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WO2007030815A1 - Procedes permettant de determiner la pathogenicite de souches virales - Google Patents

Procedes permettant de determiner la pathogenicite de souches virales Download PDF

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Publication number
WO2007030815A1
WO2007030815A1 PCT/US2006/035258 US2006035258W WO2007030815A1 WO 2007030815 A1 WO2007030815 A1 WO 2007030815A1 US 2006035258 W US2006035258 W US 2006035258W WO 2007030815 A1 WO2007030815 A1 WO 2007030815A1
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cells
dna
nuclease
population
assay
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PCT/US2006/035258
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English (en)
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Andrew J. Maniotis
Robert Folberg
Klara Valyi-Nagy
Tibor Valyi-Nagy
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The Board Of Trustees Of The University Of Illinois
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Publication of WO2007030815A1 publication Critical patent/WO2007030815A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus

Definitions

  • Methods and compositions for determining pathogenicity of viral strains in addition to detecting merely the presence of viral strains are improvements in aiding decisions involving treatment plans and diagnosis. Detecting pathogenic viral strains from biological samples otherwise requires laborious methods and long periods of culturing.
  • Chromatin condensation states vary.
  • DNA in cancer cells is tightly packaged in protein complexes that contain disulfide bonds, making the DNA less susceptible to digestion by restriction enzymes, which are site-specific proteins that cut DNA at specific nucleotides.
  • restriction enzymes which are site-specific proteins that cut DNA at specific nucleotides.
  • Detergent extraction assays cell smear assays (a modified type of Pap smear), flow cytometry, touch preparations made from comparing human lesions with their margins in the normal tissue, and comparisons between isolated chromosome sets, all demonstrate that the more invasive the cancer cells, the more protection the cells' DNA has from restriction enzyme cleavage.
  • DNA in tumor cell genomes are more sequestered than the DNA from poorly invasive or normal cells.
  • Assays measuring DNA sequestration including the three- dimensional matrix chip assay, the constricting gel assay and therapeutic patch, have shown that the most malignant cells are the ones that are also the most resistant to most current forms of chemotherapy, including many chemotherapies that are designed to interfere with DNA metabolism.
  • These assays are currently being employed to test new forms of therapeutic interventions. For example, current forms of cancer chemotherapy do not target, and will never target, the most malignant cells whose DNA is largely protected through a cytoarchitectural resistance mechanism deep within the malignant cell.
  • Methods and compositions to determine the pathogenicity of viral strains are based on the ability of pathogenic viruses to uncover sequestered sites susceptible to nuclease degradation in cells with condensed chromatin characteristics.
  • Assays to determine the pathogenicity of viral strains based on the viral strains' ability to expose nuclease sites on chromatin, including condensed chromatin in malignant cells, are disclosed. Detecting pathogenic viruses and their pathogenicity in a sample, as compared to merely detecting the presence of viruses in a sample, is an useful tool in diagnosing viral infections.
  • Methods and compositions are provided not only for detecting the presence of a pathogenic virus in a sample and but also for determining the pathogenicity of a viral strain to cells.
  • Methods for detecting the presence of a pathogenic virus in a sample include rapid infectivity and lysis assays (RILA).
  • Methods include the steps of contacting cells, such as mammalian cells, with a sample including a viral strain, to produce a population of contacted, and presumptively infected, cells. The chromatin of the contacted/infected cells is then placed in contact with a DNA nuclease for a predetermined length of time, after which the cells are screened to determine what extent, if any, the nuclear DNA has been degraded.
  • the amount of DNA present is determined through the use of a DNA binding fluorescent entity and the inference is made that that the viral strain is pathogenic based on the comparative degradation of DNA.
  • the steps of detecting and quantitating nuclear DNA includes contacting the cells with a DNA binding fluorescent moiety, removing the unbound moiety, and measuring fluorescence from the contacted cells to determine whether nuclear DNA is present. Any suitable method of DNA detection is within the scope of this disclosure. The methods also quantitate the DNA.
  • the DNA nuclease is generally an endonuclease, e.g., a restriction endonuclease that has a four base pair recognition sequence.
  • the rapid infectivity and lysis assay detects viral species and predicts their pathogenic potential based on their ability to expose, rather than sequester, the
  • Any viral strain that is capable of exposing nuclease sites on condensed chromatin is suitable for testing by RILA.
  • cytomegalovirus CMV
  • RILA is also used to determine mutants resistant to drugs that are generated, for example, in patients undergoing antiviral therapy.
  • a method for determining the pathogenicity of a viral strain from a biological sample includes the steps of:
  • Pathogenicity of viral strains are determined in cells that have condensed chromatin, for example, malignant cells.
