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Originally published In Press as doi:10.1074/jbc.M002081200 on April 7, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18503-18510, June 16, 2000
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The p53 Tumor Suppressor Stimulates the Catalytic Activity of Human Topoisomerase IIalpha by Enhancing the Rate of ATP Hydrolysis*

Young Kwon, Beom Sic Shin, and In Kwon ChungDagger

From the Department of Biology, College of Science, Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea

Received for publication, March 9, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha , as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated ~2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha -mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha . Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic DNA topoisomerase II is a nuclear enzyme that modulates the topological states of DNA via transient double-strand breaks in DNA coupled with subsequent strand passage step (1-4). The mechanism of topoisomerase II activity involves DNA cleavage, strand passage, and religation, succeeded by enzyme turnover, a process requiring ATP hydrolysis (5, 6). During this cycle, the enzyme covalently binds to DNA forming an intermediate called topoisomerase II-DNA covalent cleavable complex (1-4). Topoisomerase II is essential for cell viability (7, 8) and has been implicated in many important cellular processes such as replication, transcription, recombination, and chromosomal segregation (7-10). The enzyme also functions as a major structural component of mitotic chromosome and interphase nuclear scaffolds (11, 12).

Topoisomerase II is the intracellular target for a variety of active agents currently used in the treatment of human cancers (1, 13-15). By stabilizing the covalent enzyme-associated DNA complexes, these drugs shift the DNA cleavage/religation equilibrium of the enzyme reaction toward the cleavage state. These drugs are able to convert biological intermediate in topoisomerase II activity into a lethal one ultimately leading to cell death and thus act as cellular poisons (1, 13-16). Since the cellular level of topoisomerase II in proliferating cells is higher than that of quiescent cells, these deleterious aspects of the enzyme confer selective sensitivity of proliferative tumor cells to the cytotoxic effects of these drugs (1, 13, 15). Unlike the topoisomerase II poisons, catalytic inhibitors have been reported to inhibit topoisomerase II activity without significantly stabilizing cleavable complexes. These drugs inhibit DNA topoisomerase II activity at a step prior to the formation of the cleavable complex and thus act as antagonists of DNA topoisomerase II poisons (17-20). Such catalytic inhibition may also play a role in the cytotoxicity and anticancer activity of DNA topoisomerase II poisons. Agents identified as poisons and/or catalytic inhibitors have proven to be useful in understanding the mechanisms of the topoisomerase II-catalyzed reactions in addition to their clinical use in cancer chemotherapy.

There are two closely related isoforms of human topoisomerase II that have been designated as topoisomerase IIalpha and IIbeta , which are encoded by genes located on chromosome 17 and 3, respectively (21-23). These isoforms exist as homodimers, and their amino acid sequences show homology at regions believed to be functionally significant. Topoisomerase IIalpha and IIbeta isoforms differ in important biochemical and pharmacological properties including sensitivity to topoisomerase II-targeting drugs, thermal stability, cellular localization, and cell cycle regulation (24). This suggests that their roles in the cell may not be the same. Consistent with a cell division-specific role, the expression of topoisomerase IIalpha gene is associated with the proliferation status of cells. The level of topoisomerase IIalpha protein is very low in G1 phase, increases in S phase, and is maximal in G2/M phase, whereas the level of topoisomerase IIbeta remains relatively constant throughout the cell cycle (25).

Recently, it has been shown that expression of wild-type, but not mutant, p53 is able to decrease substantially the activity of the topoisomerase IIalpha gene promoter (26, 27). Inactivation of wild-type p53 may reduce normal regulatory suppression of topoisomerase IIalpha gene expression, resulting in accelerated cell proliferation, chromosomal rearrangements, and gene amplification seen in tumor cells. These data suggest that topoisomerase IIalpha is one of the downstream targets for p53-dependent regulation of cell cycle progression. The tumor suppressor protein p53 is a nuclear phosphoprotein that functions as a negative regulator of cell proliferation (28, 29). Inactivation of p53 by a deletion or mutation in the p53 gene or by the selective interaction with certain viral or cellular proteins leads to a selective growth advantage, resulting in tumor progression and therefore is strongly correlated with human cancer (30, 31). Although wild-type p53 protein acts as a transcriptional activator of a number of downstream effector genes containing p53-binding sites (32-34), it is also capable of repressing the activity of a variety of genes lacking the p53 consensus binding site (35-37). Genotoxic lesions leading to DNA strand breaks result in a rapid increase of p53 protein levels (38). Depending on cell type, the microenvironment of a cell, and p53 levels, the induction of wild-type, but not mutant, p53 might lead to cell cycle arrest or apoptosis (28, 29). p53-induced cell cycle arrest at the G1 phase has been associated with increased expression of p21/WAF1/CIP1 gene, which encodes a potent inhibitor of cyclin-dependent kinases (39, 40). It has been also reported that expression of p53 has been associated with growth arrest in the G2/M phase of the cell cycle (41, 42).

