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Originally published In Press as doi:10.1074/jbc.M405042200 on October 19, 2004

J. Biol. Chem., Vol. 279, Issue 53, 55618-55625, December 31, 2004
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TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II*

Hans U. Barthelmes{ddagger}, Michael Habermeyer§, Morten O. Christensen{ddagger}, Christian Mielke{ddagger}, Heidrun Interthal¶, Jeffrey J. Pouliot||, Fritz Boege{ddagger}**, and Doris Marko§

From the {ddagger}Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Medical School, Moorenstrasse 5, D-40225 Düsseldorf, Germany, the §Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger Strasse 52, 67663 Kaiserslautern, Germany, the Department of Microbiology, University of Washington School of Medicine, Seattle, Washington 98195, and the ||Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892-4034

Received for publication, May 6, 2004 , and in revised form, October 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3'-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA·topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosyl DNA phosphodiesterase 1 (TDP1)1 is an enzyme capable of hydrolyzing phosphodiester bonds between tyrosine and the 3'-phosphate of DNA (1, 2), which are typically generated in a transient manner by DNA topoisomerase I (topo I) (3). In keeping with this, yeast deletion mutations of TDP1 are deficient in the repair of DNA damage induced by camptothecin, a drug that stabilizes the transient topo I·DNA intermediate (2, 4-6). More precisely, TDP1 has been characterized in these studies as a non-exclusive effector upstream of Rad52 that removes structurally modified topo I adducts (7, 8) as well as oxidative adducts (9) from the 3'-phosphate of a DNA break prior to homologous recombination repair. In mammals, TDP1 is (in addition or instead?) involved in an XRCC-dependent single-stranded DNA repair pathway also directed at topo I·DNA adducts (10-12). Despite all the evidence of yeast deletion studies implying TDP1 in DNA repair, a familial disease caused by a mutation in the active site of the human ortholog of the enzyme exhibits a phenotype not at all typical for inadequate DNA repair, namely a slow onset of neuronal degeneration (13). This unexpected finding has prompted speculations that at least in mammals TDP1 could serve a much broader scope of functions, some of which may not even depend on catalytic activity. To further clarify the importance of TDP1 for mammalian DNA repair, we have here overexpressed GFP chimera of human TDP1 or its inactive mutant TDP1H263A (14) in human cells and studied the effects on the outcome of various types of DNA damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Cell Culture—Construction and characterization of cell lines supporting stable expression of GFP and GFP-topo I have been described previously (15, 16). To enable simultaneous visualization of topo I and topo II{alpha}, the GFP moiety in pMC-topo II{alpha}-EGFP (15) and pMC-EGFP-topo I (16) was replaced by CFP or YFP, respectively. Using these two precursor plasmids, the tricistronic expression plasmid pMC-topo II{alpha}-ECFP/EYFP-topo I was generated as described by Mielke et al. (17). In this plasmid, topo II{alpha}-ECFP is located in the first, EYFP-topo I in the second, and the selection marker in the third cistron. An internal ribosome entry site from poliovirus mediates the translational initiation of the second and third cistron. The published cDNA sequence of human TDP1 (14) differs from the corresponding sequence in the human genome (FLJ11090 National Center for Biotechnology Information, accession number NM_018319 [GenBank] ) in several positions resulting in four amino acid exchanges (D322N, M328T, P389A, and F548L). Constructs bearing all four exchanges were found to be cytotoxic, whereas constructs corrected at positions 328, 389, and 548 to coding the amino acids delineated by FLJ11090could be overexpressed. Accordingly, all experiments of this study were done with a version of full-length human TDP1, which differs from the protein encoded by FLJ11090only by the amino acid exchange D322N. This sequence was inserted into pMC-EGFPP-N (16) giving rise to GFP-TDP1, where EGFP is fused to the N terminus of full-length human TDP1. The catalytically inactive mutant GFP-TDP1H263A was made by site-directed mutagenesis PCR of the GFP-TDP1 plasmid using primer pairs encoding the His to Ala point mutation at position 263 described by Interthal et al. (14). All constructs were stably expressed in the human embryonal kidney cell line 293 (German Collection of Microorganisms and Cell Culture, Braun-schweig, Germany) as described previously (16, 18, 19). Briefly, cells grown in Dulbecco's modified Eagle's medium supplemented with Glutamax-I (Invitrogen, Karlsruhe, Germany) were transfected using Lipofectamine (Invitrogen). Stable transgenic cell lines were selected after 2 days with 0.35 µg ml-1 puromycin or with a combination of 0.35 µg ml-1 puromycin and 50 nM camptothecin (for details, see Fig. 1A). Cells were then maintained under selection. Cells with a high expression of GFP-TDP1 were enriched by fluorescence-activated cell sorting using a FACSCalibur (BD Biosciences).