  • the condensed chromatin includes sequestered restriction sites.
  • Detecting the presence of nuclear DNA includes for example, contacting the cells with a nuclear DNA binding fluorescent moiety and measuring the fluorescence from the contacted cells to determine an amount of the nuclear DNA.
  • a DNA nuclease such as, for example, an endonuclease is used to detect the exposed nuclease sites.
  • a suitable endonuclease is a restriction endonuclease that has a four base pair recognition sequence.
  • a nuclease is selected from a group of nucleases for example, EcoRI, Saul, Pstl, Hindlll, Mspl, AIuI, DNase I, micrococcal nuclease, Sau3 AI, Dpnl, and Rsa I.
  • the cells used to determine the pathogenicity of viral strains are permeablized with a detergent prior to being contacted with the restriction endonuclease.
  • the viral strain is in a concentration of about 0.001 plaque forming units
  • a rapid infectivity lysis assay (RILA) to determine the pathogenic potential of a viral strain includes the steps of:
  • Pathogenicity determination assays described herein further include detecting lysis in the population of cells.
  • the lysis in the population of cells is determined within about 2 days after contacting the population of the cells with the viral strain.
  • Pathogenicity determination assays described herein include cells, e.g., mammalian cells.
  • Pathogenicity determination assays described herein are suitable for any virus capable of decondensing the chromatin and/or exposing the nuclease sites within the nuclear DNA.
  • a suitable viral strain is a human virus.
  • a suitable viral strain is herpes simplex virus (HSV) or cytomegalovirus (CMV). BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 are photographs of nuclei of cells infected with a virus. Arrows in the 0.1 PFU/cell (Plaque-forming Units) picture point to the nuclei of cells that have been infected for 2 hours with 0.1 PFU/cell concentration of virus, whereas, the arrows in the 1.0 PFU/cell and the 10 PFU/cell point to cells still showing fluorescence whose DNA may not have been damaged, or they may indicate cells that have yet to completely exhibit the viral infection.
  • PFU/cell Plaque-forming Units
  • FIG. 2 shows infectivity level in a RILA assay.
  • the arrow in the right photograph demonstrates the presence of a few living cells among all the dead (black) cells that take up the dye.
  • FIG. 3 shows that RILA assay detects that HSV-I is capable of decondensing chromatin and inducing cell lysis.
  • FIG. 4 shows that heat inactivated HSV does not lead to chromatin decondensation and does not render it more sensitive to AIu I digestion when compared with mock infected cells (suggesting that heat treatment leads to inactivation of critical viral proteins needed for entry into host cells). UV-inactivated HSV does not lead to chromatin decondensation and does not render it more sensitive to Alul digestion when compared with mock infected cells (suggesting that de novo viral protein synthesis is required for chromatin decondensation). This suggests that the chromatin decondensation effect by Heipes simplex virus is a specific effect mediated by the virus and not due to random events caused during cell culture. DETAILED DESCRIPTION
  • compositions and methods are described for rapidly detecting pathogenic viruses and their pathogenicity in a sample, as compared to merely detecting the presence of viruses in a sample.
  • a method is provided for measuring the pathogenic potential of viruses. Even a small quantity of the virus in the sample is sufficient to determine the pathogenicity of the virus.
  • An assay is performed in vitro on cell cultures to measure the ability of a virus containing sample to induce exposure of a cell's chromatin to the enzymatic activity of nucleases. This enhanced exposure of the cells' nuclear DNA to the enzymatic activity of nucleases upon exposure and infection by a virus has been correlated with the pathogenicity (infectivity) of the virus as determined for example, by lysis of the cells. Correlations are standardized for each type of cells used in the assay, between uncovered DNA digestion sites and lysis.
  • RILA Rapid Infectivity and Lysis Assays
  • the infectivity read-out after a 2 hour incubation with virus and a 1 hour incubation with AM demonstrates that the nuclear DNA has been exposed to the enzymatic activity of AIuI resulting in digestion of the DNA (as assayed by a simple fluorescent dye reaction that detects DNA; FIG. 1).
  • the RILA assay also measures the lytic potential of viruses within a day or two because at the same time it measures the extent of DNA exposure due to as few as 0.1 PFU/cell, it documents how many cells are killed by the infection (FIG. 2).
  • the RILA assay provides a direct demonstration of the potential of any virus to expose cellular chromatin to the enzymatic activity of nucleases such as AIuI and Msp-I, and, at the same time, determine if the presence of virus is pathogenic to the cells harboring the infection. This characteristic exposure of chromatin and lysis in the RILA assay is different from all existing assays that are generally directed at identifying viruses through markers or surrogate markers thought to be associated with the virus itself, or by detecting host responses to a viral infection.
  • the RILA has the advantage of providing pathogenicity results rapidly.
  • tests assays described herein that rapidly determine if a tiny amount of virus from a patient's sera has pathogenic potential present a milestone in viral testing because the pathogenic property of a viral infection, if any, could be rapidly confirmed, or dismissed, within 3 hours using the RILA test.
