Disturbance of DNA damage recognition after UV-irradiation by nickel(II)

Carcinogenesis vol.19 no.4 pp.617–621, 1998

Disturbance of DNA damage recognition after UV-irradiation by nickel(II) and cadmium(II) in mammalian cells

Maike Hartmann and Andrea Hartwig1 Department of Biology and Chemistry, University of Bremen, D-28334 Bremen, Germany 1To

whom correspondence should be addressed Email: [email protected]

Nickel(II) and cadmium(II) have been shown previously to inhibit the incision step of nucleotide excision repair. By applying a gel-mobility-shift assay and HeLa nuclear extracts the effect of both metals on the damage recognition step of the repair process was investigated. Two proteins of 34 and 40 kDa were identified that bind with high affinity to a UV-irradiated synthetic oligonucleotide. When applying nuclear extracts from HeLa cells treated with 50 µM nickel(II) and higher, there was a dose-dependent decrease in protein binding; this effect was largely reversible by the addition of magnesium(II) to the binding reaction. In the case of cadmium(II), a dose-dependent inhibition of DNA–protein interactions was detected at 0.5 µM and higher, which was almost completely reversible by the addition of zinc(II). Therefore, compounds of both metals disturb DNA–protein interactions essential for the initiation of nucleotide excision repair most likely by the displacement of essential metal ions. Introduction Compounds of nickel and cadmium are known human carcinogens. However, their genotoxic potentials in mammalian cells are rather weak and/or restricted to cytotoxic concentrations (1,2). In contrast, compounds of both metals enhance the mutation frequency after UV-irradiation (3,4) as well as the number of mutations and cell transformations in combination with benzo[a]pyrene (5). Furthermore, both nickel(II) and cadmium(II) have been shown to interfere with the repair of UV-induced DNA damage (6–8), whereby the incision step of the repair process is affected most severely by low concentrations of both metals (7,9). However, the underlying mechanisms are not clear. UV-induced DNA lesions are removed by the nucleotide excision repair pathway, where according to the current knowledge at least 15–18 proteins are involved in the incision reaction (10). One important prerequisite for the initiation of repair events is the recognition of the DNA lesions. Even though this process is not fully understood, some proteins involved in DNA damage recognition have been identified during the last years. With respect to UV-induced DNA damage, a protein defect in patients suffering from the DNA repair disorder Xeroderma Pigmentosum complementation group A (XPA*) is absolutely required for lesion recognition; its binding to damaged DNA *Abbreviations: XPA, Xeroderma Pigmentosum complementation group A; RPA, replication protein A; UV-DRP, UV-damage recognition protein; NBT, nitroblue tetrazolium salt; X-phosphate, 5-bromo-4-chloro-3-indoyl phosphate; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. © Oxford University Press

