FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014540334> ?p ?o ?g. }

• W2014540334 endingPage "1749" @default.
• W2014540334 startingPage "1739" @default.
• W2014540334 abstract "The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic. The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic. We have shown previously that the degree of malignancy, the organization of the cytoskeleton, and the composition of the extracellular matrix (ECM) influence chromatin structure.1Maniotis AJ Valyi-Nagy K Karavitis J Moses J Boddipali V Wang Y Nunez R Setty S Arbieva Z Bissell MJ Folberg R Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.Am J Pathol. 2005; 166: 1187-1203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar We found that the DNA of cultured cell lines from malignant tumors, transformed fibroblasts harboring three oncogenes, and cells collected from human malignant tumors were more resistant to nucleases compared with DNA from normal or nonmalignant and weakly malignant cells. In addition, cells with the same genotype exhibit different degrees of DNA sequestration and exposure when cytoskeletal components were selectively disrupted or when they were cultured on different ECM components.1Maniotis AJ Valyi-Nagy K Karavitis J Moses J Boddipali V Wang Y Nunez R Setty S Arbieva Z Bissell MJ Folberg R Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.Am J Pathol. 2005; 166: 1187-1203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Without removal of oncogenes, additional deletions, or mutations, it is possible to revert cancer cells into cells that behave phenotypically normally. On two-dimensional surfaces, malignant cells can be induced by the addition of cAMP to cease blebbing, form an organized cytoskeleton, and develop contact-inhibited monolayers.2Krystosek A Puck TT The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.Proc Natl Acad Sci USA. 1990; 87: 6560-6564Crossref PubMed Scopus (43) Google Scholar, 3Puck TT Webb P Johnson R Cyclic AMP and the reverse transformation reaction.Ann NY Acad Sci. 2002; 968: 122-138Crossref PubMed Scopus (11) Google Scholar In a three-dimensional tissue culture system, it is possible to induce breast cancer cells to form polarized tissue structures resembling normal breast acini4Weaver VM Petersen OW Wang F Larabell CA Briand P Damsky C Bissell MJ Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.J Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1197) Google Scholar, 5Wang F Weaver VM Petersen OW Larabell CA Dedhar S Briand P Lupu R Bissell MJ Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.Proc Natl Acad Sci USA. 1998; 95: 14821-14826Crossref PubMed Scopus (555) Google Scholar and resume normal function,4Weaver VM Petersen OW Wang F Larabell CA Briand P Damsky C Bissell MJ Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.J Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1197) Google Scholar, 5Wang F Weaver VM Petersen OW Larabell CA Dedhar S Briand P Lupu R Bissell MJ Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.Proc Natl Acad Sci USA. 1998; 95: 14821-14826Crossref PubMed Scopus (555) Google Scholar, 6Lelièvre SA Weaver VM Nickerson JA Larabell CA Bhaumik A Petersen OW Bissell MJ Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.Proc Natl Acad Sci USA. 1998; 95: 14711-14716Crossref PubMed Scopus (211) Google Scholar, 7Weaver VM Lelievre S Lakins JN Chrenek MA Jones JC Giancotti F Werb Z Bissell MJ Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.Cancer Cell. 2002; 2: 205-216Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 8Wang F Hansen RK Radisky D Yoneda T Barcellos-Hoff MH Petersen OW Turley EA Bissell MJ Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.J Natl Cancer Inst. 2002; 94: 1494-1503Crossref PubMed Scopus (223) Google Scholar including a dramatic reduction in tumor formation, and seem to function similar to differentiated breast acini. In experimental animal models, teratocarcinoma cells placed into mammalian embryos formed normal mice,9Mintz B Illmensee K Normal genetically mosaic mice produced from malignant teratocarcinoma cells.Proc Natl Acad Sci USA. 1975; 72: 3585-3589Crossref PubMed Scopus (916) Google Scholar avian embryos transformed with Rous sarcoma virus10Dolberg DS Bissell MJ Inability of Rous sarcoma virus to cause sarcomas in the avian embryo.Nature. 