Recent studies on mammalian cells using topoisomerase II inhibitors that do not stabilize covalent cleavable complexes have shown that topoisomerase II is required for complete chromosome condensation and for entry into mitosis (43, 44). These results also suggest that exit from G2 phase is regulated by the catenation-sensitive checkpoint, which is modulated through a system different from the DNA damage-sensitive checkpoint (43). However, since p53 inhibits topoisomerase IIalpha gene expression (27), it is conceivable that the p53-induced G2 arrest could be linked to the topoisomerase IIalpha -dependent G2 arrest. In this study, we have demonstrated that p53 stimulates the catalytic activity of topoisomerase IIalpha in vitro and in vivo. The results presented indicate that p53 modulates the catalytic activity of topoisomerase IIalpha by specifically enhancing the rate of the enzyme-mediated ATP hydrolysis. Our data suggest that the p53-mediated response of cell cycle to DNA damage may, at least in part, involve activation of topoisomerase IIalpha .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymes and Chemicals-- Purified human topoisomerase IIalpha , and kinetoplast DNA (k-DNA),1 were obtained from Topogen, Inc. Etoposide was purchased from Sigma. ICRF-193 was a generous gift from Dr. R. Ishida, Aichi Cancer Center Research Institute, Nagoya, Japan. Supercoiled plasmid DNA was purified using standard methods. Restriction enzymes and other DNA-modifying enzymes were purchased from Promega. Radioactive nucleotides were from Amersham Pharmacia Biotech.

Generation and Purification of p53 Recombinant Proteins-- Full-length human p53 cDNA was cloned into the BamHI and SalI sites of pGEX-5X-3 vector (Amersham Pharmacia Biotech). The fusion protein GST-p53 was expressed in Escherichia coli DH5alpha cells by adding 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside and purified on glutathione-Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's recommendation. GST protein itself was produced from bacteria carrying an empty pGEX-5X-3 vector. To generate polyhistidine-tagged proteins, full-length p53 cDNA was ligated into pQE32 vector (Qiagen), in-frame with a 6× polyhistidine amino-terminal tag. The recombinant proteins were expressed in E. coli M15 cells by adding 1 mM isopropyl-1-thio-beta -D-galactopyranoside and purified using nickel-agarose affinity chromatography (Qiagen) according to the manufacturer's recommendation. The final protein preparation was dialyzed against TNE buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol), and stored at -80 °C.

Topoisomerase II Catalytic Activity Assays-- Topoisomerase II activity was assayed either by the ATP-dependent decatenation of k-DNA or relaxation of negatively supercoiled pBluescript in the presence or absence of recombinant p53. The decatenation reactions were performed in a total volume of 20 µl of assay buffer (50 mM Tris-HCl, pH 7.6, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, and 30 µg/ml bovine serum albumin) containing 0.2 µg of k-DNA and the indicated amounts of topoisomerase IIalpha . After incubation for 15 min at 37 °C, the reactions were stopped by the addition of 5 µl of 5% Sarcosyl, 0.025% bromphenol blue, 25% glycerol, and the products were analyzed on a 1% agarose gel containing 0.5 µg/ml ethidium bromide. Enzyme activity is expressed in units, where 1 unit is defined as amount of the enzyme required to fully decatenate 0.2 µg of k-DNA in 15 min at 37 °C. The relaxation reactions were performed in a total volume of 20 µl of assay buffer (30 mM Tris-HCl, pH 7.6, 60 mM KCl, 8 mM MgCl2, 3 mM ATP, 15 mM 2-mercaptoethanol, and 30 µg/ml bovine serum albumin) containing 0.2 µg of negatively supercoiled pBluescript, and the indicated amounts of topoisomerase IIalpha . The reactions were incubated for 15 min at 37 °C and stopped by the addition of 0.1 volume of 10% SDS. DNA samples were then analyzed on a 1.2% native agarose gel. The amounts of DNA products were quantified by densitometric analysis using the Eagle Eye II imaging system (Stratagene).

Topoisomerase II Cleavage Assay-- Topoisomerase II cleavage reactions were performed in a total volume of 20 µl of cleavage buffer (30 mM Tris-HCl, pH 7.6, 60 mM KCl, 8 mM MgCl2, 15 mM 2-mercaptoethanol, 3 mM ATP, 30 µg/ml bovine serum albumin) containing 0.2 µg of negatively supercoiled pBluescript and 50 µM etoposide in the presence or absence of recombinant p53. The reactions were initiated by adding the indicated amounts of topoisomerase IIalpha . After incubation for 15 min at 37 °C, the cleavage complexes were trapped by addition of 2 µl of 10% SDS followed by topoisomerase II digestion with proteinase K (50 µg/ml) for 30 min at 45 °C. The reaction products were purified with phenol/chloroform extraction and electrophoresed on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide. For mapping cleavage sites, topoisomerase II cleavage reactions were performed on a whole plasmid DNA, linearized with HindIII, and run into a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide. The DNA was transferred onto nylon membrane and hybridized with 32P-labeled HindIII-PvuII fragment and autoradiographed.

