Chromatin Organization Measured by AluI Restriction Enzyme Changes with Malignancy and Is Regulated by the Extracellular Matrix and the Cytoskeleton
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62338-3
ISSN1525-2191
AutoresAndrew J. Maniotis, Klara Valyi‐Nagy, John Karavitis, Jonas Moses, Viveka Boddipali, Ying Wang, Rafael Núñez, Suman Setty, Zarema Arbieva, Mina J. Bissell, Robert Folberg,
Tópico(s)Genomics and Chromatin Dynamics
ResumoGiven that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture. Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture. It is well known from pathological specimens that normal and malignant cells differ in terms of nuclear structure. We have postulated that tissue organization depends on interactions between the extracellular matrix (ECM) and the nuclear structure and chromatin.1Bissell MJ Hall HG Parry G How does the extracellular matrix direct gene expression?.J Theor Biol. 1982; 99: 31-68Crossref PubMed Google Scholar, 2Maniotis A Chen C Ingber D Demonstration of mechanical interconnections between integrins, cytoskeletal filaments, and nuclear scaffolds that stabilize nuclear structure.Proc Nat Acad Sci USA. 1997; 94: 849-854Crossref PubMed Scopus (1367) Google Scholar Observations from in vivo and culture studies have provided evidence that the ECM may indeed influence higher order chromatin structure and gene expression.3Berezney R Coffey DS Nuclear protein matrix: association with newly synthesized DNA.Science. 1975; 189: 291-293Crossref PubMed Scopus (373) Google Scholar, 4Folkman J Moscona A Role of cell shape in growth control.Nature. 1978; 273: 345-349Crossref PubMed Scopus (2010) Google Scholar, 5Ingber DE Madri JA Folkman J Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion.J In Vitro Cell Dev Biol. 1987; 23: 387-394Crossref PubMed Scopus (197) Google Scholar, 6Ingber DE Folkman J Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix.J Cell Biol. 1989; 198: 317-330Crossref Scopus (758) Google Scholar, 7Strohman RC Bayne E Spector D Obinata T Micou-Eastwood J Maniotis A Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro.In Vitro Cell Dev Biol. 1990; 25: 201-208Crossref Scopus (64) Google Scholar, 8Sjakste NI Sjakste TG The characteristics of the molecular organization of the cell nucleus in leukocytes due to the degree of their differentiation.Vestn Ross Akad Med Nauk. 1993; : 56-63PubMed Google Scholar, 9Ingber DE Dike L Hansen L Karp S Liley H Maniotis A McNamee H Mooney D Plopper G Sims J Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis.Int Rev Cytol. 1994; 150: 173-224Crossref PubMed Scopus (380) Google Scholar, 10Singhvi R Lopez GP Stephanopoulos GN Wang DI Whitesides GM Ingber DE Engineering cell shape and function.Science. 1994; 264: 696-698Crossref PubMed Scopus (1353) Google Scholar, 11Roskelley CD Srebrow A Bissell MJ A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression.Curr Opin Cell Biol. 1995; 7: 736-747Crossref PubMed Scopus (354) Google Scholar, 12Chen CS Mrksich M Huang S Whitesides GM Ingber DE Geometric control of cell life and death.Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4266) Google Scholar Furthermore, experiments on endothelial cells and fibroblasts have demonstrated that a mechanical tug to an integrin receptor at the cell surface could change the organization of both the cytoskeleton and the alignment of intranuclear molecules within 1 second, whereas force applied to non-integrin receptors such as LDL did not change nuclear organization.2Maniotis A Chen C Ingber D Demonstration of mechanical interconnections between integrins, cytoskeletal filaments, and nuclear scaffolds that stabilize nuclear structure.Proc Nat Acad Sci USA. 1997; 94: 849-854Crossref PubMed Scopus (1367) Google Scholar Using AluI restriction enzyme that cuts the widely distributed AG-CT sites, we asked five interrelated questions: 1) Do sensitivities to chromatin digestion differ in nonmalignant and malignant cells of different origins? 2) If so, can one use this information to distinguish between cells of low and high metastatic potential? 3) Does the microenvironment in general, and ECM in particular, affect chromatin structure regardless of degree of malignancy? 