WO2003044535A1 - Method, antigen and antibody for distinguishing viable and apoptotic cells - Google Patents

Abstract

This invention discloses a method for detecting apoptosis and for discriminating quiescent, proliferating, mitotic and senescent cells from apoptotic cells. This invention also relates to a novel antigen called NAPO (for negative in apoptosis), comprizing two different proteins, that is present in viable cells, but undetectable in apoptotic cells. This invention also relates to anti-NAPO antibodies. More specifically, this invention relates to a monoclonal antibody that selectively binds NAPO. This invention also relates to a hybridoma that produces anti-NAPO monoclonal antibodies. The NAPO-based detection method is useful for research into apoptosis and applications relating to diseases in which apoptosis is involved. This method can also be used for testing cellular response to apoptosis-regulating treatments, as well for drug testing for apoptosis regulating effect.

Description

METHOD, ANTIGEN AND ANTIBODY FOR DISTINGUISHING VIABLE AND APOPTOTIC CELLS

DESCRIPTION

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

A eukaryotic cell can be in proliferation, quiescence, senescence and apoptosis states, depending on its internal program that can be regulated by external effectors. When it is proliferating, 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). The phase between M and S is called Gl (gap 1), while the phase between S and M is called 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. It is essential for both biological processes such as normal tissue turnover, embryonic development, and maturation of the immune system, including pathological processes, such as hormone deprivation, thermal stress and metabolic stress. Programmed cell death is required for proper embryonic development as well as for the maintenance of homeostasis in adult tissues (Naux DL. and Korsmeyer SJ., Cell, 1999, 96:245-254; Wyllie AH. and Golstein P., Proc. Νatl. Acad. Sci. USA, 2001, 98:l l-13).Moreover, apoptosis is involved in the etiology and pathophysiology of a variety of diseases such as cancer, neurodegenerative, autoimmune, infectious and heart diseases (Reed CJ., Semin. Hematol., 2000, 37:9-16; Mattson MP., Nat. Rev. Mol. Cell Biol., 2000, 1:120129; Chervonsky AN., Curr. Opin. Immunol., 1999, 11:684688; Roulston A. et al., Annu. Rev. Microbiol, 1999, 53:577-628; Νarula J. et al., Curr. Opin. Cardiol., 2000, 15:183-188)

Apoptosis is characterized by a decrease in cell volume, a condensation of chromatin, cellular budding, and the fragmentation of DΝA 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 DΝA 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-α, 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).

Disease and shock can cause cardiac cells to induce apoptosis. For example, cells deprived of oxygen after a heart attack release signals that induce apoptosis in cells in the heart. New treatments involving apoptosis Mockers are being developed (Haunstetter A and Izumo S, Cardiovasc Res., 2000, 45:795-801; Hajjar RJ. et al., Circulation Research., 2000, 86:616-621).

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)

A failure of apoptosis in the immune system can lead to autoimmune diseases. 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)

Evidence also suggests that AIDS develops when the human immunodeficiency virus (HIN) sets off unregulated and untimely apoptosis in CD-4 and CD-8 cells, the defenders of the immune system. Inhibition of HIN-induced apoptosis is a target field for treatment of AIDS (Gougeon ML. and Montagnier L., Ann Ν Y Acad Sci., 1999, 887:199-212).

There is an enormous therapeutic potential in controlling apoptosis in these diseases. Therefore, 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. For example, 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.

Currently available techniques for apoptosis detection are based on the study of morphology of apoptotic cells (light microscopy and fluorescence microscopy coupled to nuclear staining with specific dyes, electron microscopy), DΝA fragmentation detected by terminal transferase-mediated dUTP nick-end labelling (TUΝEL) 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).

Currently, the marker most commonly used to detect apoptosis is TUNEL labelling of the 3'-OH free end of DNA fragments produced during apoptosis (Gavrieli Y. et al., J. Cell Biol., 1992, 119:493-501). 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.

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.

