WO2003044535A1 - Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables - Google Patents

Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables Download PDF

Info

Publication number
WO2003044535A1
WO2003044535A1 PCT/TR2001/000060 TR0100060W WO03044535A1 WO 2003044535 A1 WO2003044535 A1 WO 2003044535A1 TR 0100060 W TR0100060 W TR 0100060W WO 03044535 A1 WO03044535 A1 WO 03044535A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
napo
apoptosis
apoptotic
binding reagent
Prior art date
Application number
PCT/TR2001/000060
Other languages
English (en)
Inventor
Mehmet Ozturk
Berna Suat Sayan
Original Assignee
Mehmet Ozturk
Berna Suat Sayan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mehmet Ozturk, Berna Suat Sayan filed Critical Mehmet Ozturk
Priority to AU2002221261A priority Critical patent/AU2002221261A1/en
Priority to PCT/TR2001/000060 priority patent/WO2003044535A1/fr
Publication of WO2003044535A1 publication Critical patent/WO2003044535A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention is directed to detection and quantification of cell apoptosis. This invention is also directed to the characterization of the NAPO antigen that is detectable in viable, but not in apoptotic cells. This invention is also directed to the production of anti-NAPO antibodies, more particularly, a monoclonal antibody produced by a mouse-X-mouse hybridoma. A method based on the detection of NAPO for distinguishing apoptotic cells from quiescent, proliferating, mitotic and senescent cells is also disclosed.
  • This method can be used for research into apoptosis; for diagnosis of apoptosis; for distinguishing apoptosis from quiescence, proliferation, mitosis and senescence; for testing the ability of cells to undergo apoptosis upon treatment with a selected agent; and for testing the ability of a drug to induce or to inhibit apoptosis.
  • a eukaryotic cell can be in proliferation, quiescence, senescence and apoptosis states, depending on its internal program that can be regulated by external effectors.
  • a cell passes through different stages of the cell cycle. Each cycle is divided into two main alternating phases, called S (DNA synthesis) and M (mitosis).
  • S DNA synthesis
  • M mitosis
  • the phase between M and S is called Gl (gap 1)
  • G2 gap 2
  • the quiescence defines a non proliferating state of cells that can be reversible.
  • Senescence defines a physiologically irreversible state in which cells are no longer able to reenter the cell cycle, unless the senescence program is overwrote by a process called immortalization as seen in most cancer cells (Nurse P., Cell, 100:71-78, 2000; Sherr CJ. and DePinho RA., Cell, 2000, 102:407-410; Murray A. and Hunt T. "Cell Cycle", W.H. Freeman and Comp., 1st ed., 1993)
  • Apoptosis is programmed cell death, a naturally occurring process involved in both the development and aging of cells. It is the process whereby the body can rid itself of unwanted, old, or damaged cells. Apoptosis is the physiological counterpart of cell proliferation.
  • Apoptosis is characterized by a decrease in cell volume, a condensation of chromatin, cellular budding, and the fragmentation of D ⁇ A into a ladder of 180 base pair (bp) oligomers with 3'-OH free ends, a hallmark of apoptosis.
  • Cell membranes maintain their integrity through the process, and lysosomes remain intact (Saraste A., and Pulkki K., Cardiovasc. Res., 2000, 45:528-537). There is no inflammatory response from apoptosis. Affected cells undergo phagocytosis by adjacent normal cells and by some macrophages.
  • Morphological changes observed in apoptotic cells result from a series of genetically programmed biochemical changes initiated by either the activation of death receptors or by intracellular stress conditions such as D ⁇ A damage. These pro-apoptotic signals are conveyed to mitochondria to cause the release of caspase-activating factors from this organelle, followed by a cascade of caspase activation which leads to cell death (Earnshaw WC. et al., Annu. Rev. Biochem., 1999, 68:383-424; Gottlieb RA., FEBS Lett, 2000, 482:6-12).
  • Apoptosis can be activated by a number of intrinsic or extrinsic signals. These signals include the following: mild physical signals, such as ionization radiation, ultraviolet radiation, or hyperthermia, low to medium doses of toxic compounds, such as azides or hydrogen peroxides; chemotherapeutic drugs, such as etoposides and cis-platinium, death receptor activators such as tumor necrosis factor- ⁇ , fas ligand and its agonists. Apoptosis can also be activated when cells are deprived from their survival factors that maintain them in a viable condition (Zornig M. et al., Biochimica et Biophysica Acta, 2001, 1551 : F1-F37).
  • Unregulated apoptosis is involved in diseases such as cancer, heart disease, neurodegenerative disorders, autoimmune disorders, and viral and bacterial infections. Cancer, for example, not only triggers cells to proliferate but also blocks apoptosis. Cancer is partly a failure of apoptosis: the orders for the cells to kill themselves by apoptosis are blocked. New cancer treatments that involve inducing apoptosis are being researched (Huang P and Oliff A., Trends Cell Biol., 2001, 11 :343-348).
  • Apoptosis may also be involved in the destruction of neurons in people afflicted by strokes or neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. There is evidence suggesting that ischemia can kill neurons by inducing apoptosis. Thus, inhibition of apoptosis may be a therapeutic strategy for the treatment of neurodegenerative disorders such as stroke (Deigner H. et al., Expert Opin Investig Drugs, 2000, 9:747-764; Irizarry MC. and Hyman BT., J Neuropathol Exp Neurol, 2001, 60:923-928)
  • T-cells differentiate between self and nonself (foreign) cells in the body.
  • Autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis, result when a small percentage of T-cells attack the body's own tissue.
  • Drugs are being developed that protect self target cells from T-cell induced apoptosis (Naux DL. and Flavell RA., Curr Opin Immunol, 2000, 12:719-724)
  • HIN human immunodeficiency virus
  • apoptosis is a target subject for understanding cellular mechanisms of many diseases, as well as for developing new drugs that interfere with either pro-apoptotic or anti-apoptotic molecular networks.
  • Much research is now focussing on developing drugs that can either inhibit or induce apoptosis depending on the targeted disease.
  • a major difficulty with researching apoptosis and drugs to control it is that a reliable marker of apoptosis has not yet been developed. Therefore, it has become important to develop reliable assays to measure cell death.
  • a marker is also needed in order to determine whether cells are dying or have been killed by apoptosis in the diagnosis of these diseases. For example, a marker for apoptosis could be used to determine the extend of neuronal damage caused by a stroke.
  • Apoptosis drugs are being used in therapy, and a reliable marker is needed in order to evaluate the progress of the therapy.
  • a major goal of some cancer chemotherapies has become to kill cancer cells by inducing apoptosis in these cells. It is estimated, however, that almost 50 percent of cancer drug treatments fail. It would be useful to have a method to assess the performance of new treatments in a reliable and effective manner.
  • apoptosis detection is based on the study of morphology of apoptotic cells (light microscopy and fluorescence microscopy coupled to nuclear staining with specific dyes, electron microscopy), D ⁇ A fragmentation detected by terminal transferase-mediated dUTP nick-end labelling (TU ⁇ EL) and similar techniques, membrane changes detected by annexin N in vivo labelling, and on immunological assays using antibodies directed to apoptosis-related proteins (Stadelmann C. and Lassmann H., Cell Tissue Res., 2000, 301:19-31).
