ARTICLE |
Correspondence to: Josep E. Esquerda, Unitat de Neurobiologia Cellular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Avd Rovira Roure 44, E25198 Lleida, Catalunya, Spain. E-mail: josep.esquerda@cmb.udl.es
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Summary |
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Previous reports in various cells and species have shown that apoptotic cells are specifically and strongly labeled by certain c-Jun/N-terminal antibodies, such as c-Jun/sc45. This kind of immunoreactivity is confined to the cytoplasm. It is not due to c-Jun but appears to be related to c-Jun-like neoepitopes generated during apoptosis. This study was planned to gain further information about c-Jun-like immunostaining during apoptosis and to evaluate these antibodies as possible tools for characterizing cell death. Most of the experiments were performed in chick embryo spinal cord. When the apoptotic c-Jun-like immunoreactivity and caspase-3 immunostaining patterns were compared, we found that both antibodies immunostained the same dying cells in a similar pattern. In contrast to TUNEL staining, which reveals a positive reaction in both apoptotic and necrotic dying cells, active caspase-3 and c-Jun/sc45 antibodies are more selective because they stained only apoptotic cells. When cytosolic extracts from normal tissues were digested in vitro with caspase-3, c-Jun/sc45 immunoreactivity was strongly induced in several proteins, as demonstrated by Western blotting. Similar results were found when normal tissue sections were treated with caspase-3. Our results show that c-Jun/sc45 antibodies react with neoepitopes generated from cell proteins cleaved by activated caspases during apoptosis. We conclude that c-Jun/sc45 antibodies may be useful for detecting apoptosis. They can even be used in archival paraffin-embedded tissue samples. (J Histochem Cytochem 50:961972, 2002)
Key Words: apoptosis, c-Jun, caspase, immunocytochemistry, cell death, necrosis, chick embryo spinal cord, c-Jun/sc45
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Introduction |
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A KEY FEATURE OF APOPTOSIS is a sequential activation of proteolytic system machinery. Most of the morphological characteristics of apoptotic cell death are a consequence of caspase activity (
Histochemical techniques developed to visualize fragmented DNA are also commonly used for identifying apoptotic cells in situ but cannot be considered specific markers for apoptosis. The most commonly used method for this purpose is based on nick end-labeling by incubation with dUTP and deoxyribonucleotyl transferase (TdT) (TUNEL). It is now well recognized that the TUNEL assay often yields false-positives as a result of its ability to label non-apoptotic dying cells (
It has been suggested that these antibodies recognize distinct proteins of c-Jun (
In the present study we examined the localization of the cytoplasmic immunoreactivity of c-Jun/sc45 in apoptotic cells to gain further information about c-Jun-like immunostaining in apoptosis. This was then compared with immunocytochemical results obtained with an antibody against the 17-kD fragment of cleaved caspase-3, which indicates the activation of caspase-3. We conclude that, in apoptotic cells, there is a co-localization of immunostaining by the two antibodies, with the c-Jun/sc45 antibody being more sensitive than the antibody against caspase-3 in recognizing apoptotic cells. We also show that the strong c-Jun/sc45 immunoreactivity characteristic of apoptosis may be reconstructed in situ in normal cells as a consequence of caspase-3 proteolytic attack.
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Materials and Methods |
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Reagents
N-Methyl-D-aspartic acid (NMDA), ß-bungarotoxin (ß-Bgtx), normal goat serum (NGS), and protease inhibitors were purchased from Sigma (St Louis, MO). The caspase inhibitor Z-VAD-FMK was from Enzyme Systems Products (Livermore, CA). Active recombinant human caspase-3 (CPP32) was from PharMingen/Becton Dickinson (Le Pont de Claix, France). The c-Jun/sc45 polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). c-Jun/mAB monoclonal antibody was from Transduction Laboratories (Lexington, KY). The antibody against cleaved caspase-3 (17 kD) was provided by New England BioLabs (Beverly, MA). Secondary antibodies, goat anti-rabbit or goat anti-mouse and immunoglobulins, labeled with either Alexa Fluor 488 or 546, were purchased from Molecular Probes (Eugene, OR), as were 4',6-diamidino-2-phenylidole and dihydrochloride (DAPI). Biotinylated goat anti-rabbit IgG and ABC complex were from Vector (Burlingame, CA). PVDF membranes were from Millipore (Bedford, MA). Peroxidase-conjugated secondary antibodies and the ECL detection system were manufactured by Amersham (Little Chalfont, UK). Durcupan ACM was from Fluka (Buchs, Switzerland). DABCO (1-4-diazabicyclot(2-2-2) octane) was from Aldrich (Milwaukee, WI).
Tissue Processing for Light Microscopic Immunocytochemistry
Experiments were performed using tissues from chick embryos between embryonic days (E) 7.5 and 10. Samples for light microscopic immunocytochemistry were fixed by immersion in cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), for 12 hr at 4C. Some samples were later cryoprotected in 30% (w/v) sucrose in the same buffer for 24 hr before cryostat sectioning, while other samples were processed for paraffin embedding. Sections were collected on SuperFrost Plus slides (Menzel-Glaser; Freiburg, Germany), air dried at room temperature (RT), and then processed for immunocytochemistry.
Light Microscopic Immunocytochemistry
Sections were first permeabilized for 45 min in PBS containing 0.1% Triton X-100. Unspecific staining was blocked by incubating sections with 10% NGS in PBS for 1 hr. The incubation with primary antibodies c-Jun/sc45 (diluted 1:250), caspase-3 (diluted 1:70), or c-Jun/MAb (diluted 1:200) was performed overnight at 4C. After washing in PBS, sections were incubated with the appropriate secondary fluorescent-labeled Alexa-Fluor antibody (1:250) containing DAPI (3 mM), for 1 hr at RT. After washing, sections were coverslipped with an anti-fading mounting medium (0.1 M Tris-HCl buffer, pH 8.5, 20% glycerol, 10% Mowiol, and 0.1% DABCO) and images were captured by a cooled CCD camera (Life Science Resources; Cambridge, UK) coupled to a Nikon Eclipse 600 fluorescence microscope fitted with different selective filters. Immunocytochemical controls included omission of the primary antibody and preincubation with immunogen peptides, which resulted in complete abolition of immunoreactivity.
A double immunostaining method was performed for simultaneous localization of both the active caspase-3 and the antigens revealed by c-Jun/sc45 antibodies. Since both antibodies were created in rabbit, we applied a technique of antibody elution described by
Elution was carried out by immersing the slides in a mixture of 2.5% KMnO4 and 5% H2SO4 in distilled water for 10 min after 30 sec of destaining in 0.5% Na2S2O5. Sections were sequentially washed for 15 min in running tapwater, for 10 min in distilled water, and for 10 min in PBS before processing with the second immunofluorescence round. The efficiency of elution was checked before the second round of imunostaining by observing the complete elimination of the fluorescent signal.
Some immunofluorescence assays were carried out in 8-µm sections of paraffin-embedded samples. Formalin-fixed, paraffin-embedded sections of human tonsil were immunostained for c-Jun/sc45 using the avidinbiotinperoxidase detection system (Elite ABC; Vector Laboratories). Sections were then counterstained with hematoxylin.
Electron Microscopic Immunocytochemistry
For pre-embedding, specimens were fixed by immersion in a mixture of cold 4% paraformaldehyde and in 0.1% or 0.25% glutaraldehyde in 0.1 M PB, pH 7.4, for 24 hr. Transverse 50100-µm-thick sections from spinal cords were obtained using a vibratome. To block endogenous peroxidase activity, sections were incubated with 1% H2O2 in PBS for 45 min, washed in PBS, and then incubated with 10% NGS to reduce unspecific binding. Sections were then incubated overnight at 4C with c-Jun/sc45 antibody (diluted 1:200), washed in several changes of PBS, and incubated with biotinylated goat anti-rabbit IgG (diluted 1:100) for 60 min. After washing in several changes of PBS, sections were then incubated for 60 min with ABC complex, washed in PBS, and developed according to the DAB procedure. Samples were postfixed in PB 1% OsO4 for 2 hr, dehydrated in acetone, and flat-embedded in Durcupan ACM (Fluka). Ultrathin sections of selected areas containing labelled cell bodies were examined in a Zeiss EM910 electron microscope either with or without uranyl acetate and lead citrate counterstaining. Pre-embedding procedure was also performed using 1-nm gold-labeled goat anti-rabbit IgG (AuroProbe One GAR; Amersham) as a secondary antibody with ultimate silver enhancement (IntenSE M silver enhancement kit; Amersham) according to the manufacturer's instructions. Labeled samples were flat-embedded and sectioned as described above.
Pharmacological Treatment of Embryos
Drug applications were preformed in fertilized chicken eggs from COPAGA (Lleida; Catalonia, Spain) and incubated in the laboratory. A group of E8 embryos was treated with a single dose of 1 mg NMDA (Sigma). This neurotoxin was dissolved in saline and dropped directly onto the chorioallantoic membrane (CAM) through a window in the shell. Embryos were sacrificed 12 hr later (E8.5).
In another set of E7 embryos, a 1-µl dose of ß-Bgtx (100 ng) dissolved in saline solution was injected into the right leg.
After treatment, the shell window was sealed with adhesive tape and the eggs were returned to the incubator until the time of sampling.
Western and Dot-blot Assays
Fresh spinal cords from E10 embryos were homogenized using a Polytron in 2 volumes of ice-cold 50 mM Tris-HCI buffer (pH 7.4) containing 1 mM EDTA, 5 mM mercaptoethanol, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride. After ultracentrifugation at 150,000 x g for 1 hr, the supernatants corresponding to the cytosolic fraction were transferred into fresh tubes, snap-frozen in liquid nitrogen, and stored at -80C until further analysis. Before use, samples were boiled for 5 min and 20 µl of protein was loaded in SDS-polyacrylamide gels for Western blots or dotted directly onto an Immobilon PVDF transfer membrane (Millipore). For Western blotting, proteins were first separated in 7.5% SDS-PAGE gels using the Bio-Rad mini Protean II electrophoresis system and then blotted onto Immobilon PVDF transfer membranes using a semi-dry system (Pharmacia; Uppsala, Sweden). The membranes were blocked with 5% fat-free dried milk and incubated with sc45 antiserum (diluted 1:1000). Immuoreactive bands or dot-blots were detected using a horseradish peroxidase-conjugated anti-rabbit secondary antibody with a bioluminescent ECL detection system (Amersham).
In Vitro Assay for Induction of Caspase-3-mediated Proteolysis
Extracts of cytosolic fraction from normal E10 spinal cord were treated with active caspase-3 to examine whether the appearance of c-Jun7/sc45 antibody-reacting antigenic epitopes in the apoptotic neuron cytoplasm emerged as a result of caspase-3 activity. Histological sections from normal E10 spinal cord were also treated with active caspase-3. Extracts were treated according a modification of the method described by
For capase-3 in situ proteolysis assay on cryostat sections, active caspase-3 was dissolved in a buffer containing 25 mM Hepes, pH 7.4, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS, and 10% sucrose (Buffer A). Ten-µm-thick sections that were collected on SuperFrost Plus slides and air-dried overnight, were washed three times for 10 min at 37C with Buffer A and then incubated with the same buffer, which contained 2 ng/µl active caspase-3, at 37C for 4 hr. Samples were subsequently washed in PBS and processed for immunofluorescence. The specificity of caspase proteolytic activity was tested by adding Z-VAD (150 nM final).
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Results |
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c-Jun/sc45 and Caspase-3 Antibodies Displayed an Overlapping Pattern of Immunoreactivity in Dying Cells
Assays were made on developing spinal cord motor neurons (MNs). The natural programmed cell death of this neuron population has been extensively characterized in chick embryo. In this system, the MN number is adjusted employing an apoptotic programmed cell death (PCD) process that follows a well-defined temporal and spatial pattern. For lumbar segments this occurs from E6 to E10 and affects about 50% of initially differentiated MNs (
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The final common pathway in apoptotic cell death involves caspase-mediated proteolysis of several substrates whose cleavage products accumulate in dying cells. Some, like those from PARP or spectrin, can be specifically detected by antibodies and have often been used as markers for apoptosis. More recently, antibodies against the active form of caspase-3 that work on tissue sections have made it possible to carry out specific in situ labeling of apoptotic cells.
Cryostat sections of E7.5 chick embryo spinal cord were double-immunostained using both rabbit polyclonal antibodies to demonstrate the relationship between active caspase-3 labeling and c-Ju/sc45 antigens in dying cells (see Materials and Methods). This method made it possible to demonstrate that dying cells displaying cytoplasmic immunoreactivity for active caspase-3 were also immunoreactive to c-Jun/sc45 (Fig 1C1 and 1C2). Although elution by oxidation produced some tissue waste, the microscopic structure was sufficiently preserved to demonstrate the co-localization of both antibodies in dying cells.
Ultrastructural immunolabeling of c-Jun/sc45 antigens in apoptotic cells was achieved by applying pre-embedding procedures involving peroxidase or 1-nm colloidal gold particles (Fig 1D and Fig 1E). Accumulated immunoreactive deposits generated by peroxidase were mainly observed in the cytoplasm of dying cells (Fig 1D). Similar results were obtained using immunogold followed by a silver enhancement pre-embedding procedure (Fig 1E). Neurons exhibiting immunolabeling for c-Jun/sc45 showed a nuclear morphology of chromatin condensation characteristic of apoptotic cells.
c-Jun/sc45 Immunolabeling, but not TUNEL, Can Distinguish Between Apoptotic and Necrotic Cell Death
Apoptosis in chick embryo ventral horn can be dramatically stimulated by injecting ß-Bgtx. At 1224 hr after ß-Bgtx administration there was a dramatic increase in the number of apoptotic cells showing positive c-Jun/sc45 immunoreactivity, pyknotic nuclei, and TUNEL reaction. These neurons also displayed an ultrastructural apoptotic morphology (
A single application of NMDA in chick embryos older than E8 induced a massive excitotoxic lesion in spinal cord (
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Neoepitopes Emerging as a Consequence of Caspase-mediated Proteolysis Were Responsible for c-Jun/sc45 Immunoreactivity in Apoptosis: In Situ and In Vitro Generation on Healthy Cells Through Caspase-3 Digestion
Caspase-3 is a cysteine protease that plays a central role in apoptosis (
Antisera capable of specifically recognizing the active form of caspase-3 or certain neoantigenic epitopes deriving from its cleavage products have been used as histological markers for either in vivo or in vitro apoptotic cells (
Incubation of cytosolic extracts from E10 spinal cord tissues with active caspase-3 resulted in the emergence of neoantigens that reacted with c-Jun/sc45 antibody, as demonstrated by dot-blot analysis (Fig 3A). This effect was inhibited by the presence of Z-VAD, a caspase-3 inhibitor, in the incubation medium (Fig 3A-3). When these samples were analyzed by Western blotting, a large number of new immunoreactive bands appeared as a consequence of caspase-3 digestion (Fig 3A'-2). This effect was inhibited by the presence of Z-VAD (Fig 3A'-3).
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Sections from paraformaldehyde-fixed E10 chick embryo spinal cord were digested with active caspase-3 for 4 hr and processed for c-Jun/sc45 immunostaining to establish a closer correlation between caspase-3 activity and c-Jun/sc45 immunohistochemistry. Compared with undigested controls, it was clear that overall immunoreactivity increased in digested specimens (Fig 4). Enhancement of the immunoreactivity signal was especially manifest in all motor neuron somata (Fig 4B). As expected, motor neurons that became immunopositive to c-Jun/sc45 displayed normal morphology without pyknotic chromatin and also showed an extensive cytoplasmic immunostaining (Fig 4B' and 4B'''). In other words, the pattern of c-Jun/sc45 immunostaining induced in normal MNs by caspase-3 digestion closely mimicked that found in apoptotic MNs from undigested tissue. When samples were simultaneously incubated with capase-3 and Z-VAD, the induction of positive c-Jun/sc45 immunoreactivity was prevented in normal cells (Fig 4C). These samples exhibited a similar immunostaining pattern to the controls. (Fig 4C' and 4C'').
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c-Jun/sc45 Antibodies Were Capable of Detecting Apoptosis in Paraffin-embedded Specimens
In pathology laboratories, tissues are regularly embedded, processed, and stored for long periods in paraffin blocks. As a result, for retrospective studies the application of immunohistochemical procedures that can be used on paraffin sections is highly desirable. Because the c-Jun/sc45 antibody is an excellent tool for detecting apoptosis, we tested its applicability for routinely paraffin-processed sections of E7.5 embryo spinal cord samples stored for 3 years. We also compared the immunoreaction with c-Jun/sc45 in apoptotic neurons with that obtained with anti-active caspase-3 antibodies (Fig 5). Similar results were obtained with the two antibodies and specific staining of pyknotic MNs in a Golgi-like pattern was observed, as in the cryostat sections (Fig 5A and Fig 5B). However, the intensity of the immunostaining displayed by the c-Jun/sc45 antibodies was much stronger than that obtained by caspase-3 active antibodies (Fig 5A and Fig 5B).
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c-Jun/sc45 can also detect apoptotic cells in human lymphoid formalin-fixed, paraffin-embedded tissue samples, as demonstrated in sections from human tonsil (Fig 5D).
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Discussion |
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In several works, polyclonal antibodies (c-Jun/sc45 or AB-2 from Oncogene) against the peptide TPTPQFLCPKNVTD, corresponding to 91105 mouse c-Jun amino acids, have been used to study the involvement of this transcription factor in cell death and specific cytoplasmic immunostaining of apoptotic cells was also described (
Here we confirm that c-Jun/sc45 cytoplasmic immunolabeling is associated with dying pyknotic cells, and we also show by electron microscopy that these cells display ultrastructural characteristics of apoptosis. Cells immunostained with c-Jun/sc45 also exhibited positive cytoplasmic immunoreactivity for active caspase-3, which is considered a general hallmark of apoptosis (
Detection of apoptotic cells on tissue samples by light microscopy is currently achieved by TUNEL assay. However, the validity of TUNEL for identifying apoptotic cells has been seriously questioned since the observation that, in many cases, cells dying by necrosis show DNA laddering and TUNEL-positive staining (
Comparable results were obtained using a specific antibody for caspase-3-polyADP-ribose-polymerase proteolyzed product (
The ability of the sc45 antibody to detect neoepitopes emerging as a consequence of caspase-mediated proteolysis was further demonstrated by the fact that normal cells in fixed and sectioned tissue samples may acquire strong cytoplasmatic c-Jun/sc45 immunoreactivity if they are incubated with caspase-3. This staining property did not emerge when caspase-3 activity was blocked by Z-VAD. Furthermore, when cytosolic extracts from chicken spinal cord were treated with caspase-3, a marked increase in c-Jun/sc45-immunoreactive signal was observed in dot-blots. Western blotting analysis indicated that the increase in immunoreactivity was due to a broad spectrum of proteins that migrated to different molecular weights. These bands must have emerged as a consequence of caspase-3 activity because they failed to appear in the presence of Z-VAD. This confirmed our previous results in neuroblastoma cells showing that one of the neoantigens recognized by c-Jun/sc45 antibodies was the caspase-3-cleaved seryl-tRNA-synthetase (
It is not surprising that c-Jun/sc45 antibodies recognize a variety of proteins after caspase proteolysis because it is known that caspase-3 cleaves many different substrates including poly-(ADP-ribose) polymerase (PARP), fodrin, retinoblastoma protein, huntingtin, DNA-dependent protein kinase, gelsolin, ribonucleoprotein A1, vimentin and others (
Given the fact that c-Jun/sc45 antibodies are much more sensitive and specific than other immunohistochemical tools for detecting apoptosis, we suggest that they could be used as excellent and reliable markers for qualitative and quantitative studies on tissue sections from a broad diversity of tissues and species. In addition, c-Jun/sc45 antibodies may also be used for determining proteolytic activity in apoptosis and identifying new target proteins for caspase-3, which could be cleaved by caspase-3 during apoptosis.
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Acknowledgments |
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Supported by Ministerio de Educación y Ciencia (SAF97-0083), Ajuntament de Lleida and grants from the Fundació La Marató de TV3 and Fundación "La Caixa." The technical assistance of Ester Vazquez and Carmen Guerris is greatly appreciated.
Received for publication November 30, 2001; accepted January 16, 2002.
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