1984; 309: 552-556Crossref PubMed Scopus (149) Google Scholar, 11Dolberg DS Hollingsworth R Hertle M Bissell MJ Wounding and its role in RSV-mediated tumor formation.Science. 1985; 230: 676-678Crossref PubMed Scopus (155) Google Scholar did not become transformed despite viral integration, and metastatic aneuploid melanoma cells placed in chick embryos12Kulesa PM Kasemeier-Kulesa JC Teddy JM Margaryan NV Seftor EA Seftor RE Hendrix MJ Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment.Proc Natl Acad Sci USA. 2006; 103: 3752-3757Crossref PubMed Scopus (193) Google Scholar formed normal neural crest structures as they would if placed into normal adult organisms.12Kulesa PM Kasemeier-Kulesa JC Teddy JM Margaryan NV Seftor EA Seftor RE Hendrix MJ Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment.Proc Natl Acad Sci USA. 2006; 103: 3752-3757Crossref PubMed Scopus (193) Google Scholar These findings demonstrate that the phenotypes of transformed malignant or metastatic cells are highly plastic and regulated by the microenvironment into which they are placed. The mechanisms underlying these phenotypic plasticities are just beginning to be studied but are not yet understood. In addition, although there are data to indicate that changes in cell phenotype are accompanied by epigenetic changes in nuclear and chromatin organization,6Lelièvre SA Weaver VM Nickerson JA Larabell CA Bhaumik A Petersen OW Bissell MJ Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.Proc Natl Acad Sci USA. 1998; 95: 14711-14716Crossref PubMed Scopus (211) Google Scholar, 13Myers CA Schmidhauser C Mellentin-Michelotti J Fragoso G Roskelley CD Casperson G Mossi R Pujuguet P Hager G Bissell MJ Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation.Mol Cell Biol. 1998; 18: 2184-2195Crossref PubMed Scopus (91) Google Scholar, 14Plachot C Lelievre SA DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium.Exp Cell Res. 2004; 298: 122-132Crossref PubMed Scopus (80) Google Scholar, 15Pujuguet P Simian M Liaw J Timpl R Werb Z Bissell MJ Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression.J Cell Sci. 2000; 113: 849-858PubMed Google Scholar little is known about global chromatin changes as a result of alternations in the microenvironment. We used a well-characterized three-dimensional breast tumor model system4Weaver VM Petersen OW Wang F Larabell CA Briand P Damsky C Bissell MJ Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.J Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1197) Google Scholar, 16Debnath J Muthuswamy SK Brugge JS Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.Methods. 2003; 30: 256-268Crossref PubMed Scopus (1556) Google Scholar to determine whether the transition of tumor cells from disorganized clusters to organized, polar acinus-like structures is accompanied by global epigenetic changes in chromatin structure that could be detected by measuring changes in the resistance of DNA to specific DNA-digesting enzymes. With this technique, we show that the organization of chromatin in a malignant mammary epithelial cell line follows tissue architecture. Moreover, tissue phenotype, and DNA organization are unstable17Liu H Radisky DC Wang F Bissell MJ Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells.J Cell Biol. 2004; 164: 603-612Crossref PubMed Scopus (182) Google Scholar (plastic2Krystosek A Puck TT The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.Proc Natl Acad Sci USA. 1990; 87: 6560-6564Crossref PubMed Scopus (43) Google Scholar) and reversible. We take advantage of these observations to test the following question: is it possible to control reversibly DNA exposure and sequestration, cell polarity, tumor morphology, and, ultimately, tumor behavior through manipulation of the ECM? MCF10A, a nonmalignant human breast epithelial cell line, was obtained