Topoisomerase II Religation Assay-- Topoisomerase II religation reactions were performed in a total volume of 120 µl of cleavage buffer containing 1.2 µg of negatively supercoiled pBluescript, 12 units of purified topoisomerase IIalpha , 50 µM etoposide, and GST or GST-p53. After incubation for 15 min at 37 °C, the enzyme-mediated religation was induced by shifting the temperature from 37 to 65 °C. Aliquotes (20 µl) were withdrawn at various times and stopped by addition of SDS to 1%. Following topoisomerase II digestion with proteinase K (50 µg/ml) for 30 min at 45 °C, the products were phenol/chloroform extracted and electrophoresed on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide. The amounts of DNA products were quantified by densitometric analysis.

Hydrolysis of ATP by Topoisomerase IIalpha -- ATPase assays were performed in a total volume of 20 µl of relaxation assay buffer containing 0.3 µg of negatively supercoiled pBluescript, 2 units of topoisomerase IIalpha , and 0.1 mM [gamma -32P]ATP (2 µCi/reaction), and 0 or 300 nM polyhistidine-tagged p53. Reaction mixtures were incubated at 37 °C, and aliquots were removed at various intervals up to 30 min and quenched by the addition of an equal volume of 50 mM EDTA and 1% sodium dodecyl sulfate to each reaction. Each quenched sample (1 µl) was chromatographed in triplicate on polyethyleneimine-cellulose plates (Merck). Plates were developed by chromatography in fleshly made 400 mM NH4HCO3. The dried plates were exposed to imaging plates, and the exposed screens were scanned using a Bio-Imaging analyzer (Fuji, BAS-2500). The concentration of ATP hydrolyzed was determined by dividing the free inorganic phosphate counts by the total number of counts per lane and multiplying that fraction by the starting ATP concentration.

Immunoprecipitation of p53-- Forty units of purified topoisomerase IIalpha were mixed with 1.8 µM GST or GST-p53 in a total volume of 600 µl of assay buffer (30 mM Tris-HCl, pH 7.6, 60 mM KCl, 8 mM MgCl2, 15 mM 2-mercaptoethanol, 30 µg/ml bovine serum albumin) for 15 min on ice. Reaction mixtures were incubated for 1 h on ice in the presence of a monoclonal antibody against p53, Pab1801, or Pab240 (Santa Cruz Biotechnology), under gentle agitation. 10 µl of protein G-Sepharose CL-4B (Amersham Pharmacia Biotech) prepared in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 10 mM 2-mercaptoethanol, were added to the mixtures and further incubated on ice for 1 h under gentle agitation. Protein G-Sepharose was sedimented, washed three times, and resuspended in a 20 µl of assay buffer. The presence of topoisomerase IIalpha in the immunoprecipitated complexes was determined by immunoblot analysis. The supernatants and immunoprecipitants (20 µl each) were electrophoresed on a 7% SDS-polyacrylamide gel electrophoresis and transferred to Hybond-ECL membrane (Amersham Pharmacia Biotech). Topoisomerase IIalpha was detected with an antibody against human topoisomerase IIalpha (Topogen, Inc.).

Cell Culture and Transfection-- The human p53 expression plasmids and the empty vector used in this study have been described previously (30). All of the p53 constitutive expression constructs were produced with a cytomegalovirus promoter-enhancer expression vector. The Saos-2 human osteosarcoma cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units of penicillin/ml, and 100 µg of streptomycin/ml in a humidified incubator at 37 °C with 5% CO2. Transfections were carried out using LipofectAMINE (Life Technologies) as described previously (45). 5 × 106 Saos-2 cells were transfected with 5 µg of p53 expression plasmids. After 24 h, cells were trypsinized from the plate, washed once with ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, and centrifuged at 1,000 × g for 4 min. The cell pellet was resuspended in hypotonic buffer, and the nuclear proteins were extracted as described previously (46). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad). The proteins were stored in aliquots at -80 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of p53 on the Catalytic Activity of Human Topoisomerase IIalpha -- In order to determine the effects of p53 on the catalytic activity of topoisomerase IIalpha , we assayed enzyme activity by the decatenation of k-DNA in the presence of GST-p53 fusion protein. The experiment shown in Fig. 1A reveals the effect of increasing amounts of topoisomerase IIalpha on enzyme-catalyzed DNA decatenation in the presence or absence of GST-p53. The amounts of decatenated k-DNA products rose proportionally with the enzyme levels. Decatenation activity was ~2-3-fold higher in the presence of GST-p53 than in the absence of GST-p53 as determined by comparison of the band intensities of the decatenated k-DNA molecules in several enzyme dilutions. For further comparison of the stimulatory effect of GST-p53, k-DNA was incubated with topoisomerase IIalpha at a concentration where approximately 50% of catenated k-DNA molecules were converted to decatenated molecules in the absence of GST-p53 (Fig. 1B). A gradual decrease in amounts of trapped catenated k-DNA in the well and the appearance of decatenated k-DNA bands were observed by increasing the concentration of GST-p53. In order to determine whether the stimulatory effect of GST-p53 is attributed to p53, catenated k-DNA was mixed with topoisomerase IIalpha at a concentration selected to give a detectable amount of decatenated k-DNA. After incubation for 16 min, a full decatenation activity was detected by addition of GST-p53 (Fig. 1C), whereas only a partial increase in amount of decatenated k-DNA was observed by addition of either GST or buffer. Since inactivation of p53 by a mutation in the p53 gene results in tumor progression, we investigated the effects of mutant p53 proteins on the decatenation activity of topoisomerase IIalpha . When equal amounts of topoisomerase IIalpha were treated with mutant p53-22/23 containing a double amino acid mutation, decatenation activity was not significantly altered as compared with wild-type p53 (Fig. 1D). Similar results were obtained when mutant p53 proteins (p53-248 and p53-273) containing a single amino acid mutation were used for the decatenation reaction (data not shown). When the polyhistidine-tagged p53 produced in bacteria was used in the k-DNA decatenation reactions, the similar stimulatory effects were observed. Furthermore, the polyhistidine-tagged p53 concentrations needed for maximum topoisomerase IIalpha activation were comparable to those for GST-p53. None of the recombinant p53 proteins decatenated k-DNA in the absence of topoisomerase IIalpha , indicating that the observed effects were not due to bacterial enzymes copurifying with the p53 proteins.