  • the TUNEL method consists of catalytically adding a nucleotide, which has been conjugated to a chromogen system or to a fluorescent tag, to the 3'-OH end of DNA fragments as indicative of apoptosis.
  • TUNEL Apoptosis Detection Kit Procedures to detect cell death based on the TUNEL method are offered by both Roche (Cell Death Kit), Oncor (Apoptag Plus) and Koma Biotech. (TUNEL Apoptosis Detection Kit). This method involves a number of limitations. Early detection of apoptosis is not possible with this method because the DNA fragmentation is an end- point in the apoptosis pathway. False positives are often obtained when using the TUNEL method as a result of DNA damage that generates DNA fragments that have 3'-OH ends. In most TUNEL applications, mitotic cells can give false positive results. False negatives can also occur in certain cell types or situations where apoptosis does not lead to DNA laddering. Furthermore, the method is not quantitative since the amount of DNA fragments per cell is dependent upon the stage of apoptosis of the cell. In addition, this method is tedious, requires many steps and basic skills of molecular biology.
  • Annexin Another marker that is currently available is annexin, sold under the trademark APOPTEST.TM.. This marker is used in the "Apoptosis Detection Kit” offered by R&D Systems and Annexin N-PE offered by BD PharMingen. During apoptosis, a cell membrane's phospholipid asymmetry changes such that the phospholipids of the inner membrane are exposed on the outer membrane. Annexins are a homologous group of proteins that bind these phospholipids in the presence of calcium. This marker, however, suffers from a number of problems. Annexin-based tests have a strong potential for a lack of specificity due to the fact that the binding reaction is not as specific as antigen- antibody binding reactions.
  • NAPO for negative in apoptosis
  • the present invention relates to NAPO which comprises two proteins of about 60 kd and 70 kd, antibodies and fragments thereof capable of specifically reacting with an antigenic determinant of NAPO; a mouse monoclonal antibody and fragments thereof capable of specifically reacting with an antigenic determinant of NAPO, a hybridoma producing said monoclonal antibody, and apoptosis detection methods based on said NAPO, and anti-NAPO antibodies.
  • FIG. 1 shows the presence and nuclear localization of the NAPO antigen in viable cells.
  • FIG. 1A cells are immunostained with the anti-NAPO antibody.
  • FIG. IB shows the Hoechst 33258 counterstaining of these cells.
  • Cells were grown on cover-slips and fixed by paraformaldehyde and permeabilized with Triton-X-sodium citrate buffer. Following saturation for 15 minutes, fixed cells were incubated with anti-NAPO antibody, followed by FITC-conjugated goat-anti-mouse Ig. Nuclear DNA was visualized Hoechst 33258. Cover-slips were then examined under fluorescent microscope.
  • FIG. 2 shows that NAPO antigen comprises two major proteins migrating at approximately 60 kd and 70 kd, respectively.
  • Huh7 cells were metabolically labelled with 35 S Methionine and lysed in NP-40 lysis buffer.
  • the NAPO antigen was immunoprecipitated from the cell lysate with anti-NAPO antibody and Protein G sepharose, and analysed by SDS-PAGE and autoradiography.
  • Numbers (116, 66 and 45) at the left side indicate the positions and the molecular weights (kilodalton) of protein markers used as molecular weight standards; (-) line denotes the result with negative control for immunoprecipitation, (+) line is the result of immunoprecipitation with anti- NAPO antibody, showing 2 major protein bands which migrate at about 60 kd and 70 kd, respectively.
  • FIG. 3 demonstrates that the NAPO antigen is undetectable in cells undergoing spontaneous apoptosis.
  • SNU 398 hepatocellular carcinoma cells were grown on cover- slips and subjected to immunofluorescence as described in FIG. 1.
  • FIG. 3 A shows the NAPO staining of these cells and 3B shows the Hoechst 33258 counterstaining.
  • White arrows in FIG. 3B indicate cells with smaller and intensely staining nuclei of apoptotic cells which are negative for NAPO staining in FIG. 3 A.
  • FIG. 4 shows that NAPO antigen is undetectable in apoptosis induced by serum starvation.
  • FIG. 4A shows the NAPO staining of these cells and 4B shows the Hoechst 33258 counterstain.
  • White arrows in FIG. 4B indicate cells at different stages of apoptosis (with and without nuclear fragmentation), all showing negative NAPO staining when compared to viable cells. This figure was overexposed intentionally to show background fluorescence of NAPO-negative cells.
  • FIG. 5 shows that NAPO antigen is undetectable in apoptosis induced by oxidative stress.
  • Huh7 cells were grown and tested as described in figure 1, except that cells were grown in 0.1% FCS-containing culture medium for 72 hours, and treated with H 2 O 2 to induce oxidative stress and apoptosis.
  • a group of cells with small nuclei and intense Hoechst 33258 counterstaining, as shown in figure 5B, are negative for NAPO immunoreactivity, as shown in figure 5 A.
  • Figure 5 also shows that the loss of NAPO immunoreactivity in apoptotic cells is specific for this antigen.
  • the immunoreactivity of another nuclear protein, namely p53 is not lost during apoptosis in these cells.
  • FIG. 5C shows that TUNEL assay is positive in apoptosis induced by oxidative stress in Huh7 cells which were grown and treated as described for NAPO staining.
  • TUNEL assay fixed cells were stained using In Situ Cell Death Detection Kit (Roche), in stead of NAPO staining, according to supplier's instructions.
  • FIG. 6 demonstrates that NAPO is undetectable in apoptosis induced by death receptor activation.
  • Death receptor activation was carried out using two different stimuli and two different cell lines. Fas receptor was activated with an agonist anti-fas antibody, namely clone CH11 by incubating Jurkat (acute T cell leukemia) cells in a culture medium containing this antibody. Following 24 h treatment, these cells growing in suspension were attached to microscope slides by cytocentrifugation, stained for NAPO and Hoechst 33258, and examined under fluorescent microscopy, as described in FIG. 1. Arrows in FIG. 6B indicate apoptotic cells which stain negatively for NAPO as shown in FIG. 6A.
  • TNF receptor was activated in MCF-7 breast carcinoma cells by treatment with recombinant TNF- ⁇ for 72 hr, stained for NAPO and Hoechst 33258, and examined under fluorescent microscopy, as described in FIG. 1. Arrows in FIG. 6D indicate apoptotic cells which stain negatively for NAPO as shown in FIG. 6C.
  • FIG. 7 demonstrates that NAPO antigen is undetectable in apoptosis induced by UN irradiation in many cell types, including lymphoid cells (Jurkat), normal f ⁇ broblasts (MRC-5), cervical cancer epithelial cells (HeLa), colon carcinoma epithelial cells (SW480), and osteosarcoma cells (U2OS).
  • UN-C 60-120 mJ/cm 2
  • ⁇ APO immunostaining and Hoechst 33258 counterstaining as described in FIG. 1 (all except Jurkat cells) or FIG. 6 (Jurkat cells).
  • 7B, 7D, 7F, 7H and 7J indicate apoptotic Jurkat, MRC-5, HeLa, SW480 and U2OS cells, respectively. These apoptotic cells are negative for ⁇ APO, as shown respectively in FIG. 7A, 7C, 7E, 7G and 71.
