Quantitative Model-Based Image Analysis of NuMa Distribution Links Nuclear Organization with Cell Phenotype

2001 ◽  
Vol 7 (S2) ◽  
pp. 578-579
Author(s):  
David W. Knowles ◽  
Sophie A. Lelièvre ◽  
Carlos Ortiz de Solόrzano ◽  
Stephen J. Lockett ◽  
Mina J. Bissell ◽  
...  
Keyword(s):  
Image Analysis ◽  
Mammary Epithelial Cells ◽  
Nuclear Organization ◽  
Critical Role ◽  
Quantitative Model ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Proliferating Cells ◽  
Mitotic Apparatus ◽  
Model Based

The extracellular matrix (ECM) plays a critical role in directing cell behaviour and morphogenesis by regulating gene expression and nuclear organization. Using non-malignant (S1) human mammary epithelial cells (HMECs), it was previously shown that ECM-induced morphogenesis is accompanied by the redistribution of nuclear mitotic apparatus (NuMA) protein from a diffuse pattern in proliferating cells, to a multi-focal pattern as HMECs growth arrested and completed morphogenesis . A process taking 10 to 14 days.To further investigate the link between NuMA distribution and the growth stage of HMECs, we have investigated the distribution of NuMA in non-malignant S1 cells and their malignant, T4, counter-part using a novel model-based image analysis technique. This technique, based on a multi-scale Gaussian blur analysis (Figure 1), quantifies the size of punctate features in an image. Cells were cultured in the presence and absence of a reconstituted basement membrane (rBM) and imaged in 3D using confocal microscopy, for fluorescently labeled monoclonal antibodies to NuMA (fαNuMA) and fluorescently labeled total DNA.

Abstract 3035: Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells

2014 ◽  
Author(s):  
Benjamin T. Spike ◽  
Jonathan A. Kelber ◽  
Evan Booker ◽  
Madhuri Kalathur ◽  
Rose Rodewald ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Stem Cell Phenotype

Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4035-4043 ◽  
Author(s):  
Brian J. Rybarczyk ◽  
Sarah O. Lawrence ◽  
Patricia J. Simpson-Haidaris
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Wound Repair ◽  
Wound Closure ◽  
Mammary Epithelial Cells ◽  
Dermal Fibroblasts ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Matrix Assembly ◽  
And Migration

AbstractFibrinogen (FBG) assembles into matrix fibrils of fibroblasts, lung and mammary epithelial cells, but not endothelial cells. Furthermore, cryptic β15-21 residues are exposed in FBG fibrils with no evidence of thrombin or plasmin proteolysis. Herein, the effects of FBG on migration and proliferation of wounded dermal fibroblasts were investigated. FBG preassembled into matrix prior to scrape-wounding induced 3H-thymidine incorporation 8-fold and shortened the time to wound closure 1.6-fold ± 0.1-fold. FBG added immediately after wounding did not enhance either response. Fibroblast growth factor-2/platelet-derived growth factor (FGF-2/PDGF) stimulated cell proliferation 2.2-fold for FGF-2 and 3.2-fold for PDGF and wound closure 1.5-fold ± 0.1-fold in the absence of matrix-FBG. Surprisingly, exogenous growth factors had negligible effect on wound closure and cell proliferation already enhanced by matrix-FBG. Matrix-FBG-enhanced wound closure required active assembly of an FBG-fibronectin matrix, engagement of αvβ3, and FBG Aα-RGDS572-575 integrin recognition sites; Aα-RGDF95-98 sites were not sufficient for matrix-FBG assembly, enhanced wound closure, or cell proliferation. Although Bβ1-42 was not necessary for matrix assembly, it was required for matrix-FBG-enhanced cell migration. These data indicate that FBG serves as an important matrix constituent in the absence of fibrin formation to enhance wound repair and implicate Bβ1-42 as a physiologic inducer of signal transduction to promote an intermediate state of cell adhesion and a migratory cell phenotype. (Blood. 2003;102:4035-4043)

Molecular Characterization of Epithelial to Mesenchymal Transition in Human Prostatic Epithelial Cells

2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Victor K. Lin ◽  
Shih-Ya Wang ◽  
Lanxiao Wu ◽  
Smitha M. Rao ◽  
J. C. Chiao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cancer Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Mammary Epithelial Cells ◽  
Critical Role ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Mesenchymal Transition ◽  
Enhanced Mobility ◽  
E Cadherin