from American Type Culture Collection (Rockville, MD); the spontaneously transformed and malignant human breast epithelial line HMT-3522 T4-218Briand P Nielsen KV Madsen MW Petersen OW Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal.Cancer Res. 1996; 56: 2039-2044PubMed Google Scholar was isolated by one of us (M.J.B.).6Lelièvre SA Weaver VM Nickerson JA Larabell CA Bhaumik A Petersen OW Bissell MJ Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.Proc Natl Acad Sci USA. 1998; 95: 14711-14716Crossref PubMed Scopus (211) Google Scholar MCF10A cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (BioWhittaker, Inc., Walkersville, MD) containing 20 ng/ml epidermal growth factor (Calbiochem Corp., San Diego, CA), 1.4 × 10−6 mol/L hydrocortisone (BD Bioscience, San Jose, CA), 0.1 ng/ml cholera toxin (Sigma, St. Louis, MO), 10 × 10−6 g/ml human insulin (Calbiochem Corp.), 2 mmol/L l-glutamine, 5% horse serum (Fisher, Ontario, ON, Canada), and penicillin/streptomycin. HMT-3522 T4-2 cells were routinely grown in H14 medium without 10 ng/ml epidermal growth factor on Vitrogen-coated plates (BioWhittaker, Inc.) as described previously.7Weaver VM Lelievre S Lakins JN Chrenek MA Jones JC Giancotti F Werb Z Bissell MJ Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.Cancer Cell. 2002; 2: 205-216Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar Three-dimensional cultures were prepared based on previously described protocols4Weaver VM Petersen OW Wang F Larabell CA Briand P Damsky C Bissell MJ Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.J Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1197) Google Scholar, 16Debnath J Muthuswamy SK Brugge JS Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.Methods. 2003; 30: 256-268Crossref PubMed Scopus (1556) Google Scholar with some modifications. Coverslips (18 × 18 mm) were coated with 120 μl of reduced growth factor Matrigel (BD Bioscience). Single cell suspensions (0.5 to 1.0 × 105 cells per slip) were seeded on top of polymerized Matrigel, incubated for 30 minutes, and overlaid with 2.5 ml of culture medium containing no epidermal growth factor or serum. Cells were overlaid with medium containing 2% Matrigel. Cultures were grown for the number of days indicated in the figure legends, adding new medium every 3rd day. Cell smear assays were performed as described previously.1Maniotis AJ Valyi-Nagy K Karavitis J Moses J Boddipali V Wang Y Nunez R Setty S Arbieva Z Bissell MJ Folberg R Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.Am J Pathol. 2005; 166: 1187-1203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar In brief, monolayer cultures grown on 10-cm dishes (70 to 90% confluent) were mechanically dislodged or trypsinized off the plate, collected by centrifugation, and resuspended in serum-free DMEM. A drop (20 μl) of the suspension was placed on a glass slide and dried for 1 hour. DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. Nuclei were observed and photographed with a Leica inverted fluorescent microscope (Leica, Bannockburn, IL). DNA digestion was performed on cells in three-dimensional cultures grown for 14 days. In preliminary experiments, we first determined both the time and concentration of AluI required for DNA digestion, taking into account that large aggregates of T4-2 cells contain more DNA than smaller MCF10A acini. Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures. Both Triton X-100 (0.1%) and Nonidet P-40 (0.1%) were tested as detergents to permeabilize cells in three-dimensional cultures to explore if the detergent per se affected DNA digestion. No difference in sensitivity to DNA digestion was observed using either one of these detergents; however, when compared with cells treated with Triton X-100, the morphology of cells permeabilized with Nonidet P-40 was better restored to a round shape, making it easier to evaluate the results. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period. Ethidium bromide (0.5 μg/ml) was added to label DNA at the termination of the reaction. Nuclear fluorescence was photographed with a Leica inverted fluorescent microscope. Parallel cultures in each experiment were stained with trypan blue (MP Biomedicals, Aurora, OH) to confirm complete permeabilization. Cells from monolayer cultures of MCF10A and T4-2 cell lines were harvested either by scraping or trypsinization, diluted in PBS, and collected by centrifugation. The pellet was resuspended in 0.1% Nonidet P-40 and incubated for 1 minute at room temperature to permeabilize the cells, followed by a second spin to wash away detergent. Pellets were resuspended in 0.5 ml of regular DMEM with 65 U of AluI and incubated for 1 hour under rotation at 37°C. Propidium iodide (1 μg/ml; Invitrogen/Molecular Probes, Carlsbad, CA) was added directly to each reaction at its conclusion, and the cell suspension was filtered through flow tubes with a filtered cartridge before flow cytometric analysis. After treating T4-2 nonreverted and reverted cells from three-dimensional cultures with dispase (BD Bioscience) for 30 minutes at 37°C to degrade Matrigel proteins, cells were harvested into 15-ml Falcon tubes (BD Bioscience) and resuspended. To disperse the structures into single cell suspension, Trypsin (0.25%) was added (200 μl) and incubated for 5 minutes at 37°C. Single cells were collected by centrifugation and resuspended in PBS containing 1 μg/ml propidium iodide (Invitrogen/Molecular Probes). Cell suspensions were filtered through flow tubes with a filtered cartridge and taken to flow cytometric analysis. All analyses were performed on a FACSCalibur (BD Bioscience) equipped with a 488-nm laser for forward and side scatter, and 520-, 575-, and 675-nm detectors. Each run ended at 10,000 counts and was analyzed using FACS dot-plots and histograms. Propidium iodide signal represents labeled DNA, and signal intensity of undigested DNA (approximately corresponding to fluorescent intensity 102 to 104) was gated as M2. Lower signal intensity representing digested DNA (shorter DNA fragments) was gated as M1. Cells were seeded on Matrigel as described above, overlaid with 2.5 ml of growth medium containing 8 μmol/L PI3K inhibitor LY294002 (Calbiochem/EMD Biosciences, Inc., San Diego, CA) or a combination of 6 μmol/L LY294002 and 1 mmol/L N6-dibutyryl-cAMP (SigmaAldrich, St. Louis, MO). Cultures were maintained for 14 days with addition of new drugs every 3rd day. No toxicity was observed in either normal or tumorigenic three-dimensional cultures of mammary epithelial cells at these drug concentrations. Dibutyryl-cAMP analog, in addition to elevating cAMP within the cell, metabolizes to butyrate, which alone is known to have distinct biological effects. However, sodium butyrate, in concentrations ranging from 1 to 2 mmol/L, did not induce tumor reversion when added to the T4-2 cell cultures, thus eliminating a possible butyrate effect involved in tumor reversion. In experiments testing cAMP analog specificity and tumor reversion, cells were seeded as above and overlaid with medium containing either 0.5 mmol/L 8-CPT-2′-O-Me-cAMP (BioLog Life Science, Bremen, Germany) or 0.5 mmol/L N6-monobutyryl-cAMP (BioLog Life Science). Cells were seeded on Matrigel as described above and overlaid with 2.5 ml of medium containing one of the following antibodies: rabbit anti-human fibronectin (FN) (A0245, 1:100 dilution; DAKO, Carpinteria, CA), mouse anti-human laminin (Lam-89, 1:100 dilution; SigmaAldrich), mouse anti-human collagen IV (CIV22, 1:100 dilution; DAKO), rabbit anti-human collagen I (CL50111AP, 1:100 dilution; Cedarlane, Hornby, ON, Canada), and mouse anti-human γ-tubulin (Sigma-Aldrich). The cultures were fed every 3rd day by overlay with 1 ml of medium containing fresh antibody. Because the antibody solvent contains low concentrations of sodium azide, we tested for any effects that sodium azide might have on tumor reversion in a separate series of experiments and discovered that sodium azide had no effect by itself or in combination with control antibodies used in these experiments (data not shown). Cells were cultured on top of a Matrigel-covered coverslip for 14 days as described