is greatly enhanced by the replication protein A (RPA). Additionally, even though not essential for DNA repair in vitro, XPE is thought to serve as an accessory factor in damage recognition; this protein may be identical to a UV-damage recognition protein (UV-DRP) of 125 kDa purified from HeLa cells by several groups (for recent review see 11). Finally, other proteins exerting high affinities for UV-irradiated DNA have been described recently, which are not related to known Xeroderma pigmentosum complementation groups, and which may have yet unknown functions in the cellular response to DNA damage (e.g., 12,13). With respect to nickel(II) and cadmium(II), they have been shown recently to prevent the specific binding of a protein to UV-irradiated DNA when added at concentrations of 1 mM to a HeLa cell free extract (14). In a previous preliminary study we demonstrated that DNA–protein interactions between HeLa nuclear proteins and UV-irradiated DNA were interrupted when applying nickel-treated cells (9). The aim of the present study was to investigate whether nickel(II) and cadmium(II) disturb the recognition of DNA damage after treatment of intact HeLa cells with low, biologically relevant concentrations of both metals, and to define possible mechanisms of inhibition. We prepared HeLa nuclear cell-free extracts and applied the gel-mobility-shift assay to study DNA–protein interactions involved in DNA damage recognition in the absence and presence of nickel(II) and cadmium(II). We identified two proteins which bind with high affinity to a UV-damaged synthetic oligonucleotide; this binding was substantially diminished by nickel(II) and cadmium(II). The inhibition of DNA– protein interactions by nickel(II) and cadmium(II) were reversible by the addition of magnesium(II) and zinc(II), respectively. Materials and methods Cell culture and treatment with metal compounds HeLa cells were grown as monolayers in minimal essential medium (MEM, Gibco, Karlsruhe, Germany), containing 10% fetal bovine serum (Gibco), 100 units penicillin/ml and 100 µg streptomycin/ml. The cultures were incubated at 37°C with 5% CO2 in air and 100% humidification. Logarithmically growing cells were treated with NiCl236 H2O or CdCl23H2O (Merck, Darmstadt, Germany) as described for the respective experiments. Gel-mobility-shift assay Nuclear protein extracts were prepared from HeLa cells essentially as described by Schreiber et al. (15). The total protein concentration was determined according to Bradford (16) with bovine serum albumin as a standard. To detect damage-specific DNA–protein interactions, a digoxygenin-endlabeled synthetic oligonucleotide (48 bp; MWG Biotech, Ebersberg, Germany) with the following sequence was applied together with its complementary strand: 59-CCAGGAATTGGAGCCTTTTTTTTTTTTCTCAGCAAGGGCGATGCTATC-39. For the binding reaction, 2 µl protein extract (1 µg/µl) were pretreated with 2 µl of the unlabeled and unirradiated oligonucleotide (600 fmol/µl) in a gel shift buffer (final concentration: 13.3 mM Hepes, 9.6% glycerol, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 89 µg/ml bovine serum albumin, 0.4 mM dithiothreitol, pH 7.9) for 10 min at room temperature. Afterwards, 1 µl poly(dIdC) (0.5 µg/µl) as well as 2 µl of the digoxygenin-labeled oligonucleotide (120 fmol/µl), either unirradiated or irradiated with 18 kJ/m2 UVC by applying a General Electric germicidal lamp, were added for another 35 min. The binding mixture was loaded on a 6% polyacrylamide gel (37.5 acrylamide:1.0

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Fig. 1. Specific binding of proteins to UV-irradiated DNA. HeLa nuclear cell-free extracts were incubated with a digoxygenin-endlabeled oligonucleotide either unirradiated or irradiated with 18 kJ/m2 UV and DNA–protein interactions were analysed by a gel-mobility-shift assay as described in Materials and methods. Poly(dI-dC) and an excess of unlabeled, unirradiated oligonucleotide of the same sequence (specific competitor) were added where indicated. The experiment has been independently conducted three times; shown is one representative experiment.

bisacrylamide; 45 mM Tris–HCl, 45 mM boric acid, 1 mM EDTA, pH 8.0) and electrophoresis was conducted at 110 V for 2.5 h. Southern blot was done in a semi-dry electro-blotting apparature applying a positively charged nylon membrane (Hybond-N1, Amersham, Braunschweig, Germany). The detection of the digoxygenin-labeled oligonucleotide was performed colorimetrically by the alkaline phosphatase conjugated to an anti-digoxygenin antibody using nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (X-phosphate) as substrates (Boehringer, Mannheim, Germany). Molecular weight analysis of the DNA binding proteins To determine the molecular weight of the damage-specific DNA binding proteins, we applied anti-digoxygenin antibodies attached to magnetic DNA affinity beads (Boehringer, Mannheim, Germany) based on a procedure described in detail by Gabrielsen et al. (17). Fifty pmol of the digoxygeninlabeled oligonucleotide, either unirradiated or irradiated with 18 kJ/m2 UV, were incubated with 250 µg magnetic DNA affinity beads loaded with Antidigoxygenin antibodies for 30 min at room temperature in TEN100 (10 mM Tris–HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5). Magnetic separation of attached oligonucleotides from free oligonucleotides was achieved by a strong neodynium–iron–boron permanent magnet. In order to isolate the UV damagespecific DNA binding proteins, 200 µg HeLa nuclear extracts were incubated with the attached oligonucleotides as well as with poly(dI-dC) and a 5-fold excess of the specific DNA competitor as described above for the gel-mobilityshift assay. Afterwards, the proteins bound specifically to the UV-irradiated oligonucleotide were eluted with 6 M guanidine-HCl, pH 1.5. The samples were desalted by using centricon-10 microconcentrators (Amicon, Witten, Germany) and precipitated with acetone. Finally, the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and silver-stained as described by Heukeshoven and Dernick (18).