4) If so, is there some specificity with regards to the nature of the ECM molecules used? And finally, 5) Is the cytoskeleton involved? We find that answers to the above questions are affirmative. We used primary uveal melanoma cell lines of low (OCM1a) and high (M619) invasive potential, and a highly invasive metastatic uveal melanoma cell line (MUM2B); the characteristics of these cell lines have been described in detail previously.13Maniotis AJ Folberg R Hess A Seftor EA Gardner LMG Pe'er J Trent JM Meltzer PS Hendrix MJC Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.Am J Pathol. 1999; 155: 739-752Abstract Full Text Full Text PDF PubMed Scopus (1692) Google Scholar Determination of the modal chromosome number revealed the OCM1a and M619 lines to be in the tetraploid range while the MUM2B line was pseudo-diploid. These cell lines were also characterized by their differential ability to form laminin-rich patterns that appear to control melanoma morphogenesis.14Maniotis AJ Chen X Garcia C DeChristopher PJ Wu D Pe'er J Folberg R Control of melanoma morphogenesis, endothelial survival, and perfusion by extracellular matrix.Lab Invest. 2002; 82: 1031-1043Crossref PubMed Scopus (86) Google Scholar OCM1a uveal melanoma cells were a generous gift from Dr. June Kan-Mitchell, Karmanos Cancer Institute, Wayne State University, Detroit, MI, and UM54 normal uveal melanocytes were a generous gift from Dr. J. William Harbour, Washington University, St Louis, MO. Melanoma cells and uveal melanocytes were plated in EMEM (BioWhittaker, Inc., Walkersville, MD), and supplemented with heat inactivated 15% fetal bovine serum (Fisher, Ontario, Canada) without the addition of exogenous extracellular matrix molecules or growth factors. Normal WI-38 human fibroblasts were obtained from the ATTC (Rockville, MD), and were maintained in 10% serum in DMEM. Telomerase, large-T, and ras transfected fibroblasts were developed by Hahn et al15Hahn WC Counter CM Lundberg AS Beijersbergen RL Brooks MW Weinberg RA Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468Crossref PubMed Scopus (2001) Google Scholar and maintained according to their protocols, and were a generous gift from Dr. Igor Roninson (University of Illinois at Chicago). HT1089 fibrosarcoma cells were obtained from the ATTC. No antibacterial or antifungal drugs were used in the maintenance of cell lines or in experiments, as their chronic use has been shown to interfere with the differentiation potential of other primary cell types.7Strohman RC Bayne E Spector D Obinata T Micou-Eastwood J Maniotis A Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro.In Vitro Cell Dev Biol. 1990; 25: 201-208Crossref Scopus (64) Google Scholar MCF10A breast epithelial cells and MDA-MB231 breast carcinoma cells were obtained from the ATTC, and were maintained on DMEM plus heat inactivated calf serum. The isolation and phenotypes of HMT-3522 human mammary epithelial cells16Briand P Petersen OW van DB A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium.In Vitro Cell Dev Biol. 1987; 23: 181-188Crossref PubMed Scopus (210) Google Scholar, 17Briand 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 both nonmalignant (S1) and their tumorigenic counterparts designated T4–218Weaver 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 (1220) Google Scholar were described previously. All cell cultures were determined to be free of mycoplasma contamination using the GenProbe rapid detection system (Fisher, Itasca, IL). We used three assays to compare sequestration and exposure of AluI binding sites in interphase cells of different invasive behaviors. In the permeabilized cell assay, we exposed cells in monolayer cultures to AluI restriction enzyme after permeabilization with Triton X-100. In the cell smear assay, we mechanically dislodged cells from culture, applied them to glass slides, allowed the cells to air dry, and exposed the cells to AluI restriction enzyme, thus simulating a diagnostic cytological examination performed in a clinical setting (eg, the examination of cells extracted by fine needle aspiration biopsy, brush biopsy, or scraping). In the flow cytometry assay, we quantified differential digestion of chromatin between cell lines by AluI restriction enzyme over time, again using methodology that could be easily adapted to a clinical diagnostic setting. Cells grown as monolayer