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  		Fig. 1.   Effects of GST-p53 on the k-DNA decatenation activity of topoisomerase IIalpha . Decatenation reactions were performed in a total volume of 20 µl and analyzed on a 1% agarose gel containing 0.5 µg/ml ethidium bromide, as described under "Experimental Procedures." The positions of catenated k-DNA, decatenated open circular (oc), and decatenated relaxed DNA (rel) are indicated. A, titration of topoisomerase IIalpha against a fixed concentration of GST-p53. Decatena- tion reactions contained 0.2 µg of k-DNA, the indicated amounts of topoisomerase IIalpha , and 0 or 300 nM GST-p53. Lane 1 contained catenated k-DNA alone. B, titration of GST-p53 against a fixed amount of topoisomerase IIalpha . Decatenation reactions contained 0.2 µg of k-DNA, 0.5 unit of topoisomerase IIalpha , and the indicated concentrations of GST-p53. C, a large reaction mixture (100 µl) containing 1 µg of k-DNA and 300 nM GST or GST-p53 or buffer were incubated with 2.5 unit of topoisomerase IIalpha . Aliquots (20 µl) were withdrawn at various times, and analyzed on an agarose gel. D, decatenation reactions contained 0.2 µg of k-DNA, 0.5 unit of topoisomerase IIalpha , and wild-type p53 or mutant p53-22/23 as indicated.

To ensure that the stimulatory effects of p53 were not confined to the decatenation activity of topoisomerase IIalpha , DNA relaxation assays were performed. Fig. 2A shows the effects of GST-p53 on increasing amounts of topoisomerase IIalpha in a supercoiled DNA relaxation assay. When identical amounts of topoisomerase IIalpha were assayed, the fraction of relaxed DNA was greater in the presence of GST-p53 than in the absence of GST-p53. This was also manifested by a decrease in the supercoiled DNA band intensities. For example, 1 unit of topoisomerase IIalpha was only minimally active in DNA relaxation in the absence of GST-p53. However, at this enzyme concentration, input supercoiled DNA was fully relaxed in the presence of GST-p53. When increasing amounts of GST-p53 were incubated with a fixed concentration of enzyme, the relaxation activity was also stimulated in a dose-dependent manner (Fig. 2B).


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  		Fig. 2.   Effects of GST-p53 on the relaxation activity of topoisomerase IIalpha . Relaxation reactions were performed in a total volume of 20 µl and analyzed on a 1.2% native agarose gel, as described under "Experimental Procedures." The positions of supercoiled DNA (sc) and relaxed DNA (rel) are indicated. A, titration of topoisomerase IIalpha against a fixed concentration of GST-p53. Relaxation reactions contained 0.2 µg of negatively supercoiled pBluescript, the indicated amounts of topoisomerase IIalpha , and 0 or 300 nM GST-p53. Lane 1 contained supercoiled DNA alone. B, titration of GST-p53 against a fixed amount of topoisomerase IIalpha . Relaxation reactions contained 0.2 µg of negatively supercoiled pBluescript, 1 unit of topoisomerase IIalpha , and the indicated concentrations of GST-p53.

