SWI/SNF Chromatin Remodeling Enzyme ATPases Promote Cell Proliferation in Normal Mammary Epithelial Cells

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Journal of Cellular Physiology (Impact Factor: 3.84). 06/2010; 223(3):667-78. DOI: 10.1002/jcp.22072
Source: PubMed


The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma-related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre-disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF-10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three-dimensional rBM culture, although some BRM-depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells.

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Available from: Jeffrey A Nickerson, Oct 05, 2015
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    • "EdU assay revealed an increase in EdU positive cells after doxycycline-induced silencing of GAS5 in shGAS5 clones (Figure 3A and B) while no effect was observed in control cells (data not shown). Using a linear regression method [29], we determined the time taken for 100% EdU incorporation, which corresponds to the time needed for single cell cycle length (Figure 3B). The cell cycle length of uninduced shGAS5 clones was 55–77 hours while the GAS5 silenced cells exhibited a shorter cell cycle of 40 to 47 hours (Figure 3C). "
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    ABSTRACT: Background Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA “sponge”. Our aim was to investigate the possible role of the GAS5 in the growth of MPM. Methods Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry. Results GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression. Conclusions The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.
    Molecular Cancer 05/2014; 13:119. DOI:10.1186/1476-4598-13-119 · 4.26 Impact Factor
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    • "MCF-10A cells inducibly expressing a non-specific shRNA or shRNA against BRG1 or BRM (BRG1i and BRMi) have been previously described [8]. Western analysis for BRG1 and BRM expression confirmed the alteration in expression of these proteins (Figure 1A). "
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    ABSTRACT: Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.
    PLoS ONE 02/2013; 8(2):e55628. DOI:10.1371/journal.pone.0055628 · 3.23 Impact Factor
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    • "The morphological similarity between fibrocystic and metastatic cells may not have been expected, since MCF10A cells have been used as controls in several research studies [39], [40]. Our statistical analysis revealed that diagnostically relevant descriptors such as number of nucleoli, nucleolar size or nucleolar margination toward the periphery may not be reliable parameters to distinguish malignant from fibrocystic cells. "
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    ABSTRACT: Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations. Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
    PLoS ONE 01/2012; 7(1):e29230. DOI:10.1371/journal.pone.0029230 · 3.23 Impact Factor
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