cultures in the absence of exogenous matrix molecules were exposed to 0.1% Triton X-100 (Sigma, St. Louis, MO) for 90 seconds followed by a rinse of DMEM. The preparation was exposed to AluI restriction enzyme (0.5 μl in 40 μl DMEM; Promega, Madison, WI) for 60 minutes to 24 hours. The permeabilized cultures were then exposed to ethidium bromide (25 μl, 1 μg/ml; Sigma) and photographed with a Leica inverted microscope (Leica, Bannockburn, IL). After mechanically dislodging cells grown under monolayer conditions, the slurry was suspended in 1X PBS or serum-free DMEM, and a drop containing 15 μl of the suspension was placed onto a glass slide. The drops were allowed to evaporate over 30 minutes to 1 hour. AluI restriction enzyme (0.5 μl in 40 μl) or MspI restriction enzyme (0.5 μl in 40 μl DMEM; Promega) was applied to the dried cells, and the preparation was placed in a humidified 37°C chamber to optimize enzyme activity and minimize enzyme evaporation. Endonuclease digestions were terminated at pre-designated time points (30 minutes and hourly increments thereafter up to 24 hours) to determine the optimum digestion time that would allow for discrimination of differential chromatin digestion between cell lines. Ethidium bromide was added to terminate the digestion, and the preparation was photographed immediately. Touch preparations of normal human tissue and human tumor tissue were made and air dried. The preparation was incubated with AluI restriction enzyme and the reaction was terminated with ethidium bromide at 5 and 24 hours. To test the influence of different soluble ECM molecules on AluI sensitivity, we mechanically dislodged cells that had been growing under monolayer conditions from flasks and suspended the slurry of dislodged cells in 1X PBS or serum-free DMEM. Two rows of 15 μl drops of the suspension were applied to a glass slide, 3 drops per row. In some experiments as described below, we added soluble laminin, Matrigel, FBS, RGD-C, collagen Type I, bFGF, EGF (all reagents from Clontech, Palo Alto, CA), circularized RGD (RGD-C, a kind gift from Renata Pasquallini) to one of the two rows of drops. RGD-C is known to bind to integrin receptors.2Maniotis A Chen C Ingber D Demonstration of mechanical interconnections between integrins, cytoskeletal filaments, and nuclear scaffolds that stabilize nuclear structure.Proc Nat Acad Sci USA. 1997; 94: 849-854Crossref PubMed Scopus (1367) Google Scholar The test and control drops were then permitted to evaporate at room temperature for at least 1 hour, leaving “smears” of dried cells that had or had not been incubated with a test molecule. For each assay, cellular permeability was checked using the trypan blue exclusion method. The AluI restriction enzyme (0.5 μl in 40 μl DMEM; Promega) was applied to each drop in a humidified 37° incubator for up to 24 hours. The buffers used in these assays did not contain either DTT or mercaptoethanol to avoid removing any proteins within the cytoplasm or nucleus that might sequester AluI binding sites from enzyme digestion. Incubation with AluI was terminated by adding ethidium bromide (Sigma; 250 μl, 100 ng/ml) to each drop per slide at 30 minutes and at hourly intervals thereafter up to 24 hours of incubation. In the cell smear assay and the permeabilized cell assay, samples were photographed with a Leica inverted microscope and were scored qualitatively from the micrographs as follows: 1) Nuclei in which no fluorescence was detected except for nucleoli were scored as complete digestion; 2) Nuclei in which fluorescence intensity was equal to that detected at time before application of restriction enzyme was scored as no digestion; and 3) Detection of non-nucleolar nuclear fluorescence that was less intense than detected before application of restriction enzyme was scored as partial digestion. To provide for a quantitative measure of DNA digestion, we performed flow cytometry. Cells from monolayer cultures of UM54 normal uveal melanocytes, poorly invasive OCM1a primary uveal melanoma, highly invasive M619 primary uveal melanoma, and highly invasive MUM2B metastatic uveal melanoma were suspended in DMEM and spun down at 1400 rpm for 5 minutes. The pellet was re-suspended in 0.1% Triton X-100, incubated for 1 minute at room temperature, and spun down again at 1400 rpm for 5 minutes. After re-suspension in DMEM, 0.5 μl of AluI restriction enzyme in 40 μl of DMEM was added and the preparation was incubated at following time points: 0 (baseline), 1, 3, and 5 hours at 37°C. Propidium iodide (10 μl/ml; Molecular Probes, Eugene, OR) was added at the conclusion of each digestion period. The cells were analyzed in a FACS Calibur (BD Bioscience, San Jose, CA) equipped with a 488 laser, detectors for forward and side scatter, and 520, 575, and 675 nm detectors. 