Effects of p53 on the DNA Cleavage Activity of Topoisomerase IIalpha -- Since the formation of covalent cleavable complexes between topoisomerase II and DNA is one of the crucial steps in enzyme activity, the effects of p53 on the cleavage of supercoiled DNA mediated by topoisomerase IIalpha were determined. Topoisomerase II cleavage reactions were performed in the presence of GST or GST-p53 by using the antitumor drug etoposide, which stabilizes the covalent topoisomerase II-associated DNA complexes. The resulting DNA samples were analyzed on an agarose gel containing ethidium bromide. As shown in Fig. 3A, increasing amounts of topoisomerase IIalpha in the presence of a fixed concentration (50 µM) of etoposide induced a dose-dependent formation of double-stranded breaks in DNA. Addition of GST-p53 to cleavage reactions did not alter the amount of linear DNA at all enzyme dilutions. However, relaxation activity of topoisomerase IIalpha was stimulated by GST-p53 as compared with the reactions containing GST. To examine the effects of GST-p53 on the site-specific DNA cleavage mediated by topoisomerase IIalpha , the cleavage sites were mapped in pBluescript DNA using indirect end labeling (47). Topoisomerase IIalpha induced dose-dependent DNA cleavage, as indicated by the appearance of specific DNA cleavage bands (Fig. 3B). In the presence of GST-p53, no significant increase in topoisomerase IIalpha cleavage was observed for all dilutions of enzyme tested as compared with GST or buffer containing reactions. Furthermore, no new cleavage site was observed by the inclusion of GST-p53 in the cleavage reactions. Taken together, these data indicate that the stimulation of the topoisomerase IIalpha catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes.


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  		Fig. 3.   Effects of GST-p53 on the ability of topoisomerase IIalpha to mediate DNA cleavage and religation. A, DNA cleavage reactions contained 0.2 µg of negatively supercoiled pBluescript, 50 µM etoposide, and 300 nM GST or GST-p53. The reactions were initiated by addition of the indicated amounts of topoisomerase IIalpha . The positions of open circular (oc), linear (lin), supercoiled (sc), and relaxed DNA (rel) are indicated. Lanes 1 and 8 contained supercoiled and linear DNA, respectively. B, cleavage reactions were performed on a whole pBluescript in the presence of GST or GST-p53 or buffer. The cleavage products were linearlized with HindIII and anaylzed on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide. The DNA was transferred onto nylon membrane and hybridized with radiolabeled probe as described under "Experimental Procedures." C, a large reaction mixture (120 µl) containing 50 µM etoposide and 300 nM GST or GST-p53 was incubated with 12 units of topoisomerase IIalpha . The reaction mixture was then heated to 65 °C, and aliquotes (20 µl) were withdrawn at various times after heat treatment. The reactions were terminated by addition of SDS and proteinase K as described under "Experimental Procedures."

Effects of p53 on the DNA Religation Activity of Topoisomerase IIalpha -- The formation of covalent cleavable complexes can be readily reversed by adding EDTA or salt to the reaction mixture or by elevating the temperature prior to the addition of SDS (48-52). To determine whether p53 has an effect on the topoisomerase IIalpha -mediated DNA religation activity, we performed DNA religation reactions using a heat-induced religation assay. DNA religation induced by temperature shift relies on the fact that religation activity of topoisomerase II remains less sensitive to variations in temperature than DNA cleavage activity. By shifting the temperature from 37 °C to 65 °C before termination with SDS and proteinase K, linear DNA molecules generated by topoisomerase IIalpha -mediated DNA cleavage in the presence of etoposide were reconverted to covalently closed circular DNA in a time-dependent manner (Fig. 3C). These data revealed that GST-p53 did not alter the ability of the enzyme to religate linear DNA. Similar effects were observed when the polyhistidine-tagged p53 was used (data not shown).

Effects of p53 on the Catalytic Inhibition of Topoisomerase IIalpha by ICRF-193-- Unlike the topoisomerase II poison, some antitumor drugs such as ICRF-193, merbarone, aclarubicin, quinobenoxazines, and staurosporine have been shown to inhibit topoisomerase II activity without significantly stabilizing cleavable complexes (17-20, 53). The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using ICRF-193 (Fig. 4A). In the absence of GST-p53, ICRF-193 partially inhibited decatenation activity of topoisomerase IIalpha at 1 µM concentration and strongly inhibited at higher concentrations in a dose-dependent manner under the conditions employed in this assay. However, GST-p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193 as compared with the reactions without GST-p53. For example, in the presence of GST-p53, catenated k-DNA was almost completely converted to decatenated products at 1 µM concentration of ICRF-193. Similar results were obtained when DNA relaxation activity was assayed (Fig. 4B). These results suggest that p53 may enhance the catalytic activity of topoisomerase IIalpha through the mechanism by which ICRF-193 inhibits enzyme activity.


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  		Fig. 4.   Effects of GST-p53 on the catalytic inhibition of topoisomerase IIalpha by ICRF-193. A, decatenation reactions contained 0.2 µg of k-DNA, 1.5 unit of topoisomerase IIalpha , the indicated concentrations of ICRF-193, and 0 or 300 nM GST-p53. The positions of catenated k-DNA, decatenated open circular (oc), and decatenated relaxed DNA (rel) are indicated. Lane 1 contained catenated k-DNA alone. B, relaxation reactions contained 0.2 µg of negatively supercoiled pBluescript, 1.5 unit of topoisomerase IIalpha , and the indicated concentrations of ICRF-193, and 0 or 300 nM GST-p53. The positions of supercoiled DNA (sc) and relaxed DNA (rel) are indicated.