above. Coverslips were washed three times with cytoskeleton extraction buffer (50 mmol/L HEPES, 300 mmol/L sucrose, 100 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L ethylenediaminetetraacetic acid, and 0.5% Triton X-100) containing 1 mmol/L sodium orthovanadate, 20 mmol/L sodium fluoride, and 20 μl/ml protease inhibitor cocktail from Sigma-Aldrich for 3 seconds, rinsed with 1× PBS, and fixed in 100% ice-cold methanol (5 minutes at −20°C) followed by 5 minutes of incubation in ice-cold 100% acetone. Cells were rinsed three times in 1× PBS and incubated overnight at 4°C with mouse anti-human β4 integrin (clone E31) at a 1:200 dilution (Chemicon, Temecula, CA) or mouse anti-β-catenin (clone 14) at 5 μg/ml dilution (BD Transduction Laboratories, San Jose, CA). Slides were washed three times for 2 to 5 minutes, incubated with a 1:200 dilution of goat anti-mouse secondary antibody conjugated to Alexa Fluor 546 (Molecular Probes) for 30 minutes, washed three times (as above), and mounted on glass slides in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Control cultures for all experiments were treated with the same concentration of nonspecific rabbit IgG (catalog no. ab27478; Abcam, Cambridge, MA). The cultures were fed every 3rd day by overlay of 1 ml of medium containing fresh antibody. Confocal images were taken on a Zeiss LSM510 laser-scanning microscope (Carl Zeiss Micro Imaging Inc., Thornwood, NY) using incident light fluorescence and differential interference contrast (DIC) with ×25 and ×63 water immersion objectives. An argon laser was applied for red fluorescence (Alexa Fluor 546 and EtBr2 at 543-nm wavelength) and a UV laser for blue fluorescence (4,6-diamidino-2-phenylindole at 405-nm wavelength). Images were captured using LSM Software Release 2.5 (Carl Zeiss, Thornwood, NY) on a standard high-end Pentium PC (Fujistu-Siemens, Maarssen, The Netherlands). The behavior of T4-2 tumorigenic cells in three-dimensional culture conditions was compared with MCF10A breast epithelial cells. After 10 to 14 days, MCF10A cells formed polarized acini with the nuclei arranged circumferentially around the hollow interior.16Debnath J Muthuswamy SK Brugge JS Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.Methods. 2003; 30: 256-268Crossref PubMed Scopus (1556) Google Scholar β4-Integrin was distributed in a circular ring, facing the ECM environment, and β-catenin was identified internally on cell surfaces between different cells comprising the multicellular acini (Figure 1A). By contrast, within a few days, T4-2 cells developed into disorganized aggregates that were considerably larger than polarized structures formed by MCF10A cells (note differences in scale bar sizes, Figure 1). In the disorganized T4-2 aggregates, β4-integrin distribution was observed on cell surfaces facing all directions, both between cells and on surfaces facing the extracellular environment. In addition, in the disorganized T4-2 aggregates, β-catenin was distributed in and between most of the cells (Figure 1B). Compared with the architecture of MCF10A acini, the disorganized T4-2 aggregates seemed to have completely lost polarization. Tumorigenic T4-2 cells can be induced to develop into architecturally normal acinus-like structures in three-dimensional cultures when treated with a PI3K inhibitor.8Wang F Hansen RK Radisky D Yoneda T Barcellos-Hoff MH Petersen OW Turley EA Bissell MJ Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.J Natl Cancer Inst. 2002; 94: 1494-1503Crossref PubMed Scopus (223) Google Scholar, 17Liu H Radisky DC Wang F Bissell MJ Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells.J Cell Biol. 2004; 164: 603-612Crossref PubMed Scopus (182) Google Scholar We confirmed these results for reversion of tumorigenic T4-2 cells by culturing on Matrigel in the presence of 8 μmol/L PI3K inhibitor LY294002 (not shown).4Weaver VM Petersen OW Wang F Larabell CA Briand P Damsky C Bissell MJ Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.J Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1197) Google Scholar, 7Weaver VM Lelievre