10,000 cells were counted and the results were analyzed with FACS dot-plots and histograms. Three AluI digestions monitored by flow cytometry were performed for each of the three melanoma cell lines. The percentage of cells in M2–M4 (roughly corresponding to 102 to 104 fluorescence intensity) was calculated and compared between cell lines at 5 hours of digestion with the t-test. The PI signal, representing stoichometrically the amount of labeled undigested DNA,19Darzynkiewicz Z Juan G DNA content measurement for DNA ploidy and cell cycle analysis.in: Robinson JP Current Protocols in Cytometry. John Wiley & Sons, Inc., New York1997Google Scholar was recorded immediately after cell permeabilization and labeling with PI and used as a baseline (0 time point), and was then measured for each cell line after 1, 3, and, 5 hours of exposure to AluI restriction enzyme. CellQuest software (BD Bioscience) was used to generate overlays of the histograms for each cell line at each time point in the digestion experiment. We have previously described a method by which the entire chromatin contents of nuclei can be extracted, producing a complete chromosome set for each cell thus manipulated.20Maniotis A Bojanowski K Ingber D Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells.J Cell Biochem. 1997; 65: 114-130Crossref PubMed Scopus (123) Google Scholar, 21Bojanowski K Maniotis AJ Plisov S Larsen AK Ingber DE DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA.J Cell Biochem. 1998; 69: 127-142Crossref PubMed Scopus (29) Google Scholar We removed chromatin sets from cells that had not been pretreated with any agent. All chromosome extractions and digestions were carried out under isotonic culture conditions in DMEM at pH 7.4; similar results also were obtained using complete cell culture medium containing FBS. Chromosome sets removed from metaphase cells of varying invasive potentials were studied under buffered media conditions that do not disturb the native structure of chromosomes.20Maniotis A Bojanowski K Ingber D Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells.J Cell Biochem. 1997; 65: 114-130Crossref PubMed Scopus (123) Google Scholar, 21Bojanowski K Maniotis AJ Plisov S Larsen AK Ingber DE DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA.J Cell Biochem. 1998; 69: 127-142Crossref PubMed Scopus (29) Google Scholar, 22Hoskins GC Electron microscopic observations of human chromosomes isolated by micrurgy.Nature. 1965; 207: 1215-1216Crossref PubMed Scopus (12) Google Scholar, 23Hoskins GC Sensitivity of micrurgically removed chromosomal spindle fibres to enzyme disruption.Nature. 1968; 217: 748-750Crossref PubMed Scopus (42) Google Scholar, 24Diacumakos EG Holland S Pecora P Chromosome displacement in and extraction from human cells at different mitotic stages.Nature. 1971; 232: 33-36Crossref PubMed Scopus (16) Google Scholar, 25Korf BR Diacumakos EG Random arrangement of mitotic chromosomes in radial metaphases of the Indian muntjac.Cytogenet Cell Genet. 1977; 19: 335-343Crossref PubMed Scopus (15) Google Scholar, 26Korf BR Diacumakos EG Absence of true interchromosomal connectives in microsurgically isolated chromosomes.Exp Cell Res. 1980; 130: 377-385Crossref PubMed Scopus (12) Google Scholar, 27Houchmandzadeh B Marko JF Chatenay D Libchaber A Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration.J Cell Biol. 1997; 139: 1-12Crossref PubMed Scopus (125) Google Scholar, 28Bojanowski K Ingber DE Ionic control of chromosome architecture in living and permeabilized cells.Exp Cell Res. 1998; 244: 286-294Crossref PubMed Scopus (12) Google Scholar, 29Poirier MG Marko JF Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold.Proc Natl Acad Sci USA. 2002; 99: 15393-15397Crossref PubMed Scopus (133) Google Scholar Cells