  • FIG. 8 shows the presence of ⁇ APO in viable quiescent cells.
  • MRC-5 human embryonic lung fibroblast cells (passage 18) were grown to confluency and serum starved for 3 days to induce quiescence as described previously (Campisi J. et al., Exp Cell Res, 1984, 152: 459-466).
  • the BrdU incorporation test was used for verification. For this, cells were exposed to BrdU for lhr prior to fixation and permeabilization.
  • ⁇ APO and Hoechst 33258 staining was performed as described in FIG.l. BrdU staining was done with FITC-conjugated anti-BrdU antibody from DAKO.
  • FIG. 8A Quiescent MRC-5 cells are totally negative for BrdU (FIG. 8A), but totally positive for ⁇ APO (FIG. 8C). In control experiment, about 15% of asynchronizely growing cells are positive for BrdU staining (FIG. 8E) and all of asynchronizely growing cells are positive for ⁇ APO (FIG. 8G).
  • FIG. 8B, 8D, 8F and 8H show Hoechst 33258 counterstaining.
  • FIG. 9 demonstrates that NAPO antigen is present in cells at each phase of the cell cycle including Gl, S, G2 and mitotic phases. Huh7 cells were synchronized by nocodazole treatment, followed by mitotic shake-off, as described (Zieve GW. et al., Exp.
  • FIG. 9B shows NAPO staining of nocodazole-treated cells where 2 of 8 cells are in mitosis, as indicated by the pattern of Hoechst 33258 staining, shown with arrows in FIG. 9C. These mitotic cells show diffuse but strongly positive NAPO staining (arrows in FIG.
  • FIG. 9B shows Other cells are also strongly positive, but NAPO staining is not diffused.
  • FIG. 9D, 9F and 9H show positive NAPO staining at times 8h, 24h and 36h, indicating that NAPO is positive at respectively Gl, S, and G2 phases of the cell cycle, according to the data shown in figure 9A.
  • FIG. 9E, 9G, 91 show Hoechst 33258 counterstaining of the same samples.
  • FIG. 10 shows that NAPO antigen is present in senescent cells.
  • Senescence was induced by serial passage of MRC-5 cells from passage 18 to passage 40. Passage 18, and passage 40 cells were defined as, pre-senescent and senescent cells, respectively.
  • Pre-senescent and senescent MRC-5 cells were grown on cover-slips and subjected to immunofluorescence with the anti-NAPO antibody, as described in FIG.l.
  • Pre-senescent (FIG. 10A, C, E) and senescent (FIG. 10B, D, F) MRC-5 cells were also stained for senescence-associated ⁇ -galactosidase activity (FIG. 10A, B).
  • FIG. 10A senescence-associated ⁇ -galactosidase-negative (FIG. 10A) and senescence-associated ⁇ - galactosidase-positive cells (FIG. 10B) were positive for NAPO immunoreactivity (FIG. 10C and 10D, respectively).
  • FIG. 10E and 10F show Hoechst 33258 counterstaining of cells shown in FIG. 10C and 10D, respectively.
  • FIG. 11 shows that NAPO can be used to test the ability of compounds to modulate apoptosis.
  • the ability of H 2 O 2 to activate apoptosis was tested by treatment of Huh7 cells with this compound, as described in FIG. 5.
  • the ability of sodium selenite to inhibit H 2 O 2 -activated apoptosis was tested by co-treatment of Huh7 cells with both compounds.
  • the ability of H 2 O 2 to induce apoptosis was tested by adding this compound into culture medium of Huh7 cells as described in FIG. 5. NAPO staining showed that this compound induces apoptosis in these cells (FIG. 11 A), as compared to untreated cells (FIG. 11C).
  • FIG. 11B, 11D, and 1 IF show Hoechst 33258 counterstaining of these cells.
  • FIG 12. shows that NAPO can be used in combination with another apoptotic marker for better definition of apoptotic cells.
  • Apoptosis was induced in Huh7 cells as described in FIG.5. Unfixed cells were first stained with PE-conjugated Annexin V to mark the membranes of apoptotic cells, following supplier's instructions. Cells were then fixed, permeabilized, stained for NAPO, and counterstained with Hoechst 33258, as described in FIG.l.
  • FIG. 12 A under fluorescent microscope with appropriate filters, apoptotic cells show red membrane staining (annexin V-positive, NAPO-negative), while viable cells show green nuclear staining (annexin N-negative, ⁇ APO-positive).
  • FIG. 12B the same cells also show positive Hoechst 33258 staining, apoptotic cells being more intensely stained.
  • ⁇ APO marker can also be used as an apoptotic marker using indirect immunoperoxidase assay.
  • HepG2 and Huh7 cells were grown in culture medium with 0.2 % FCS for 72h, and fixed in methanol. Coverslips were first incubated with anti- ⁇ APO antibody, followed by HRP-conjugated anti- mouse Ig. Peroxidase activity was tested by incubating the samples with diaminobenzidine and H 2 0 2 which generate a brown color. Cells were then counterstained with haematoxylin which provides purple blue nuclear staining.
  • FIG. 13 A and 13B show respectively, Huh7 and HepG2 cells.
  • NAPO means a nuclear antigen comprising two proteins migrating at about 60 kilodalton (kd) and 70 kd, respectively, and present in viable cells, but undetectable or present at significantly decreased levels in apoptotic cells, when tested with an anti-NAPO antibody.
  • anti-NAPO antibodies means any antibodies or fractions thereof that have been produced using a source of NAPO antigen such as Colo 320DM human cancer cell line. Alternatively, anti-NAPO antibodies can also be produced by a purified NAPO antigen as an immunogen.
  • anti-NAPO antibodies includes recombinant, chimeric, and affinity modified forms made by techniques of molecular biology well known in the art.
  • Fab means an antigen binding fragment which is obtained by cleaving an antibody with papain in the hinge region yielding two Fab fragments, each having the heavy and light chain domains of an antibody, plus an Fc portion.
  • Fc means the antibody fragment which may activate complement.
  • Fv fragments means heterodimers of the heavy and light chain variable domains of an antibody. These variable domains may be joined by a peptide linker or by an engineered disulphide bond.
  • PI propidium iodide
  • TUNEL terminal transferase-mediated dUTP nick-end labelling
  • PAGE polyacrylamide gel electrophoresis
  • SDS sodium dodecyl sulfate
  • TNF tumor necrosis factor
  • BrdU 5-Bromo-2'-deoxyuridine
  • DMEM Dulbecco's modified minimal essential medium
  • FCS fetal calf serum
  • rpm revolutions per minute
  • AIDS acquired immunodeficiency syndrome
  • HIV human immunodeficiency virus
  • BSA bovine serum albumin
  • PBS phosphate-buffered saline
  • PBS-T PBS containing 0.1 % Tween-20
  • kd kilodalton
  • MW molecular weight
  • HRP Hexeradish peroxidase
  • the present invention resides in the discovery that NAPO antigen is present in viable cells at different states such as quiescence, proliferation, mitosis, senescence, but becomes immunologically undetectable upon induction of apoptosis.
  • the lack of NAPO immunoreactivity can therefore serve as a marker for detection of apoptosis.
  • the presence of NAPO can serve as a marker for any viable cell independent of its state.
  • a monoclonal antibody, designated anti-NAPO, that defines a unique epitope exposed in viable but not in apoptotic cells is provided by the invention.