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  		FIG. 1.
Constitutive overexpression of GFP-TDP1 in 293 cells. A, cellular green fluorescence was measured by flow cytometry in untransfected 293 cells (top), cells stably transfected with GFP-TDP1 and selected with 0.35 µg ml-1 puromycin (middle top), a subfraction generated thereof by fluorescence activated cell sorting (middle bottom), and cells stably transfected with GFP-TDP1 and selected with a combination of 0.35 µg ml-1 puromycin and 50 nM camptothecin (CPT) (bottom). B, immunoblotting directed against GFP (top) or TDP1 (bottom). Lanes 1-4 were loaded with whole cell lysates equivalent to 5 x 105 cells that was made of untransfected 293 cells (lane 1), cell lines stably transfected with and selected for high expression of GFP-TDP1 (lane 2) or GFP-TDP1H263A (lane 3), and cells stably transfected with GFP alone (lane 4). For a quantitative evaluation, lanes 5-7 were loaded with 10, 30, and 90 ng, respectively, of recombinant GFP protein. The arrow points at the band representing endogenous TDP1.

 
Immunoblotting—Whole cell lysates were prepared by adding to cells suspended in phosphate-buffered saline an equal volume of 2-fold lysis buffer (25 mM Tris-HCl, pH 6.8, 10% SDS, 8 M urea, 20% glycerol, 0.04% bromphenol blue, 10 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM phenylmethylsulfonyl fluoride, 20 µg ml-1 aprotinin, 10 µg ml-1 pepstatin A). Material equivalent to 5 x 105 cells was then applied to each slot of an SDS gel. After electrophoresis, proteins were electroblotted onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MD). The membranes were subsequently blocked with phosphate-buffered saline, containing 2% hydrolyzed casein, 2% bovine serum albumin, and 0.05% Tween 20, and then incubated for 1 h with the primary antibodies diluted with the same buffer. Topo I, topo II{alpha}, and topo II{beta} were stained with rabbit peptide antibodies against C-terminal residues of the human enzymes (20). TDP1 was stained with a rabbit antiserum antibody obtained after immunization with full-length bacterially expressed human TDP1. GFP, YFP, and CFP were stained with mouse monoclonal antibodies (Clontech, Heidelberg, Germany). After washing, the filters were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies diluted with phosphate-buffered saline containing 2% bovine serum albumin and 0.1% Tween 20. Following extensive washing with the same buffer, labeled protein bands were finally visualized by chemoluminescence (ECL Plus, Amersham Biosciences, Freiburg, Germany). For immunoband depletion, cells were first cultured with camptothecin (1-20 µM, 20 min to 1 h, as detailed in the legends to Figs. 5 and 6) or etoposide (10-50 µM, 20 min to 1 h, as detailed in the legends to Figs. 5 and 6), and the same concentration of drug was also added to the lysis buffer.



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  		FIG. 5.
Expression, activity, and drug susceptibility of topo I and topo II in 293 cells overexpressing TDP1, as compared with 293 cells not transfected or expressing GFP not fused to TDP1. A, expression of topo I and topo II. Whole cell lysates of 293 cells not transfected (lanes 1, 4, and 7), overexpressing GFP-TDP1 (lanes 2, 5, and 8), or expressing GFP alone (lanes 3, 6, and 9) were subjected to Western blotting. The equivalent of 5 x 105 cells was loaded to each lane. Tree identical blots were then probed with antibodies against topo I (left section), topo II{alpha} (middle section), and topo II{beta} (right section), respectively. B, topo I activity was determined by relaxation of supercoiled pUC18 plasmid DNA (400 ng) in the presence of 0.1 mM orthovanadate, which completely inhibits topo II activity (see C, lanes 2, 7, and 12). Nuclear extracts equivalent to 106 cells not transfected (left), overexpressing GFP-TDP1 (middle), or expressing GFP alone (right) were reacted with 400 ng of pUC18 DNA at 37 °C for the time intervals indicated. The outmost lane on the left shows DNA not exposed to nuclear extract. DNA electrophoresis was performed in the absence of ethidium bromide to separate supercoiled (superc.) and relaxed plasmid forms. C, topo II activity was determined by decatenation of kinetoplast DNA (400 ng). Nuclear extract equivalent to 106 cells not transfected (lanes 2-6), overexpressing GFP-TDP1 (lanes 7-11), or expressing GFP alone (lanes 12-16) was added, and the reactions were carried out for 60 min at 37 °C. Wedges indicate 1:3, 1:9, and 1:18 dilutions of extracts. Lanes 2, 7, and 12 (+) show reactions of undiluted nuclear extracts in the presence of 10 µM orthovanadate. Lane 1 shows substrate not reacted with extract. DNA electrophoresis was performed in the presence of ethidium bromide to separate catenated DNA network (CN) and free DNA circles (FC). D, immunoband depletion. 293 cells overexpressing GFP-TDP1 were treated for 30 min with camptothecin (CPT) (left) or VP-16 (middle and right) at concentrations ranging from 0.1 to 10 µM (wedges). Subsequently, the equivalent of 5 x 105 cells was subjected to Western blotting, and blots were probed with antibodies against topo I (left), topo II{alpha} (middle), or topo II{beta} (right). The first lane on the left of each section shows cells kept without drugs.