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. As well, its use is often limited to cells grown in suspension, however, most cells are adherent and are grown on a matrix. The method also requires the use of live or unpreserved cells. There is therefore a great need for a specific, antigenic, versatile marker for the rapid detection of cell death by apoptosis, which can be used for research, diagnostics, and therapeutics. This marker must be able to distinguish between cell death by apoptosis and other known states of cells which includes quiescence, proliferation (composed of Gl, S, G2 and mitosis phases), and senescence. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptosis and other forms of cellular states. However, we are facing a relative paucity for simple techniques fulfilling these requirements, and furthermore allowing quantitative analysis (van Heerde WL. et al., Cardiovasc. Res., 2000, 45:549-559). Immunological detection of apoptosis-related proteins is probably the best approach to overcome this obstacle, but there are only a few known apoptosis marker antigens (Stadelmann C. and Lassmann H., Cell Tissue Res., 2000, 301:19-31).

SUMMARY OF THE INVENTION

To detect apoptotic cells and to differentiate apoptosis from other cell states, namely quiescence, proliferation (composed of Gl, S, G2 and mitosis phases) and senescence, we identified a novel marker antigen termed NAPO (for negative in apoptosis), and developed a method based on the detection of NAPO using specific anti-NAPO antibodies. NAPO antigen is detectable in all known states of cells except apoptosis.

In summary, 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.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating embodiments of the present invention, there are shown in the drawings certain features. It should be understood, however, that this invention is not limited to the precise embodiments shown.

FIG. 1 shows the presence and nuclear localization of the NAPO antigen in viable cells. In 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 ³⁵S 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. SNU 398 cells tested as described in figure 3, except that cells were grown additionally for 72 hours in serum-free culture medium to induce apoptosis. Immunostaining and counterstaining was performed as described in FIG. 1. 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 H₂O₂ 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. As shown in figure 5C, the immunoreactivity of another nuclear protein, namely p53 is not lost during apoptosis in these cells. Indeed, the immunoreactivity of p53, when tested with an anti-p53 antibody termed 6B10 (Yolcu E. et al., Oncogene, 2001, 20:1398-1401). is stronger in apoptotic cells (FIG. 5C), indicated with arrows in FIG. 5D showing Hoechst 33258 counterstaining. FIG. 5 also 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. For TUNEL assay, fixed cells were stained using In Situ Cell Death Detection Kit (Roche), in stead of NAPO staining, according to supplier's instructions. Apoptotic cells indicated by arrows in FIG 5F (Hoechst 33258 counterstaining) are positive for TUNEL, as shown in FIG. 5E, whereas such apoptotic cells are negative for NAPO (FIG. 5A). These results indicate that NAPO is a novel antigen whose immunoreactivity is lost in apoptotic cells.

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. Note that cells at different stages of apoptosis (with or without nuclear fragmentation) are negative for NAPO. TNF receptor was activated in MCF-7 breast carcinoma cells by treatment with recombinant TNF-α 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 fϊbroblasts (MRC-5), cervical cancer epithelial cells (HeLa), colon carcinoma epithelial cells (SW480), and osteosarcoma cells (U2OS). Different cell lines were exposed to different doses of UN-C (60-120 mJ/cm²) and subjected to ΝAPO immunostaining and Hoechst 33258 counterstaining, as described in FIG. 1 (all except Jurkat cells) or FIG. 6 (Jurkat cells). Arrows in FIG. 7B, 7D, 7F, 7H and 7J indicate apoptotic Jurkat, MRC-5, HeLa, SW480 and U2OS cells, respectively. These apoptotic cells are negative for ΝAPO, as shown respectively in FIG. 7A, 7C, 7E, 7G and 71.

FIG. 8 shows the presence of ΝAPO 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). As quiescent cells are totally negative for DΝA synthesis, the BrdU incorporation test was used for verification. For this, cells were exposed to BrdU for lhr prior to fixation and permeabilization. ΝAPO and Hoechst 33258 staining was performed as described in FIG.l. BrdU staining was done with FITC-conjugated anti-BrdU antibody from DAKO. Quiescent MRC-5 cells are totally negative for BrdU (FIG. 8A), but totally positive for ΝAPO (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 ΝAPO (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. Cell Res., 1980, 126:397-405). Freshly collected cells were then grown in culture for up to 36 h. The S phase was identified by BrdU incorporation assay as described in FIG. 8. The cells at time points between 4 — 16 h and 20-32 h, were identified as cells in Gl and S phases, respectively, while cells at 36 h were mostly at G2 phase, as indicated by the ratio of the BrdU-positive cells in FIG. 9A. 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. 9B). Other cells are also strongly positive, but NAPO staining is not diffused. For the study of NAPO during other phases of the cell cycle, cells were stained at 4 h intervals, following the release from mitotic arrest. 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 β-galactosidase activity (FIG. 10A, B). Both senescence- associated β-galactosidase-negative (FIG. 10A) and senescence-associated β- 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 H₂O₂ 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 H₂O₂-activated apoptosis was tested by co-treatment of Huh7 cells with both compounds. The ability of H₂O₂ 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). The ability of sodium selenite to inhibit H₂O -induced apoptosis was tested by pretreating Huh7 cells with this compound prior to H₂O₂ treatment. NAPO staining of H₂O₂ and sodium selenite treated cells (FIG. HE), when compared to H₂O₂-treated cells (FIG.11 A) shows that sodium selenite inhibits H₂O₂-induced apoptosis in Huh7 cells. 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. As shown in 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, ΝAPO-positive). In FIG. 12B, the same cells also show positive Hoechst 33258 staining, apoptotic cells being more intensely stained.