Stimulation of Topoisomerase IIalpha -catalyzed ATP Hydrolysis by p53-- ICRF-193 has been shown to inhibit the catalytic activity of topoisomerase II by stabilizing the closed-clamp form of the enzyme and preventing its conversion to the open-clamp form (54). ICRF-193 was found to inhibit the ATPase activity of topoisomerase II. Since p53 reduced the enzyme catalytic inhibition by ICRF-193, we examined the effects of p53 on the ATPase activity of topoisomerase IIalpha . In the ATP hydrolysis assays, polyhistidine-tagged p53 was used because unwanted bacterial ATPase activity was copurified with GST-p53 on glutathione-Sepharose affinity chromatography. As shown by data in Fig. 5, polyhistidine-tagged p53 stimulated the ATPase activity of topoisomerase IIalpha ~2-fold as compared with the reactions without p53. This increase in the rate of ATP hydrolysis is comparable to the stimulation of the enzyme's overall catalytic activity by GST-p53 (see Fig. 1). Reactions containing only polyhistidine-tagged p53 without topoisomerase IIalpha gave near base-line levels of ATPase activity. These results strongly suggest that p53 modulates the catalytic activity of topoisomerase IIalpha by specifically enhancing the ATPase activity of the enzyme.


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  		Fig. 5.   Stimulation of topoisomerase IIalpha catalyzed ATP hydrolysis by p53. Reactions contained 0.3 µg of negatively supercoiled pBluescript, 0 or 2 units of topoisomerase IIalpha , and 0 or 300 nM polyhistidine-tagged p53. The data are plotted as picomole of ATP hydrolyzed versus time. , with enzyme, with p53; black-square, with enzyme, no p53; black-down-triangle  without enzyme, with p53; black-triangle, without enzyme, no p53. Results represent an average from three independent experiments.

Physical Interaction between p53 and Topoisomerase IIalpha -- Previously, it was reported that topoisomerase II can form a molecular complex with wild-type p53 without a double-stranded DNA intermediary (55). However, it is unclear whether other proteins could mediate the binding between topoisomerase II and p53. To address this issue directly, purified topoisomerase IIalpha was incubated with GST or GST-p53 in relaxation assay buffer, and the reaction mixtures were immunoprecipitated with p53-specific monoclonal antibody. The resulting proteins in supernatants (S) and immunoprecipitants (IP) were resolved on a 7% SDS-polyacrylamide gel electrophoresis and were subjected to Western blot using an affinity-purified antibody specific for topoisomerase IIalpha . As shown in Fig. 6A, most of the topoisomerase IIalpha was found in the supernatant fraction, but some of the topoisomerase IIalpha was detected in the immunoprecipitants recovered by a p53-specific monoclonal antibody Pab1801. However, topoisomerase IIalpha was absent in the immunoprecipitants recovered from reaction mixtures containing topoisomerase IIalpha and GST protein. When another p53-specific monoclonal antibody Pab240 was used for immunoprecipitation, similar results were obtained. Supernatant and immunoprecipitant fractions were also tested for the presence of k-DNA decatenation activity (Fig. 6B). The results clearly indicate that decatenation activity was present in the p53-immunoprecipitants but absent in the immunoprecipitants recovered from the reaction mixtures containing topoisomerase IIalpha and GST protein. These results suggest that p53 binds directly to topoisomerase IIalpha in the absence of DNA.


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  		Fig. 6.   Binding of GST-p53 to topoisomerase IIalpha . Reaction mixtures containing purified topoisomerase IIalpha and GST or GST-p53 were precipitated with a monoclonal antibody against p53, Pab1801, or Pab240 and protein G-Sepharose, as described under "Experimental Procedures." A, the supernatants (S) and immunoprecipitants (IP) (20 µl each) were separated on a 7% SDS-polyacrylamide gel electrophoresis and immunostained with an antibody against topoisomerase IIalpha . B, the supernatant and immunoprecipitatants (10 µl each) were assayed for k-DNA decatenation activity. The positions of catenated k-DNA, decatenated open circular (oc), and decatenated relaxed DNA (rel) are indicated.