 



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  		FIG. 6.
Impact of VP-16 on topo I. A, immunoband depletion. Cells expressing GFP-TDP1 (left), GFP-TDP1H263A (middle), and untransfected (untransf.) cells (right) were treated for 1 h with 10 µM VP-16 (lanes 2, 5, and 8), 1 µM camptothecin (CPT) (lanes 3, 6, and 9), or left untreated (lanes 1, 4, and 7). Subsequently, the equivalent of 5 x 105 cells was subjected to Western blotting, and blots were probed with antibodies against topo I (bottom) or topo II{alpha} (top). B, immunoblotting and immunoband depletion of 293 cells coexpressing YFP-topo I and topo II{alpha}-CFP. Cells coexpressing YFP-topo I and topo II{alpha}-CFP were treated for 20 min with 50 µM VP-16 (lane 4), 10 µM camptothecin (CPT) (lane 5) or were kept without drug (lanes 1-3). Western blots were probed with antibodies against topo I (left), topo II{alpha} (middle), or YFP/CFP (right). C, impact of VP-16 on localization of YFP-topo I and topo II{alpha}-CFP. Cells coexpressing YFP-topo I and topo II{alpha}-CFP were cultured under the fluorescence microscope and imaged by phase contrast (top), yellow fluorescence (middle), or cyan fluorescence (bottom) before (left) and after treatment for 1 h with 10 µM VP-16 (middle), and after subsequent additional exposure for 1 min to 10 µM camptothecin (CPT) (right).

 
DNA Relaxation, Decatenation, and Cleavage Assays—DNA relaxation, decatenation, and cleavage activities were assessed at 37 °C in a final volume of 40 µl of assay buffer (10 mM Tris-HCl, pH 7.9, 3 mM MgCl2, 0.03% bovine serum albumin, 0.3 mM EDTA, 0.5 mM dithiothreitol, and 50 mM KCl). The plasmid pUC18 or catenated kinetoplast DNA from Crithidia fasciculata (400 ng per assay) served as substrates. For the assessment of topo II activity, the buffer was supplemented with 1 mM ATP. Nuclear extracts were prepared as described previously (19, 20) and added to the assays in various amounts and for various time periods as specified in the legend to Fig. 5. Reactions were terminated with 1% SDS, 0.1 mg ml-1 proteinase K. Reaction products were analyzed by submarine agarose gel electrophoresis. It was carried out in the presence of ethidium bromide (2 µg ml-1), when cleaved (open circular or linear) and uncleaved (closed circular) plasmid forms were to be separated. It was carried out in the absence of ethidium bromide when relaxed and supercoiled plasmid forms were to be separated.

Life Cell Imaging—Images of epifluorescence were acquired with a Zeiss Axiovert 100 inverted microscope, whereas confocal imaging and measurements of fluorescecence recovery after photobleaching (FRAP) were done with a Zeiss LSM 510 inverted confocal laser-scanning microscope. A heated cell chamber (Bioptechs Inc., Butler, PA) and a heated 63x/1.4 NA oil-immersion objective were used with both microscopes to allow for culturing of the cells under the microscope precisely at 37 °C. For FRAP measurements, fluorescent images of a single optical section were taken at 1.6-s time intervals before (n = 5) and after bleaching of a circular area at 20 milliwatts nominal laser power with three iterations. Imaging scans were acquired with the laser power attenuated to 0.1-1% of the bleaching intensity. For a quantitative analysis of FRAP, fluorescence intensities of the bleached region and the entire cell nucleus were measured at each time point. Data were corrected for extracellular background intensity and for the overall loss in total intensity as a result of the bleach pulse itself and of the imaging scans. The relative intensity of the bleached area Irel was calculated according to Phair and Misteli (21).