Epithelial to mesenchymal transition (EMT) has been believed to play a critical role in cancer metastasis. TGFβ has been described as an inducer of EMT in normal mammary epithelial cells by signaling through receptor serine/threonine kinase pathways to regulate epithelial cell plasticity and invasion. In this study, we investigated the EMT cellular responses, including morphologic changes, phenotype switches, invasiveness enhancement, and cellular contraction alteration, in TGFβ stimulated human prostate normal epithelial cells (PZ-HPV-7). Migration of TGFβ treated PZ-HPV-7 cells across matrigel was measured in invasion chambers (8 μm pore size). The cells were treated with or without TGFβ (2 ng/ml) in PrEGM media for 3 days. Immunoblot assay was conducted and it was demonstrated that the induction of vimentin when stimulated by TGFβ was accompanied by a downregulation of E-cadherin, though p-cadherin level was not altered. It was also observed that there was a decrease in cytokaretin 5/6 expression associated with the downregulation of E-cadherin during the induction of EMT. In order to study the cell contraction, three-dimensional collage lattice assay was performed. It was demonstrated that TGFβ-stimulated PZ-HPV-7 cells gained contractility. Our results showed that TGFβ stimulation induced PZ-HPV-7 cells to undergo epithelial to mesenchymal transition. EMT characteristics such as acquisition of mesenchymal markers and loss of epithelial markers were evident in the induction of vimentin and downregulation of E-cadherin and cytokeratins, as well as phenotypic alterations including increased contraction and enhanced mobility were detected.

scholarly journals NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

2007 ◽  
Vol 18 (2) ◽  
pp. 348-361 ◽  
Author(s):  
Patricia C. Abad ◽  
Jason Lewis ◽  
I. Saira Mian ◽  
David W. Knowles ◽  
Jennifer Sturgis ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Mammary Epithelium ◽  
Chromatin Organization ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Deoxyribonuclease I ◽  
C Terminus

The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in three-dimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin.

BRCA1 exon 11 alternative splicing, multiple functions and the association with cancer

2012 ◽  
Vol 40 (4) ◽  
pp. 768-772 ◽  
Author(s):  
Claudia Tammaro ◽  
Michela Raponi ◽  
David I. Wilson ◽  
Diana Baralle
Keyword(s):  
Breast Cancer ◽  
Alternative Splicing ◽  
Cancer Biology ◽  
Mammary Epithelial Cells ◽  
Splice Variants ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Exon 11 ◽  
The Impact ◽  
Brca1 Exon

BRCA1 (breast cancer early-onset 1) alternative splicing levels are regulated in a cell-cycle- and cell-type-specific manner, with splice variants being present in different proportions in tumour cell lines as well as in normal mammary epithelial cells. The importance of this difference in the pathogenesis of breast cancer has yet to be determined. Developing an understanding of the impact of BRCA1 isoform ratio changes on cell phenotype will be of value in breast cancer and may offer therapeutic options. In the present paper, we describe the splicing isoforms of BRCA1 exon 11, their possible role in cancer biology and the importance of maintaining a balanced ratio.

scholarly journals 42 Quantifying mammary growth and proliferative effects of estradiol in Holstein heifer calves

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 42-43
Author(s):  
Nicole R Hardy ◽  
Kellie Enger ◽  
Benjamin Enger
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Cellular Proliferation ◽  
Corn Oil ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Epithelial Tissue ◽  
Proliferating Cells ◽  
Tissue Area ◽  
The Right

Abstract Growth of the bovine mammary gland is influenced by estradiol during a heifer’s life. The objective of this study was to determine the effect of estradiol administration on two different mammary parenchymal tissue regions and quantify the response in cellular proliferation between different estradiol treatments and regions. Treatments were administered daily during the 12 days prior to euthanasia at 82 days of age. Holstein heifer calves (n = 12) were divided amongst 3 treatments, control (n = 4, CON), short term (n = 4, SHORT), and long term (n = 4, LONG). CON calves were administered corn oil injections while SHORT calves received 9 injections of corn oil followed by 3 injections of estradiol; LONG calves received 12 injections of estradiol. BrdU was administered 2 hours prior to harvest to label proliferating mammary epithelial cells. Mammary parenchyma was dissected from the right rear gland and separated into two regions, center and edge parenchyma, for further examination. Tissues were examined using brightfield microscopy and fluorescent immunohistochemistry. LONG calves had a greater percentage (P < 0.05) of epithelial tissue area (34.0% ± 1.51) than CON (21.4% ± 1.5) and SHORT (23.0% ± 1.51) calves, and a lower stromal tissue area percentage (63.6% vs 73.9% and 74.0% ± 2.10, respectively; P < 0.05). There was a treatment by region interaction in the cellular proliferation. LONG calves had a greater percentage of proliferating epithelial cells than CON calves in the center (P < 0.05) and edge (P < 0.05) parenchymal regions, and a greater percentage of proliferating cells in the center parenchyma than SHORT calves (P < 0.05). These results indicate that duration of estradiol administration elicits different effects on mammary growth and that mammary epithelial cell proliferation is specific to mammary gland region.