S Lakins JN Chrenek MA Jones JC Giancotti F Werb Z Bissell MJ Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.Cancer Cell. 2002; 2: 205-216Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 8Wang F Hansen RK Radisky D Yoneda T Barcellos-Hoff MH Petersen OW Turley EA Bissell MJ Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.J Natl Cancer Inst. 2002; 94: 1494-1503Crossref PubMed Scopus (223) Google Scholar, 17Liu H Radisky DC Wang F Bissell MJ Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells.J Cell Biol. 2004; 164: 603-612Crossref PubMed Scopus (182) Google Scholar We also obtained phenotypic reversion using dibutyryl-cAMP at a concentration of 1 mmol/L (not shown), consistent with previous observations of phenotypic tumor reversion in monolayer cultures.2Krystosek A Puck TT The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.Proc Natl Acad Sci USA. 1990; 87: 6560-6564Crossref PubMed Scopus (43) Google Scholar A combination of 6 μmol/L LY294002 PI3K inhibitor and 1 mmol/L dibutyryl-cAMP induced the formation of reverted structures most similar to the acini formed by MCF10A cells in terms of size, polarity, and lumen formation (Figure 1C). In the acini-like structures formed by the reverted T4-2 cells, a dense ring of β4-integrin surrounded each spheroid, similar to the ring of integrin formed around MCF10A acini. In addition, β-catenin was redistributed from a disorganized haphazard pattern in the nondrug-treated T4-2 aggregates to a lace-like or stellar pattern of the drug-treated structures that closely resembled β-catenin distribution in MCF10A cells in three-dimensional culture conditions (Figure 1, compare B and C). Moreover, acinus-like structures formed by T4-2 cells exposed to the combination of LY294002 PI3K inhibitor and dibutyryl-cAMP were hollow, similar to the MCF10A acini. Therefore, because of the degree of fidelity we observed in the architecture of the revertants compared with normal MCF10A structures when using the combination of 6 μmol/L LY294002 PI3K inhibitor and 1 mmol/L dibutyryl-cAMP, this treatment was used as our standard protocol to consistently induce revertants exhibiting acinus-like structures with size and phenotype most similar to MCF10A acini. In the cell-smear assay we developed previously,1Maniotis AJ Valyi-Nagy K Karavitis J Moses J Boddipali V Wang Y Nunez R Setty S Arbieva Z Bissell MJ Folberg R Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.Am J Pathol. 2005; 166: 1187-1203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar the DNA of MCF10A cells was extensively digested after a 1.5-hour exposure to AluI restriction enzyme. By contrast, DNA in the nuclei of T4-2 cells appeared to be mostly undigested after 1.5 hours of exposure to AluI. To confirm these qualitative observations, DNA digestion was quantified by flow cytometry. Under these conditions, a shift in the DNA profile of MCF10A cells was dramatically more than that observed for T4-2 cells after 6" @default.
• W2014540334 created "2016-06-24" @default.
• W2014540334 creator A5024887006 @default.
• W2014540334 creator A5039002471 @default.
• W2014540334 creator A5040694583 @default.
• W2014540334 creator A5072917052 @default.
• W2014540334 creator A5081617275 @default.
• W2014540334 creator A5087756204 @default.
• W2014540334 date "2007-05-01" @default.
• W2014540334 modified "2023-10-16" @default.
• W2014540334 title "Epigenetic Reversion of Breast Carcinoma Phenotype Is Accompanied by Changes in DNA Sequestration as Measured by AluI Restriction Enzyme" @default.
• W2014540334 cites W1534148261 @default.
• W2014540334 cites W1556421161 @default.
• W2014540334 cites W1576399086 @default.
• W2014540334 cites W1597673292 @default.
• W2014540334 cites W1885709862 @default.
• W2014540334 cites W1964596167 @default.
• W2014540334 cites W1967120404 @default.
• W2014540334 cites W1968807484 @default.
• W2014540334 cites W1980039397 @default.
• W2014540334 cites W1981912870 @default.
• W2014540334 cites W1982903815 @default.
• W2014540334 cites W1984699863 @default.
• W2014540334 cites W1995996876 @default.