FIG 13. ΝAPO 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-ΝAPO antibody, followed by HRP-conjugated anti- mouse Ig. Peroxidase activity was tested by incubating the samples with diaminobenzidine and H₂0₂ which generate a brown color. Cells were then counterstained with haematoxylin which provides purple blue nuclear staining. With this test, the nuclei of viable cells are brown (ΝAPO-positive), whereas apoptotic cells show purple blue nuclear staining (ΝAPO-negative), when analyzed under light microscopy. FIG. 13 A and 13B show respectively, Huh7 and HepG2 cells. DESCRIPTION OF THE INVENTION

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The following common abbreviations are used throughout the specification and in the claims:

The abbreviation " 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.

The term "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. The term "anti-NAPO antibodies" includes recombinant, chimeric, and affinity modified forms made by techniques of molecular biology well known in the art.

The term "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. The term "Fc" means the antibody fragment which may activate complement.

The term "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.

Other abbreviations are 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) and HRP (Horseradish 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. At the same time, 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. As analyzed by indirect immunofluorescence microscopy, 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. These findings indicate that NAPO is a novel epitope exposed on viable cells, but its immunoreactivity decreases during apoptosis to reach the limits of detection by anti-NAPO antibodies. TABLE 1 : NAPO Antigen in Viable and Apoptotic Cells

Cell Line Origin Morphology Viable Apoptotic

Huh 7 HCC Epithelial (+) (-)

HepG2 Hepatoblastoma Epithelial (+) (-)

SNU 398 HCC Epithelial (+) (-)

MCF-7 Breast Cancer Epithelial (+) (-)

HeLa Cervix Cancer Epithelial (+) (-)

SW480 Colon Cancer Epithelial (+) (-)

Colo 320 DM Colon Cancer Epithelial (+) Not tested

LNCaP Prostate Cancer Epithelial (+) (-)

U2OS Osteosarcoma Epithelial (+) (-)

A375 Melanoma Epithelial (+) (-)

Jurkat TCL Lymphoid (+) (-)

MRC-5 Lung Fibroblastic (+) (-)

293 Embryonal Kidney Epithelial (+) (-)

1HCC: hepatocellular carcinoma; TCL: acute T cell leukemia

The methods and materials used to produce the monoclonal antibodies and define the antigen of the invention are described in detail below. In summary, a panel of monoclonal antibodies was raised against viable cells by immunizing mice with a lysate prepared from asynchronously growing Colo 320 DM cells (ATCC No: CCL-220). One of these antibodies, anti-NAPO, was found to react strongly with a nuclear antigen in Colo 320

DM. Other antibodies displayed weaker immunoreactivity with this nuclear antigen. The anti-NAPO monoclonal antibody described herein is the one that reacted strongly with

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 ΝAPO (-) cells, but not

ΝAPO (+) 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).

This is a characteristic feature of cells that have entered the program of apoptosis (Fadok NA. et al., J. Immunol, 1992, 149:4029-4035; van Heerde WL. et al, Cardiovasc. Res., 2000, 45:549-559).

The methods described herein can be used to generate additional monoclonal antibodies with the characteristics of the anti-ΝAPO 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-ΝAPO antibody provided by the hybrid cell line CL52 (Harlow E. and Lane D., "Antibodies: A laboratory manual", Cold Spring Harbor ed.l, 1988).

Also included within the scope of the present invention are antibody fragments and derivatives which comprise at least the functional portion of the antigen binding domain of an anti-ΝAPO antibody molecule.

Antibody fragments which contain the binding domain of the molecule can be generated by known techniques. For example, 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).

