PolyADP-ribose-mediated regulation of p53 complexed with topoisomerase I following ionizing radiation
Heather M. Smith and
Andrew J. Grosovsky1
Environmental Toxicology Program, University of California, 5419 Boyce Hall, Riverside, CA 92521, USA
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Abstract
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This investigation demonstrates that the p53 and topoisomerase I (topo I) proteins which form a molecular complex in vivo are polyADP ribosylated following 1 Gy of
irradiation. Immunoprecipitations using a topo I monoclonal antibody were performed on protein extracts from
-irradiated TK6 human lymphoblastoid cells. Western blots on topo I immunoprecipitations probed with a polyADP-ribose polymer antibody demonstrated that several proteins, including p53, are co-immunoprecipitated with topo I. Furthermore, p53 and topo I are ADP ribosylated within 15 min following
irradiation. Unlike the other proteins within the complex, p53 is polyADP ribosylated at low levels in non-irradiated cells, and it is also the most heavily polyADP ribosylated following irradiation. Radiation induced polyADP ribosylation persists for at least 48 h following exposure. The DNA damage response does not involve the recruitment of free p53 to complex with topo I; the amount of p53 protein complexed with topo I was found to be independent of radiation exposure. It has recently been reported that p53 acts to catalytically stimulate the activity of topo I in the absence of DNA damage. We hypothesize that the rapid modification of the complex by polyADP ribosylation following radiation is a regulatory response to diminish topo I cleavage in the presence of DNA damage.
Abbreviations: PARP, polyADP-ribose polymerase; topo I, topoisomerase I
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Introduction
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Several DNA-damage-induced cellular responses have been elucidated in mammalian cells. The level and activity of the gene products involved in these responses are regulated through a variety of transcriptional and post-transcriptional mechanisms. For example, DNA damage initiates a p53-dependant regulatory cascade involving increased rate of transcription at GADD45, and other genes that are critical for cell cycle regulation and DNA repair (13). After damage occurs, intracellular levels of p53 protein increase in the cell through de novo protein synthesis (4,5) and possibly post-translational stabilization of p53 (6). Although not completely characterized, several post-translational modifications of p53 which affect protein activity have been observed in vitro and in vivo. Phosphorylation of p53 by a variety of protein kinases such as casein kinase II, cdc2 kinase and DNA-PK can occur at multiple sites and has been shown to augment protein activity (7). Degradation of the p53 protein occurs via ubiquitin-dependent proteolysis which provides an important mechanism for restoration of constitutive p53 protein levels following cellular response to DNA damage (8). However, the possibility remains that other post-translational modifications may play an additional role in regulation of the level and activity of p53 protein.
Recently, several lines of evidence have suggested that interaction of p53 with polyADP-ribose polymerase (PARP) is critical for cellular response to DNA damage. PARP and p53 form a complex in primary rat embryo cells (9), and in irradiated and non-irradiated human cells (10), as demonstrated by reciprocal co-immunoprecipitation. Interaction of p53 with free ADP-ribose polymers, or with ADP-ribose polymers bound to automodified PARP, interfered with p53 function, as shown by inhibition of consensus sequence binding and by diminishment of binding to DNA single strand ends (11). Synthesis of polyADP-ribose polymers has also been reported to be required for regulation of basal and damage-induced levels of p53 activity in V79 cells (12). Despite the interaction of PARP and p53 in vivo, and the apparent importance of this interaction in mobilizing the p53 response to DNA damage, modification of p53 by polyADP ribosylation has not been fully investigated. Although ADP ribosylation of p53 in vitro using exogenously added PARP and NAD has been shown (9,13), it remains to be determined whether p53 is polyADP ribosylated in vivo by endogenous cellular processes, and in response to DNA damage.
It has been reported that p53 and topoisomerase I (topo I) form a molecular complex which enhances the catalytic activity of topo I (14,15). The activity of topo I is modified as part of the cellular DNA damage response, although unlike p53, topo I activity is down-regulated (16). Studies with metabolic inhibitors of PARP such as 3-aminobenzamide (3-AB) suggest that regulation of topo I activity after DNA damage is attributable to polyADP ribosylation (16). However, the effect of 3-AB may be partially or wholly attributable to polyADP ribosylation of the complexed p53. This study was designed to determine directly whether the inhibition of topo I activity in the DNA damage response was associated with post-translational modification by PARP, and whether the p53 component of the complex is also modified by ADP ribosylation. Additionally, we wished to determine whether the formation of the p53topo I complex is influenced by DNA damage. Co-immunoprecipitation results indicate that topo I and p53 form a tight complex in a human B-lymphoblastoid cell line independent of radiation exposure. Furthermore, each of these proteins is polyADP ribosylated with differential kinetics following the induction of DNA damage by
irradiation.