from which chromosomes were to be removed were grown on small coverslips and placed in the center of a 35-mm plastic dish lid containing 2 ml of gassed DMEM. This preparation was allowed to equilibrate at 37°C in a 5% CO2 buffered incubator before micromanipulation. For optimal micromanipulation conditions, cells were grown to near confluence to produce strong attachments to each other and ECM needed to resist the pulling forces associated with rapid (within 1 second) micropuncture and removal of chromosomes without causing cell detachment or death of the manipulated cell. Chromosome sets were obtained either in the presence or absence of colchicine and cytochalasin-B as reported previously.20Maniotis A Bojanowski K Ingber D Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells.J Cell Biochem. 1997; 65: 114-130Crossref PubMed Scopus (123) Google Scholar All chromosome set extractions and digestions were carried out under isotonic culture conditions in DMEM at pH 7.4; similar results also were obtained using complete cell culture medium containing serum. Chromosome sets were extracted from living metaphase cells by rapidly piercing the cell directly on the side of a mitotic plate with a glass microneedle and then laterally drawing out the chromosomes through the hole on the cell surface created by the micropuncture using a Leitz micromanipulator (Leica Microsystems). In certain studies which used treatments that cause cell detachment (mercaptoethanol, DTT, and proteinase K), chromosome sets were removed from the cell with a pipette and placed on culture substrata. The chromosome sets were then deposited to pre-designated areas defined by scratching the Petri dish lid with a fine needle. Narishige micropipettes (Narishige Scientific Instruments, Tokyo, Japan) were pulled with a Sutter micropipette puller (Sutter Instrument Company, Novato, CA) adjusted to produce long barrels approximately 1 to 5 μm wide along a length of 40 to 100 μm (tip widths were consistently less than 0.5 μm). In this isolated chromosome set assay, it is important to use restriction enzymes and proteases in the range of specific ion concentrations if they are to function efficiently. We used buffered media conditions that preserve physiological ion concentrations to maintain the extent of chromatin compaction observed in living cells while providing the appropriate concentrations of NaCl and MgCl2 to permit enzymes to work.20Maniotis A Bojanowski K Ingber D Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells.J Cell Biochem. 1997; 65: 114-130Crossref PubMed Scopus (123) Google Scholar The restriction enzymes were used in the absence of DTT or β-mercaptoethanol (which are typically included in commercially available kits used to digest DNA) to avoid interfering with DNA-associated proteins. Restriction enzymes were dissolved in 2 ml of DMEM applied directly to 35 mm tissue culture dish lids on which the chromosome preparations were attached in the concentrations listed below. Changes in chromosome morphology were photographed by time-lapse video microscopy in real time. All microsurgical procedures were observed at 63× by phase and fluorescence microscopy with a Leica inverted microscope, and captured using a time-lapse video recorder (Sony) and Pinnacle Image software (Pinnacle Systems, Modesto, CA). We tested the following enzymes: AluI (5–50 units), Msp-I, (75 units, Promega), DNase I (1–20 units/droplet; Promega,), micrococcal nuclease (1–10 units; Promega), EcoRI (5–50 units; Promega), HindIII (5–10 units; Promega), BamHI (5–10 units; New England Biolabs, Beverly, MA), RNase A (1–100 units; Sigma), RNase I (1–100 units; Promega), EMBO (3 units; Promega), Sau-III (20 units; Promega), PstI (30 units; Promega), XHO-1 (40 units; Promega). Ethidium bromide (25 μl, 1 μg/ml; Sigma) was added to the medium after 1 to 3 hours of digestion with restriction enzyme and the preparations were photographed with a Leica inverted microscope. We designed a method to place different polymerized ECM molecules on glass slides to screen for changes in the sensitivity of AluI-sensitive sites in normal cells and tumor cells or tumor cells with varying degrees of invasive behavior. First, a coating of FBS or Type I collagen was applied to the glass slide and allowed to dry