Monoclonal Antibody Development

Ten millions of COLO 320 DM (ATCC No: CCL-220) cells were lysed in 2 ml PBS and

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.).

Cell Culture

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 μg/ml penicillin/streptomycin at 37°C and 5% CO₂.

Induction of apoptosis

Apoptotic cell death was induced by either serum starvation or treatment with H₂0₂, UV- C, cisplatin, anti-Fas antibody or TNF-α treatment. SNU 398 hepatocellular carcinoma cells were induced in serum-free medium for 3 days and tested for apoptosis. For oxidative stress-induced apoptosis, Huh7 cells were incubated in a culture medium containing 0.1% FCS for 72 hours, and treated with freshly prepared 100 μM H₂O₂ for at least 4 hours prior to apoptosis assay. 293 cells were treated with 200 μM H₂O₂ or 100 μM cisplatin. MCF-7, HeLa, U2OS, A375, SW480, LNCaP, Jurkat and MRC-5 cells were treated with UN-C irradiation (60-120 mJ/cm²) using Bio-Rad GS Gene Linker.TM UV Chamber. For physiologically induced apoptosis studies, TΝF-α-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.

Induction of quiescence

Pre-senescent 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.

Mitotic arrest and cell cycle synchronization

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.

Immunoprecipitation

Huh7 cells grown to 70% confluency were starved in DMEM lacking methionine (Sigma) and labelled with 200 μCi ³⁵S-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

Cells were grown on coverslips and fixed with 70% ice-cold ethanol, 100% ice-cold acetone, or 100% ice-cold methanol for 1 minute, or with 4% paraformaldehyde for 1 hour. When paraformaldehyde was used, cells were permeabilized for 3 minutes with 0.1% Triton X-100 in 0.1% sodium citrate. Following saturation with 3% Bovine Serum Albumin (BSA, Sigma) in PBS containing 0.1% Triton X-100 (Sigma) for 15 minutes, fixed cells were incubated with anti-NAPO antibody (1:2 to 1:10 diluted hybridoma supernatant or 1-10 μg/ml purified antibody) for 1 hour at room temperature. FITC- conjugated goat-anti-mouse Ig antibody (DAKO) 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 μg/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.

Immunoperoxidase

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

The TUNEL (Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labelling) assay was performed using In Situ Cell Death Detection Kit (Roche), according to manufacturer's recommendations.

Annexin V staining

The 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 labelling and identification of S phase cells

For BrdU incorporation, cells were incubated with 30 μM 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.

Senescence associated β-galactosidase assay

MRC-5 cells were grown to passage 40 and subjected to senescence-associated β- galactosidase (SA β-gal) assay, as described by Dimri et al. (Dimri GP. et al., Proc. Natl. Acad. Sci. USA, 1995, 92:9363-9367). Briefly, cells were fixed in 3% formaldehyde for 5 minutes and incubated with senescence-associated β-galactosidase solution (40 mM citric acid/sodium phosphate buffer (pH: 6.0), 5 mM potassium ferro cyanide, 5 mM potassium ferric cyanide, 150 mM NaCl, 2 mM MgCl₂ and 1 mg/ml 5-Bromo-4-Chloro- 3-Indolyl-β-D-galactopyranoside) for up to 12 hours, and examined under light microscope. Anti-NAPO identifies an epitope exposed in a nuclear protein complex whose expression appears to be restricted to viable cells. 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. When using 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. Alternatively, 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 μg 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, IgGι_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).

In addition, the 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. A wide variety of 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. Typically, diagnostic assays entail the detection of the formation of a complex through the binding of the monoclonal antibody to NAPO. When unlabelled, the antibodies find use, for example, in agglutination assays. In addition, 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. Alternatively, the antibodies can be directly labelled.

NAPO immune complexes can be detected using any procedure known in the art. For in situ detection of apoptotic cells, 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. Following staining with Annexin V, 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.

