- Email: [email protected]
Multiparametric Staining to Identify Apoptotic Human Cells
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
234, 174–177 (1997)
EX973591
SHORT NOTE Multiparametric Staining to Identify Apoptotic Human Cells Claudia Negri, Maddalena Donzelli, Rosa Bernardi, Laura Rossi, Alexander Bu¨rkle,* and A. Ivana Scovassi1 Istituto di Genetica Biochimica ed Evoluzionistica C.N.R., Via Abbiategrasso 207, I-27100 Pavia, Italy; and *Deutsches Krebsforschungszentrum, Abteilung 0610, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
To analyze relevant features of HeLa and HL60 cells driven into apoptosis by etoposide, we have developed a new ‘‘tricolor’’ assay, based on the simultaneous analysis in the single cell of chromatin condensation, DNA degradation, and cellular poly(ADP-ribose) synthesis. The latter reaction is catalyzed by poly(ADP-ribose)polymerase (E.C. 2.4.2.30), an enzyme which is activated by the presence of DNA free ends. The protocol consists in the visualization of apoptotic cells by Hoechst staining, TUNEL assay, and immunoreaction with anti-poly(ADP-ribose) antibody. We thus provide the first evidence that endogenous poly(ADP-ribose) production is indeed stimulated in cells undergoing apoptosis after treatment with antitumoral drugs, and that the monitoring of this endogenous enzymatic reaction, combined with morphological and other biochemical parameters, should facilitate the detection of apoptotic cells. q 1997 Academic Press
INTRODUCTION
Cell death by apoptosis is characterized by a number of morphological and biochemical features which, however, are not universal [1]. Therefore, the definition of the apoptotic phenotype requires the evaluation of more than one parameter in order to be generally accepted. In previous work, we characterized morphological and biochemical events occurring during apoptosis induced in human tumoral cell lines by chemotherapeutic drugs. In HeLa cells incubated with etoposide or longterm cultured, we have observed several typical features of apoptosis, such as chromatin condensation, nuclear fragmentation, and DNA and protein degradation [2, 3]. Furthermore, we have characterized poly(ADPribosylation), a posttranslational modification modulated during apoptosis, and we have observed an in1 To whom correspondence and reprint requests should be addressed. Fax: 39-382-422286. E-mail: [email protected].
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MATERIALS AND METHODS Cell cultures and treatments. HeLa cells were grown as monolayer in Dulbecco’s modified Eagle’s medium (D-MEM, GIBCO-BRL). HL60 cells were grown in suspension in RPMI 1640 medium (Dutch mod.; HyClone, Cramlington, NE). Media were supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT), 4 mM glutamine, 2 mM sodium pyruvate, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from GIBCO-BRL). Cells were grown at 377C in a humidified atmosphere containing 5% CO2 . HeLa cells were seeded on coverslips at a concentration of 105 cells/ ml, maintained in culture for 2 days and then treated with 50 mM etoposide (Bristol Ital., Sermoneta, Italy) for 3 h followed by 3 h of incubation in fresh medium. For some experiments, 1 mM 3-aminobenzamide (Sigma, St. Louis, MO) was administered for 3 h before addition of etoposide. HL60 cells were seeded at 5 1 105 cells/ml, maintained in culture for 2 days, and treated with 68 mM etoposide for increasing time periods (0, 2, and 6 h). Multiparametric fluorescence analysis (Tricolor protocol). HeLa cell monolayers were rinsed in PBS and fixed with ice-cold 10% trichloroacetic acid for 15 min, followed by successive washes in cold 70%, 90%, and absolute ethanol for 3 min each (6). HL60 cell suspensions washed in PBS were resuspended at a concentration of 2 1 106 cells/ml in PBS containing 10% FCS, cytocentrifuged on microscope slides, and fixed as HeLa cells. Samples were processed sequentially as follows: (a) DNA apoptotic fragments were end-labeled using the Apoptosis Detection System (Promega, Madison, WI). This protocol allows the evaluation of the extent of DNA degradation by incorporating fluorescein-12-dUTP at the 3*-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase (TdT), which forms a polymeric tail by the principle of the TUNEL (TdT-mediated dUTP nickend labeling) assay [5]. Briefly, after fixation samples were rinsed in PBS and incubated for 10 min at RT with equilibration buffer
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AID
crease in the activity of the enzyme poly(ADP-ribose)polymerase (PARP) in extracts of apoptotic cells [2–4]. In the present study, we have analyzed different markers of apoptosis in HeLa and HL60 cells after etoposide treatment in order to achieve a multiparametric identification of apoptotic cells. At the single cell level, we have visualized chromatin changes by Hoechst staining and DNA fragments by the TUNEL assay [5]. Moreover, we have followed the endogenous cellular synthesis of poly(ADP-ribose) by the immunoreaction with monoclonal antibody 10H against this polymer.
