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WO1992017610A1 - Procede et kit de detection de l'apoptose - Google Patents

Procede et kit de detection de l'apoptose Download PDF

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Publication number
WO1992017610A1
WO1992017610A1 PCT/NL1992/000059 NL9200059W WO9217610A1 WO 1992017610 A1 WO1992017610 A1 WO 1992017610A1 NL 9200059 W NL9200059 W NL 9200059W WO 9217610 A1 WO9217610 A1 WO 9217610A1
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Prior art keywords
modified
deoxynucleotide
detectable label
cells
antibody
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PCT/NL1992/000059
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English (en)
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Richard Robert Jonker
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Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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Application filed by Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno filed Critical Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Publication of WO1992017610A1 publication Critical patent/WO1992017610A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the invention relates to a method and a kit for detecting apoptosis in mammalian cells. More particularly, the invention relates to a new technique for detecting apoptosis in individual cells.
  • Apoptosis is an important phenomenon in embryogenesis, cell differentiation and tumor regression.
  • Apoptosis is a general form of negative cell selection which can be induced by factors in the environment of the cells. These factors include: cytokines (e.g. Tumor Necrosis Factor, TNF) , T-lymphocytes, specific glucocorticosteroids and radiation.
  • cytokines e.g. Tumor Necrosis Factor, TNF
  • TNF Tumor Necrosis Factor
  • TNF Tumor Necrosis Factor
  • apoptosis The most important characteristics of apoptosis are break-down of the chromatin into nucleosome-sized DNA fragments (ca. 160 base pairs long) , segmentation of the nucleus, condensation of the cytoplasm and flaking of the cell membrane.
  • Sellins and Cohen Gene induction by ⁇ -irradiation leads to DNA fragmentation in lymphocytes, 1987, The Journal of Immunology 139, 3199-3206 have demonstrated that apoptosis is an active process in which RNA synthesis and protein synthesis take place before the process that leads to apoptosis can begin. If either of these is brought to a standstill, no apoptosis develo s.
  • apoptosis in connection with medical treatments where apoptosis is induced, occurs or is a relevant side-effect, can be detected.
  • cancer treatment strategies in development which are aimed at specifically inducing apoptosis in tumors. Evaluations of these methods of treatment require a good, objective method for detecting apoptosis both in tumour tissue and in healthy tissue.
  • interphase Death a specific form of apoptosis which can for instance be induced with irradiation, is an important phenomenon for which a good quantification is desirable.
  • the present invention provides a new method for detecting apoptosis in mammalian cells, wherein the cells to be examined are incubated in a medium suitable for in situ nick translation, while at least one of the deoxynucleotides present in the medium has been modified such that it can be detected, and the cells, after the nick translation medium has been removed, are examined for the presence of DNA containing modified deoxynucleotide.
  • the invention further provides a kit for detecting apoptosis in m_-it ⁇ malian cells, comprising (a) components from which a medium suitable for in situ nick translation can be composed, including at least one deoxynucleotide modified such that it can be detected, and (b) means with which the cells, after removal of the nick translation medium, can be examined for the presence of DNA containing modified deoxynucleotide.
  • apoptosis is detected by labelling breaks in the DNA in apoptotic cells and thereby making them observable.
  • Labelling the DNA breaks can be realized using enzymes which specifically incorporate deoxynucleotides (conveniently referred to herein as nucleotides) into DNA from a break in the DNA helix. This takes place according to the nick translation method which is known as such. This method is used in molecular biology for labelling DNA for hybridization uses (see: Molecular Cloning, A Laboratory Manual, J. Sambrook et al. , Cold Spring Harbor Laboratory Press, 1989, page 10.7-10.10).