Uses

Reference is made to the articles by Vaux and Korsmeyer (Vaux DL. and Korsmeyer SJ., Cell, 1999, 96:245-254) and Wyllie and Golstein (Wyllie AH. and Golstein P., Proc. Natl. Acad. Sci. USA, 2001, 98:11-13) which describe in detail the role of apoptosis in embryonic development as well as for the maintenance of homeostasis in adult tissues. Moreover, apoptosis is involved in the etiology and pathophysiology of a variety of diseases such as cancer, neurodegenerative, autoimmune, infectious and heart diseases, as described in detail in articles by Reed CJ.(Reed CJ., Semin. Hematol., 2000, 37:9-16), Mattson MP. (Mattson MP., Nat. Rev. Mol. Cell Biol., 2000, 1:120-129), Chervonsky AV. (Chervonsky AV. Curr. Opin. Immunol., 1999, 11:684-688), Roulston A. et al. (Roulston A. et al, Annu. Rev. Microbiol., 1999, 53:577-628) and Narula J. et al. (Narula J. et al., Curr. Opin. Cardiol., 2000, 15:183-188). As stated in these articles, homeostasis is maintained through a balance between cell proliferation and cell death. 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. For example, it is within the scope of the invention that 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.

Uses in Research

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.

These 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. For example, 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.

Additionally, 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. For example, 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.

Use in Diagnostic Assays

The method of the present invention could also be used to diagnose the extend of damage caused by a particular disease. For example, there is an enormous use for 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. With anti-NAPO antibodies, however, 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.

Advantages

The use of 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.

In addition, 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.

Some of the protocols used by current products are time consuming and require sophisticated laboratory equipment and expertise. 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. For example, 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.

While the present invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth.

The present invention will be illustrated by, but is not intended to be limited to, the following examples.

Example 1

Demonstration that 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 μg/ml penicillin/streptomycin at 37°C and 5% CO₂, 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 μg/ml purified antibody) for 1 hour at room temperature. FITC-conjugated goat-anti-mouse antibody (DAKO) was used as the secondary antibody and diluted in the ratio of 1:100. Nuclear DNA was visualized by incubation with 3 μg/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).

Example 2

Demonstration that 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 μCi/100 mm plate ³⁵S-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. Example 3

Demonstration that NAPO antigen is undetectable in spontaneously induced apoptotic cells

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).

Example 4

Demonstration that NAPO antigen is undetectable in apoptosis induced by serum starvation

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. Cells displaying morphological characteristics of apoptosis (nuclear condensation and fragmentation) displayed negative NAPO staining in contrast to positive nuclear staining of all non- apoptotic cells (FIG. 4A). FIG. 4B shows nuclear staining of both viable and apoptotic cells with Hoechst 33258.

Example 5

Demonstration that NAPO antigen is undetectable in apoptosis induced by oxidative stress

To confirm the loss of NAPO antigen during apoptosis in another cellular system, hepatocellular carcinoma-derived Huh7 cells were used. H₂O₂ (100 μM) 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 ρ53 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 ΝAPO 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 TUΝEL assay. In contrast to ΝAPO, TUΝEL stained apoptotic cell nuclei positively, and viable cells were negative (FIG. 5 E). FIG. 5D and 5F illustrate Hoechst 33258 counterstaining.

Example 6

Demonstration that NAPO is undetectable in apoptosis induced by death receptor activation

In order to show whether ΝAPO antigen is lost during death-receptor mediated apoptosis, TΝF-α-treated MCF-7 and anti-Fas antibody-treated Jurkat cells were used. MCF-7 breast carcinoma cells were treated with 50 ng/ml TΝF-α-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 ΝAPO as described in example 1. Jurkat cells were stained similarly, except that they were first attached onto frosted microscope slides (S/P brand colorfrost microslides from Allegiance, USA) by Cytospin (Shandon) centrifugation. ΝAPO was undetectable in both apoptotic Jurkat (FIG. 6A) and apoptotic MCF-7 cells (FIG. 6C). FIG. 6B and 6D illustrate Hoechst 33258 counterstaining. Example 7

Demonstration that NAPO is undetectable in apoptosis induced by exposure to UV- irradiation

Jurkat T-cell leukemia, MRC-5 normal fibroblast, HeLa cervical carcinoma, SW480 colon carcinoma and U2OS osteosarcoma cells were treated with 120 mJ/cm² 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 ΝAPO staining. FIG. 7B, 7D, 7F, 7H and 7J illustrate Hoechst 33258 counterstaining.

Example 8

Demonstration that NAPO is present in viable quiescent cells

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). In parallel to these experiments, both quiescent and proliferating cell populations were stained for ΝAPO as described in example 1. Under both conditions, all cells displayed a similarly positive nuclear staining for ΝAPO (FIG 8C and 8G). FIG. 8B, 8D, 8F and 8H illustrate Hoechst 33258 counterstaining. These observations indicated that ΝAPO expression is not lost in non dividing quiescent cells.

