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J Biol Chem, Vol. 274, Issue 45, 32122-32126, November 5, 1999

Selective Loss of Poly(ADP-ribose) and the 85-kDa Fragment of Poly(ADP-ribose) Polymerase in Nucleoli during Alkylation-induced Apoptosis of HeLa Cells^*

Rafael Alvarez-Gonzalez^§¶, Herbert Spring, Marcus Müller, and Alexander Bürkle

From the  Division of Tumor Virology and the  Biomedical Structural Analysis Unit, German Cancer Research Center, Heidelberg, Germany and the ^§ Department of Molecular Biology and Immunology and The Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, Texas 76107-2699

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alkylation treatment of HeLa cells results in the rapid induction of apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H. & Alvarez-Gonzalez, R. (1998) Cancer Res. 58, 5075-5078). Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Higher eucaryotic organisms have developed a sophisticated signaling system to eliminate nonfunctional cells in a highly coordinated sequence of "suicidal" events. This tightly regulated process is known as "programmed cell death" or "apoptosis" (1-3). Superfluous cell populations that undergo apoptosis include genetically damaged and/or aging cells. The death program is executed in three chronologically distinct phases (4). First to occur is "the condemned phase" where a fully reversible sequence of metabolic and cell cycle adaptation(s) take place with mitochondrial components, such as cytochrome c and the bcl-2 gene produced, playing a decisive role. The second phase of "commitment" is irreversible and derives from unleashing a cascade of proteolytic signals emanating from the mitochondrion to the nucleus. Finally, the "execution phase" of apoptosis is manifested by the macromolecular degradation of chromosomal DNA catalyzed by caspase (cysteine-aspartase)-activated death-factor (5, 6). In this phase, key nuclear proteins are degraded by caspase-3, the main "executioner" of nuclear disassembly (7), and this leads to the disintegration of the nucleus. One of the primary targets for caspase-3 is poly(ADP-ribose)polymerase (PARP)¹ (E.C. 2.4.2.30) (8-10). PARP is a nuclear DNA-binding protein of 1,014 amino acid residues (113 kDa) (11, 12) that is constitutively expressed in eucaryotes and comprises up to 1% of the total nuclear protein. This enzyme displays a multimodular domain structure that can be dissected into three functionally distinct domains from the amino terminus to the carboxyl terminus (13). The first module is the "DNA-binding domain" (DBD), a fragment of 46-kDa that contains two zinc fingers, which mediate binding to DNA strands breaks as well as a bipartite nuclear localization signal (14). The two karyophilic regions of the nuclear localization signal are separated by the DEVD sequence that is cleaved by caspase-3 during apoptosis, resulting in the formation of two proteolytic fragments of PARP, a 29-kDa amino terminus, and an 85-kDa carboxyl terminus. The carboxyl-terminally located 54-kDa domain of PARP represents the NAD⁺-binding domain with the characteristic "PARP signature," a highly conserved sequence comprising the catalytically crucial amino acid residue Glu-988 (15). The catalytic activity of PARP is dramatically stimulated by noncovalent contact of the DNA-binding domain with DNA strand breaks and results in the post-translational modification of various "acceptor" proteins, including PARP itself, with poly(ADP-ribose). Finally, in between the DNA-binding domain and the NAD⁺-binding domain, there is also a 22-kDa "automodification domain" (16, 17) which comprises the modules that facilitate the homo- (18) and/or heterodimerization (19) of PARP with other chromatin proteins. Recently, a substantial effort has been invested to elucidate the physiological function of the protein-poly(ADP-ribosyl)ation pathway in cellular recovery from DNA damage (11, 20-22). In general, it appears that an early enzymatic activation of PARP occurs upon DNA-strand break formation (23, 24). However, when the genetic damage cannot be repaired, the cell decides to trigger apoptosis, which leads to the proteolytic cleavage of PARP (8-10). These opposite roles played by PARP create a functional paradox where protein-poly(ADP-ribosyl)ation (11, 20-22) appears to play an initial "protective role" by facilitating DNA base excision repair (25), although also becoming a pivotal protein target for caspase-3 during apoptotic execution (8-10). To shed light on the biochemical function of PARP in the different stages of apoptosis, we have now performed a detailed time-course study of poly(ADP-ribose) metabolism in HeLa cells as PARP goes from initial enzymatic activation to caspase-3 catalyzed cleavage and concomitant poly(ADP-ribose) glycohydrolase (PARG) activation following MNNG treatment. By integrating these data with our previous biochemical characterization of poly(ADP-ribosyl)ated PARP in MNNG-treated cells (26, 27), we derive a provocative, yet plausible explanation for the bifunctional role of PARP in the chronology of apoptosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Treatment-- HeLa cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ in Dulbecco`s minimal Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Sigma). MNNG (Serva, Heidelberg, Germany) was dissolved in PBS (10 mM solution) and added to the culture medium at a final concentration of 50 µM. Cells were incubated at 37 °C following addition of MNNG and were harvested at 0, 5, 10, 15, 30, 60, 90, and 120 min and in some experiments, 2.5 and 3 h after treatment.