  • anti-NAPO stains viable, permeabilized normal and cancerous human cells including epithelial, lymphoid and fibroblastic cells (see FIG. 4- 13, for examples). Indeed, all human-derived viable cells tested so far (Table 1) stain positively with anti-NAPO antibody, indicating that NAPO antigen is ubiquitously expressed in all types of human viable cells.
  • the antibody labels all viable cells regardless their state, as examplified by positive staining of quiescent, proliferating (all phases of the cell cycle including Gl, S, G2 and mitosis) and senescent cells (FIG. 8-10). In contrast, the antibody fail to label cells undergoing apoptosis regardless the apoptosis- inducing stimuli (FIG. 4-7).
  • the antigen defined by anti-NAPO is mostly localized in the nucleus and it comprises two proteins of about 60 kd and 70 kd.
  • 1HCC hepatocellular carcinoma
  • TCL acute T cell leukemia
  • NAPO antigen As determined by indirect immunofluorescence microscopy, no reactivity of anti-NAPO was observed in apoptotic cells generated by different treatments known to induce apoptosis. Annexin N staining experiments revealed that ⁇ APO (-) cells, but not
  • ⁇ APO (+) cells had extracellularly exposed phosphatidyl serine which can be detected by a reagent such as annexin N having high affinity for phosphatidyl serine (FIG. 12).
  • the methods described herein can be used to generate additional monoclonal antibodies with the characteristics of the anti- ⁇ APO antibody described in the examples set forth below, or by methods well-known to those skilled in the art (Harlow E. and Lane D., “Antibodies: A laboratory manual”, Cold Spring Harbor ed.l, 1988). Screening procedures to identify antibodies with the desired characteristics are also described herein. In addition, the identification of antibodies and immunoreactive fragments thereof within the scope of the invention can be accomplished using standard competitive binding assays known to the skilled artisan using the anti- ⁇ APO antibody provided by the hybrid cell line CL52 (Harlow E. and Lane D., "Antibodies: A laboratory manual”, Cold Spring Harbor ed.l, 1988).
  • antibody fragments and derivatives which comprise at least the functional portion of the antigen binding domain of an anti- ⁇ APO antibody molecule.
  • Antibody fragments which contain the binding domain of the molecule can be generated by known techniques.
  • such fragments include, but are not limited to: The F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment; and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Antibody fragments also include Fv fragments, i.e. antibody products within which there are not constant region amino acid residues (Coligan et al., "Current Protocols In Immunology", Wiley Interscience ed. 2.8, 2.10, 1991 or Harlow E. and Lane D., "Antibodies: A laboratory manual", Cold Spring Harbor ed.1, 1988).
  • COLO 320 DM (ATCC No: CCL-220) cells were lysed in 2 ml PBS and
  • mice 0.5 ml of lysate was injected into tail vein of Balb/c mice. One month later, mice were immunized twice more at one week intervals, hybridomas were prepared from splenic cells, and antibody-producing clones were selected as described previously (Ozturk et al., Cancer Res., 1989, 49:6764-6773). Briefly, monoclonal antibody anti-NAPO was screened and cloned from a hybridoma generated by fusing a mouse myeloma cell line with splenocytes from a mouse immunized with Colo 320 DM cell line lysate.
  • Splenocytes from the immunized mouse were fused with myeloma cells using polyethylene glycol by the method previously described (Kohler G. and Milstein C, Nature, 1975, 256:495-497).
  • Anti-NAPO was screened against Colo 320 DM cell lysate. It was shown to be an IgG isotype using antibodies against mouse IgG such as FITC- conjugated goat anti-mouse IgG (Sigma F2012 or Sigma F2883). Hybridoma supematants were used for most assays. When needed, ascites for anti-NAPO were produced in mice and the antibody was purified from ascites fluid by protein A or protein G affinity column (Pharmacia, Piscataway, N.J.).
  • Huh7, SNU 398, COLO 320 DM, MCF-7, HeLa, U2OS, SW480, A375, 293, MRC-5 and U2OS cells were grown in DMEM (Biochrome or Gibco).
  • Jurkat and LNCaP cells were grown in RPMI 1640. All cells were grown in media supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, 100 ⁇ g/ml penicillin/streptomycin at 37°C and 5% CO 2 .
  • FCS fetal calf serum
  • Apoptotic cell death was induced by either serum starvation or treatment with H 2 0 2 , UV- C, cisplatin, anti-Fas antibody or TNF- ⁇ treatment.
  • SNU 398 hepatocellular carcinoma cells were induced in serum-free medium for 3 days and tested for apoptosis.
  • Huh7 cells were incubated in a culture medium containing 0.1% FCS for 72 hours, and treated with freshly prepared 100 ⁇ M H 2 O 2 for at least 4 hours prior to apoptosis assay. 293 cells were treated with 200 ⁇ M H 2 O 2 or 100 ⁇ M cisplatin.
  • MCF-7, HeLa, U2OS, A375, SW480, LNCaP, Jurkat and MRC-5 cells were treated with UN-C irradiation (60-120 mJ/cm 2 ) using Bio-Rad GS Gene Linker.TM UV Chamber.
  • UN-C irradiation 60-120 mJ/cm 2
  • Bio-Rad GS Gene Linker.TM UV Chamber a UV Chamber.
  • T ⁇ F- ⁇ -treated (Boehringer Mannheim, 50 ng/ml for 72h) MCF-7 and anti-fas antibody-treated (Upstate Biotechnology-clone CHI 1, 25 ng/ml for 24h) Jurkat cells were used.
  • MRC-5 cells (passage 18) were grown to confluency on coverslips and serum starved for 3 days. At the end of 3 days, one set of cells was tested for BrdU labelling and the other set was subjected to immunofluorescence for the expression of the NAPO antigen as described later. Asynchronisely growing MRC-5 cells of the same passage were used as a control.
  • Huh7 cells were grown to 60% confluency and incubated with 50 ng/ml nocodazole (Sigma) for 18 hours. Mitotic cells were collected by mitotic shake-off and replated onto coverslips. At indicated time points (between 4 h and 36 h), one set of cells was tested for BrdU labelling, and the other set was subjected to immunofluorescence for the expression of the NAPO antigen.
  • Huh7 cells grown to 70% confluency were starved in DMEM lacking methionine (Sigma) and labelled with 200 ⁇ Ci 35 S-methionine (Amersham) per 4 ml medium for two hours.
  • Cells were scraped in ice-cold PBS and lysed in NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris pH 8.0, protease inhibitor cocktail-Roche), and centrifuged at 13,000 rpm at 4°C for 30 minutes.
  • the cell lysate was incubated with anti- NAPO antibody (1:10 diluted hybridoma supernatant) for 2 hours and the NAPO antigen was immunoprecipitated by using Protein G Sepharose (Pharmacia). Immunofluorescence
  • FITC- conjugated goat-anti-mouse Ig antibody was used as the secondary antibody and diluted as recommended by the supplier.