  • a method for determining the presence and/or pathogenicity of a viral strain includes the steps of contacting a population of cells with a sample known to contain, or suspected of containing, a viral strain and determining if that contact resulted in modifying the cell's nuclear DNA/cliromatin.
  • the method allows for the determination of whether the virus has induced a change in the cell's DNA/chromatin in a manner that makes the DNA/chromatin more susceptible to nucleases, relative to the nuclear DNA/cliromatin of cells not exposed to the virus.
  • the cells are eukaryotic cells, for example, are mammalian cells including human cells. Suitable cells include primary cell cultures or established cell lines.
  • Malignant cells such as melanoma cells are suitable. However, because cells may vary in their response to viral infection, controls need to use the same cells, and quantitation is standardized for specific cell types.
  • the cells contacted with the virus are typically cultured in vitro using standard techniques known to those skilled in the art. Cells are contacted with the virus at low viral titers, including titers as low as 10 PFU/cell, 5 PFU/cell, 1 PFU/cell, 0.5 PFU/cell or 0.1 PFU/cell. The cells and the virus are incubated together for a predetermined length of time under conditions suitable for attachment of the virus to the cells.
  • the cells' DNA is contacted with a DNA nuclease.
  • the cells are subjected to a wash to remove any non-bound material from said cells prior contacting the cells with the DNA nuclease.
  • the nuclear DNA/chromatin of the cells is contacted with a nuclease for a predetermined length of time.
  • a nuclease for a predetermined length of time.
  • the enzymatic activity of the nuclease is inhibited after the desired length of exposure of the cellular DNA/chromatin to the nuclease by the addition of a DNA binding fluorescent moiety such as ethidium bromide.
  • the length of time that the cellular DNA/chromatin is exposed to nuclease activity is dependent on the nuclease used, and the manner in which the nuclear DNA/chromatin is contacted with the nuclease.
  • the cell nuclear DNA/chromatin may be contacted with the nuclease (in the form of a solution) after the cell membranes have been permeabilized.
  • the cells are permeabilized using standard reagents and techniques known to those skilled in the art using organic solvents such as alcohols and acetone, detergents or other agents.
  • the cells are permeabilized by contacting the cells with a detergent such as Triton X-100.
  • the cell membranes are mechanically disrupted to allow the solution containing the nuclease, access to the cell's nuclear DNA/chromatin.
  • the cells are removed from the cell culture plate and allowed to dry on a solid support as a means of disrupting the cells membranes and allowing access of the nuclease to the cell's nuclear DNA/chromatin.
  • a solution comprising the nuclease is then applied to the dried cells.
  • the nuclease used to treat the viral contacted cells is selected from a group of any DNA nuclease including exonucleases and endonucleases.
  • the nuclease may be an endonuclease, such as EcoRI, Saul, Pstl, Hindlll, Mspl, AIuI, Mspl, DNase I, or micrococcal nuclease.
  • the nuclease is a restriction endonuclease that has a four base pair recognition site, for example, AIuI.
  • the cells After the cells have been contacted with a solution containing a virus, and have been subsequently treated with a solution containing a nuclease, the cells are then analyzed to determine the extent the cell's nuclear DNA has been degraded. Any of the techniques known to those skilled in the art for detecting the presence of DNA can be used in accordance with this invention.
  • the amount of intact DNA remaining after the nuclease treatment is measured by the use of a DNA binding fluorescent entity.
  • the DNA binding fluorescent entity is one that specifically binds to double stranded DNA, such as the DNA intercalating agents ethidium bromide and theophyllinea.
  • the amount of fluorescence emitted from the viral infected cells is compared to a control set of cells that were mock infected, exposed to the nuclease and then contacted with the DNA binding fluorescent entity.
  • a detected decrease in fluorescence from the virus contacted cells relative to the control cells is indicative of enhanced DNA degredation and thus is indicative of the pathogenicity of the viral strain.
  • RILA allows for the systematical testing a variety of different kinds of viruses, as well as allowing the determination of the pathogenicity of various strains of the same virus.
  • the procedures disclosed herein can be used to test whether a particular drug or therapeutic regiment has generated any " pathogenic mutants.”
  • Example 1 Chromatin Organization Measured by AIuI Restriction Enzyme Changes with Malignancy.
  • RILA Rapid Infectivity and Lysis Assays
  • fibroblasts 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.
  • MCFlOA breast epithelial cells and MDA- MB231 breast carcinoma cells were obtained from the ATTC, and were maintained on DMEM plus heat inactivated calf serum.
  • HMT-3522 human mammary epithelial cells both nonmalignant (Sl) and their tumorigenic counterparts designated T4-2 were described previously.
  • AU cell cultures were determined to be free of mycoplasma contamination using the GenProbe rapid detection system (Fisher, Itasca, IL). Restriction Enzyme Assays of Interphase Cells
  • the slurry was suspended in IX PBS or serum-free DMEM, and a drop containing 15 ⁇ l of the suspension was placed onto a glass slide. The drops were allowed to evaporate over 30 minutes to 1 hour.
  • AM restriction enzyme 0.5 ⁇ l in 40 ⁇ l
  • Mspl restriction enzyme 0.5 ⁇ l in 40 ⁇ l DMEM; Promega