Epifluorescence-- Nonconfluent HeLa cell cultures were grown on coverslips for 48-72 h, exposed to 50 µM MNNG for the times indicated above, and fixed for 10 min in ice-cold 10% (w/v) trichloroacetic acid (28). After washes in 70, 90, and 96% ethanol, cells were air dried, rehydrated in PBS, and incubated with 5 µg/ml monoclonal antibody 10H (kind gift of M. Miwa and T. Sugimura, Tokyo, Japan) diluted in PBS, 5% nonfat dry milk, 0.05% Tween 20 for 30 min at 37 °C. This antibody is specifically directed against poly(ADP-ribose) (29). The coverslips were washed 4 times for 5 min each in PBS and subsequently incubated with FITC-conjugated secondary antibody (diluted 1:50 in PBS, 5% nondry fat milk, 0.05% Tween 20) for 30 min at 37 °C. After 4 washes in PBS (5 min each) the slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing 1 µg/ml 4',6-diamidino-2-phenylindole, and viewed with either a Leica epifluorescent microscope or a Zeiss LSM 510 confocal microscope (vide infra). Similar procedures were used with mouse monoclonal antibodies F-I-23 and C-II-10 (kind gift of G. G. Poirier, Quebec, Canada), except that cells were fixed in 5% formaldehyde in PBS for 30 min at room temperature, followed by permeabilization in 0.4% Triton X-100 in PBS and rinsing in PBS.

Confocal Microscopy-- Confocal microscopy was performed with a Carl Zeiss LSM 510 UV confocal laser scanning microscope (Jena, Germany). For fluorescence excitation, an argon ion laser with 488-nm wavelength and an appropriate combination of beam splitter and barrier filter were used. The images were simultaneously taken in the fluorescence confocal mode (depth of focus 700 nm) as well as in the transmitted light mode of the instrument using differential interference contrast according to Nomarski. It should be noted that hundreds of cells were visualized under the confocal microscope for each condition with identical morphology. However, only a small number of cells from three independent experiments were selected to illustrate the results shown in Figs. 1-4.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The dose-dependent accumulation of poly(ADP-ribose) in the nucleus of DNA-damaged cells has previously been visualized as granular immunofluorescence (28) generated with the antibody 10H, a monoclonal immunoglobulin highly specific for ADP-ribose polymers (29). Here, we have followed the time-dependent subnuclear distribution of ADP-ribose polymers synthesized in HeLa cells in response to treatment with the alkylating agent MNNG as well as of PARP and its proteolytic fragments by epifluorescence and confocal laser scanning microscopy.