completely. Then a stencil was positioned on top of the dried FBS or collagen and clamped so that it would not move. The stencil was fashioned in such a way that it allowed application of a second ECM molecule over the dried FBS or collagen in square patches. Laminin or Matrigel were then applied to areas of the slide exposed by the stencil and the entire assemblage was baked in an oven for 1 hour at 90°C. The stencil was gently removed under sterile conditions, and cells were then seeded at subconfluent density onto the entire surface. The cultures were then exposed to 0.1% Triton X-100 (Sigma) for 90 seconds followed by a rinse of DMEM. AluI restriction enzyme (0.5 μl in 40 μl DMEM) was applied for 30 to 60 minutes after which time ethidium bromide (250 μl, 100 ng/ml) was placed on the preparation. Nuclei were photographed immediately and scored for digestion as described above. As reported previously, highly invasive primary and metastatic melanoma cells grow in monolayers on tissue culture plastic but form looping patterns when placed in three-dimensional (3D) culture conditions identical to those seen in tissue sections of primary aggressive and metastatic lesions resected from patients.14Maniotis AJ Chen X Garcia C DeChristopher PJ Wu D Pe'er J Folberg R Control of melanoma morphogenesis, endothelial survival, and perfusion by extracellular matrix.Lab Invest. 2002; 82: 1031-1043Crossref PubMed Scopus (86) Google Scholar Three-dimensional cultures were established by growing cells on Matrigel30Petersen OW Ronnov-Jessen L Howlett AR Bissell MJ Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.Proc Natl Acad Sci USA. 1992; 89: 9064-9068Crossref PubMed Scopus (886) Google Scholar (Collaborative Biomedical, Bedford, MA) or Type I collagen31Howlett AR Bailey N Damsky C Petersen OW Bissell MJ Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma.J Cell Sci. 1995; 108: 1945-1957PubMed Google Scholar (Collaborative Biomedical) placed onto plastic tissue culture dishes to a depth of about 0.2 mm followed by polymerization for 1 hour at 37°C. The plates were then returned to the incubator and allowed to completely polymerize for several hours. Cells were then seeded at saturating densities (400 million cells/dish) on the polymerized 3D gel coatings, and the cultures were checked daily for the presence of looping patterns that are characteristic for highly invasive melanoma cells.13Maniotis AJ Folberg R Hess A Seftor EA Gardner LMG Pe'er J Trent JM Meltzer PS Hendrix MJC Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.Am J Pathol. 1999; 155: 739-752Abstract Full Text Full Text PDF PubMed Scopus (1692) Google Scholar Techniques for growing mammary cells on 3D gels have been described30Petersen OW Ronnov-Jessen L Howlett AR Bissell MJ Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.Proc Natl Acad Sci USA. 1992; 89: 9064-9068Crossref PubMed Scopus (886) Google Scholar, 31Howlett AR Bailey N Damsky C Petersen OW Bissell MJ Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma.J Cell Sci. 1995; 108: 1945-1957PubMed Google Scholar (see review by Schmiechle and Bissell32Schmeichel KL Bissell MJ Modeling tissue-specific signaling and organ function in three dimensions.J Cell Sci. 2003; 116: 2377-2388Crossref PubMed Scopus (483) Google Scholar). Cells were fixed by adding in 4% buffered glutaraldehyde to the culture medium, followed by post fixation in osmium tetroxide. After dehydrations in ethanol, cultures were embedded in Epon. Ultra-thin sections were cut perpendicular and parallel to the culture, stained with uranyl acetate and lead citrate, and were examined with a JEOL 1011 transmission electron microscope (Peabody, MA). The sequestration and exposure of AluI binding sites was tested qualitatively by exposing permeabilized cultured cells on glass slides to restriction enzyme (permeabilized cell assay, Figure 1, A–F) to maintain the structural integrity of the nucleus and cytoplasm. We also smeared cultured cells on slides and allowed them to dry (cell smear assay, Figure 1, G–J) to rapidly assess a large number of samples. Each assay was repeated 18 times with identical results. In both the permeabilized cell assay and the cell smear assay, we detected striking difference in the
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