  • cytometric techniques As the invention relates to a method of labelling individual cells, it is possible to measure them fast using cytometric techniques. Cytometry is often used for the study of mammalian cells. The two relevant techniques " are:
  • the principle of flow cytometry is that particles which are suspended in a liquid stream are passed through a laser beam. The laser light is scattered during this passage and for each individual cell the fluorescence that develops is measured. Thus, it is possible to obtain information on the size of the cell, structure and chemical composition of a number of cells of the order of some thousands of cells per second (see: M.R. Melamed, et al.: Flow Cytometry and Sorting, 1979, pp. 1-9 and 11-37, John Wiley & Sons, New York) .
  • the microscopic image of (a number of) cells are processed by a computer and analyzed using pattern recognition techniques. Thus, in a rapid manner, cells can be identified and characteristics of these cells can be measured.
  • the label used can also be detected in different manners, for instance, be visualized under a microscope, directly or after use of a suitable staining technique, or, in the case of a radioactive label, be observed using autoradiography.
  • the method according to the invention is based on the labelling of DNA breaks which are characteristic of cells in apoptosis.
  • the chromatin of the cells is cut into oligonucleosome-sized fragments.
  • Endonucleases cut the DNA between two nucleosome ⁇ . The breaks formed during this DNA fragmentation are the target for the in si tu nick translation.
  • the invention makes it possible to specifically label breaks in the DNA of cells in apoptosis.
  • the formation of DNA breaks is an early characteristic of apoptosis, so that the invention also enables detection of cells in the initial stage of apoptosis.
  • the cells can be distinguished clearly and objectively from cells that are not in apoptosis. Because the present method is an in si tu method, it is possible to accurately determine the percentage of cells in apoptosis.
  • the invention also enables assessment of apoptosis within a subpopulation of all cells. A clear distinction based on labelling technique enables rapid screening using flow and image cytometry.
  • Nucleotides can be modified in different ways in such a manner that they can be provided with a detectable label: - Modified nucleotides which can be recognized by an antibody, for instance Bromo-deoxyuridin (BrdU) and Iodo-deoxyuridin (IdU) . These are thymidin analogues which can be detected with antibodies which specifically bind to BrdU and IdU. These antibodies can be conjugated for instance to a fluorescent group or to an enzyme that permits optical detection. It is also possible to detect these antibodies by means of a second layer of fluorescently labelled antibodies.
  • BrdU Bromo-deoxyuridin
  • IdU Iodo-deoxyuridin
  • These antibodies can be conjugated for instance to a fluorescent group or to an enzyme that permits optical detection. It is also possible to detect these antibodies by means of a second layer of fluorescently labelled antibodies.
  • nucleotides which contain a group that can specifically bind to other molecules, for instance a biotin group or a digoxigenin group, bound to dUTP or dATP.
  • the DNA nucleotides can be coupled to biotin via a so-called spacer arm.
  • Biotin can be detected using avidin or streptavidin.
  • Biotin and (strept)avidin have a very high affinity for each other.
  • (Strept)avidin in turn can be simply coupled to a fluorescent substance, another dye, or an enzyme. It is for instance possible to utilize commercially available conjugates of streptavidin and avidin with FITC and Cascade Blue.
  • the hapten digoxigenin can be detected using antibodies.
  • Radioactively labelled nucleotides for instance nucleotides labelled with tritium.
  • Incorporation can be implemented with all enzymes that can incorporate nucleotides from a single or double-stranded break in the DNA, such as _____ coli DNA polymerase I, terminal deoxynucleotidyl-transferase and Klenow polymerase.
  • all fluorescent substances can be used which can be coupled to a protein molecule.
  • a dye that has an emission spectrum that can be distinguished well from the dye which the DNA breaks are labelled with it is preferred to use a dye that has an emission spectrum that can be distinguished well from the dye which the DNA breaks are labelled with.
  • FITC, Bodipy and Cascade Blue® are very suitable as dyes for the DNA breaks to be labelled, while DAPI, Hoechst 33258 and Propidium Jodide can be used for the counterstaining of the DNA.