scholarly journals C-terminal domain small phosphatase-like 2 promotes epithelial-to-mesenchymal transition via Snail dephosphorylation and stabilization

Open Biology ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 170274 ◽  
Author(s):  
Yulan Zhao ◽  
Jinquan Liu ◽  
Fenfang Chen ◽  
Xin-Hua Feng
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Epithelial To Mesenchymal Transition ◽  
Mammary Epithelial Cells ◽  
Critical Role ◽  
Mesenchymal Cell ◽  
Cellular Reprogramming ◽  
Mammary Epithelial ◽  
Mesenchymal Transition ◽  
Proteasome Degradation

The epithelial-to-mesenchymal transition (EMT) is a cellular reprogramming process converting epithelial cells into mesenchymal cell morphology. Snail is a critical regulator of EMT by both suppressing epithelial gene expression and promoting mesenchymal gene expression. Expression and activity of Snail are tightly controlled at transcriptional and post-translational levels. It has previously been reported that Snail undergoes phosphorylation and ubiquitin-dependent proteasome degradation. Here, we report nuclear phosphatase SCP4/CTDSPL2 acts as a novel Snail phosphatase. SCP4 physically interacts with and directly dephosphorylates Snail. SCP4-mediated dephosphorylation of Snail suppresses the ubiquitin-dependent proteasome degradation of Snail and consequently enhances TGFβ-induced EMT. The knockdown of SCP4 in MCF10A mammary epithelial cells leads to attenuated cell migration. Collectively, our finding demonstrates that SCP4 plays a critical role in EMT through Snail dephosphorylation and stabilization.

Essential role of biliary glycoprotein (CD66a) in morphogenesis of the human mammary epithelial cell line MCF10F

1999 ◽  
Vol 112 (23) ◽  
pp. 4193-4205 ◽  
Author(s):  
J. Huang ◽  
J.D. Hardy ◽  
Y. Sun ◽  
J.E. Shively
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Epithelial Cells ◽  
Cell Line ◽  
Myoepithelial Cell ◽  
Mammary Epithelial Cells ◽  
Tumor Cell Line ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Epithelial Cell Line

Normal mammary epithelial cells express the cell surface protein biliary glycoprotein (BGP or CD66a) in a polarized manner, suggesting that this protein may play a role in the formation of mammary acini. In order to test this hypothesis, we interrupted the expression of BGP in the mammary epithelial line MCF10F when cultured in or on Matrigel, a source of extracellular matrix (ECM). When analyzed by immunofluorescence confocal microscopy, the BGP staining is confined to the lumenal surface and colocalizes with actin. Sequential scanning electron microscopy demonstrates that the MCF10F cells migrate to form clusters, followed by apoptotic cell death within the center, resulting in lumen formation. Transmission electron micrographs reveal the presence of tight junctions and desmosomes between the cells, microvilli along the lumenal surface, and typical apoptotic bodies within the lumen. When the MCF10F cells are transfected with the BGP antisense gene and grown in Matrigel, they exhibit reduced acini formation (12% and 20%) compared to untransfected cells (52%) or to cells transfected with vector only (62%). Acini formation is also significantly reduced when MCF10F cells grown in Matrigel are treated with anti-BGP antibody (18% at 100 microgram/ml), or recombinant soluble BGP (18% at 0.4 microM). In contrast, the BGP-negative MCF7 breast tumor cell line, which does not form acini when grown in matrigel, exhibits >60% cell death with the occasional formation of acini, when transfected with the BGP sense gene and grown in Matrigel. These results support the hypothesis that BGP plays a role in the normal differentiation program of mammary epithelial cells, indicating that its expression is essential to the formation of the lumen. Furthermore, and as shown by others, the differentiation program depends on the presence of ECM. The lack of expression of BGP in the MCF7 breast cancer cell line suggests that the downregulation of BGP expression confers a growth advantage to these cells in ECM. In addition, we found that the MCF10F cells could be separated into a BGP-positive epithelial fraction (MCF10F-e), and a BGP-negative myoepithelial fraction (MCF10F-m). When the myoepithelial cell-enriched fraction is grown on Matrigel, web-like structures are formed. These cells have a typical spindle shape cell morphology and express keratin, alpha-smooth muscle actin and vimentin, markers of the myoepithelial cell phenotype. When MCF10F-m cells are treated with IFNgamma, they express CEA (carcinoembryonic antigen) but not BGP. Since breast carcinomas, especially in situ carcinomas, express CEA, this finding may suggest a heretofore unappreciated relationship between myoepithelial cells and breast cancer.

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