Analysis of DNA Damage by Single Cell Gel Electrophoresis (Comet Assay)—Alkaline single cell gel electrophoresis was performed according to (22) with slight modifications. 106 cells ml-1 were incubated for 1 h in serum-containing medium in the presence or absence of the test compounds. Thereafter, aliquots (70 µl = 70,000 cells) were centrifuged (5 min, 200 x g), and the resulting cell pellet was resuspended in 65 µl of low melting agarose and distributed onto a frosted glass microscope slide precoated with a layer of normal melting agarose. The slides were covered with a glass slide and kept at 4 °C for 10 min to allow for solidification of the agarose. After removing the cover glass, slides were immersed for 1 h at 4 °C in lysis solution (89 ml of lysis stock solution, 1 ml of Triton X-100, and 10 ml of Me2SO). Subsequently, DNA was allowed to unwind (pH 13.5, 20 min, 4 °C), and then horizontal gel electrophoresis was performed at 4 °C for 20 min (25 V, 300 mA). Thereafter, the slides were washed three times with 0.4 M Tris-HCl, pH 7.5, and stained with ethidium bromide (50 µl, 10 µg ml-1). Fluorescence microscopy was performed with a Zeiss Axioskop 20 (excitation: {lambda} = 546 ± 12 nm; emission: {lambda} ≥ 590 nm). Slides were subjected to computer aided image analysis (Comet Assay II System, Perceptive Instruments, Suffolk, Great Britain), scoring 50 images per slide randomly picked from each electrophoresis. For each drug concentration, two slides were processed and analyzed independently. The results were parameterized with respect to tail intensity (intensity of the DNA in the comet tail calculated as percentage of overall DNA intensity of the same cell). Such quantitative data were always derived from four independent sets of experiments and from the evaluation of 100 individual cells (50/slide) for each drug concentration and each experiment. For graphical representation, the local distribution of staining intensity was coded by false colors using in decreasing order yellow, dark blue, petrol, black, light blue, and red.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive Expression of GFP-TDP1 or the Active Site Mutant GFP-TDP1H263A Is Tolerated at High Levels—Stable transfection with the chimeric cDNA of GFP-TDP1 gave rise to a large number of transgenic cell clones with GFP fluorescence of the cells ranging from 2-1000-fold above background (Fig. 1A, middle top). From these we enriched cells with a GFP fluorescence of 100-1000-fold above background (Fig. 1A, middle bottom). These cells could easily be maintained overextended periods of time without loss of fluorescence. It had a similar morphology and doubling time as the parental cell line, indicating that GFP-TDP1 was not toxic and did not interfere with cell proliferation. It should be noted that a population of transgenic cell clones with much higher expression levels of GFP-TDP1 could be selected by a combination of puromycin and camptothecin (Fig. 1A, bottom), suggesting that TDP1 overexpression counteracts topo I-mediated cell killing, which supports recent data obtained with yeast TDP1 (23). A cell population expressing the catalytically inactive mutant (14) GFP-TDP1H263A established in a similar fashion did not profit from co-selection with puromycin and camptothecin (data not shown) suggesting that the mutant was indeed inactive in the cell. To control expression levels and integrity of GFP-TDP1 and GFP-TDP1H263A, Western blots of whole cell lysates were probed with antibodies against TDP1 or GFP. When probed with GFP antibodies (Fig. 1B, top), cells transfected with GFP-TDP1 (lane 2) or GFP-TDP1H263A (lane 3) showed a single band of expected size, whereas such a band was absent in untransfected cells (lane 1), and a much smaller band became apparent in cells expressing GFP alone (lane 4). Expression levels of GFP-TDP1 and GFP-TDP1H263A were similar (compare lanes 2 and 3), and by comparison with pure recombinant GFP (lanes 5-7), they were calculated in the range of 106 molecules per cell, whereas endogenous TDP1 expression is estimated in the range of 104 molecules per cell. When blots were probed with TDP1 antibodies (Fig. 1B, bottom), GFP-TDP1 and GFP-TDP1H263A became visible as bands of 100 kDa, which were at least 100-fold stronger than endogenous TDP1, visible as a faint band of about 70 kDa (compare upper and lower band in lanes 2 and 3). Summing up the data presented in Fig. 1, we ascertained that the chimeric transgenes were not rearranged in the cells, that green fluorescence of the transgenic cell lines was unambiguously due to GFP-TDP1 or GFP-TDP1H263A, respectively, and that constitutive expression of the biofluorescent TDP1 varieties exceeded that of the endogenous enzyme by a factor of at least 100.

GFP-TDP1 and GFP-TDP1H263A Have a Similar Nuclear Pattern That Differs Markedly from Topo I—Fig. 2A shows cells cultured under the fluorescence microscope and imaged at interphase by phase contrast (left) or GFP fluorescence (right). Upon comparing cells expressing GFP-topo I (top), GFP-TDP1 (middle top), or GFP-TDP1H263A (middle bottom) to a cell expressing non-fused GFP (bottom), it becomes apparent that these proteins are exclusively localized in the cell nucleus, whereas GFP is localized in the nucleus and the cytosol. In contrast to GFP, GFP-TDP1, and the active site mutant GFP-TDP1H263A are uniformly distributed in the entire nuclear space and not excluded from the nucleoli. The similarity of the pattern suggests that the point mutation in GFP-TDP1H263A does not disrupt general properties such as folding or cellular targeting of the protein. Localization of both TDP1 varieties differs markedly from GFP-topo I, which is mostly nucleolar. Fig. 2B shows time-lapsed images of cells expressing GFP-TDP1 (left) or GFP-topo I (right) as they go through mitosis. Again, GFP-TDP1 differs from GFP-topo I, in as much as it does not associate with the chromatin until late telophase, whereas GFP-topo I remains DNA-bound during the entire mitotic cycle. Thus, GFP-topo I behaves like a DNA-associated protein, whereas GFP-TDP1 and GFP-TDP1H263A (data not shown) behave like diffusible nuclear passengers.