Example 9

Demonstration that NAPO antigen is present in cells at each phase of the cell cycle including Gl, S, G2 and mitosis We also analyzed the expression pattern of NAPO in synchronized cells in order to follow its presence during different phases of the cell cycle. For this purpose Huh7 cells were treated with 50 ng/ml Nocodazole (Fluka), mitotic cells were collected by mitotic shake-off, and plated onto coverslips. Synchronized Huh7 cells were tested every 4 hours for 36 h of culture for both BrdU incorporation and NAPO staining. BrdU incorporation was minimal until 16 h after the release from mitotic arrest with a maximum of BrdU incorporation at 24 h, followed by a significant decrease at 36 h (figure 4A). According to BrdU incorporation index, 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). Except in mitosis, 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. Thus, 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.

Example 10

Demonstration that NAPO antigen is present in senescent cells

To test whether NAPO antigen expression is modified during senescence, 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 β-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. Then, cells at passages 18 and 40 were fixed in 3% formaldehyde for 5 minutes and incubated with senescence-associated β-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 MgCl₂ and 1 mg/ml 5-Bromo-4-Chloro-3-Indolyl-β-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 β-galactosidase-positive cells at passage 40 (FIG. 10B) also showed positive for NAPO staining (FIG. 10D). Cells at passage 18 served as negative control for senescence-associated β-galactosidase assay (FIG. 10A), which showed positive NAPO staining (FIG.10C) as expected. FIG.10E and 10F are Hoechst 33258 counterstaining pictures.

Example 11

Demonstration that NAPO can be used to test the ability of compounds to modulate apoptosis

We provide an example to demonstrate that NAPO can be used to test the ability of compounds to modulate apoptosis. The ability of H O₂ to activate apoptosis was tested by treatment of Huh7 cells with this compound, as described in example 5. The ability of sodium selenite to inhibit H₂O₂-activated apoptosis was tested by co-treatment of Huh7 cells with both compounds. One hundred nM sodium selenite (Sigma) was added to culture medium 48h prior to H₂O₂ treatment. NAPO staining showed that H₂O₂ compound induces apoptosis in these cells (FIG. 11 A), as compared to untreated cells (FIG. 11C). The pretreatment of Huh7 cells with sodium selenite compound prior to H₂O₂ treatment showed that sodium selenite protects Huh7 cells from H₂O₂-induced apoptosis (FIG. HE). FIG. 11B, 11D, and 11F show Hoechst 33258 DNA staining of these cells. Thus, as examplified here, NAPO can serve as a marker to test chemical compounds for both apoptosis inducing and apoptosis inhibiting activities.

Example 12

Demonstration that NAPO can be used in combination with another apoptotic marker for better definition of apoptotic cells To demonstrate the use of NAPO in combination with another apoptotic marker for better definition of apoptotic cells, apoptosis was induced in Huh7 cells as described in example 5. Unfixed cells were first stained with PE-coηjugated 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. 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, ΝAPO-positive). In FIG. 12B, the same cells also show positive Hoechst 33258 staining, apoptotic cells being more intensely stained.

Example 13

Demonstration that NAPO can also be used as an apoptotic marker using indirect immunoperoxidase assay

To demonstrate the use of ΝAPO as an apoptosis marker, we used mostly indirect immunofluorescence assay. However, this antigen can be tested with any known immunoassay under user preferred conditions. For demonstration, we provide here the example of 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-ΝAPO antibody, followed by HRP-conjugated anti-mouse Ig (DAKO). Peroxidase activity was tested by incubating the samples with diaminobenzidine and H 0₂ which generate a brown color, as described in Immunochemistry in Practice (Johnstone A. and Thorpe R. "Immunochemistry in Practice" Blackwell scientific Publications, 2nd ed. 1987). Cells were then counterstained with haematoxylin which provides purple blue nuclear staining. FIG. 13A and 13B show respectively, Huh7 and HepG2 cells. With this test, the nuclei of viable cells are brown (ΝAPO-positive), whereas apoptotic cells show purple blue nuclear staining (ΝAPO- negative) when analyzed under light microscopy.