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Materials and methods
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Cell line and cell culture
TK6 is a human B lymphoblastoid cell line (17,18) which is p53 wild-type (19) and shows an increase in p53 protein following irradiation (20). TK6 can be grown in logarithmic suspension cultures at densities of up to 106 cells/ml. The cultures are maintained in RPMI 1640 (Mediatech, Herndon, VA) supplemented with L-glutamine, penicillin/streptomycin and 10% iron supplemented calf serum (HyClone, Logan, UT).
Topo I activity assays
Nuclear extracts from
irradiated and non-irradiated TK6 human lymphoblastoid cells were isolated (21) and assayed for topo I activity by conversion of supercoiled plasmid to a relaxed form. Irradiated samples were exposed to 1 Gy of 137Cs
irradiation, a dose which reduces clonogenic cell survival to 20% (22). Supercoiled pBR322 plasmid (1.6 µg/20 µl) in 10x topo I assay buffer (TopoGEN, Columbus, OH) was treated with nuclear extract normalized for equal amounts of protein (2.5 µg), as determined using the Bio-Rad Protein Assay (Bio-Rad, Cambridge, MA). Topological relaxation of the plasmid proceeded by incubation at 37°C for 1, 5, 10, 30, 60 or 120 min. Stop solution (TopoGEN) was added at the conclusion of each incubation period. Reaction products of pBR322 form I (supercoiled) and form II (open circular) were separated by electrophoresis in 1% agarose for 18 h at 15 V. Images of ethidium bromide stained gels were scanned and analyzed using Scion Image for Windows 95 Beta 3b available at http://www.scion.com/.
Protein extracts
Protein extracts were made of 2x107 TK6 cells at selected time points up to 48 h after exposure to 0 or 1 Gy irradiation. Prior to protein extraction, cells were incubated at 37°C for 1 h in RPMI 1640 without methionine, glutamine and cysteine (ICN Biomedicals, Costa Mesa, CA). Tran35S-Label (ICN Biomedicals), which contains 35S-labled L-methionine, was added 2 h prior to harvest at a concentration of 20 µCi 35S per 106 cells. For protein extraction, cells were washed in sterile cold PBS and centrifuged at 13 000 g for 10 min. Aliquots of 150 µl X-buffer (25 mM TrisHCl pH 8, 10 mM EDTA, 1 mM PMSF, 0.006% bromophenol blue, and 50 mM glucose as a gentle cell lysis reagent) were added to each sample. Samples were stored at 80°C until use. Parallel samples were processed without bromophenol blue and 35S label for protein concentrations as determined using the Bio-Rad Protein Assay (Bio-Rad).
Immunoprecipitations using topo I and p53 monoclonal antibodies
Total protein (30 µg) from each extract was diluted 40x with double distilled H2O and incubated overnight on a rotating shaker at 4°C with topo I monoclonal antibody (1:500 dilution) generously provided by Dr Yung-chi Cheng (Yale University School of Medicine, New Haven, CT). Rabbit anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA) was added to each sample for an additional overnight incubation at 4°C (2.4x103 mg per sample). Then, 25 µl of Protein Aagarose (Oncogene, Uniondale, NY) were added for a third overnight incubation at 4°C. After the final incubation, samples were centrifuged at 13 000 g for 35 min, washed with 0.5 ml NET-gel buffer (50 mM TrisHCl pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA pH 8.0, 0.25% gelatin, 0.02% sodium azide), and centrifuged again at 13 000 g for 35 min. Supernatant was removed and 10 µl of sample buffer (0.625 M TrisHCl pH 6.8, 2% SDS, 10% glycerol, 5% BME, 0.001% bromophenol blue) were added to each sample. Samples were boiled for 5 min and centrifuged at 13 000 g for 10 min. Western blot analysis (data not shown) of total cellular protein and topo I immunoprecipitates demonstrated that ~25% of the total cellular topo I is immunoprecipitated by this procedure. Immunoprecipitations with p53 monoclonal antibody Ab-6 (Oncogene) were performed as described above using a 1:500 dilution.