  • the immunofluorescence staining of Huh7 cells for p53 protein was tested using 6B10 monoclonal antibody (Yolcu E. et al., Oncogene, 2001, 20:1398). Nuclear DNA was visualized by incubation with 3 ⁇ g/ml Hoechst 33258 (Sigma) for 5 minutes in dark. Cover-slips were then rinsed with distilled water, mounted on glass microscopic slides in 50% glycerol and examined under fluorescent microscope (Zeiss). Jurkat cells were cytospinned (Shandon) for 3 minutes at 200 rpm. before immunofluorescence procedures.
  • Huh7 and HepG2 cells were grown in culture medium containing 0.2 % FCS for 72h to induce apoptosis. Next, cells were fixed and incubated with anti-NAPO antibody, followed by HRP-conjugated anti mouse Ig antibody (DAKO). Immune complexes were visualized by adding hydrogen peroxide and 3, 3'diaminobenzidine solution (5 mg 3, 3'diaminobenzidine in 10 ml 0.2 M Tris-HCI buffer; pH 7.6, and 0.1 ml freshly added 1% v/v hydrogen peroxide). The reaction was stopped by adding PBS, cells were counterstained with haematoxylin and analyzed under light microscope. TUNEL staining
  • TUNEL Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labelling
  • Annexin V assay was perfonned by Annexin V-PE reagent (PharMingen), according to manufacturer's recommendations, and cells were fixed. After TUNEL and Annexin V assays, cells were counterstained with Hoechst 33258 and examined as described.
  • BrdU incorporation cells were incubated with 30 ⁇ M BrdU (Sigma) for 1 h prior to fixation with ice-cold 70% ethanol for 10 minutes. Following DNA denaturation in 2 N HC1 for 20 minutes, cells were incubated with FITC-conjugated anti-BrdU antibody (DAKO) in the dilution as recommended by the supplier, cells were counterstained with Hoechst 33258 and examined as described.
  • DAKO FITC-conjugated anti-BrdU antibody
  • MRC-5 cells were grown to passage 40 and subjected to senescence-associated ⁇ - galactosidase (SA ⁇ -gal) assay, as described by Dimri et al. (Dimri GP. et al., Proc. Natl. Acad. Sci. USA, 1995, 92:9363-9367).
  • SA ⁇ -gal senescence-associated ⁇ - galactosidase
  • the monoclonal antibodies and immunoreactive fragments of the invention can be used to distinguish apoptotic cells from normal cells, to study the molecular mechanisms of apoptosis, to diagnose samples from apoptosis- related diseases and to identify novel agonists or antagonists of apoptosis.
  • Antibodies to NAPO can be generated using techniques similar to those described in this invention.
  • human cells such as Colo 320 DM cell line
  • they may be physically lysed in a physiological buffer such as PBS with repeated freeze-thawing, and suspended or diluted in an appropriate physiological carrier for immunization, or may be coupled to an adjuvant.
  • nuclear fractions can be prepared from such cells using protocols that are well known and can vary considerably yet remain effective (Mills JC. et al., Methods in Cell Biology, 1995, 46:217-242).
  • Other alternatives for antigen preparation is semipurification or purification of NAPO by gel chromatography, ion exchange chromatography, of affinity purification methods.
  • Immunogenic amounts of antigenic preparations containing NAPO proteins, or antigenic portions thereof, are injected, generally at concentrations in the range of 1 ⁇ g to 100 mg/kg of host. Administration may be by injection, such as intramuscularly, peritoneally, subcutaneously, or intravenously. Administration may be one or a plurality of times, usually at one to four week intervals.
  • the immortalized cell lines may be cloned and screened in accordance with conventional techniques, and antibodies in the cell supematants detected that are capable of binding to NAPO.
  • the appropriate immortalized cell lines may then be grown in vitro or injected into the peritoneal cavity of an appropriate host for production of ascites fluid.
  • Immortalized hybridoma cell lines can be readily produced from a variety of sources. Alternatively, these cell lines may be fused with other neoplastic B-cells, where such other B-cells may serve as recipients for genomic DNA coding for the antibody.
  • the monoclonal antibody secreted by the transformed or hybrid cell lines may be of any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgG ⁇ _ 4 , or IgF. As IgG is the most common isotype utilized in diagnostic assays, it is often preferred.
  • the anti-NAPO antibodies may be used intact, or as fragments, such as Fv, Fab, and F(ab') 2 . Such antibody fragments provide better diffusion characteristics than the whole anti-NAPO antibody, due to their smaller size.
  • the means for chemical modification methods are considered well-known in the art (Harlow E. and Lane D., "Antibodies: A laboratory manual”, Cold Spring Harbor ed.l, 1988).
  • the anti-NAPO antibodies are fragmented to obtain highly immunoreactive F(ab') 2 , F(ab'), and Fab fragments using the enzyme pepsin by methods well known in the art (Harlow E. and Lane D., "Antibodies: A laboratory manual”, Cold Spring Harbor ed.l, 1988).
  • antibodies and fragments thereof may be altered to an affinity modified form, avidity modified form, or both, by altering binding sites or altering the hinge region using recombinant DNA techniques well known in the art as described in the above cited references.
  • the anti-NAPO antibodies of this invention, or fragments thereof, may be used without modification or may be modified in a variety of ways, for example, by labelling.
  • Labelling is intended to mean joining, either covalently or non-covalently, a label which directly or indirectly provides for a means of detection.
  • a label can comprise any material possessing a detectable chemical or physical property.
  • labels are known, including radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), fluorescers, chromophores, luminescers, and magnetic particles.
  • These labels are detectable on the basis of either their own physical properties (eg., fluorescers, chromophores and radioisotopes), or their reactive or binding properties (eg., enzymes, substrates, cofactors and inhibitors). These materials are well known to one skilled in the art.
  • Anti-NAPO antibodies or fragments thereof can be used to detect apoptosis in various biological samples by combining them with the sample in question.
  • Biological samples can include, but are not limited to, normal and pathological tissue biopsy samples (heart, skeletal muscle, brain, skin, lymph nodes, spleen, breast, liver, prostate, colon, etc.) and cell cultures. Presence of apoptosis is tested for by incubating the anti-NAPO antibody with the biological sample under conditions conductive to NAPO immune complex formation, followed by the detection of complex formation.
  • the antibodies or fragments of the present invention can be either labelled or unlabelled for this purpose.
  • diagnostic assays entail the detection of the formation of a complex through the binding of the monoclonal antibody to NAPO.
  • the antibodies find use, for example, in agglutination assays.
  • unlabelled antibodies can be used in combination with other labelled antibodies (second antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for immunoglobulin.
  • the antibodies can be directly labelled.
  • NAPO immune complexes can be detected using any procedure known in the art.
  • direct and indirect immunofluorescence assays direct and indirect immunostaining techniques using enzymes such as peroxidases and alkaline phosphatases which can catalyse the production of visible dyes, can be used.
  • Numerous types of immunoassays are also available, including enzyme immune assay (EIA), enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), radioimmune assay (RIA), fluorescence immune assay, either single or double antibody techniques, and other techniques where either the peptides or antibodies of this invention are labelled with some detectable tag. (See Maggio E., "Enzyme Immunoassay", CRC Press, 1981).
  • the methods of the present invention can be combined with either the TUNEL method or with the use of propidium iodide (PI) in order to identify viable and apoptotic cells in the same sample.