Enzymatic Activation of PARP Following Genotoxic Treatment of HeLa Cells-- Fig. 1 displays the immunofluorescent signal generated with antibody 10H after 15 min of alkylating damage. Fig. 1, top left corner, shows the fluorescent signal generated in conjunction with the FITC-conjugated secondary antibody. The top right corner in Fig. 1 represents the same cell shown in the top left corner, under transmitted confocal light according to Nomarski. Fig. 1, bottom left corner, was generated by superimposing the two images described above. This latter presentation form of confocal micrographs was the style that we selected to describe all subsequent experiments (vide infra). Fig. 2 (panel a) displays the expected absence of poly(ADP-ribose) in control HeLa cells, because PARP is only activated by DNA-strand breaks (23) following DNA damage. Panels b, c, and d illustrate the time-dependent increase in the intensity of the immunofluorescent signal in the nucleus, with a maximum level reached after 15 min (panel d), consistent with previously published results (30). We also observed reproducibly that after 15 min of MNNG treatment, nucleoli did not immunostain for poly(ADP-ribose). Therefore, we next proceeded to evaluate the biological significance of the peculiar karyoplasmic localization of ADP-ribose polymers once proteolysis of PARP had begun (26).

View larger version (91K): [in this window] [in a new window]   Fig. 1.   Enzymatic activation of PARP in the nucleoplasm of HeLa cells following exposure to 50 µM MNNG for 15 min, as detected with antibody 10H. The top left corner shows the FITC signal specific for antibody 10H. The top right corner displays transmitted light only (Nomarski). The bottom left corner shows images superimposed. The bar represents 20 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Time-dependent enzymatic activation of PARP and PARG in the nucleoplasm of HeLa cells following exposure to 50 µM MNNG, as detected with antibody 10H. HeLa cells were treated with MNNG for 0, 5, 10, 15, 30, 60, 90, 120, 150, and 180 min at 37 °C, fixed with 10% trichloroacetic acid, and subsequently processed with antibody 10H. Panels a, b, c, d, e, f, g, h, i, and j, display the immunofluorescence observed under the confocal microscope after 0, 5, 10, 15, 30, 60, 90, 120, 150, and 180 min of MNNG treatment, respectively. The bar represents 20 µm.

Enzymatic Activation of PARG Following Genotoxic Treatment of HeLa Cells-- In view of the reproducible quantitative proteolytic degradation of PARP in MNNG-treated cells between 15 and 120 min (26), we also studied poly(ADP-ribose) catabolism, which is catalyzed mostly by PARG in MNNG-treated HeLa cells between 15 and 180 min of incubation at 37 °C. Fig. 2 also shows that the levels of nucleoplasmic poly(ADP-ribose) steadily decreased between 30 and 180 min post-DNA damage (panels e-j). Interestingly, panels i and j, which do not show detectable levels of poly(ADP-ribose) anymore, also reveal some of the characteristic morphological changes of apoptotic cells. Thus, during the period of incubation in which PARP is proteolytically degraded by caspase-3 during apoptotic execution (26), there is still poly(ADP-ribose) catabolism going on (panels e-j). Moreover, under the chosen experimental conditions, this time period coincides with genomic DNA fragmentation (27). Also, the results described here indicate that there is still ongoing degradation of protein-bound poly(ADP-ribose) at a time when NAD⁺ and ATP pools have already been depleted (31) and PARP has been proteolytically cleaved (26).

The highly dynamic nature of the poly(ADP-ribosyl)ation pathway (synthesis and turnover) in cells with damaged DNA implies the transient existence of protein-bound polymers. Indeed, the half-life of this highly polyanionic, nucleic acid-like molecule, can be very short (less than 1 min) in DNA-damaged cells (30, 32). Therefore, the time-dependent disappearance of the poly(ADP-ribose)-specific 10H antibody fluorescent signal that we observed between 30 and 180 min post-MNNG treatment (Fig. 2, panels e-i) indicates that the intranuclear levels of this polymer dramatically decrease concomitantly with the proteolysis of PARP. For this reason, we can also conclude that the activity of PARG remains mostly active between 30 and 180 min of MNNG treatment. However, it is important to note that our experiments do not completely rule out a possible contribution by phosphodiesterase activity to poly(ADP-ribose) catabolism. Nevertheless, because PARG is the main catabolic enzyme for poly(ADP-ribose) in DNA-damaged cells (30, 32, 33), our data support the notion that PARG is mostly active during apoptotic execution.