  • the in situ nick translation procedure was carried out in so-called 'nick translation buffer' . It consisted of 5 mM MgCl 2 , 10 mM ⁇ -mercapto-ethanol, 50 mM Tris-pH 7.8 and 10 ⁇ g/ml Bovine Serum Albumin (BSA) . There were used 200,000 cells in 12.5 ⁇ l buffer. To this buffer were added: l unit polymerase (E. __i) , 0.2 nmol biotin-16-dUTP, 0.2 nmol unlabelled dATP, dCTP and dGTP. This mixture was incubated for 90 min at 15°C. Then the cells were washed and incubated in a staining buffer at room temperature for 30 min.
  • l unit polymerase E. _i
  • This colour buffer consisted of 2.5 ⁇ g/ml of an avidin-FITC conjugate, DCS grade in 4*SSC, 0.1% Triton-X-100 and 5% Nonfat Dry Milk. Then the cells were washed with Hepes buffered Hank's Salt Solution (HH) containing 0.1% Triton-X-100 and counterstained with 1 ⁇ g/ml of the fluorescent DNA dye DAPI for 30 minutes at 4°C in HH.
  • HH Hepes buffered Hank's Salt Solution
  • Fig. l shows the detection of apoptosis after irradiation, represented as dot plots of flow cytometry data. The measurements of 2,000 individual cells are shown. Individual cells are shown as dots in a two-dimensional space. Two bivariate distributions are shown of the DAPI fluorescence (x- axis) against the FITC fluorescence (y-axis) . The-DAPI fluorescence is a measure for the DNA contents. The FITC fluorescence is a measure for the amount of biotin-11-dUTP incorporated, and hence an indicator for apoptosis. Cells having a high FITC fluorescence are in apoptosis. The cells in Fig.
  • mice mouse thymocytes, isolated from BCBA mice. They were irradiated and then incubated at 37°C for 6 h. Then the cells were fixed with formaldehyde, followed by ethanol fixation. Then the in situ nick translation was carried out. The dots in the square with the arrow are the cells in apoptosis. These cells have an increased FITC fluorescence.
  • Fig. 2 shows the increase of apoptosis in DA-1 cells as a function of the radiation dose.
  • DA-1 cells were cultured with medium that had been enriched with IL-3.
  • Samples of 1.10 6 cells were irradiated and incubated with and without IL-3. After 6 h the samples were fixed.
  • the in situ nick translation procedure was carried out and the cells were measured. It is clear from this Figure that the in situ nick translation is a sensitive technique. From 0.25 Gy already an increased number of cells in apoptosis is measured. This figure also shows that the effect of growth factors on apoptosis can be studied with this method: IL-3 prevents this radiation-induced apoptosis virtually completely.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé de détection de l'apoptose dans les cellules mammifères. Il consiste à soumettre les cellules à examiner à l'incubation dans un milieu adapté à la translation de coupure in situ, l'un au moins des désoxynucléotides présents dans le milieu ayant été modifié de manière à le rendre détectable, et, après avoir enlevé le milieu de translation par coupure, à examiner les cellules pour la présence d'ADN contenant un désoxynucléotide modifié. On a également prévu un kit de détection de l'apoptose dans les cellules mammifères. Il comporte (a) des constituants permettant la composition d'un milieu adapté à la translation de coupure in situ, notamment au moins un désoxynucléotide que l'on a modifié de manière à le rendre détectable; et (b) un dispositif permettant, après l'enlèvement du milieu de translation de coupure, d'examiner les cellules pour la présence d'ADN contenant un désoxynuléotide modifié.
PCT/NL1992/000059 1991-03-27 1992-03-26 Procede et kit de detection de l'apoptose WO1992017610A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9100550A NL9100550A (nl) 1991-03-27 1991-03-27 Werkwijze en kit voor het detecteren van apoptose.
NL9100550 1991-03-27

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WO1992017610A1 true WO1992017610A1 (fr) 1992-10-15

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AU (1) AU1570192A (fr)
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0629856A3 (fr) * 1993-06-12 1996-07-17 Boehringer Mannheim Gmbh Méthode pour la detection d'ADN métaboliquement marqué.