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  		FIG. 2.
In vivo localization of GFP-TDP1, GFP-TDP1H263A and GFP-topoI, and GFP. A, 293 cells cultured under a fluorescence microscope were imaged at interphase by phase contrast (left) and green fluorescence (right). Cells expressed GFP-topo I (top), GFP-TDP1 (middle-top), GFP-TDP1H263A (middle bottom), or GFP alone (bottom). B, cells cultured under the microscope were monitored while going through mitosis. Cells expressing GFP-topo I and GFP-TDP1 are shown in double columns on the right and left, respectively. Images in each double column were obtained by phase contrast (left) and green fluorescence (right) and show (from top to bottom) the same cell as it goes from metaphase to early G1 phase.

 
The Impact of Camptothecin on Localization and Mobility of GFP-TDP1—The drug camptothecin binds and stabilizes covalent catalytic DNA-intermediates of topo I (24) and attenuates the mobility of GFP-linked topo I in the cell, which can be measured by FRAP. Recently, such experiments have revealed that camptothecin targets topo I preferentially in the nucleoplasm, thus shifting the nuclear distribution equilibrium of the enzyme away from the nucleoli in a very rapid manner (16). Since DNA-linked topo I becomes eventually a substrate for TDP1 (1, 2, 7, 8), we expected GFP-TDP1 to follow topo I to the nucleoplasm and to also become less mobile when processing its immobilized substrate. However, the data shown in Fig. 3 lend only partial support to this hypothesis. Upon exposure to camptothecin, GFP-topo I (Fig. 3A, left) and GFP-TDP1 (Fig. 3A, middle) were both depleted from the nucleoli, and FRAP kinetics of both proteins obtained in the nucleoplasm became slower (Fig. 3B, left, compare open and closed triangles; Fig. 3B, right, compare open circles with closed boxes). However, nucleolar depletion of GFP-TDP1 was not as rapid as that of GFP-topo I itself (Fig. 3A, compare left and middle at 1' and 5'). Moreover, the inactive mutant GFP-TDP1H263A was also cleared from nucleoli upon prolonged exposure to camptothecin (data not shown). These findings suggest that nucleolar clearance of TDP1 upon prolonged exposure to camptothecin does not stem from a comigration with topo I to the nucleoplasm but rather from the general disintegration and redistribution of nucleolar structures upon prolonged inhibition of rDNA transcription by camptothecin (25). This notion is also supported by the observation that exposure to the topo II-poison VP-16 did not induce nucleolar depletion of GFP-TDP1 (Fig. 3A, right). It should also be noted that FRAP kinetics of GFP-TDP1 were 10 times faster than those of GFP-topo I (Fig. 3B, compare open triangles in left diagram with open circles in right diagram), indicating TDP1 to be more freely diffusible than topo I.



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  		FIG. 3.
Impact of camptothecin (CPT) on localization and mobility of GFP-TDP1 and GFP-topo I. A, cells expressing GFP-topo I (double column on the left) or GFP-TDP1 (double columns in the middle and on the right) were cultured under a confocal laser scanning microscope. Each double column shows from top to bottom the same set of cells before (0') and at various time points after addition of 20 µM camptothecin (left, middle) or 10 µM VP-16 (right) to the medium. Times given in the column on the left apply to all three columns. At each time point, images were recorded by transmitted light (left) and confocal scanning of green fluorescence in midplane (right). B, FRAP curves determined in the nucleoplasm of 293 cells expressing GFP-topo I (left) or GFP-TDP1 (right), which either were exposed for 20 min to 20 µM camptothecin (closed triangles, closed squares) or 10 µM VP-16 (closed triangles) or were kept without drugs (open symbols). Drawn out lines represent all data points, some of which are highlighted by symbols. The actual bleach spots are indicated in corresponding confocal images of GFP fluorescence of the cells shown at the top of the curve plots.