Expression of Wild-type p53 in Saos-2 Cells Stimulates Topoisomerase II Activity-- All the results described above are obtained with p53 produced in bacteria. It is well known that there is no post-translational modification of p53 in bacteria. Moreover, the structural conformation of p53 produced in bacteria might differ from that of the protein synthesized in mammalian cells. In order to investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we transiently expressed wild-type p53 and mutant p53-22/23 in Saos-2 human osteosarcoma cells which lack functional p53 and compared the topoisomerase II activity in nuclear extracts by decatenation of k-DNA (Fig. 7A). As indicated by amounts of trapped catenated k-DNA in the well, decatenation activity was ~2-fold higher in nuclear extracts of cells expressing wild-type p53 (lanes 2-4) as compared with extracts from cells expressing mutant p53-22/23 (lanes 5-7) or mock-transfected cells (lanes 8-10). Immunoblots of nuclear extracts with topoisomerase IIalpha -specific antibody showed that a level of topoisomerase IIalpha protein was slightly decreased in Saos-2 cells transfected with wild-type p53 as compared with cells transfected with mutant p53-22/23 or mock-transfected cells (Fig. 7B, upper). This experiment was consistent with previous findings that wild-type p53 functions as a transcriptional repressor of topoisomerase IIalpha (27). Finally, immunoblots of protein from transfected cell lysates were performed to confirm that wild-type and mutant p53 proteins were being produced in the cells transfected with the p53 expression vectors. As shown in Fig. 7B, bottom, similar levels of wild-type and mutant p53 proteins were detected. These results clearly indicate that wild-type, but not mutant, p53 stimulates the catalytic activity of topoisomerase II in vivo. Stimulation of topoisomerase II activity by wild-type p53 in vivo was underestimated since topoisomerase IIalpha protein level was slightly decreased in cells transfected with wild-type p53.


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  		Fig. 7.   Expression of wild-type p53 in Saos-2 cells stimulates topoisomerase II activity. A, topoisomerase II activity was measured by decatenation of k-DNA in nuclear extracts of cells expressing wild-type p53 (lanes 2-4) or mutant p53-22/23 (lanes 5-7) or of mock-transfected cells (lanes 8-10). Shown is a representative decatenation reaction of three separate experiments. Lane 1 contained no nuclear extract. The positions of catenated k-DNA, decatenated open circular (oc) and decatenated relaxed DNA (rel) are indicated. B, immunoblots of nuclear extracts from Saos-2 cells transfected with wild-type p53 or mutant p53-22/23 or from mock-transfected cells showing topoisomerase IIalpha (upper) and p53 (bottom) expression. The immunoblots represent three separate experiments. mock-tran, mock-transfected.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic DNA topoisomerase II is the essential enzyme to remove the catenations formed between the DNA molecules of sister chromatids during replication and hence permit their faithful segregation during mitosis (43, 44). Here, we report that p53 has a pronounced stimulatory effect of DNA catalysis by topoisomerase IIalpha as measured by decatenation of k-DNA and by relaxation of supercoiled DNA. The results of the ATP hydrolysis assays revealed that p53 stimulates the catalytic activity of topoisomerase IIalpha by specifically enhancing the ATPase activity of the enzyme. We also show that p53 physically associates with topoisomerase IIalpha without a double-stranded DNA intermediary in vitro. Transient transfection experiments with Saos-2 cells showed that topoisomerase II activity was stimulated by ectopically expressed wild-type p53 but not by mutant p53-22/23. In this report, we propose that p53 could be a catalytic regulator of topoisomerase IIalpha .

A number of mechanistic studies have shown that the catalytic cycle of topoisomerase II can be divided into several discrete reaction steps (56). The physiological regulation of DNA topology by topoisomerase II requires a coordinated function of the all steps of the enzymes catalytic cycle. To determine the mechanism by which p53 activates topoisomerase IIalpha , the effects of p53 on the DNA cleavage/religation reaction steps of the catalytic cycle were examined. The results showed that GST-p53 had no effect on the formation of covalent cleavable complexes in the presence of etoposide. Cleavage sites induced by topoisomerase IIalpha remained unaffected by GST-p53. Furthermore, the religation step, during which the double-stranded break is resealed, was not altered by GST-p53. These results provide the evidence that p53 does not enhance the rate of enzyme catalysis at cleavage/religation steps. Previous studies showed that p53 also physically associates with topoisomerase I and enhances its DNA relaxation activity (57, 58). Unlike topoisomerase IIalpha , both cleavage and religation steps in the catalytic cycle of topoisomerase I were stimulated by p53.

A specific catalytic inhibitor of topoisomerase II, ICRF-193, blocks the final step of the catalytic cycle, ATP hydrolysis (54). This drug traps topoisomerase II on the DNA in its closed clamp form and prevents both enzyme release and regeneration. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. In conjunction with the results of the ATPase assays, these findings strongly suggest that ATP hydrolysis is the control step for the modulation of topoisomerase IIalpha catalytic activity by p53. Although topoisomerase II has been known to be an essential enzyme for proliferation of eukaryotic cells and play important roles in many aspects of DNA processes, little is understood concerning the mechanism by which its catalytic activity is regulated in eukaryotic cells except for a few examples. The catalytic activity of topoisomerase II is stimulated ~2-3-fold following phosphorylation by either casein kinase II or protein kinase C (56, 59). Like p53 action on topoisomerase II activity, both protein kinases enhance enzyme activity by specifically stimulating the ability of topoisomerase II to hydrolyze its ATP cofactor. More recently, it was reported that casein kinase II increases the activity of topoisomerase IIalpha by stabilization against thermal inactivation of the enzyme (60). This activation of topoisomerase IIalpha is apparently independent of any phosphorylation. However, the functional significance of this regulation in vivo is unclear. Topoisomerase IIalpha appeared to physically interact with Rb protein, and wild-type, but not mutant, Rb inhibited topoisomerase II activity (61).