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(200 mM potassium cacodylate, pH 6.6, 25 mM Tris–HCl, pH 6.6, 0.2 mM DTT, 0.25 mg/ml BSA, and 2.5 mM CoCl2). Thereafter, the enzymatic reaction was performed for 60 min at 377C in the dark in the equilibration buffer supplemented with the following reagents: 10 mM dATP, 1 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, 5 mM fluorescein-12-dUTP and 25 U of TdT (final concentrations). To remove unincorporated dUTP, slides were washed three times for 15 min at RT with 21 SSC and three times for 5 min with PBS. (b) Poly(ADP-ribose) synthesis was evaluated by immunofluorescence. After PBS washing of TUNEL-processed samples, cells were incubated for 30 min at RT with PTN (PBS containing 0.1% Tween 20 and 10% newborn calf serum) and then for 1 h at RT with 101 concentrated hybridoma supernatant containing monoclonal antibody 10H against poly(ADP-ribose) provided by Professor Miwa [7]. Thereafter, cells were washed five times for 5 min with PBS and incubated for 30 min at RT with a Texas red-linked sheep anti-mouse Ig antibody (Amersham Ital.) diluted 1:50 in PTN. Aspecific binding was avoided by three washes in PBS. (c) After washing in PBS, chromatin condensation and nuclear morphology were visualized by staining DNA with 0.1 mg/ml Hoechst 33258 (Sigma) for 3 min at RT in the dark. Samples were then washed three times with PBS and mounted with PBS containing 10% glycerol and 0.02% sodium azide. Microscopic observation was carried out using a Leitz Orthoplan microscope equipped with a 501 objective. Light emissions of Texas red, fluorescein, and Hoechst were detected using specific filters. Cells were photographed using Scotch Chrome (640 ASA) film.
RESULTS
Etoposide-treated HeLa and HL60 cells were monitored for DNA fragmentation with the TUNEL assay, immunostained with monoclonal antibody 10H to reveal poly(ADP-ribose) production, and countercoloured with Hoechst 33258. The use of reagents with different light emission, such as blue for Hoechst, green (fluoresceine isothiocyanate) for the TdT substrate dUTP, and Texas red for the anti-mouse secondary antibody, allowed the visualization of the three different parameters in the same samples. In Fig. 1 (top) it is evident that an apoptotic HeLa cell exhibits typical morphological features by Hoechst staining and shows also a TUNEL-positive green fluorescence. Furthermore, the same cell is positive for the red fluorescence from immunoreaction with 10H monoclonal antibody against ADP-ribose polymers. The ‘‘tricolor’’ procedure was applied to HL60 cells treated for increasing times with a single dose of etoposide (68 mM). Results are shown in Fig. 1 (bottom). Control cells appeared to be morphologically normal, with intact DNA and negative 10H staining (Figs. 1a, 1b, and 1c, respectively). After 2 and 6 h of incubation, as shown by Hoechst morphology (Figs. 1d and 1g, re-
spectively) and by TUNEL assay (Figs. 1e and 1h, respectively), the number of cells with chromatin fragmentation increased. As observed for HeLa cells, also in HL60 cells the positivity for 10H antibody is mainly restricted to cells showing apoptotic features (Figs. 1f and 1i). Compared to HeLa cells, HL60 revealed a higher background for TUNEL assay, possibly due to the cytocentrifugation step. To confirm the specificity of the 10H immunofluorescence signals, HeLa cells were treated with the ADPribosylation inhibitor 3-aminobenzamide (3-AB). As it is shown in Fig. 2, this compound, administered before etoposide incubation, inhibits the appearance of 10Hpositive cells after etoposide treatment. However, 3-AB did not alter the induction of apoptosis visualized by Hoechst DNA staining. DISCUSSION