Claims

CLAIMSWhat is claimed is:

1. A method for identifying viable cells and apoptotic cells, said method comprising the steps of:

(a) providing a first binding reagent specific for a first cellular component present only in viable cells, said first binding reagent labelled with a first detectable label,

(b) providing a second binding reagent specific for a second cellular component, said second binding reagent is labelled with a second detectable label;

(c) contacting a sample of permeabilized cells with said first binding reagent (a) wherein said first binding reagent (a) labels viable cells leaving apoptotic cells unlabelled by said first label;

(d) contacting said sample of (c) with said second binding reagent (b), wherein said second binding reagent (b) labels both viable and apoptotic cells with said second label;

(e) analysing said cells of (d) by an instrument allowing analysis of both first and second labels and identifying the cells by the pattern of labelling, wherein viable cells are labelled with said first label and said second label and apoptotic cells are labelled with only second label.

2. A method according to claim 1, wherein the second binding reagent labels only apoptotic cells, and accordingly cells are identified by the pattern of labelling wherein viable cells are labelled with only said first label, and apoptotic cells are labelled with only said second label in step (e).

3. A method according to claim 1, wherein step (d) is performed first, followed by steps (c) and (e).

4. A method according to claim 1, wherein steps (c) and (d) are performed simultaneously.

5. A method according to claim 1, wherein the first cellular component of (a) is a nuclear antigen termed NAPO which is present in viable cells, but undetectable in apoptotic cells,

6. A cellular antigen termed NAPO, said antigen showing the following characteristics:

(1) present in both normal and cancer cells,

(2) present in many cell types including but not limited to epithelial, lymphoid and fibroblastic cells,

(3) comprises two proteins with molecular weights of about 60 kd and about 70 kd, respectively,

(4) present in quiescent, proliferating and senescent cells,

(5) present in Gl, S, G2 and M phases of the cell cycle in proliferating cells,

(6) undetectable in apoptotic cells,

(7) localised in the nucleus of viable cells, except during mitosis where it shows a diffused intracellular presence,

(8) its antigen epitope is resistant to short treatment with acetone, methanol, 70% ethanol or 4 % paraformaldehyde.

7. A method according to claim 1 wherein the first binding reagent is an antibody or fragment thereof specific for NAPO;

8. A method according to claim 1 wherein the first binding reagent is a monoclonal antibody^' or fragment thereof which is specific for NAPO;

9. The monoclonal antibody of claim 8 having mouse subtype IgG which is produced by the cell line CL52;

10. A hybrid cell line termed CL52 producing the monoclonal antibody of claim 9.

11. An antibody according to claim 7, which forms an immune complex with the same epitope as the monoclonal antibody of claim 9.

12. A method of claim 1, wherein first binding reagent is produced as a monoclonal antibody to NAPO, by injecting whole lysates of viable cells into mice, fusing spleen cells from said mice with mouse plasmocytoma cells, screening for hybridomas producing antibodies reacting with viable but not with apoptotic cells, further selecting those reacting with NAPO antigen, and obtaining antibodies from said hybridomas;

13. A method according to claim 1, wherein the first binding reagent is labelled indirectly via a protein specific for said binding reagent;

14. A method according to claim 1, wherein the second binding reagent is labelled indirectly via a protein specific for said binding reagent;

15. A method according to claim 1, wherein the first and second labels are selected from the group consisting of enzymes, chromophores, fluorophores, radio-labelled materials, a metal, a dye, a detectable immunoglobulin, a protein and a detectable part of a protein;

16. A method according to claim 1, wherein the instrument described in step (e) is selected from the group consisting of light microscope, fluorescent microscope and flow cytometer.

17. A method for differentiating apoptotic cells from senescent, quiescent, proliferating and mitotic cells, according to claim 1, wherein:

(a) cell samples described in steps (c) and (d) contain senescent, quiescent, proliferating and/or mitotic cells;

(b) in the analysis step of (e), senescent, quiescent, proliferating and mitotic cells are labelled with said first label and said second label and apoptotic cells are labelled with only first label,

18. A method for differentiating apoptotic cells from senescent, quiescent, proliferating and mitotic cells, according to claim 2, wherein the senescent, quiescent, proliferating and mitotic cells are labelled with only said first label, and apoptotic cells are labelled with only said second label.

19. A method for differentiating apoptotic cells from senescent, quiescent, proliferating and mitotic cells, according to claim 3,

20. A method for differentiating apoptotic cells from senescent, quiescent, proliferating and mitotic cells, according to claim 4.