WO1996028571A1 (fr) * 1995-03-15 1996-09-19 Societe Francaise De Recherches Et D'investissements (S.F.R.I.) Procede de detection qualitative et quantitative de lesions de l'adn
DE19544333A1 (de) * 1995-11-28 1997-06-05 Deutsches Krebsforsch Verfahren zur Beurteilung der Aktivität von Arzneistoffen
US5858694A (en) * 1997-05-30 1999-01-12 Cell Pathways, Inc. Method for identifying compounds for inhibition of cancerous lesions
EP0851939A4 (fr) * 1995-08-30 2001-02-28 New York Medical College Methodes de marquage des extremites d'adn par des nucleotides halogenes et de detection de ceux-ci a l'aide d'anticorps
CN111579538A (zh) * 2020-04-22 2020-08-25 山东第一医科大学(山东省医学科学院) 一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002064A1 (fr) * 1985-10-04 1987-04-09 Institut National De La Sante Et De La Recherche M Recuperation et dosage d'adn dans un liquide biologique acellulaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002064A1 (fr) * 1985-10-04 1987-04-09 Institut National De La Sante Et De La Recherche M Recuperation et dosage d'adn dans un liquide biologique acellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMERICAN J. PATHOLOGY vol. 139, no. 2, 1991, HAGERSTOWN, MD US pages 251 - 254; K. FEHSEL ET AL.: 'Analysis of TNFalpha-induced DNA strand breaks at the single cell level' *
JOURNAL OF IMMUNOLOGICAL METHODS. vol. 136, 24 January 1991, NEW YORK US pages 125 - 131; A. FACCHINETTI ET AL.: 'An improved method for the detection of DNA fragmentation' *
JOURNAL OF IMMUNOLOGICAL METHODS. vol. 137, 3 March 1991, NEW YORK US pages 79 - 87; W. SWAT ET AL: 'Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry' *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0629856A3 (fr) * 1993-06-12 1996-07-17 Boehringer Mannheim Gmbh Méthode pour la detection d'ADN métaboliquement marqué.
WO1996028571A1 (fr) * 1995-03-15 1996-09-19 Societe Francaise De Recherches Et D'investissements (S.F.R.I.) Procede de detection qualitative et quantitative de lesions de l'adn
FR2731711A1 (fr) * 1995-03-15 1996-09-20 Rech Investissements Sfri Soc Procede de detection qualitative et quantitative de lesions de l'adn
US6261767B1 (en) 1995-03-15 2001-07-17 Societe Francaise De Recherches Et D'investissements (Sfri) Method for the qualitative and quantitative detection of DNA damage
EP0851939A4 (fr) * 1995-08-30 2001-02-28 New York Medical College Methodes de marquage des extremites d'adn par des nucleotides halogenes et de detection de ceux-ci a l'aide d'anticorps
DE19544333A1 (de) * 1995-11-28 1997-06-05 Deutsches Krebsforsch Verfahren zur Beurteilung der Aktivität von Arzneistoffen
DE19544333C2 (de) * 1995-11-28 1998-12-10 Deutsches Krebsforsch Verfahren zur Beurteilung der Aktivität von Arzneistoffen
US5858694A (en) * 1997-05-30 1999-01-12 Cell Pathways, Inc. Method for identifying compounds for inhibition of cancerous lesions
CN111579538A (zh) * 2020-04-22 2020-08-25 山东第一医科大学(山东省医学科学院) 一种应用于循环肿瘤细胞检测用凋亡试剂盒及其检测方法
CN111579538B (zh) * 2020-04-22 2022-12-30 山东第一医科大学(山东省医学科学院) 一种利用凋亡试剂盒检测循环肿瘤细胞的方法

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NL9100550A (nl) 1992-10-16
EP0581801A1 (fr) 1994-02-09
AU1570192A (en) 1992-11-02

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