 
Enhanced TDP1 Expression Decreases DNA Damage by Poisons of Topo I and Topo II—In summary, the data in Figs. 2 and 3 suggest that TDP1 and topo I are unlikely to be in constant association because they have a different mobility and different localization patterns in the living cell nucleus. However, upon exposure to camptothecin they become both less mobile in the nucleoplasm (Fig. 3B). This could reflect recruitment of GFP-TDP1 to multiprotein complexes dedicated to the repair of DNA-linked topo I. Since the cells express GFP-TDP1 at least 100-fold in excess of endogenous TDP1 (see Fig. 1), their capacity of repairing such lesions should be enhanced if TDP1 was a limiting factor of the respective repair pathway. To test this hypothesis, we measured the damage inflicted by camptothecin on the genome of cells overexpressing GFP-TDP1 in comparison with cells expressing similar levels of the active site mutant GFP-TDP1H263A (see Fig. 1B) or unfused GFP. This was done by a standardized procedure based on the quantification of DNA strand breaks via the size and intensity of the comet-shaped DNA tail emerging upon single cell gel electrophoresis from a cell harboring a damaged genome (22). In the absence of DNA-damaging drugs DNA comets were not apparent in any of the three cell lines (Fig. 4, left), indicating that overexpression of GFP, GFP-TDP1, or GFP-TDP1H263A per se did not increase the fragility of the genome. Camptothecin-induced DNA tails were significantly smaller in cells overexpressing GFP-TDP1 than in control cells or in cells expressing GFP-TDP1H263A (Fig. 4, middle left). Thus, the capacity for repairing topo I·DNA adducts seemed to correlate with the expression of catalytically active TDP1, which fits the previous observation (Fig. 1A, bottom) that cells supporting a high expression of GFP-TDP1 can be selected with camptothecin. Taken together, these findings suggest TDP1 activity to be a limiting factor of the repair pathway. To our utter astonishment, a similar effect was seen upon exposure to the topo II poison etoposide (VP-16) initially meant as a negative control (Fig. 4, middle right). In cells expressing GFP alone, VP-16 created very large DNA tails, and DNA staining intensity in the nucleus decreased by 2 orders of intensity, indicating a more or less complete fragmentation of the genome. In contrast, in cells overexpressing GFP-TDP1 DNA staining intensity was hardly affected and much smaller DNA tails emerged from the cells (Fig. 4, middle right column, compare pictures at the top and in the middle). This protective effect was not obtained with the active site mutant GFP-TDP1H263A (Fig. 4, middle right column, compare pictures at the bottom and in the middle), indicating that it depended on the catalytic activity of the enzyme. Prima facie, this unexpected result seemed to suggest that overexpression of active TDP1 confers some sort of a general resistance to DNA-damaging agents. However, this hypothesis had to be discarded because treatment of the cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a chemical compound that damages DNA in a manner independent of topo I or topo II, produced DNA comets of similar size in all three cell lines (Fig. 4, right). These findings were corroborated by a quantitative analysis of several hundred DNA comets (Fig. 4, diagrams at the bottom), which confirmed that overexpression of GFP-TDP1 significantly (p < 0.01) reduced DNA damage by camptothecin and VP-16 (by 40 ± 10 and 42 ± 12%, respectively), whereas it did not affect DNA damage by MNNG. The effect was clearly dependent on the overexpression of active GFP-TDP1 and not seen with the inactive mutant GFP-TDP1H263A (Fig. 4, bottom, compare white and gray bars).



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  		FIG. 4.
DNA damage by camptothecin (CPT), VP-16, and MNNG. 293 cells were treated with drugs and subjected to single cell gel electrophoresis, and the DNA tails emerging from the cells were visualized by ethidium bromide staining and fluorescence microscopy. The top half gives a graphical representation of typical results obtained with cells expressing GFP not fused to TDP1 (top) as compared with cells overexpressing GFP-TDP1 (middle) and 293 cells overexpressing GFP-TDP1H263A (bottom). Cells were cultured either without drug (left) or in the presence of 100 µM camptothecin (middle left), 10 µM VP-16 (middle right), or 10 µM MNNG (right) for 1 h. The local distribution of staining intensity was color-coded in decreasing order yellow, dark blue, petrol, black, light blue, and red. The bottom half shows the corresponding quantitative assessment of DNA damage by computer aided integration of the DNA tails with respect to staining intensity and area. DNA damage is expressed as the tail intensity of the DNA in the comet in percentage of overall DNA intensity of the same cell. Mean values (boxes) and standard deviations (error bars) were in each case calculated from measurements of 300 individual cells, randomly picked from three independent assays. Data of cells overexpressing unfused GFP, GFP-TDP1, or GFP-TDP1H263A are shown by black, white, or gray boxes, respectively. Data were subjected to Wilcoxon's signed rank test. Values significantly (p < 0.01) different from each other are marked by brackets.

 
Overexpression of GFP-TDP1 Does Not Affect Endogenous Topo I and Topo II—The only plausible interpretation of the data shown in Fig. 4 seemed to be that overexpression of active TDP1 increases the repair of DNA adducts of both types of topoisomerases, provided that their endogenous expression, activity, and DNA-damaging potential were not affected by the genetic modification. To exclude this, we carried out a series of control experiments summarized in Fig. 5. By probing Western blots with antibodies against topo I, topo II{alpha}, and topo II{beta}, we ascertained that the protein levels of all three enzymes were the same in cells overexpressing GFP-TDP1 (Fig. 5A, middle) as in cells not transfected (Fig. 5A, left), or expressing GFP not fused to TDP1 (Fig. 5A, right). Moreover, we ascertained that nuclear extracts of the three cell lines contained a similar catalytic activity of topo I (Fig. 5B) or topo II (Fig. 5C). Thus, we excluded alterations of expression or activity of the drug targets as a possible cause of the decrease in DNA damage. Finally, we ascertained by immunoband depletion that drug sensitivity of topo I, topo II{alpha}, and topo II{beta} inside the cells was not compromised by the overexpression of GFP-TDP1. The assay makes use of the fact that topoisomerases will be trapped in large DNA·protein complexes upon addition of topo poisons, provided that they are capable of interacting with DNA and sensitive to the drugs. Thus, the active and drug-sensitive fraction of the enzymes will be prevented from entering an SDS-polyacrylamide gel and consequently will not be detectable in subsequent immunoblotting. Fig. 5D attests to the fact that upon exposure of cells overexpressing GFP-TDP1 to the appropriate drugs, topo I, topo II{alpha}, and topo II{beta} were depleted in the normal fashion. Thus, drug sensitivity of the enzymes in vivo was not altered by the enhanced expression of TDP1, and the decrease in DNA damage apparent in the comet assay could be unambiguously assigned to an increase in repair capacity.

TDP1-mediated Decrease in DNA Damage by Poisons of Topo II Does Not Involve Topo I—It has been clearly shown that TDP1 removes adducts from the 3'-end of DNA only (1), whereas topo II attaches itself to the 5'-end. Thus, our finding of TDP1 counteracting DNA damage by poisons of topo II can hardly be explained by a direct action of TDP1 on DNA-linked topo II. A more feasible explanation would be that etoposide triggers a series of events that eventually lead to an enhanced DNA-linkage of topo I, which then is acted upon by TDP1. To test this hypothesis, we performed another series of control experiments summarized in Fig. 6. By immunoband depletion, we ascertained that upon prolonged exposure to etoposide topo I was not trapped in DNA·protein complexes (Fig. 6A). In cells overexpressing GFP-TDP1 (left), GFP-TDP1H263A (middle), and untransfected cells (right), exposure to 10 µM etoposide for 1 h (i.e. the same conditions as used for the comet assays shown in Fig. 4) did not diminish the topo I band, as compared with cells kept without drugs (Fig. 6A, bottom, compare lanes 2, 5, and 8 with 1, 4, and 7), whereas topo II was notably depleted (i.e. trapped on DNA) (Fig. 6A, top, compare lanes 2, 5, and 8 with 1, 4, and 7). As a positive control, exposure to 1 µM camptothecin for 1 h resulted in a clear depletion of topo I (Fig. 6A, bottom, lanes 3, 6, and 9). To corroborate these control data with a more sensitive assay, we coexpressed biofluorescent topo II{alpha} (linked to CFP) and topo I (linked to YFP) in the same cell and studied the impact of etoposide on the localization of the two proteins (Fig. 6, B and C). A brief characterization of the cell clone used for this experiment is shown in Fig. 6B. Both fusion proteins were not overexpressed as shown by probing immunoblots with antibodies against topo I, topo II{alpha}, and YFP/CFP (Fig. 6B, lanes 1-3). Activity of the chimeric enzymes in the cells was demonstrated by immunoband depletion of the fused proteins upon exposure to poisons of topo II or topo I (Fig. 6B, lanes 4 and 5). Fig. 6C shows time-lapsed images of the same cell before (left column) and 1 h after (middle column) addition of 10 µM etoposide. The drug induced redistribution of topo II from the nucleoli to nucleoplasmic foci (bottom row) as expected (15). It can clearly be seen that topo I present in the same cell (middle row) was not at all affected by the topo II poison whereas it exhibited a rapid relocation upon subsequent addition of camptothecin (right column). In summary these data rule out that DNA cleavage by topo I could play a role in bringing about DNA damage by topo II poisons and thus cannot explain why TDP1 overexpression counteracts the DNA-damaging action of such drugs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our report provides for the first time information about the localization and mobility of human TDP1 in living human cells. Not unexpectedly, the enzyme imposes as a nuclear protein. It appears to be more mobile and diffusible than topo I, and it has a different distribution in the nucleus. During interphase, TDP1 is equally distributed in the entire nuclear space, whereas topo I is mainly a nucleolar protein. At the early stages of mitosis TDP1 is not bound to DNA, whereas topo I is. Upon exposure to camptothecin, the mobility of both proteins is attenuated in the nucleoplasm, but nucleolar clearance of TDP1 is much slower than that of topo I excluding a comigration of the two proteins. These findings conform to a concept derived from structural (8) and biochemical analysis (7) as well as genetic manipulation of yeast (2, 4) all suggesting that TDP1 only acts on topo I covalently linked to DNA and, even then, not at the earliest occurrence of such complexes but rather after they have been converted to DNA double-strand breaks (4) and subjected to proteolytic processing (7) and other structural alterations (8). It should be noted, however, that the cell lines used in this study are not an ideal tool for gaining a more detailed insight into this mechanism because they were created with the intention of studying a molar excess of TDP1 over topo I.