  • the TUNEL method is used in fixed-dead cells. Cells in which a decrease or loss of NAPO immunoreactivity is observed combined with either a positive TUNEL or a positive PI reading indicated apoptotic cells; cells in which NAPO levels are not deceased/not lost in combination with either a negative TUNEL or a positive PI reading indicates viable cell.
  • the methods of the present invention can also be combined with Annexin V method in order to identify viable and apoptotic cells in the same sample. The Annexin V method is used with viable and unfixed cells.
  • cells can be fixed and permeabilized and stained with anti-NAPO antibody.
  • Cells in which a decrease or loss of NAPO immunoreactivity is observed combined with a positive Annexin V staining indicates apoptotic cells; cells in which NAPO levels are not decreased/not lost in combination with a negative annexin V reading indicates viable cell.
  • Physiologic cell death occurs primarily through "cell suicide” or apoptosis. Alterations in cell survival contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders and AIDS, thus treatments designed to specifically alter the apoptotic threshold may have the potential to change the natural progression of these related diseases.
  • the anti-NAPO antibodies can be used in methods to detect, distinguish, monitor or quantify apoptotic cells in diagnostic applications for the treatment of both cancer and AIDS.
  • the physiological and pathological implications of apoptosis render applications of anti- NAPO monoclonal antibody and fragments thereof far reaching, including use in research and diagnostics.
  • the present invention comprises a specific marker for apoptosis, which discriminates between cells dying by apoptosis and those viable. This marker could be used by scientists who are working on determining mechanisms of apoptosis.
  • markers of apoptosis could also be used in research relating to diseases in which apoptosis is involved, both to determine the mechanisms of the diseases and method of treatment.
  • anti-NAPO antibodies could be used in cancer research, where a potential chemotherapeutic drug could be tested for its ability to induce apoptosis. This could be done by exposing a cell sample to different concentrations of the test drug. The cells would then be analyzed for the presence of NAPO using the anti-NAPO antibodies or fragments thereof of the present invention. The ability of the chemotherapeutic test drug to induce apoptosis could be determined and compared to the apoptosis induction of well known drugs.
  • anti-NAPO antibodies or fragments thereof could be used as markers to assess the dose response of cells to chemotherapeutic drugs in order to determine ideal dosages for treatment.
  • the present invention could also be used in the basic research of and drug development for neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, including neuronal post-ischemic damage in stroke.
  • cells could be treated with an apoptosis inhibitor test drug at different concentrations and at different times post-apoptosis induction. Cells would then be collected at chosen times after the introduction of the test drug.
  • the anti-NAPO antibodies and fragments thereof would provide a method of assessing the drug's apoptosis inhibitory potency. It would also allow determination of the stage of apoptosis at which the test drug has an inhibitory effect and the stage at which the drug is not longer effective.
  • the method of the present invention could also be used to diagnose the extend of damage caused by a particular disease.
  • anti-NAPO antibodies as a diagnostic marker for post-ischemia neuronal damage by apoptosis.
  • the TUNEL method has proven to be an inadequate marker for estimation of neuronal damage by necrosis versus apoptosis within in vivo and in vitro models.
  • anti-NAPO antibodies it would be possible not only to quantify the severity of the damage caused by ischemia but also the proportion of cell death that was caused by apoptosis at various time points. This knowledge is important for designing and monitoring apoptosis inhibitor drug therapies, especially in terms of effectiveness, doses, and treatment schedule of the drugs. The same strategy could also be applied to neurodegenerative diseases.
  • anti-NAPO antibodies or fragments thereof to detect apoptosis has numerous advantages over other methods currently on the market. Firstly, the use of anti-NAPO antibodies or fragments thereof can distinguish cells that are dead or dying by apoptosis from those that are viable, quiescent, proliferating, mitotic or senescent; thus, the present invention provides reliable results. Most cell death kits currently on the market are based on markers present in apoptotic, but absent in viable cells. NAPO is a marker with opposite features meaning that it is a marker present in viable, but absent in apoptotic cells. It can easily be used in combination with currently available kits to increase test specificity and for confirmation.
  • the present invention as opposed to cell death kits currently available, offers the advantages of immune tests, versatile, enabling the detection of apoptosis in most cell types, and distinguishing it from other states of cell life such as quiescence, proliferation, mitosis and senescence. It can be used with both tissue samples, primary cell cultures and cell lines.
  • a further advantage of the present invention is that it can be used with a choice of qualitative and quantitative protocols adaptable to various laboratory equipment and expertise.
  • the present invention can be applied using common laboratory techniques such as ELISA and immunochemistry where no specialized laboratory equipment is required. It can also be applied using specialized equipment such as a flow cytometer.
  • NAPO is a nuclear antigen that is present in viable cells
  • Huh 7 hepatocellular carcinoma cells were grown on coverslips, using DMEM (Biochrome or Gibco), supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, 100 ⁇ g/ml penicillin/streptomycin at 37°C and 5% CO 2 , for 24h and fixed with 4% paraformaldehyde for 1 hour. Cells were permeabilized for 3 minutes with 0.1% Triton X-100 in 0.1% sodium citrate. Following saturation with 3% BSA in PBS-T (0.1%) for 15 minutes, fixed cells were incubated with anti-NAPO antibody (1:2 to 1:10 diluted hybridoma supernatant or 1-10 ⁇ g/ml purified antibody) for 1 hour at room temperature.
  • FCS fetal calf serum
  • FITC-conjugated goat-anti-mouse antibody was used as the secondary antibody and diluted in the ratio of 1:100. Nuclear DNA was visualized by incubation with 3 ⁇ g/ml Hoechst 33258 (Sigma) for 5 minutes in dark. Cover-slips were then rinsed with distilled water, mounted on glass microscopic slides in 50% glycerol and examined under fluorescent microscope. As shown in FIG. 1A NAPO is localized to nucleus in Huh7 cells. FIG. IB shows the Hoechst 33258 counterstaining of these cells in which only the nucleus of the cells is stained with the dye. To control the specificity of the antibody reactivity, cell were incubated with the secondary antibody in the absence of the anti-NAPO antibody (data not shown).
  • NAPO antigen comprises 2 major proteins migrating at approximately 60 kd and 70 kd, respectively
  • Huh7 hepatocellular carcinoma cells were grown as described in example 1, and starved for 2 hours in methionine free medium and metabolically labelled with 200 ⁇ Ci/100 mm plate 35 S-methionine for 2 hours.
  • Cells were scraped in ice-cold PBS and lysed in NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris pH 8.0, protease inhibitor cocktail- Roche), and centrifuged at 13,000 rpm at 4°C for 30 minutes. The cell lysate was incubated with anti-NAPO antibody for 2 hours and the NAPO antigen was immunoprecipitated by using Protein G Sepharose (Pharmacia).
  • Immunoprecipates were denatured in SDS-sample buffer and loaded on 10% SDS-polyacrylamide gel. Following electrophoresis, gel was fixed in a solution containing 50% distilled water, 40% methanol and 10% glacial acetic acid. Fixed gel was soaked into Amplify TM (Amersham) for amplification of radioactive signal for 30 min, and dried under heated vacuum. Dried gel was exposed to autoradiography for 1-7 days to visualize radioactive bands on X-ray films. As shown in figure 2 lane (+), anti-NAPO antibody recognized two major proteins migrating at approximately 60 kd and 70 kd, respectively. Immunoprecipitation in FIG.2 line (-) was performed in the absence of the anti-NAPO antibody to observe the background produced by Protein G Sepharose.
