METHOD AND KIT FOR DETECTING APOPTOSIS

Abstract: A method is disclosed 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. Further disclosed is a kit for the detection of apoptosis in mammalian cells, comprising (a) components from which a medium suitable for in situ nick translation can be composed, including at least one deoxynucleotide that has been 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.

Description: Title: Method and kit for detecting apoptosis. 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. Other names for apoptosis are "interphase death" and "prograiπmed cell death" . The importance of detecting apoptosis in cells has been recognized, among others, by Kerr at al. (Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics, 1972, British Journal of Cancer 26: 239-257). 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. 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 γ-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. It is of great importance that apoptosis in connection with medical treatments where apoptosis is induced, occurs or is a relevant side-effect, can be detected. There are 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. Also, 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. Different methods for detecting apoptosis are known. These can be subdivided as follows: - Isolation of oligonucleosome-sized DNA fragments from apoptotic cells. However, for this method a relatively large number of cells are required and moreover this method does not provide any exact information on the percentage of cells in apoptosis. - Morphological evaluation. The morphological characteristics of cells in apoptosis are clearly distinguishable from other cells. During apoptosis both a shrinkage of the cytoplasm and a segmentation of the nucleus develop. For this type of analysis, however, a pathologist or trained analyst is required. Moreover, it is not easy to automate this process. Also, a good morphological evaluation is mainly limited to the later stages of apoptosis. - Methods based on differential permeability to certain fluorescent dyes of living and dead cells. In a later stage of apoptosis the cell membrane becomes permeable. The cells thereby become accessible to dyes. Stained cells can be counted using a microscope or a flow cytometer. This method is simple but not really specific for apoptosis and requires a fairly long time interval (ca. 6-24 h) between the induction of apoptosis and detection. 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_-itιmalian 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. According to the invention, 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). 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: - Flow cytometry. 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) . - Image cytometry. 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, however, 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. During the apoptotic DNA fragmentation, the chromatin of the cells is cut into oligonucleosome-sized fragments. Endonucleases cut the DNA between two nucleosomeε. 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. - Derivatives of 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. - Nucleotides which are coupled directly to a fluorescent group or molecule, such as fluorescein, coumarin, rhodamine and their derivatives. 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. As a label, all fluorescent substances can be used which can be coupled to a protein molecule. For optional counterstaining of the DNA, 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 invention will be further explained and illustrated in and by an Example and with reference to Figs 1 and 2. Example The in situ nick translation procedure was carried out in so-called 'nick translation buffer' . It consisted of 5 mM MgCl 2 , 10 mM β-mercapto-ethanol, 50 mM Tris-pH 7.8 and 10 μg/ml Bovine Serum Albumin (BSA) . There were used 200,000 cells in 12.5 μl 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. This colour buffer consisted of 2.5 μg/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 μg/ml of the fluorescent DNA dye DAPI for 30 minutes at 4°C in HH. Figures 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. l are 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.

Claims: 1. A method for detecting apoptosis in mammalian cells, wherein the cells to be examined are incubated in a medium suitable for in si tu 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. 2. A method as claimed in claim 1, wherein a nick translation medium is used in which at least one of the deoxynucleotides carries a detectable label. 3. A method as claimed in claim 2, wherein as the detectable label a fluorescent group or molecule, or a radioactive element is used. 4. A method as claimed in claim 1, wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified such that it can be detected using a substance that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 5. A method as claimed in claim 4,'wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified such that it can be detected using an antibody that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 6. A method as claimed in claim 5, wherein a nick translation medium is used that contains a modified deoxynucleotide selected from the group consisting of bromo- deoxyuridin (BrdU) and iodo-deoxyuridin (IdU) , which modified deoxynucleotide can be detected using an antibody that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 7. A method as claimed in claim 5, wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified with a hapten which can be detected using an antibody that can specifically bind to the haptenized deoxynucleotide and has been provided with a detectable label. 8. A method as claimed in claim 7, wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified with digoxigenin which can be detected using an antibody that can specifically bind to digoxigenin and carries a detectable label. 9. A method as claimed in claim , wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified with biotin which can be detected using (strept) vidin carrying a detectable label. 10. A method as claimed in claim 1, wherein a nick translation medium is used in which at least one of the deoxynucleotides has been modified such that it can be detected using a first antibody that can specifically bind to the modified deoxynucleotide and a second antibody that can specifically bind to the first antibody and has been provided with a detectable label. 11. A method as claimed in any one of claims 4-10, wherein as the detectable label a fluorescent substance, a dye, or a detectable enzyme is used. 12. A method as claimed in any one of claims 1-11, wherein the cells are examined for the presence of DNA containing modified deoxynucleotide, using flow cytometry or image cytometry- 13. A kit for the detection of apoptosis in mammalian cells, comprising (a) components from which a medium suitable for in situ nick translation can be composed, including at least one deoxynucleotide that has been 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. 14. A kit as claimed in claim 13, comprising at least one deoxynucleotide that carries a detectable label. 15. A kit as claimed in claim 14, wherein the detectable label is a radioactive element or a fluorescent group or molecule. 16. A kit as claimed in claim 13, comprising at least one modified deoxynucleotide as well as a substance that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 17. A kit as claimed in claim 16, comprising at least one modified deoxynucleotide as well as an antibody that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 18. A kit as claimed in claim 17, comprising a modified deoxynucleotide selected from the group consisting of bromo- deoxyuridin (BrdU) and iodo-deoxyuridin (IdU) , as well as an antibody that can specifically bind to the modified deoxynucleotide and has been provided with a detectable label. 19. A kit as claimed in claim 17, comprising at least one hapten-modified deoxynucleotide, as well as an antibody that can specifically bind to the haptenized deoxynucleotide and has been provided with a detectable label. 20. A kit as claimed in claim 19, comprising at least one digoxigenin-modified deoxynucleotide, as well as an antibody that can specifically bind to digoxigenin and carries a detectable label. 21. A kit as claimed in claim 16, comprising at least one biotin-modified deoxynucleotide, as well as (strept)avidin carrying a detectable label. 22. A kit as claimed in claim 13, comprising at least one modified deoxynucleotide, a first antibody that can specifically bind to the modified deoxynucleotide, as well as a second antibody that can specifically bind to the first antibody and has been provided with a detectable label. 23. A kit as claimed in any one of claims 16-22, wherein the detectable label is a fluorescent substance, a dye, or a detectable enzyme is used.

Inventors: JONKER RICHARD ROBERT, JONKER, RICHARD, ROBERT
Applicants: TNO, NEDERLANDSE ORGANISATIE VOOR TOEGEPAST-NATUURWETEN
Application year: 1992
Main classification: C12Q1
IPC codes: C12Q1