Western blot analysis
To ensure equal loading for western blot analysis, scintillation counting of 35S was performed using 2 µl aliquots from each immunoprecipitation. The use of 35S counts to normalize protein loading provides the needed resolution for working with immunoprecipitates, where the yield of proteins is often too small to permit accurate determinations by a Bradford assay. After counting, samples were loaded on a 415% gradient Trisglycine gel for use in a Mini-Protean II Gel Electrophoresis Apparatus (Bio-Rad) and were run for ~45 min at 200 V. Western blots for analysis of polyADP-ribose modification of immunoprecipitated proteins were incubated with a 1:2000 dilution of anti-polyADP-ribose polyclonal antibody (Trevigen, Gaithersburg, MD) overnight at 4°C. Membranes were washed three times in TBST for 15 min at room temperature and then incubated with HRP-conjugated goat anti-guinea pig IgG at a 1:5000 dilution for 1 h at room temperature. The blotting procedure was completed using the ECL detection system (Amersham, Arlington Heights, IL). Kodak X-OMAT AR film was placed over the membrane for visualization of results. Blots of p53 protein were treated similarly using the p53 monoclonal antibody Ab-6 (Oncogene) at a 1:10 000 dilution and HRP-conjugated goat anti-mouse IgG (Amersham) at a 1:6600 dilution.
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Results
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Topo I activity assays
Topo I activity was measured by the ability of nuclear extracts to convert supercoiled plasmid (form I) to open circular plasmid (form II) at 37°C. Topological relaxation of plasmid, following incubation with nuclear extract, was visualized by altered migration in agarose gel electrophoresis (Figure 1
). Densitometry analysis of forms I and II was then performed to quantify the kinetics and extent of relaxation. Supercoiled plasmid was completely converted to open circular form within 1 min of incubation with nuclear extract from non-irradiated cells (Figure 1A
). In contrast, topo I activity was significantly inhibited in cells exposed to 1 Gy
irradiation; supercoiled pBR322 was incompletely relaxed even after incubation for 2 h with irradiated nuclear extracts (Figure 1B
). The radiation-induced inhibition of topo I activity diminished rapidly within the first 1 h post-irradiation, but topo I activity remained inhibited for >24 h following exposure (Figure 2
). These data resemble similar observations in fibroblastic cell lines (16).

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Fig. 1. Relaxation assay for topoisomerase I activity. (A) Topoisomerase I activity in non-irradiated TK6 cells. Nuclear extracts from non-irradiated cells were incubated with pBR322 at 37°C for 1, 5, 10, 30 and 60 min. Topo I activity was measured by the conversion of supercoiled plasmid (Form I) to open circular (Form II). Lane 1 (far left), migration of pBR322 without nuclear extract treatment; lanes 26, relaxation following incubation with nuclear extract for 1, 5, 10, 30 and 60 min, respectively; lane 7 (far right), positive control which shows the migration of pBR322 following incubation with 10 U purified topo I at 37°C for 60 min. (B) Topo I activity in TK6 cells 24 h post 1 Gy irradiation. Nuclear extracts from cells irradiated with 1 Gy were incubated with pBR322 at 37°C for 1, 5, 10, 30, 60 and 120 min. Topo I activity was measured by the conversion of supercoiled plasmid (Form I) to open circular (Form II). Lane 1 (far right), migration of pBR322 without nuclear extract treatment; lanes 27, relaxation following incubation with nuclear extract for 1, 5, 10, 30, 60 and 120 min, as indicated.
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Fig. 2. Topo I activity in irradiated and non-irradiated TK6 human lymphoblastoid cells. Supercoiled plasmid pBR322 was incubated for 1 min with nuclear extracts from irradiated cells receiving 1 Gy irradiation (open symbols) or non-irradiated control cells (closed symbols). Extracts were prepared at 1 min, and 1, 6, 24 and 48 h post-irradiation. Results were visualized in a 1% agarose gel stained with ethidium bromide. Densitometric analysis of the bands in each lane was used to quantify the percent of plasmid which remained incompletely relaxed after each incubation period.
