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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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No discernable effect of rat strain on apoptosis in the mammary gland. A, Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to cleaved forms (19 kDa and 17 kDa fragments) of caspase 3 (n = 3). B, The amount of cleaved caspase 3 was quantified using a Bio-Rad ChemiDoc XRS + imaging system and normalized to the amount of β-actin. Each data bar represents the mean ± SEM, n = 3 biological replicates. C, Cells isolated from mammary glands of ACI and BN rats, treated with E2 for 3 weeks, were stained with Annexin V and propidium iodide and analyzed by flow cytometry. Cells isolated from an involuting mammary gland (3 days post-lactation) were stained and analyzed as a positive control. D, Each data bar represents the number of apoptotic cells (positive for annexin V, negative for PI) expressed as a percentage of total PI negative cells (mean ± SEM, n = 3).
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Figure 3: No discernable effect of rat strain on apoptosis in the mammary gland. A, Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to cleaved forms (19 kDa and 17 kDa fragments) of caspase 3 (n = 3). B, The amount of cleaved caspase 3 was quantified using a Bio-Rad ChemiDoc XRS + imaging system and normalized to the amount of β-actin. Each data bar represents the mean ± SEM, n = 3 biological replicates. C, Cells isolated from mammary glands of ACI and BN rats, treated with E2 for 3 weeks, were stained with Annexin V and propidium iodide and analyzed by flow cytometry. Cells isolated from an involuting mammary gland (3 days post-lactation) were stained and analyzed as a positive control. D, Each data bar represents the number of apoptotic cells (positive for annexin V, negative for PI) expressed as a percentage of total PI negative cells (mean ± SEM, n = 3).

Mentions: Apoptosis within the mammary gland was evaluated using two independent methods. In the first, the levels of the activated 17 and 19 kDa forms of caspase 3 were quantified by western blotting. No significant differences in the levels of cleaved caspase 3 were observed when mammary glands from E2 treated ACI and BN rats were compared (Figure 3A and 3B). Binding of Annexin V to dispersed mammary cells was quantified by flow cytometry as a second indicator of apoptosis. Approximately 20% of cells isolated from mammary glands of ACI and BN rats that were treated with E2 for 3 weeks stained positive for Annexin V and negative for PI (Figure 3C and 3D). When an involuting mammary gland from an ACI rat (3 days post-lactation) was evaluated as a positive control, approximately 80% of cells isolated cells stained positive for Annexin V. Together, these data suggest that the levels of apoptosis in the mammary glands of E2 treated ACI and BN rats did not differ significantly.


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)

No discernable effect of rat strain on apoptosis in the mammary gland. A, Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to cleaved forms (19 kDa and 17 kDa fragments) of caspase 3 (n = 3). B, The amount of cleaved caspase 3 was quantified using a Bio-Rad ChemiDoc XRS + imaging system and normalized to the amount of β-actin. Each data bar represents the mean ± SEM, n = 3 biological replicates. C, Cells isolated from mammary glands of ACI and BN rats, treated with E2 for 3 weeks, were stained with Annexin V and propidium iodide and analyzed by flow cytometry. Cells isolated from an involuting mammary gland (3 days post-lactation) were stained and analyzed as a positive control. D, Each data bar represents the number of apoptotic cells (positive for annexin V, negative for PI) expressed as a percentage of total PI negative cells (mean ± SEM, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: No discernable effect of rat strain on apoptosis in the mammary gland. A, Representative western blots of protein lysates prepared from mammary glands of ACI and BN rats, treated with E2 for 1, 3 or 12 weeks, and probed with antibody to cleaved forms (19 kDa and 17 kDa fragments) of caspase 3 (n = 3). B, The amount of cleaved caspase 3 was quantified using a Bio-Rad ChemiDoc XRS + imaging system and normalized to the amount of β-actin. Each data bar represents the mean ± SEM, n = 3 biological replicates. C, Cells isolated from mammary glands of ACI and BN rats, treated with E2 for 3 weeks, were stained with Annexin V and propidium iodide and analyzed by flow cytometry. Cells isolated from an involuting mammary gland (3 days post-lactation) were stained and analyzed as a positive control. D, Each data bar represents the number of apoptotic cells (positive for annexin V, negative for PI) expressed as a percentage of total PI negative cells (mean ± SEM, n = 3).
Mentions: Apoptosis within the mammary gland was evaluated using two independent methods. In the first, the levels of the activated 17 and 19 kDa forms of caspase 3 were quantified by western blotting. No significant differences in the levels of cleaved caspase 3 were observed when mammary glands from E2 treated ACI and BN rats were compared (Figure 3A and 3B). Binding of Annexin V to dispersed mammary cells was quantified by flow cytometry as a second indicator of apoptosis. Approximately 20% of cells isolated from mammary glands of ACI and BN rats that were treated with E2 for 3 weeks stained positive for Annexin V and negative for PI (Figure 3C and 3D). When an involuting mammary gland from an ACI rat (3 days post-lactation) was evaluated as a positive control, approximately 80% of cells isolated cells stained positive for Annexin V. Together, these data suggest that the levels of apoptosis in the mammary glands of E2 treated ACI and BN rats did not differ significantly.

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