  • SNU 398 hepatocellular carcinoma cells were grown for 24 hr, fixed and stained as described in example 1. Some SNU 398 cells undergo spontaneous apoptosis under these conditions. The apoptotic cells have smaller nuclei intensely staining with Hoechst 33258, as shown in FIG. 3B. The same cells are stained negatively with anti-NAPO antibody (FIG. 3 A).
  • SNU 398 cells which undergo apoptosis when grown under serum-free conditions were first grown as described in example 1 for 24 hr, and serum starved for three days in the same culture medium, except that no FCS was added, Cells were fixed and tested for NAPO antigen immunoreactivity, as described in example 1.
  • FIG. 4B shows nuclear staining of both viable and apoptotic cells with Hoechst 33258.
  • hepatocellular carcinoma-derived Huh7 cells were used. H 2 O 2 (100 ⁇ M) treatment of these cells induce apoptosis under serum-deficient (0.1% FCS) conditions. As shown in FIG. 5A, NAPO antigen was negative in apoptotic Huh7 cells that are identified as cells with small nuclei by Hoechst 33258 counterstaining (FIG. 5B). To test whether the loss of NAPO expression is specific to this antigen, rather than a common feature shared by nuclear proteins, we also tested Huh7 cells for p53 protein immunoreactivity, under similar conditions.
  • Huh7 cells express a mutant ⁇ 53 protein that accumulate in their nuclei (Nolkmann M. et al., Oncogene, 1994, 9:195-204). Both apoptotic and non apoptotic Huh7 cells displayed positive staining for p53 protein. Indeed, apoptotic cells displayed a stronger p53 immunoreactivity when compared to non apoptotic cells (FIG. 5C). This indicated that the loss of ⁇ APO immunoreactivity in apoptotic Huh7 cells was specific to this antigen rather than a common feature of nuclear proteins. In addition, the same cells were tested for apoptosis using TU ⁇ EL assay. In contrast to ⁇ APO, TU ⁇ EL stained apoptotic cell nuclei positively, and viable cells were negative (FIG. 5 E). FIG. 5D and 5F illustrate Hoechst 33258 counterstaining.
  • T ⁇ F- ⁇ -treated MCF-7 and anti-Fas antibody-treated Jurkat cells were used.
  • MCF-7 breast carcinoma cells were treated with 50 ng/ml T ⁇ F- ⁇ -treated (Boehringer Mannheim) for 72h.
  • Jurkat acute T-cell leukemia cells were treated with 25 ng/ml agonist anti-fas antibody (Upstate Biotechnology-clone CHI 1) for 24h.
  • MCF-7 cells were stained for ⁇ APO as described in example 1.
  • FIG. 6A apoptotic Jurkat
  • FIG. 6C apoptotic MCF-7 cells
  • Jurkat T-cell leukemia, MRC-5 normal fibroblast, HeLa cervical carcinoma, SW480 colon carcinoma and U2OS osteosarcoma cells were treated with 120 mJ/cm 2 UN-C, and analyzed for apoptosis 24h later. All cells except Jurkat were stained as described in example 1. Jurkat cells were stained as described in example 6. As shown in FIG. 7 A, 7C, 7E, 7G and 71 respectively, Jurkat, MRC-5, HeLa, SW480 and U2OS cells under apoptosis following UV irradiation displayed undetectable ⁇ APO staining. FIG. 7B, 7D, 7F, 7H and 7J illustrate Hoechst 33258 counterstaining.
  • MRC-5 human fibroblast cells at passage 18 were grown to confluency and serum starved for 3 days to induce quiescence. To show that these cells are indeed quiescent, BrdU incorporation was also tested. Our results indicate that no BrdU labelling is observed in quiescent cells (FIG. 8 A), whereas approximately 15 % of asynchronously growing MRC-5 cells are positive for BrdU i.e. in S phase (FIG. 8E).
  • both quiescent and proliferating cell populations were stained for ⁇ APO as described in example 1. Under both conditions, all cells displayed a similarly positive nuclear staining for ⁇ APO (FIG 8C and 8G).
  • FIG. 8B, 8D, 8F and 8H illustrate Hoechst 33258 counterstaining. These observations indicated that ⁇ APO expression is not lost in non dividing quiescent cells.
  • FIG. 9A cells at time points 4-16 h were evaluated as Gl phase cells, cells at time points 20-24 h as S phase cells, and those at time point 36 h as G2 phase cells (FIG. 9A).
  • Mitotically arrested cells showed a diffusely positive (nuclear and cytoplasmic) NAPO staining (FIG 9B).
  • NAPO staining pattern was nuclear throughout the cell cycle, at all time points (time points 8 h, 24 h and 36 h are shown in FIG. 9D, 9F and 9H, respectively).
  • FIG. 9C, 9E, 9G and 91 illustrate Hoechst 33258 counterstaining.
  • NAPO staining was always positive during the cell cycle, the only noticeable change being a diffuse staining during mitosis, in contrast to strictly nuclear staining in other phases of the cell cycle.
  • MRC-5 cells were grown until passage 40 at which point they remain alive and attached to cell plate, but they stop dividing, a characteristic feature of senescence (Fulder SJ. and Holliday RA., Cell, 1975, 6:67-73). The senescence is often accompanied by a positive senescence associated ⁇ -galactosidase activity, which is negative in pre-senescent cells (Dimri GP. et al., Proc. Natl. Acad. Sci. USA, 1995, 92:9363-9367). MRC-5 fibroblast cells at passage 18 were passaged until they stop growing at passage 40.
  • cells at passages 18 and 40 were fixed in 3% formaldehyde for 5 minutes and incubated with senescence-associated ⁇ -galactosidase assay solution containing 40 mM citric acid/sodium phosphate buffer (pH: 6.0), 5 mM potassium ferro cyanide, 5 mM potassium ferricsyanide, 150 mM NaCl, 2 mM MgCl 2 and 1 mg/ml 5-Bromo-4-Chloro-3-Indolyl- ⁇ -D-galactopyranoside for up to 12 hours, and examined under light microscope to demonstrate senescence at passage 40. Similarly, cells at passages 18 and 40 were stained for NAPO immunoreactivity, as described in example 1.
  • Senescence-associated ⁇ -galactosidase-positive cells at passage 40 also showed positive for NAPO staining (FIG. 10D).
  • Cells at passage 18 served as negative control for senescence-associated ⁇ -galactosidase assay (FIG. 10A), which showed positive NAPO staining (FIG.10C) as expected.
  • FIG.10E and 10F are Hoechst 33258 counterstaining pictures.
  • FIG. 11B, 11D, and 11F show Hoechst 33258 DNA staining of these cells.
  • NAPO can serve as a marker to test chemical compounds for both apoptosis inducing and apoptosis inhibiting activities.
  • apoptosis was induced in Huh7 cells as described in example 5. Unfixed cells were first stained with PE-co ⁇ jugated Annexin V (BD PharMingen) to mark the membranes of apoptotic cells, following supplier's instructions. Cells were then fixed in 70% ethonol, stained for NAPO, and counterstained with Hoechst 33258, as described in example 1. As shown in FIG.