Functional Localization of PARP and Its Fragments in Apoptosis-- Previously, it has been demonstrated that PARP protein functions as the main covalent target for poly(ADP-ribosyl)ation in cultured cells exposed to alkylating DNA damage (34). Very recently, it has also been observed that the automodification reaction of PARP, at least initially, involves 4 (35) of the 16 Glu residues localized in the automodification domain. Furthermore, it has been shown that, while the 85-kDa proteolytic fragment of PARP specifically interacts with p53 in HeLa cells within 30 min of MNNG treatment (26), the 29-kDa amino-terminal fragment keeps DNA loose ends together early in apoptosis (36). Therefore, we next proceeded to monitor the fate of these two proteolytic fragments in HeLa cells after MNNG treatment. We accomplished this goal by using two highly specific monoclonal antibodies, namely, F-I-23 and C-II-10 (37), which recognize the amino-terminal and the carboxyl-terminal apoptotic fragments of PARP, respectively. Figs. 3 and 4 display the time-dependent distribution of the immunofluorescent signal generated with these monoclonal antibodies under the confocal microscope. As expected, control cells (panel a of Figs. 3 and 4) displayed a homogeneous staining of the nucleus (including all nucleoli) before the enzymatic activation of PARP (compare with Fig. 2, panel a). These results are consistent with the notion that PARP is a constitutive "house-keeping" protein, whereas polymers of ADP-ribose are synthesized only in response to DNA damage (11, 20, 21). Also shown in the confocal micrographs of Fig. 3 is that throughout the time of incubation (panels b-f), immunofluorescent signals generated with the F-I-23 antibody were visible in all nucleoli. By contrast, the results obtained with the C-II-10 antibody (Fig. 4) showed that the immunofluorescent signal decreased in the nucleoli to undetectable levels while evenly staining over the entire karyoplasm between 15 min (panel b) and 120 min (panels c-f) of MNNG treatment. Strikingly, this disappearance of the 85-kDa fragment of PARP from the nucleoli paralleled the selective karyoplasmic distribution of ADP-ribose polymers (Fig. 2). It should be noted, however, that while the F-I-23 signal immunostained the entire nucleus throughout 2 h of MNNG treatment, a slight decrease in the level of immunofluorescent intensity was noticed, perhaps indicating further proteolytic processing of the 29-kDa fragment late in apoptosis.