The critical question that remains to be answered is how p53 modulates the catalytic activity of topoisomerase IIalpha . The most obvious mechanism is via direct association of p53 with topoisomerase IIalpha . Our data showing the co-immunoprecipitation of purified topoisomerase IIalpha with p53 and the presence of k-DNA decatenation activity in the immunoprecipitated complexes are consistent with this suggestion. Furthermore, it is unlikely that other factors could mediate the binding between topoisomerase IIalpha and p53. Yuwen and co-workers (55) reported that topoisomerase II can form a complex with wild-type p53. Interaction between p53 and topoisomerase II was confirmed by co-immunoprecipitation of p53 protein by a monoclonal antibody to topoisomerase II in p53-overexpressed HeLa cell lysates. This binding was shown to occur in the absence of DNA. These data, together with our results presented in this study, strongly suggest that the two proteins may form molecular complexes in vivo. Recently, Cowell et al. (62) reported that both topoisomerase IIalpha and IIbeta interact with p53 in vivo and in vitro, and the regulatory COOH-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with topoisomerase II (62). These data suggest that mutant p53 proteins (p53-22/23, p53-248, and p53-273) used in this study can physically associate with topoisomerase IIalpha , resulting in similar levels of stimulatory effect in vitro on the decatenation activity with wild-type p53. However, in vivo transient transfection experiments revealed that decatenation activity was higher in nuclear extracts of cells expressing wild-type p53 as compared with extracts from cells expressing mutant p53-22/23 or mock-transfected cells. These results suggest that in vivo stimulation of topoisomerase II catalytic activity by p53 may additionally require leucine and tryptophan residues at amino acids 22 and 23.

Since decatenation function of topoisomerase IIalpha is required at mitosis for high chromosome condensation and chromosome segregation, the level and activity of the enzyme are tightly controlled during cell cycle. mRNA levels of topoisomerase IIalpha are virtually absent in the G1 phase and accumulate to high levels during late S phase (63). The protein levels are maximal in the G2/M phase. It would appear that topoisomerase IIalpha is activated only when its decatenation function is required over the cell cycle. Our data propose a new role for p53 as a regulator of topoisomerase IIalpha . Modulation of the catalytic activity of topoisomerase IIalpha by p53 might contribute to tight regulation of cell cycle progression when DNA has been damaged. Increased expression of wild-type p53 in response to DNA damage would arrest cells in G1 phase by stimulating the p21 gene expression, to allow time for damaged DNA to be repaired before continuation of cell cycle (64). A significant G2 arrest function has also been reported for p53 (41, 42). p53 can lead to arrest of cell growth at a G2/M phase of the cell cycle in the absence of DNA-damaging treatments. In the experiments using a topoisomerase II inhibitor, ICRF-193, that does not stabilize cleavable complexes, Downes and co-workers (43) showed that topoisomerase II-dependent G2 checkpoint is distinct from the G2-damage checkpoint. This topoisomerase II-dependent G2 checkpoint could be sensitive to catenation state of DNA or the decatenation activity of topoisomerase II. Thus, the main function of the G2 phase is to allow adequate decatenation of replicated DNA. A previous report has indicated that expression of wild-type p53 severely inhibits topoisomerase IIalpha gene promoter activity (27). Inactivation of p53 may reduce normal regulatory repression of topoisomerase IIalpha gene expression and contribute to abortive G2 cycle checkpoints. These results also suggest that the p53-induced G2 arrest could be linked to the topoisomerase II-dependent G2 cycle checkpoint through transcriptional repression of topoisomerase IIalpha by p53. Our data provide strong evidence that p53 stimulates the catalytic activity of topoisomerase IIalpha by specifically enhancing the ATPase activity. We therefore propose that wild-type p53 contributes to the proper regulation of topoisomerase IIalpha levels required in the G2 checkpoint by at least two modes: via repression of topoisomerase IIalpha gene expression (27) and/or via stimulation of topoisomerase IIalpha catalytic activity by the formation of molecular complexes. Future experiments will be required to understand the functional significance of p53 regulation in topoisomerase IIalpha activity in vivo.

    ACKNOWLEDGEMENTS

We thank Drs. J. H. Lee, H. S. Koo, and J. M. Suh for critical comments on the manuscript.

    FOOTNOTES

* This work was supported in part by Grant 98-MM-02-01-A-01 from the Molecular Medicine Research Group Program, MOST, and Grant 95K2-0401-00-01-5 from the Korea Science and Engineering Foundation through the Bioproducts Research Center at Yonsei University.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.

Dagger To whom correspondence should be addressed: Dept. of Biology, College of Science, Yonsei University, 134 Shinchon-dong, Seoul 120-749, Korea. Tel.: 822-361-2660; Fax: 822-312-5657; E-mail: topoviro@yonsei.ac.kr.

Published, JBC Papers in Press, April 7, 2000, DOI 10.1074/jbc.M002081200

    ABBREVIATIONS

The abbreviations used are: k-DNA, kinetoplast DNA; GST, glutathione S-transferase.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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