We developed a new protocol for the simultaneous analysis of three parameters of apoptosis, namely, (i) the changes in nuclear morphology visualized by Hoechst 33258 staining; (ii) the appearance of DNA breaks, evaluated by the TUNEL assay; and (iii) the endogenous cellular poly(ADP-ribose) synthesis monitored by indirect immunofluorescence, using the monoclonal antibody 10H raised against this polymer. This antibody has been previously used for the in situ detection of poly(ADP-ribosylation) in living cells treated with alkylating agents and then fixed with trichloroacetic acid [6, 8, 9], and it has been recently utilized to detect polymer synthesis on nitrocellulose-immobilized PARP undergoing automodification, during the activity blot procedure [10], and for Western blot analysis [11]. In HeLa cells, polymer-specific fluorescence was localized in nuclei characterized by apoptotic morphology when counterstained with Hoechst 33258, and by extensive DNA degradation, an event producing 3*-OH free ends, the substrate for the TdT reaction. In HL60 cells, we have observed that after a 2-h treatment with 68 mM etoposide, about 15% of cells showed typical apoptotic features and that this percentage reached more than 80% after 6 h of incubation with the drug. Under these conditions, poly(ADP-ribose) synthesis appeared to be increased with the time of treatment and to be confined to TUNEL-positive cells with few exceptions, represented by apoptotic cells brilliant for poly(ADP-ribose) staining but with a weak TUNEL fluo-
FIG. 1. Tricolor assay in HeLa and HL60 cells. (Top) HeLa cells treated with 50 mM etoposide for 3 h followed by 3 h of incubation in drug-free medium. (Bottom) HL60 cells treated with 68 mM etoposide for increasing times up to 6 h. (a–c) Control cells, (d–f) cells treated with 68 mM etoposide for 2 h, (g–i) cells treated with 68 mM etoposide for 6 h. Blue fluorescence, Hoechst staining; green fluorescence, TUNEL assay; red fluorescence, immunoreactivity of 10H antibody. Experiments were performed as described under Materials and Methods. FIG. 2. Effect of 3-aminobenzamide on poly(ADP-ribose) synthesis and apoptosis. HeLa cell treatments: 50 mM etoposide for 3 h followed by a 3-h incubation in drug-free medium; 1 mM 3-aminobenzamide for 3 h followed or not by etoposide treatment.
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SHORT NOTE
rescence. The nature of this morphology is currently under investigation. The endogenous activation of the catalytic function of PARP during the process of apoptosis may seem surprising in view of the literature describing quantitative and specific proteolytic cleavage of this enzyme by apoptosis-activated ICE-like enzymes [12–14]. The two observations may be reconciled by assuming that PARP activation precedes PARP proteolysis or that a minor fraction of uncleaved PARP is responsible for the observed poly(ADP-ribose) formation or both the events. By applying the ‘‘tricolor’’ procedure we demonstrated that, after induction of apoptosis with the chemotherapeutic agent etoposide, an apoptotic cell can be visualized simultaneously by the three parameters chromatin condensation, DNA fragmentation, and high-level poly(ADP-ribose) synthesis. This convenient multiparametric procedure should facilitate the identification of apoptotic cells at the single-cell level.
The financial support of Telethon-Italy (Grant E.550) is gratefully acknowledged. We thank Professor Masanao Miwa (Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan; Fax: 81-298-53-3039) for the use of monoclonal antibody 10H. M.D. was recipient of a FIRC (Fondazione Italiana per la Ricerca sul Cancro) fellowship; R.B. was a student from Scuola di Dottorato in Scienze Genetiche, and L.R. is a student from Scuola di Dottorato in Fisiopatologia Sperimentale, Universita` degli Studi di Pavia.
REFERENCES 1. Zakeri, Z., Bursch, W., Tenniswood, M., and Lockshin, R. A. (1995) Cell Death Differ. 2, 87–96. 2. Negri, C., Bernardi, R., Braghetti, A., Astaldi Ricotti, G. C. B., and Scovassi, A. I. (1993) Carcinogenesis 14, 2559–2564. 3. Negri, C., Bernardi, R., Donzelli, M., Prosperi, E., and Scovassi, A. I. (1996) Cell Death Differ. 3, 425–429. 4. Bernardi, R., Negri, C., Donzelli, M., Guano, F., Torti, M., Prosperi, E., and Scovassi, A. I. (1995) Biochimie 77, 378–384. 5. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493–501. 6. Bu¨rkle, A., Chen, G., Ku¨pper, J.-H., Grube, K., and Zeller, W. J. (1993) Carcinogenesis 14, 559–561. 7. Kawamitsu, H., Hoshino, H., Okada, H., Miwa, M., Momoi, H., and Sugimura, T. (1984) Biochemistry 23, 3771–3777. 8. Ku¨pper, J.-H., de Murcia, G., and Bu¨rkle, A. (1990) J. Biol. Chem. 265, 18721–18724. 9. Ku¨pper, J.-H., Van Gool, L., Mu¨ller, M., and Bu¨rkle, A. (1996) Histochem. J. 28, 391–395. 10. Shah, G. M., Kaufmann, S. H., and Poirier, G. G. (1995) Anal. Biochem. 232, 251–254. 11. Simbulan-Rosenthal, C. M., Rosenthal, D. S., Hilz, H., Hickey, R., Malkas, L., Applegren, N., Wu, Y., Bers, G., and Smulson, M. E. (1996) Biochemistry 35, 11622–11633. 12. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346–347. 13. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37– 43. 14. Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801–809.
Received February 10, 1997 Revised version received April 4, 1997
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ECR 3591
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6i23$$$162
06-24-97 13:54:49
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234, 174–177 (1997)
EX973591
SHORT NOTE Multiparametric Staining to Identify Apoptotic Human Cells Claudia Negri, Maddalena Donzelli, Rosa Bernardi, Laura Rossi, Alexander Bu¨rkle,* and A. Ivana Scovassi1 Istituto di Genetica Biochimica ed Evoluzionistica C.N.R., Via Abbiategrasso 207, I-27100 Pavia, Italy; and *Deutsches Krebsforschungszentrum, Abteilung 0610, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
To analyze relevant features of HeLa and HL60 cells driven into apoptosis by etoposide, we have developed a new ‘‘tricolor’’ assay, based on the simultaneous analysis in the single cell of chromatin condensation, DNA degradation, and cellular poly(ADP-ribose) synthesis. The latter reaction is catalyzed by poly(ADP-ribose)polymerase (E.C. 2.4.2.30), an enzyme which is activated by the presence of DNA free ends. The protocol consists in the visualization of apoptotic cells by Hoechst staining, TUNEL assay, and immunoreaction with anti-poly(ADP-ribose) antibody. We thus provide the first evidence that endogenous poly(ADP-ribose) production is indeed stimulated in cells undergoing apoptosis after treatment with antitumoral drugs, and that the monitoring of this endogenous enzymatic reaction, combined with morphological and other biochemical parameters, should facilitate the detection of apoptotic cells. q 1997 Academic Press
INTRODUCTION
Cell death by apoptosis is characterized by a number of morphological and biochemical features which, however, are not universal [1]. Therefore, the definition of the apoptotic phenotype requires the evaluation of more than one parameter in order to be generally accepted. In previous work, we characterized morphological and biochemical events occurring during apoptosis induced in human tumoral cell lines by chemotherapeutic drugs. In HeLa cells incubated with etoposide or longterm cultured, we have observed several typical features of apoptosis, such as chromatin condensation, nuclear fragmentation, and DNA and protein degradation [2, 3]. Furthermore, we have characterized poly(ADPribosylation), a posttranslational modification modulated during apoptosis, and we have observed an in1 To whom correspondence and reprint requests should be addressed. Fax: 39-382-422286. E-mail: [email protected].
ECR 3591
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6i23$$$161
MATERIALS AND METHODS Cell cultures and treatments. HeLa cells were grown as monolayer in Dulbecco’s modified Eagle’s medium (D-MEM, GIBCO-BRL). HL60 cells were grown in suspension in RPMI 1640 medium (Dutch mod.; HyClone, Cramlington, NE). Media were supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT), 4 mM glutamine, 2 mM sodium pyruvate, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from GIBCO-BRL). Cells were grown at 377C in a humidified atmosphere containing 5% CO2 . HeLa cells were seeded on coverslips at a concentration of 105 cells/ ml, maintained in culture for 2 days and then treated with 50 mM etoposide (Bristol Ital., Sermoneta, Italy) for 3 h followed by 3 h of incubation in fresh medium. For some experiments, 1 mM 3-aminobenzamide (Sigma, St. Louis, MO) was administered for 3 h before addition of etoposide. HL60 cells were seeded at 5 1 105 cells/ml, maintained in culture for 2 days, and treated with 68 mM etoposide for increasing time periods (0, 2, and 6 h). Multiparametric fluorescence analysis (Tricolor protocol). HeLa cell monolayers were rinsed in PBS and fixed with ice-cold 10% trichloroacetic acid for 15 min, followed by successive washes in cold 70%, 90%, and absolute ethanol for 3 min each (6). HL60 cell suspensions washed in PBS were resuspended at a concentration of 2 1 106 cells/ml in PBS containing 10% FCS, cytocentrifuged on microscope slides, and fixed as HeLa cells. Samples were processed sequentially as follows: (a) DNA apoptotic fragments were end-labeled using the Apoptosis Detection System (Promega, Madison, WI). This protocol allows the evaluation of the extent of DNA degradation by incorporating fluorescein-12-dUTP at the 3*-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase (TdT), which forms a polymeric tail by the principle of the TUNEL (TdT-mediated dUTP nickend labeling) assay [5]. Briefly, after fixation samples were rinsed in PBS and incubated for 10 min at RT with equilibration buffer
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
crease in the activity of the enzyme poly(ADP-ribose)polymerase (PARP) in extracts of apoptotic cells [2–4]. In the present study, we have analyzed different markers of apoptosis in HeLa and HL60 cells after etoposide treatment in order to achieve a multiparametric identification of apoptotic cells. At the single cell level, we have visualized chromatin changes by Hoechst staining and DNA fragments by the TUNEL assay [5]. Moreover, we have followed the endogenous cellular synthesis of poly(ADP-ribose) by the immunoreaction with monoclonal antibody 10H against this polymer.
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SHORT NOTE
(200 mM potassium cacodylate, pH 6.6, 25 mM Tris–HCl, pH 6.6, 0.2 mM DTT, 0.25 mg/ml BSA, and 2.5 mM CoCl2). Thereafter, the enzymatic reaction was performed for 60 min at 377C in the dark in the equilibration buffer supplemented with the following reagents: 10 mM dATP, 1 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, 5 mM fluorescein-12-dUTP and 25 U of TdT (final concentrations). To remove unincorporated dUTP, slides were washed three times for 15 min at RT with 21 SSC and three times for 5 min with PBS. (b) Poly(ADP-ribose) synthesis was evaluated by immunofluorescence. After PBS washing of TUNEL-processed samples, cells were incubated for 30 min at RT with PTN (PBS containing 0.1% Tween 20 and 10% newborn calf serum) and then for 1 h at RT with 101 concentrated hybridoma supernatant containing monoclonal antibody 10H against poly(ADP-ribose) provided by Professor Miwa [7]. Thereafter, cells were washed five times for 5 min with PBS and incubated for 30 min at RT with a Texas red-linked sheep anti-mouse Ig antibody (Amersham Ital.) diluted 1:50 in PTN. Aspecific binding was avoided by three washes in PBS. (c) After washing in PBS, chromatin condensation and nuclear morphology were visualized by staining DNA with 0.1 mg/ml Hoechst 33258 (Sigma) for 3 min at RT in the dark. Samples were then washed three times with PBS and mounted with PBS containing 10% glycerol and 0.02% sodium azide. Microscopic observation was carried out using a Leitz Orthoplan microscope equipped with a 501 objective. Light emissions of Texas red, fluorescein, and Hoechst were detected using specific filters. Cells were photographed using Scotch Chrome (640 ASA) film.
RESULTS
Etoposide-treated HeLa and HL60 cells were monitored for DNA fragmentation with the TUNEL assay, immunostained with monoclonal antibody 10H to reveal poly(ADP-ribose) production, and countercoloured with Hoechst 33258. The use of reagents with different light emission, such as blue for Hoechst, green (fluoresceine isothiocyanate) for the TdT substrate dUTP, and Texas red for the anti-mouse secondary antibody, allowed the visualization of the three different parameters in the same samples. In Fig. 1 (top) it is evident that an apoptotic HeLa cell exhibits typical morphological features by Hoechst staining and shows also a TUNEL-positive green fluorescence. Furthermore, the same cell is positive for the red fluorescence from immunoreaction with 10H monoclonal antibody against ADP-ribose polymers. The ‘‘tricolor’’ procedure was applied to HL60 cells treated for increasing times with a single dose of etoposide (68 mM). Results are shown in Fig. 1 (bottom). Control cells appeared to be morphologically normal, with intact DNA and negative 10H staining (Figs. 1a, 1b, and 1c, respectively). After 2 and 6 h of incubation, as shown by Hoechst morphology (Figs. 1d and 1g, re-
spectively) and by TUNEL assay (Figs. 1e and 1h, respectively), the number of cells with chromatin fragmentation increased. As observed for HeLa cells, also in HL60 cells the positivity for 10H antibody is mainly restricted to cells showing apoptotic features (Figs. 1f and 1i). Compared to HeLa cells, HL60 revealed a higher background for TUNEL assay, possibly due to the cytocentrifugation step. To confirm the specificity of the 10H immunofluorescence signals, HeLa cells were treated with the ADPribosylation inhibitor 3-aminobenzamide (3-AB). As it is shown in Fig. 2, this compound, administered before etoposide incubation, inhibits the appearance of 10Hpositive cells after etoposide treatment. However, 3-AB did not alter the induction of apoptosis visualized by Hoechst DNA staining. DISCUSSION
We developed a new protocol for the simultaneous analysis of three parameters of apoptosis, namely, (i) the changes in nuclear morphology visualized by Hoechst 33258 staining; (ii) the appearance of DNA breaks, evaluated by the TUNEL assay; and (iii) the endogenous cellular poly(ADP-ribose) synthesis monitored by indirect immunofluorescence, using the monoclonal antibody 10H raised against this polymer. This antibody has been previously used for the in situ detection of poly(ADP-ribosylation) in living cells treated with alkylating agents and then fixed with trichloroacetic acid [6, 8, 9], and it has been recently utilized to detect polymer synthesis on nitrocellulose-immobilized PARP undergoing automodification, during the activity blot procedure [10], and for Western blot analysis [11]. In HeLa cells, polymer-specific fluorescence was localized in nuclei characterized by apoptotic morphology when counterstained with Hoechst 33258, and by extensive DNA degradation, an event producing 3*-OH free ends, the substrate for the TdT reaction. In HL60 cells, we have observed that after a 2-h treatment with 68 mM etoposide, about 15% of cells showed typical apoptotic features and that this percentage reached more than 80% after 6 h of incubation with the drug. Under these conditions, poly(ADP-ribose) synthesis appeared to be increased with the time of treatment and to be confined to TUNEL-positive cells with few exceptions, represented by apoptotic cells brilliant for poly(ADP-ribose) staining but with a weak TUNEL fluo-
FIG. 1. Tricolor assay in HeLa and HL60 cells. (Top) HeLa cells treated with 50 mM etoposide for 3 h followed by 3 h of incubation in drug-free medium. (Bottom) HL60 cells treated with 68 mM etoposide for increasing times up to 6 h. (a–c) Control cells, (d–f) cells treated with 68 mM etoposide for 2 h, (g–i) cells treated with 68 mM etoposide for 6 h. Blue fluorescence, Hoechst staining; green fluorescence, TUNEL assay; red fluorescence, immunoreactivity of 10H antibody. Experiments were performed as described under Materials and Methods. FIG. 2. Effect of 3-aminobenzamide on poly(ADP-ribose) synthesis and apoptosis. HeLa cell treatments: 50 mM etoposide for 3 h followed by a 3-h incubation in drug-free medium; 1 mM 3-aminobenzamide for 3 h followed or not by etoposide treatment.
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rescence. The nature of this morphology is currently under investigation. The endogenous activation of the catalytic function of PARP during the process of apoptosis may seem surprising in view of the literature describing quantitative and specific proteolytic cleavage of this enzyme by apoptosis-activated ICE-like enzymes [12–14]. The two observations may be reconciled by assuming that PARP activation precedes PARP proteolysis or that a minor fraction of uncleaved PARP is responsible for the observed poly(ADP-ribose) formation or both the events. By applying the ‘‘tricolor’’ procedure we demonstrated that, after induction of apoptosis with the chemotherapeutic agent etoposide, an apoptotic cell can be visualized simultaneously by the three parameters chromatin condensation, DNA fragmentation, and high-level poly(ADP-ribose) synthesis. This convenient multiparametric procedure should facilitate the identification of apoptotic cells at the single-cell level.
The financial support of Telethon-Italy (Grant E.550) is gratefully acknowledged. We thank Professor Masanao Miwa (Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan; Fax: 81-298-53-3039) for the use of monoclonal antibody 10H. M.D. was recipient of a FIRC (Fondazione Italiana per la Ricerca sul Cancro) fellowship; R.B. was a student from Scuola di Dottorato in Scienze Genetiche, and L.R. is a student from Scuola di Dottorato in Fisiopatologia Sperimentale, Universita` degli Studi di Pavia.
REFERENCES 1. Zakeri, Z., Bursch, W., Tenniswood, M., and Lockshin, R. A. (1995) Cell Death Differ. 2, 87–96. 2. Negri, C., Bernardi, R., Braghetti, A., Astaldi Ricotti, G. C. B., and Scovassi, A. I. (1993) Carcinogenesis 14, 2559–2564. 3. Negri, C., Bernardi, R., Donzelli, M., Prosperi, E., and Scovassi, A. I. (1996) Cell Death Differ. 3, 425–429. 4. Bernardi, R., Negri, C., Donzelli, M., Guano, F., Torti, M., Prosperi, E., and Scovassi, A. I. (1995) Biochimie 77, 378–384. 5. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493–501. 6. Bu¨rkle, A., Chen, G., Ku¨pper, J.-H., Grube, K., and Zeller, W. J. (1993) Carcinogenesis 14, 559–561. 7. Kawamitsu, H., Hoshino, H., Okada, H., Miwa, M., Momoi, H., and Sugimura, T. (1984) Biochemistry 23, 3771–3777. 8. Ku¨pper, J.-H., de Murcia, G., and Bu¨rkle, A. (1990) J. Biol. Chem. 265, 18721–18724. 9. Ku¨pper, J.-H., Van Gool, L., Mu¨ller, M., and Bu¨rkle, A. (1996) Histochem. J. 28, 391–395. 10. Shah, G. M., Kaufmann, S. H., and Poirier, G. G. (1995) Anal. Biochem. 232, 251–254. 11. Simbulan-Rosenthal, C. M., Rosenthal, D. S., Hilz, H., Hickey, R., Malkas, L., Applegren, N., Wu, Y., Bers, G., and Smulson, M. E. (1996) Biochemistry 35, 11622–11633. 12. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346–347. 13. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37– 43. 14. Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801–809.
Received February 10, 1997 Revised version received April 4, 1997
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ECR 3591
/
6i23$$$162
06-24-97 13:54:49
eca