The most relevant finding of this study regards the interplay of TDP1 and topoisomerase poisons. We show that cells having a very high expression level of TDP1 gain resistance to camptothecin and can be selected by the drug (Fig. 1A). We also demonstrate that an enhanced expression of active TDP1 reduces DNA damage inflicted by the topo I poison camptothecin, whereas overexpression of the inactive mutant TDP1H263A does not (Fig. 4). These finding suggest that the activity of TDP1 is a limiting factor of the DNA repair pathway(s), in which it is involved, a notion also supported by observations made with overexpression of yeast TDP1 in two different rodent cell lines (23) and in recent complementation studies, showing that an inactive mutant of yeast TDP1 is unable to rescue the camptothecin-sensitive phenotype of a TDP1 deletion strain (26). This makes sense in the light of the molar excess of topo I over TDP1 normally prevailing in a cell. However, our data suggest that TDP1 is not only a decisive factor in the known pathways dedicated to repairing topo I·DNA complexes (27) but also in those repairing topo II·DNA complexes, since it decreases DNA damage by the topo II poison VP-16 to a similar extent as that inflicted by camptothecin. This finding was entirely unexpected because TDP1 has clearly been shown to remove adducts from the 3'-end of DNA only (1), whereas topo II attaches itself to the 5'-end. Therefore it seems unlikely that TDP1 acts directly on DNA·topo II intermediates. Based on the data shown in Fig. 6, we can also exclude a down stream involvement of topo I in the DNA-damaging effect of topo II poisons that otherwise would explain why TDP1 overexpression ameliorates the impact of such drugs on the nuclear genome. Consequently, we have to consider an even more indirect role of TDP1 in the repair of topo II-mediated damage. One explanation would be that TDP1 might affect the formation or targeting of a complex specifically dedicated to repairing topo II breaks. Such a scenario is not difficult to envision, since it is known that yeast TDP1 acts in a RAD52-dependent repair pathway (4-6), that enzymes of the RAD52 epistasis group are involved in the repair of DNA breaks caused by topo II poisons in yeast (28) and mammals (29), and that during the repair of DNA double-strand breaks the same enzymes form complexes (30) that agglomerate to large repair centers capable of coprocessing multiple DNA lesions (31, 32). In this scenario, overexpression of TDP1 could promote the formation of active repair complexes and more or larger repair centers, which in summary would allow a more rapid repair of both topo I- and topo II-mediated damage by homologous recombination. This scenario is the more credible, since in mammals the default mechanism for the repair of topo II-mediated DNA breaks seems to be non-homologous endjoining (33). Thus, it could well be that overexpression of TDP1 increases the capacity of a mammalian cell for the repair of topo II-mediated DNA strand breaks by a supportive up-regulation of the RAD52 pathway. Yet, it is strange that this is not supported by the inactive TDP1 mutant, which should be just as capable of forming repair complexes as the active enzyme. To explain the requirement of activity, several hypotheses can be formed: (i) in the living cell, accessory proteins could render TDP1 capable of cleaving 5'-tyrosyls or of catalyzing some alternate reactions, leading indirectly to the removal of a 5'-phosphotyrosyl. (ii) The repair of topo II·DNA adducts could involve intermediate modifications of free DNA 3'-phosphates that need to be resolved by TDP1. (iii) TDP1 could have a second function as a signaling enzyme that catalyzes a reaction on an unknown diffusible molecule that launches a signal cascade leading to the up-regulation of other repair enzymes or of pro-apoptotic proteins.

The testing of these hypotheses goes beyond the scope of this study, but the data obtained so far give a clear indication that the function of TDP1 in DNA repair of mammals is somewhat different from that in yeast and more complex. In yeast, deletion mutations of TDP1 do not enhance the sensitivity to topo II poisons (4), whereas in mammalian cells overexpression of active TDP1 significantly reduces the DNA damage inflicted by such drugs, and we have excluded the possibility that this could be due to an altered drug sensitivity of topo II{alpha} or topo II{beta} itself or to a generalized resistance of the cells to any type of DNA damage. Thus, we have to accept expressional induction of TDP1 as a potent mechanism by which mammalian cells can counteract DNA damage caused by poisoning of topo I and topo II. We find this particularly disquieting because we observed here also that TDP1 expression can increase by up to 1000-fold without interfering with cell proliferation. Therefore, it seems entirely feasible for a human cancer cell to induce TDP1 expression when challenged with topo I and/or topo II poisons, and it might be quite meaningful to study expression levels of the enzyme in specimen of human tumors subsequent to chemotherapy with such drugs. It might even be worthwhile to consider TDP1 as a therapeutic cotarget of topo I and topo II poisons.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (Grants Bo 910/3-1, Bo 910/4-1, Bo 910/5-1, GRK 639, GRK 1033, and HA 1434/13-1); the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie, Germany (Grant 0310938); and the Deutsche Krebshilfe e.V. (Grant 10-1822-Bo I). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed. Tel.: 49-211-81-18290; Fax: 49-211-81-18021; E-mail: Boege{at}med.uni-duesseldorf.de.

1 The abbreviations used are: TDP1, tyrosyl DNA phosphodiesterase 1; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; topo I, DNA topoisomerase I; topo II, DNA topoisomerase II; VP-16, etoposide; FRAP, fluorescecence recovery after photobleaching. Back


   ACKNOWLEDGMENTS
 
We are grateful to Jörg Hacker and Hilde Merkert, Research Center for Infectious Diseases, University of Würzburg, Germany for generously providing access to a confocal laser-scanning microscope.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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