View larger version (96K): [in this window] [in a new window]   Fig. 3.   Time-dependent fluctuations in the subnuclear distribution of PARP and its 29-kDa apoptotic fragment in HeLa cells following exposure to 50 µM MNNG, as detected with antibody F-I-23. HeLa cells were treated with MNNG for 0, 15, 30, 60, 90, and 120 min at 37 °C, fixed with formaldehyde, and subsequently processed with F-I-23 antibody as indicated under "Experimental Procedures." Panels a, b, c, d, e, and f display the immunofluorescence observed under the confocal microscope after 0, 15, 30, 60, 90, and 120 min of incubation with MNNG. The bar represents 20 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 4.   Time-dependent fluctuations in the subnuclear distribution of PARP and its 85-kDa apoptotic fragment in HeLa cells following exposure to 50 µM MNNG, as detected with antibody C-II-10. HeLa cells were treated with MNNG for 0, 15, 30, 60, 90, and 120 min at 37 °C, fixed, and subsequently processed with C-II-10. Panels a, b, c, d, e, and f display the immunofluorescence observed under the confocal microscope after 0, 15, 30, 60, 90, and 120 min of incubation with MNNG, respectively. The bar represents 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this paper, we show for the first time in a detailed time-course study, the visualization of poly(ADP-ribose) metabolism as well as the subnuclear distribution of native PARP and its apoptotic fragments following DNA damage with MNNG. We show the specific karyoplasmic and nucleolar staining of the 29- and 85-kDa proteolytic fragments of PARP (Figs. 3 and 4, respectively), as well as the preferential karyoplasmic distribution of protein-bound ADP-ribose polymers coincident with the onset of PARP proteolysis (Fig. 2). The biological significance of our confocal observations is further underlined by a recent report (38) indicating that apoptotic proteolysis of key nuclear proteins may take place in the nucleolus just before apoptotic execution. Therefore, it is tempting to speculate that the absence of C-II-10 immunofluorescent staining in nucleoli (Fig. 4) after proteolysis of poly(ADP-ribosyl)ated-PARP is due to the fact that the protein-bound ADP-ribose chains direct the transfer of this apoptotic fragment from the nucleolus to the karyoplasm. This interpretation is further substantiated by the following observations: first, it has recently been demonstrated that the auto-poly(ADP-ribosyl)ation reaction of PARP mainly occurs near the amino terminus of the 85-kDa proteolytic fragment (35); and second, no 10H antibody signal (specific for polymeric ADP-ribose) is observed in nucleoli after 15 min of MNNG treatment (Figs. 1 and 2). Our data, together with previous results (26), suggest that the poly(ADP-ribosyl)ated 85-kDa fragment of PARP may be localized to the karyoplasm during apoptotic execution to modulate the activity(ies) of proteins, such as p53 (26), by either protein-protein interactions and/or noncovalent poly(ADP-ribose)-protein interactions (39). This interpretation is also supported by data shown in Fig. 3 revealing that F-I-23 immunofluorescence remained evenly distributed over the entire nucleoplasm throughout the 120 min of confocal microscopic observation. Therefore, it also appears that the 29-kDa fragment is not poly(ADP-ribosyl)ated in MNNG-treated HeLa cells. This notion is also fully consistent with our recent biochemical characterization of mono(ADP-ribosyl)ated-PARP (35). A second alternative that might also explain the DNA damage-dependent pattern of immunostaining for the apoptotic fragments of PARP and ADP-ribose polymers after 15 min of MNNG treatment is the possibility that, upon cleavage of the DEVD domain by caspase-3 (40), the bipartite nuclear localization signal of this protein is irreversibly dissected into putative karyoplasmic- and nucleolar-specific targeting sequences. Further studies with mutants carrying specific deletions in the nuclear localization signal region of PARP need to be performed to test this hypothesis.

In conclusion, we describe the rapid and selective disappearance of poly(ADP-ribose) and the 85-kDa fragment of PARP, but not the 29-kDa fragment, from nucleoli of HeLa cells following alkylation damage. In light of the recent results by Stegh et al. (38), our data underscore the notion that nucleoli are remarkable organelle centers for proteolytic processing of nuclear proteins early in apoptosis. The precise definition of the roles of apoptotic fragments of PARP will significantly enhance our understanding of the dramatic structural and functional changes of the nucleus in the chronology apoptosis.

	ACKNOWLEDGEMENTS

We thank Prof. Harald zur Hausen, Director of the Deutsches Krebsforschungszentrum, for strong support of this project.

	FOOTNOTES

^* This research project was supported in part by a Cancer Research grant from Bank One of Texas, Grant GM45451 from the National Institutes of Health (to R. A. G.), and Grant Bu698/2-4 from the Deutsche Forschungsgemeinschaft (to A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ On sabbatical leave from the Dept. of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107-2699. To whom correspondence should be sent: Dept. of Molecular Biology and Immunology and Associate Director for Basic Research, The Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107-2699. Tel.: 817-735-2117; Fax: 817-735-2133; E-mail: ralvarez@hsc.unt.edu.

	ABBREVIATIONS

The abbreviations used are: PARP, poly(ADP-ribose) polymerase; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; caspase, cysteine-dependent aspartase; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PARG, poly(ADP-ribose) glycohydrolase.

	REFERENCES

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