  • apoptotic cells show red membrane staining (annexin V-positive, NAPO-negative), while viable cells show green nuclear staining (annexin N-negative, ⁇ APO-positive).
  • FIG. 12B the same cells also show positive Hoechst 33258 staining, apoptotic cells being more intensely stained.
  • NAPO can also be used as an apoptotic marker using indirect immunoperoxidase assay
  • FIG. 13A and 13B show respectively, Huh7 and HepG2 cells.
  • the nuclei of viable cells are brown ( ⁇ APO-positive), whereas apoptotic cells show purple blue nuclear staining ( ⁇ APO- negative) when analyzed under light microscopy.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode permettant de détecter une apoptose et de différencier des cellules quiescentes, prolifératives, mitotiques et sénescentes de cellules apoptotiques. L'invention concerne également un nouveau antigène, appelé NAPO (pour négatif dans une apoptose), comprenant deux protéines différentes présentes dans des cellules viables mais indécelables dans des cellules apoptotiques. L'invention concerne en outre des anticorps anti-NAPO, et plus spécifiquement, un anticorps monoclonal qui se lie sélectivement à NAPO. L'invention concerne enfin un hybridome produisant des anticorps monoclonaux anti-NAPO. On utilise une méthode basée sur la détection de NAPO pour rechercher une apoptose ainsi que des applications associées à des maladies dans lesquelles une apoptose est impliquée. On peut également utiliser cette méthode pour tester une réponse cellulaire à des traitements de régulation d'apoptose et pour tester des médicaments à effet régulateur d'apoptose.
PCT/TR2001/000060 2001-11-22 2001-11-22 Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables WO2003044535A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002221261A AU2002221261A1 (en) 2001-11-22 2001-11-22 Method, antigen and antibody for distinguishing viable and apoptotic cells
PCT/TR2001/000060 WO2003044535A1 (fr) 2001-11-22 2001-11-22 Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/TR2001/000060 WO2003044535A1 (fr) 2001-11-22 2001-11-22 Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables

Publications (1)

Publication Number Publication Date
WO2003044535A1 true WO2003044535A1 (fr) 2003-05-30

Family

ID=21619337

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/TR2001/000060 WO2003044535A1 (fr) 2001-11-22 2001-11-22 Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables

Country Status (2)

Country Link
AU (1) AU2002221261A1 (fr)
WO (1) WO2003044535A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110672499A (zh) * 2019-09-29 2020-01-10 杭州联科生物技术股份有限公司 一种Annexin V凋亡检测的阳性质控液及其制备方法
CN110850067A (zh) * 2018-08-21 2020-02-28 上海恒润达生生物科技有限公司 嵌合抗原受体亲和力检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998049325A1 (fr) * 1997-04-28 1998-11-05 Mcgill University Proteines hybrides de marquage, vecteurs d'expression et lignees cellulaires transfectees de ces proteines destines a l'analyse du transport du noyau
US6054437A (en) * 1996-01-26 2000-04-25 Hitachi Chemical Research Center, Inc. Method for inducing death of neoplastic cells using piperazine derivatives
US6310247B1 (en) * 1998-12-17 2001-10-30 Hoffmann-La Roche Inc. 7-aryl-6(Z)-heptatrienoic acid retinamides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054437A (en) * 1996-01-26 2000-04-25 Hitachi Chemical Research Center, Inc. Method for inducing death of neoplastic cells using piperazine derivatives
WO1998049325A1 (fr) * 1997-04-28 1998-11-05 Mcgill University Proteines hybrides de marquage, vecteurs d'expression et lignees cellulaires transfectees de ces proteines destines a l'analyse du transport du noyau
US6310247B1 (en) * 1998-12-17 2001-10-30 Hoffmann-La Roche Inc. 7-aryl-6(Z)-heptatrienoic acid retinamides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GOPING GERTRUD ET AL: "Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.", JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 47, no. 4, April 1999 (1999-04-01), pages 561 - 568, XP002235827, ISSN: 0022-1554 *
NEGRI CLAUDIA ET AL: "Multiparametric staining to identify apoptotic human cells.", EXPERIMENTAL CELL RESEARCH, vol. 234, no. 1, 1997, pages 174 - 177, XP002235828, ISSN: 0014-4827 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110850067A (zh) * 2018-08-21 2020-02-28 上海恒润达生生物科技有限公司 嵌合抗原受体亲和力检测方法
CN110850067B (zh) * 2018-08-21 2023-08-15 上海恒润达生生物科技股份有限公司 嵌合抗原受体亲和力检测方法
CN110672499A (zh) * 2019-09-29 2020-01-10 杭州联科生物技术股份有限公司 一种Annexin V凋亡检测的阳性质控液及其制备方法
CN110672499B (zh) * 2019-09-29 2021-12-10 杭州联科生物技术股份有限公司 一种Annexin V凋亡检测的阳性质控液及其制备方法

Also Published As

Publication number Publication date
AU2002221261A1 (en) 2003-06-10

Similar Documents

Publication Publication Date Title
JP3190042B2 (ja) 核マトリックス蛋白液アッセー
Primakoff et al. A map of the guinea pig sperm surface constructed with monoclonal antibodies
Van Eyken et al. A cytokeratin immunohistochemical study of cholestatic liver disease: Evidence that hepatocytes can express “bile duct‐type” cytokeratins
US8673593B2 (en) Antibodies to alpha-synuclein
CN104777307B (zh) 先兆子痫检测和治疗的方法和组合物
JP5847755B2 (ja) 細胞を検出する方法、およびこれに有用な薬剤
US6518033B1 (en) Method of detecting the presence of CD155 for diagnosis of cancer and to determine treatment
Vitadello et al. Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes
JP2004503747A (ja) 結合物質の同定するための試薬と方法
Jing et al. Beta2-GPI: a novel factor in the development of hepatocellular carcinoma
US20090221004A1 (en) Caspase-cleavage anti-keratin antibodies for detection of apoptosis
KR101836929B1 (ko) Gdf15 단백질의 발현 수준을 측정하는 제제를 포함하는 돌발성 난청 예후 분석용 키트
Fischer et al. Nuclear envelope organization in papillary thyroid carcinoma
WO2003044535A1 (fr) Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables
Hardiyanto et al. The N-linked carbohydrate moiety of male reproductive tract CD52 (mrt-CD52) interferes with the complement system via binding to C1q
Whittaker et al. Plasma membrane orientation of simian virus 40 T antigen in three transformed cell lines mapped with monoclonal antibodies
Schmelz et al. PEAZ‐1: A new human prostate neoplastic epithelial cell line
Bouic et al. Immunohistological localization of alpha 1-microglobulin in normal rat tissues.
JP4610149B2 (ja) ラット先体反応後精子に対する抗体およびその利用
KR101416777B1 (ko) 방사선 피폭에 의한 심장 손상 또는 폐 손상 예측용 바이오마커 및 그 예측방법
US7157286B2 (en) Method for immunologically measuring RCAS1 and kit for measuring the same
JP2000002703A (ja) ヒト補体制御因子の検出方法及びその用途
JPH05304983A (ja) ヒト尿中蛋白質分解酵素阻害蛋白質の定量のための物質および定量方法
Armitage Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation
JPWO2006059712A1 (ja) ヒトステロイドサルファターゼに結合するモノクローナル抗体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP