Report |
Address correspondence to Mehmet Ozturk, Department of Molecular Biology and Genetics, Bilkent University, 06533 Ankara, Turkey. Tel.: (90) 312-266-50-81. Fax: (90) 312-266-50-97. E-mail: ozturk{at}fen.bilkent.edu.tr
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Abstract |
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Key Words: apoptosis; apoptotic cell death; apoptotic marker; quiescence; senescence
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Introduction |
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Apoptosis, as a critical component of life in multicellular organisms, is a target subject for understanding cellular mechanisms of many diseases, as well as for developing new drugs that interfere with either proapoptotic or antiapoptotic molecular networks. Consequently, it has become important to develop reliable assays to measure cell death. Techniques currently available for apoptosis detection are based on the study of morphology of apoptotic cells (light and fluorescence microscopy coupled to nuclear staining with specific dyes and electron microscopy), DNA fragmentation detected by terminal transferase-mediated dUTP nick-end labeling (TUNEL)* and similar techniques, membrane changes detected by annexin V in vivo labeling, and on immunological assays using antibodies directed to apoptosis-related proteins (Stadelmann and Lassmann, 2000). Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptosis and other forms of cellular changes, as well as distinction between different stages of the cell death process. However, we are facing a relative paucity for simple techniques fulfilling these requirements, and furthermore allowing quantitative analysis (van Heerde et al., 2000). 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 and Lassmann, 2000).
Here we describe a mouse monoclonal antibodydefined nuclear antigen composed of two polypeptides that we call NAPO (for negative in apoptosis), which is strongly expressed in cells under many conditions (proliferation, quiescence, mitosis, and senescence) except apoptosis. The immunoreactivity of the antigen, as tested by immunofluorescence technique, is lost in apoptotic cells in a way opposite to TUNEL and annexin V staining. Thus, NAPO antigen may serve as a reliable marker for apoptosis.
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Results and discussion |
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Identification of NAPO as an apoptotic marker
Hepatocellular carcinomaderived SNU 398 cells, which undergo apoptosis when grown under serum-free conditions were serum starved for three days and tested for NAPO antigen immunoreactivity. Cells displaying morphological characteristics of apoptosis (cell shrinkage, nuclear condensation, and fragmentation) displayed negative NAPO staining in contrast to positive nuclear staining of all nonapoptotic cells (Fig. 1, F and G).
To confirm the loss of NAPO antigen during apoptosis in another cellular system, hepatocellular carcinomaderived Huh7 cells were used. H2O2 (100 µM) treatment of these cells induce apoptosis under serum-deficient (0.1% FCS) conditions (unpublished data). As shown in Fig. 2 A, NAPO antigen was negative in apoptotic Huh7 cells that are identified as cells with small nuclei by Hoechst 33258 counterstaining (Fig. 2 B). 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 p53 protein that accumulate in their nuclei (Volkmann et al., 1994). Both apoptotic and nonapoptotic Huh7 cells displayed positive staining for p53 protein. Indeed, apoptotic cells displayed a stronger p53 immunoreactivity when compared with nonapoptotic cells (unpublished data). This indicated that the loss of NAPO immunoreactivity in apoptotic Huh7 cells was specific to this antigen rather than a common feature of nuclear proteins.
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Expression of the NAPO antigen in quiescent cells
MRC-5 human embryonic lung fibroblast cells (passage 18) were grown to confluency and serum starved for 3 d to induce quiescence. To show that these cells are indeed quiescent, BrdU incorporation was also tested. Our results indicate that 15% of asynchronously growing MRC-5 cells are positive for BrdU i.e., in S phase (Fig. 3 A), whereas no BrdU labeling was observed in quiescent cells (Fig. 3 E). Under both conditions, all cells displayed a similarly positive nuclear staining for NAPO (Fig. 3, C and G). These observations indicated that NAPO expression is not lost in nondividing quiescent cells.
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Materials and methods |
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Tissue culture
Huh7, SNU 398, COLO 320, MCF-7, HeLa, U2OS, SW480, A375, 293, MRC-5, COS7, IAR-6, and CHOK-I cells were grown in DME (Biochrome or GIBCO BRL). HC11 was grown in RPMI 1640 (Biological Industries) supplemented with 10 ng/ml EGF (Sigma-Aldrich) and 5 µg/ml insulin (Sigma-Aldrich). Jurkat and LNCaP cells were grown in RPMI 1640. All cells were grown in media supplemented with 10% FCS, 1% nonessential 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 H2O2, UV-C, cisplatin, anti-Fas antibody or TNF- treatment. SNU 398 hepatocellular carcinoma cells were induced in serum-free medium for 3 d and tested for apoptosis. For oxidative stressinduced apoptosis, Huh7 cells were incubated in a culture medium containing 0.1% FCS for 72 h, and treated with freshly prepared 100 µM H2O2 for at least 4 h before 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 UV-C irradiation (60120 mJ/cm2). For physiologically induced apoptosis studies, TNF-
treated (Boehringer; 50 ng/ml for 72 h) MCF-7 and anti-fas antibodytreated (Upstate Biotechnology; clone CH11, 25 ng/ml for 24 h) Jurkat cells were used.
Induction of quiescence
Presenescent MRC-5 cells (passage 18) were grown to confluency on coverslips and serum starved for 3 d. At the end of 3 d, one set of cells was tested for BrdU labeling and the other set was subjected to immunofluorescence for the expression of the NAPO antigen as described later. Asynchronously 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-Aldrich) for 18 h. Mitotic cells were collected by mitotic shake-off and replated onto coverslips. At indicated time points (between 4 and 36 h), one set of cells was tested for BrdU labeling, 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 DME lacking methionine (Sigma-Aldrich) and labeled with 200 µCi [35S]methionine (Amersham Pharmacia Biotech) per 4 ml medium for 2 h. 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 min. The cell lysate was incubated with anti-NAPO antibody for 2 h and the NAPO antigen was immunoprecipitated by using protein G sepharose (Amersham Pharmacia Biotech).
Immunofluorescence
Cells were grown on coverslips and fixed with 100% ice-cold acetone for 1 min or by 4% paraformaldehyde for 1 h. When paraformaldehyde was used, cells were permeabilized for 3 min with 0.1% Triton X-100 in 0.1% sodium citrate. After saturation with 3% BSA in PBS-T (0.1%) for 15 min, fixed cells were incubated with anti-NAPO antibody for 1 h at room temperature. FITC-conjugated goat antimouse 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 et al., 2001). Nuclear DNA was visualized by incubation with 3 µg/ml Hoechst 33258 (Sigma-Aldrich) for 5 min in the 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 min at 200 rpm before immunofluorescence procedures.
TUNEL and annexin V stainings
The TUNEL assay was performed using an in situ cell death detection kit (Roche), according to manufacturer's recommendations. The annexin V assay was performed by annexin V-PE reagent (PharMingen), according to manufacturer's recommendations, and cells were fixed in ethanol. After TUNEL and annexin V assays, cells were counterstained with Hoechst 33258 and examined as described.
BrdU labeling and identification of S phase cells
For BrdU incorporation, cells were incubated with 30 µM BrdU for 1 h before fixation with ice-cold 70% ethanol for 10 min. After DNA denaturation in 2 N HCl for 20 min, 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 ß-galactosidase assay
MRC-5 cells were grown to passage 40 and subjected to senescence-associated ß-galactosidase (SA ß-gal) assay, as described by Dimri et al. (1995). Briefly, cells were fixed in 3% formaldehyde for 5 min and incubated with SA ß-gal 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 X-Gal) for up to 12 h, and examined under light microscope.
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Footnotes |
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* Abbreviations used in this paper: TUNEL, terminal transferasemediated dUTP nick-end labeling.
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Acknowledgments |
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The initial stage of this project (monoclonal antibody production) was performed at Massachusetts General Hospital Cancer Center (Charlestown, MA) and supported by a grant (CA-54567) from the National Institutes of Health to M. Ozturk.
Submitted: 6 August 2001
Revised: 14 September 2001
Accepted: 17 October 2001
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