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ADP ribosylation of p53 and topo I
It has been proposed that radiation-induced inhibition of topo I activity is due to damage-induced polyADP ribosylation of the enzyme (16). In order to test this hypothesis, an aliquot of protein (30 µg) from irradiated and non-irradiated TK6 cells was immunopreciptated by incubation with topo I monoclonal antibody. Equal amounts of immunoprecipitated proteins were then electrophoresed, blotted and probed using a polyADP-ribose polymer polyclonal antibody (Figure 3
). Relative band intensity was determined by densitometry, and protein loading was controlled by pre-labeling cells with 35S. This procedure demonstrated a radiation-induced ADP ribosylation of topo I. Additionally, an ADP-ribosylated protein at 53 kDa was co-immunoprecipitated. Association of p53 and topo I has been reported previously using both a protein fusion approach (15) and co-immunoprecipitation using a p53 antibody (14). However, to confirm that the 53 kDa band was p53 complexed with topo I, western blot analysis was performed using a p53 monoclonal antibody probe on proteins immunoprecipitated with the topo I monoclonal antibody (Figure 4A
). The 53 kDa peptides were identified as p53 since they are detected by the p53 monoclonal antibody, and co-migrate with purified p53 protein which was used as a size standard (Figure 4A
). A second 55 kDa band was observed and represents cross reactivity between an anti-mouseHRP conjugated secondary antibody and the topo I monoclonal antibody heavy chain from the immunoprecipitation reaction.

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Fig. 3. Immunoprecipitations of ADP-ribosylated proteins in TK6. Immunoprecipitations using a topo I monoclonal antibody were performed on total protein extracts from TK6 cells exposed to 0 or 1 Gy 137Cs -rays. Equal amounts of immunoprecipitated proteins, determined from 35S labeling, were then electrophoresed in a 415% Trisglycine polyacrylamide gel, transferred to a PVDF membrane, and probed with an antibody to ADP-ribose polymers. Control cells receiving no irradiation are shown in lane 1 (far left). The polyADP ribosylation status of topo I monoclonal antibody precipitated proteins was monitored at various times from 1 min to 48 h following irradiation as indicated at the top of each lane.
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Fig. 4. (A) Western blot using p53 monoclonal antibody probe. Western blot analysis of topo I monoclonal antibody immunoprecipitations using p53 monoclonal antibody probe. Immunoprecipitations using a topo I monoclonal antibody were performed on total protein extracts from TK6 cells exposed to 0 or 1 Gy 137Cs -rays. Equal amounts of immunoprecipitated proteins, determined from 35S labeling, were then electrophoresed in a 415% Trisglycine polyacrylamide gel, transferred to a PVDF membrane, and probed with a p53 monoclonal antibody. Anti-mouse-HRP conjugated secondary antibody was used to visualize results. Cross reactivity between this antibody and the topo I monoclonal antibody heavy chain from the immunoprecipitation was observed as a slightly larger band than p53, and is indicated. Baculovirus expressed human p53 was used as a migration control as indicated. The recovery of p53 by immunoprecipitation with the topo I monoclonal antibody was determined in control non-irradiated cells (far right lane) and in cells receiving 1 Gy 137Cs -rays at times up to 48 h following radiation (as indicated). Densitometric analysis demonstrated that the amount of p53 protein recovered by co-immunoprecipitation was similar in irradiated and non-irradiated cells, and also does not vary with time following irradiation. (B) Induction of cellular p53 following irradiation. Extracts of total cellular protein were made 1, 6, 24 and 48 h following 1 Gy 137Cs -ray exposure as well as with no irradiation. Equal amounts of protein, as determined by Bradford assay, were then electrophoresed in a 415% Trisglycine polyacrylamide gel, transferred to a PVDF membrane, and probed with the same p53 monoclonal antibody used for the analysis shown in (A). Anti-mouseHRP conjugated secondary antibody was used to visualize the results. In contrast to the unchanging levels of p53 recovered by co-precipitation with a topo I monoclonal antibody (A), the anticipated increase in overall cellular levels of p53 following irradiation is observed.