  • 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 AIuI restriction enzyme and the reaction was terminated with ethidium bromide at 5 and 24 hours.
  • RGD-C is known to bind to integrin receptors.
  • 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.
  • cellular permeability was checked using the trypan blue exclusion method.
  • the AIuI restriction enzyme (0.5 ⁇ l in 40 ⁇ l 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 AM binding sites from enzyme digestion.
  • Incubation with AIuI was terminated by adding ethidium bromide (Sigma; 250 ⁇ l, 100 ng/ml) to each drop per slide at 30 minutes and at hourly intervals thereafter up to 24 hours of incubation.
  • 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.
  • 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 0 C in a 5% CO 2 buffered incubator before micromanipulation.
  • Chromosome sets were obtained either in the presence or absence of colchicine and cytochalasin-B. 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).
  • Leitz micromanipulator Leica Microsystems
  • 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 ⁇ m wide along a length of 40 to 100 ⁇ m (tip widths were consistently less than 0.5 ⁇ m).
  • restriction enzymes and proteases are important to use restriction enzymes and proteases in the range of specific ion concentrations if they are to function efficiently.
  • Buffered media conditions that preserve physiological ion concentrations were used to maintain the extent of chromatin compaction observed in living cells while providing the appropriate concentrations of NaCl and MgCl 2 to permit enzymes to work.
  • the restriction enzymes were used in the absence of DTT or ⁇ -mercaptoethanol (which are typically included in commercially available kits used to digest DNA) to avoid interfering with DNA-associated proteins.
  • HSV-I Herpes Simplex Virus Type 1
  • RILA Rapid Infectivity and Lysis Assay
  • HSV-I herpes simplex virus type 1
  • AIuI herpes simplex virus type 1
  • the (RILA) assay is based on the measurement of changes in the sensitivity of MUM-2 DNA to AM digestion following exposure to biologic material (body fluids) and comparison of the detected changes to the effect of positive controls (HSV-I infection) and negative controls (mock infection).
  • In vitro MUM-2 cultures are either exposed to the test body fluid, or known amounts of HSV-I (positive control) or sterile PBS (negative control) for 2 hours. This is followed by preparation of cell smears from the melanoma cultures, exposure of the smears to the AM restriction enzyme, ethidium bromide staining of DNA, microscopic visualization, photography and data analysis (cell smear assay).
  • the RILA assay is intended for the rapid detection of infectious HSV-I in body fluids.
  • RILA assays were conducted as follows: MUM-2 cells were grown to approximately 70% confluency on 6-well tissue culture plates. Cell numbers per were counted. The tissue culture medium was then removed from the wells and the cells were exposed for 1 hour at 37 C to one of the following inocula: A. 0.5 ml of sterile PBS (mock infection, negative control) B. HSV-I with a calculated multiplicity of infection (MOI) ranging from 0.001 plaque forming units (PFU) per cell to 10 PFU/cell diluted in PBS to a final volume of 0.5 ml.
  • MOI multiplicity of infection
  • test body fluid diluted to in PBS to a final volume of 0.5 ml.
  • fresh tissue culture medium (1 ml) was added to each well and the cultures were either incubated for another hour at 37 0 C and then processed for the cell smear assay or further incubated in repeatedly refreshed culture medium for 2 weeks for traditional virus detection by culture.
  • AM restriction enzyme Promega (0.5 ul in 40 ul of DMEM) was applied to the dried cells, and the preparation was placed in a humidified 37 0 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 allows for discrimination of differential chromatin digestion. Ethidium bromide was added to terminate the digestion and the preparation was photographed immediately using an inverted microscope and was scored qualitatively as follows:
  • Results The Rapid Infectivity and Lysis Assay can be performed on samples from body fluids in less than 2 hours and detects very small quantities of virus (as little as 0.1 PFU/cell compared with a mock viral infection).
  • the read-out after a 2 hour incubation with virus and a 1 hour incubation with AIuI is the demonstration of digested DNA (as assayed by a simple fluorescent dye reaction that detects DNA see FIG. 1).
  • the RILA assay can also measure the lytic potential of viruses within a day or two because at the same time it measures the extent of DNA exposure due to as few as 0.1 PFU/cell, it documents how many cells are killed by the infection (FIG. 2).
  • One advantage that this assay provides is directly demonstrating the potential of any virus to expose cellular DNA to AIuI, and at the same time determining if the presence of virus is pathogenic to the cells harboring the infection. This ability stands in stark contrast to all existing assays that are principally directed at identifying viruses, typically through markers thought to be associated with the virus itself, or by detecting surrogate markers, or by detecting host responses to a viral infection.
  • the RILA assay was repeated using various PFU concentrations using virology's most rigorous kinds of controls to demonstrate how the RILA assay detects that HSV-I is capable of decondensing chromatin, and inducing cell lysis. For example, HSV-I has been consistently observed to decondense chromatin, and with clarity of differential signal intensity (see FIG. 3A, High Magnification; and FIG. 3B, Low Magnification).