• W2014540334 cites W1998709931 @default.
• W2014540334 cites W2004845206 @default.
• W2014540334 cites W2009507555 @default.
• W2014540334 cites W2010035589 @default.
• W2014540334 cites W2010585577 @default.
• W2014540334 cites W2017092245 @default.
• W2014540334 cites W2026309875 @default.
• W2014540334 cites W2027754859 @default.
• W2014540334 cites W2032842750 @default.
• W2014540334 cites W2033763775 @default.
• W2014540334 cites W2036145275 @default.
• W2014540334 cites W2043366026 @default.
• W2014540334 cites W2047833892 @default.
• W2014540334 cites W2052599502 @default.
• W2014540334 cites W2062565240 @default.
• W2014540334 cites W2075752842 @default.
• W2014540334 cites W2078037416 @default.
• W2014540334 cites W2082067280 @default.
• W2014540334 cites W2086336448 @default.
• W2014540334 cites W2089040409 @default.
• W2014540334 cites W2114013728 @default.
• W2014540334 cites W2121096799 @default.
• W2014540334 cites W2134086869 @default.
• W2014540334 cites W2165022280 @default.
• W2014540334 cites W2166269000 @default.
• W2014540334 cites W2166287438 @default.
• W2014540334 cites W2167284102 @default.
• W2014540334 cites W2167517334 @default.
• W2014540334 cites W2171892966 @default.
• W2014540334 cites W2188422153 @default.
• W2014540334 cites W2402460376 @default.
• W2014540334 cites W2604911295 @default.
• W2014540334 cites W4291234170 @default.
• W2014540334 doi "https://doi.org/10.2353/ajpath.2007.060922" @default.
• W2014540334 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1854967" @default.
• W2014540334 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17456778" @default.
• W2014540334 hasPublicationYear "2007" @default.
• W2014540334 type Work @default.
• W2014540334 sameAs 2014540334 @default.
• W2014540334 citedByCount "30" @default.
• W2014540334 countsByYear W20145403342012 @default.
• W2014540334 countsByYear W20145403342013 @default.
• W2014540334 countsByYear W20145403342014 @default.
• W2014540334 countsByYear W20145403342015 @default.
• W2014540334 countsByYear W20145403342017 @default.
• W2014540334 countsByYear W20145403342019 @default.
• W2014540334 countsByYear W20145403342020 @default.
• W2014540334 countsByYear W20145403342021 @default.
• W2014540334 crossrefType "journal-article" @default.
• W2014540334 hasAuthorship W2014540334A5024887006 @default.
• W2014540334 hasAuthorship W2014540334A5039002471 @default.
• W2014540334 hasAuthorship W2014540334A5040694583 @default.
• W2014540334 hasAuthorship W2014540334A5072917052 @default.
• W2014540334 hasAuthorship W2014540334A5081617275 @default.
• W2014540334 hasAuthorship W2014540334A5087756204 @default.
• W2014540334 hasBestOaLocation W20145403341 @default.
• W2014540334 hasConcept C104317684 @default.
• W2014540334 hasConcept C121608353 @default.
• W2014540334 hasConcept C127716648 @default.
• W2014540334 hasConcept C128319531 @default.
• W2014540334 hasConcept C142724271 @default.
• W2014540334 hasConcept C150194340 @default.
• W2014540334 hasConcept C190727270 @default.
• W2014540334 hasConcept C2777546739 @default.
• W2014540334 hasConcept C3018521938 @default.
• W2014540334 hasConcept C41091548 @default.
• W2014540334 hasConcept C502942594 @default.
• W2014540334 hasConcept C530470458 @default.
• W2014540334 hasConcept C54355233 @default.
• W2014540334 hasConcept C552990157 @default.
• W2014540334 hasConcept C70883133 @default.
• W2014540334 hasConcept C71924100 @default.
• W2014540334 hasConcept C86803240 @default.
• W2014540334 hasConceptScore W2014540334C104317684 @default.

• next