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The p53 protein is ADP-ribosylated at low levels in non-irradiated cells, and heavily ADP ribosylated as early as 15 min post-irradiation (Figure 3
). In contrast, topo I is not detectably ADP ribosylated in non-irradiated cells. However, following irradiation ADP-ribosylation of topo I occurs even more quickly than p53, and appears to be complete within 1 min of exposure (Figure 3
). The extent of polyADP ribosylation remains stable on both p53 and topo I for at least 48 h post-irradiation (Figure 3
). The amount of p53 protein complexed with topo I, as judged by band intensity in western blot analysis, appears similar in non-irradiated and irradiated samples and also remains unchanged throughout a 48 h post-irradiation period (Figure 4A
). In contrast, overall levels of cellular p53 are increased as expected following
irradiation, as demonstrated by western blot analysis of cellular protein extracts (Figure 4B
). These data indicate that p53 is not recruited to form a complex with topo I after radiation-induced DNA damage, but the pre-existing complex is quickly and extensively modified by polyADP ribosylation.
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Discussion
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It has been reported recently that p53 and topo I form a molecular complex which enhances the catalytic activity of topo I (14,15). This investigation demonstrates that the p53 and topo I proteins within this complex are polyADP ribosylated following 1 Gy of
irradiation. ADP ribosylation of topo I and p53 would seem to account for the inhibition of topo I activity observed immediately following radiation exposure (16; Figure 2
). Additional functions may be unique to this p53topo I complex, which would also probably be regulated by polyADP ribosylation.
Ionizing-radiation-induced DNA damage response does not involve the recruitment of free p53 to complex with topo I. Rather, the amount of p53 protein complexed with topo I was found to be independent of radiation exposure (Figure 4
). However, in response to radiation-induced damage, the p53 which is tightly associated with topo I is heavily modified by polyADP ribosylation. Minutes after irradiation, ADP ribosylation of topo-associated p53 rapidly increases from low levels observed in non-irradiated cells, and persists at a high level for at least 48 h post-irradiation (Figure 3
). Since the polyADP ribosylated form of p53 does not demonstrate a detectable shift in electrophoretic mobility (Figure 3
), the data suggest that the polyADP-ribose chains which are added to p53 are short, but they must be at least two ADP-ribose units in length as required for antibody recognition (23). These results pertain only to topo I associated p53 and do not indicate that polyADP ribosylation is a generalized mechanism for regulation of all cellular p53.
It has been suggested that the physiological functions for the p53topo I complex may include roles in transcription and repair (15). However, the results presented here may be inconsistent with this hypothesis; ADP ribosylation is generally found to be an inactivating modification (24), and would be expected to inhibit the p53topo I complex from activating or facilitating transcription or repair. Preliminary results (H.M.Smith and A.J.Grosovsky, unpublished data) suggest that cellular p53 which is not complexed with topo I remains unmodified by polyADP ribosylation, even following
irradiation, and this unribosylated p53 may perform DNA-damage-induced trans-activation functions.
We alternatively hypothesize that p53 may function in the complex to regulate the induction of strand breakage by topo I activity. This hypothesis does not exclude the likely possibility that topo I activity in some cellular contexts may function independently of p53 regulation. Topo I is a nuclear enzyme which creates a transient break in one strand of DNA and, while rotating about the DNA axis, passes one strand through the break and religates the broken strand thereby relieving topological stress along the DNA helix (25). Unmodified topo I activity, including strand breakage, following DNA damage may exacerbate the effect of nearby strand breakage, leading to additional cell death or mutation. The rapid modification of the complex by polyADP ribosylation following radiation-induced DNA damage may act to diminish this possibility. The suggestion that topo I activity is regulated by p53 is consistent with reports that unmodified p53 enhances the catalytic activity of topo I (14,15), and may account for the association of p53 and topo I in non-irradiated cells observed here (Figure 4A
).
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Acknowledgments
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The authors would like to acknowledge and thank Drs Sarjeet Gill, David Eastmond and David Boothman for support and insightful discussions, Dr Xuan Liu for providing baculovirus-expressed human p53, and Dr Yung-chi Cheng of Yale University School of Medicine for generously providing topoisomerase I monoclonal antibody for use in these experiments.
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Notes
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1 To whom correspondence should be addressed Email: grosovsky{at}ucr.edu 
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Received January 19, 1999;
revised April 30, 1999;
accepted May 4, 1999.