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Abstract

L'invention concerne des procédés et compositions permettant de détecter rapidement la présence et le potentiel lytique d'un virus pathogène dans un prélèvement et de déterminer la pathogénicité d'une souche virale de cellules, et contrairement à simplement détecter la présence de virus dans un prélèvement. Des essais biologiques permettent de déterminer la pathogénicité de souches virales sur l'aptitude des souches virales à exposer des sites de nucléase sur l'ADN nucléaire de la cellule hôte (par ex., de chromatine condensée dans des cellules malignes) à une nucléase d'ADN .
PCT/US2006/035258 2005-09-09 2006-09-11 Procedes permettant de determiner la pathogenicite de souches virales WO2007030815A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004108951A1 (fr) * 2003-06-06 2004-12-16 The Board Of Trustees Of The University Of Illinois Methodes d'evaluation du potentiel invasif d'une cellule faisant appel a l'analyse de chromatine
US20050142534A1 (en) * 2003-10-14 2005-06-30 The Board Of Trustees Of The University Of Illinois Methods and compositions related to a matrix chip

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004108951A1 (fr) * 2003-06-06 2004-12-16 The Board Of Trustees Of The University Of Illinois Methodes d'evaluation du potentiel invasif d'une cellule faisant appel a l'analyse de chromatine
US20050142534A1 (en) * 2003-10-14 2005-06-30 The Board Of Trustees Of The University Of Illinois Methods and compositions related to a matrix chip

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BENYOUCEF S ET AL: "A MICROASSAY FOR DETERMINATION OF THE CYTOPATHOGENICITY OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 ISOLATES", MICROBIOLOGY AND IMMUNOLOGY, TOKYO, JP, vol. 40, no. 5, 1996, pages 381 - 388, XP009037321, ISSN: 0385-5600 *
BENYOUCEF SAMIRA ET AL: "Combination of whole blood culture and a rapid and sensitive cell assay for the determination of the cytopathogenicity of human immunodeficiency virus type-1 isolates", JOURNAL OF VIROLOGICAL METHODS, AMSTERDAM, NL, vol. 71, no. 1, March 1998 (1998-03-01), pages 123 - 131, XP002298650, ISSN: 0166-0934 *
MANIOTIS ANDREW J ET AL: "Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.", THE AMERICAN JOURNAL OF PATHOLOGY APR 2005, vol. 166, no. 4, April 2005 (2005-04-01), pages 1187 - 1203, XP002415346, ISSN: 0002-9440 *

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