of luminal epithelial cells (K8 positive) in S phase (BrdU positive) was quantified using a VectraTM multispectral fluorescence imaging system and illustrated as the percentage of total luminal epithelial cells. C, The number of luminal epithelial cells per field was quantified as an indicator of epithelial density. Each data bar in Panels B and C represents the mean ± standard error of the mean (SEM, n = 3). 1, p < 0.05 for comparison of E2 treated vs. sham treated rats of same strain. 2, p < 0.05 for comparison of E2 treated BN vs. treated ACI rats.
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Related In: Results  -  Collection

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Figure 2: Rat strain-specific effects of 17β-estradiol on mammary epithelial cell proliferation. A, Representative fluorescent images of mammary tissues from ACI and BN rats, either sham treated (Ctrl) or treated with E2 for 1 week (n = 3). Column 1, nuclei identified by staining DNA with 4′,6-diamidino-2-phenylindole (DAPI, blue). Column 2, basal epithelial cells were identified by immunostaining for cytokeratin 5 (K5, green). Column 3, luminal epithelial cells were identified by immunostaining for cytokeratin 8 (K8, red). Colum 4, cells transiting S phase were identified by immunostaining for BrdU (yellow). Column 5, merged images from columns 1 through 4. Scale bars, 50 μm. B, The number of luminal epithelial cells (K8 positive) in S phase (BrdU positive) was quantified using a VectraTM multispectral fluorescence imaging system and illustrated as the percentage of total luminal epithelial cells. C, The number of luminal epithelial cells per field was quantified as an indicator of epithelial density. Each data bar in Panels B and C represents the mean ± standard error of the mean (SEM, n = 3). 1, p < 0.05 for comparison of E2 treated vs. sham treated rats of same strain. 2, p < 0.05 for comparison of E2 treated BN vs. treated ACI rats.
Mentions: Proliferation in defined mammary cell populations was quantified by IHC using antibodies to K5, a marker of basal epithelium, K8, a marker of luminal epithelium, and BrdU, a marker for cells that transited the S phase of the cell cycle within the 4 hours preceding euthanasia. Representative images from ACI and BN rats treated for 1 week with E2 and age matched, sham treated, control rats are illustrated in Figure 2A. Images generated at the 3 week and 12 week time points are appended as Additional file 2: Figure S1A and S1B, respectively. The mammary epithelia of both control and E2 treated ACI and BN rats were comprised of an outer layer of basal cells surrounding the inner luminal cells. Quantification by Vectra system demonstrated that the fraction of BrdU positive cells in the luminal epithelium of sham treated ACI and BN rats was below 1.0% at each of the time points and did not differ between strains (Figure 2A and 2B). Treatment with E2 dramatically induced proliferation within the luminal epithelium of ACI rats. The fraction of luminal cells staining positive for BrdU was increased to 10.6%, 8.2% and 5.8% in ACI rats treated with E2 for 1, 3 and 12 weeks, respectively. By contrast, E2 treatment increased the fraction of luminal cells staining positive for BrdU in BN rats to only 3.2% following 1 week and 1.8% following 3 weeks of treatment, and no significant increase was observed in BN rats treated with E2 for 12 weeks (Figure 2A and 2B). The fraction of S phase cells in the luminal epithelium of E2 treated ACI rats was significantly greater than in treated BN rats at each of the three time points. The difference in induction of luminal epithelial cell proliferation in these two rat strains was clearly reflected in the morphological and histological differences described above (Figure 1B, 1C and 2A), as well as in differences in epithelial density measured by quantifying the number of luminal epithelial cells per microscopic field. This indicator of epithelial density did not differ between sham treated ACI and BN rats at any of the time points examined (Figure 2C). The number of luminal epithelial cells per field was increased more than 6-fold in ACI rats treated with E2 for 1, 3 or 12 weeks, relative to age-matched control ACI rats. By contrast, the number of luminal epithelial cells per field was increased 1.7-, 2.4- and 3.2-fold in BN rats treated for 1, 3 and 12 weeks, respectively, relative to control BN rats. Together, these data demonstrate that the proliferative response of the luminal epithelium of ACI rats to E2 is markedly greater than that of BN rats. Proliferation in the basal epithelium was not quantified because the basal cells in E2 treated rats assumed an elongated morphology that made it difficult to assign a specific nucleus to the cells staining positive for K5.

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