Results To identify proteins which bind with high affinity to UVirradiated DNA we established a non-radioactive gel-mobilityshift assay by applying a 48 bp digoxygenin-labeled oligonucleotide, either unirradiated or irradiated with 18 kJ/m2 UV, and HeLa nuclear cell free extracts (Figure 1). In the absence of competitor DNA, no difference is observed between proteins binding to both oligonucleotides (lanes 1 and 2), indicating unspecific DNA binding. However, when poly(dI-dC) was added as unspecific competitor DNA, a preferential binding 618

Fig. 2. Effect of UV-irradiated competitor DNA on the binding of the UVdamaged-DNA binding proteins. HeLa nuclear cell-free extracts were incubated with a digoxygenin-endlabeled oligonucleotide either unirradiated or irradiated with 18 kJ/m2 UV and DNA–protein interactions were analysed by a gel-mobility-shift assay as described in Materials and methods. In addition to poly(dI-dC) and a 5-fold excess of the unlabeled, unirradiated oligonucleotide, different amounts of a UV-irradiated, unlabeled oligonucleotide of the same sequence were added where indicated. The experiment has been independently conducted three times; shown is one representative experiment.

to the UV-irradiated oligonucleotide was observed (lanes 3 and 4), even though protein binding still occurs to the unirradiated oligonucleotide. To eliminate these interactions we preincubated the nuclear extracts for 10 min with a 5- or 10-fold excess of the unirradiated, unlabeled oligonucleotide of the same sequence as a specific competitor; under these conditions, we still observe the protein binding to UV-irradiated DNA, whereas the unspecific binding to the non-damaged oligonucleotide is diminished completely (lanes 5 to 8). The binding reaction was further investigated by adding an excess of UV-irradiated, unlabeled oligonucleotide. As shown in Figure 2, the protein binding was suppressed completely with increasing amounts of the competitor, adding further evidence that the protein(s) interact(s) specifically with UV-irradiated DNA. To analyse the molecular weight of the protein(s) binding to the UV-irradiated oligonucleotide we applied a magnetic DNA affinity purification procedure. The principle of this method is based on the specific binding of nuclear proteins to the UV-irradiated digoxygenin-labeled oligonucleotide attached to magnetic beads loaded with anti-digoxygenin antibodies. After coupling of the oligonucleotides to the magnetic beads as described in Materials and methods, HeLa nuclear extracts prepared exactly as described for the gelmobility-shift assay were incubated with the magnetic beads, thereby allowing the DNA damage recognition protein(s) to bind. After magnetic separation of the bound and non-bound components of the nuclear extracts and the elution of the specific binding proteins with 6 M guanindine-HCl, an SDS– PAGE was conducted (Figure 3). While the stronger bands appearing at the positions of 56 and 70 kDa are derived from the antibodies detached from the magnetic beads during the elution procedure (Figure 3A), two specific bands were detected, corresponding to two proteins with molecular weights of 34 and 40 kDa (Figure 3B). In a next approach we investigated whether the specific binding of the proteins to the UV-irradiated oligonucleotide is disrupted by nickel(II) and/or cadmium(II). According to incubation protocols established in previous studies, HeLa cells were preincubated with NiCl2 for 24 h at concentrations between 50 and 600 µM, or with cadmium(II) for 2 h at concentrations between 0.5 and 5 µM, which are not cytotoxic

Nickel(II) and cadmium(II) UV-irradiation in mammalian cells

Fig. 3. Molecular weight analysis of the DNA damage recognition proteins. HeLa nuclear cell-free extracts were incubated with a UV-irradiated digoxygenin-labeled oligonucleotide and the UV-damage binding proteins were isolated by a magnetic affinity purification procedure as described in Materials and methods. Molecular weights were determined by SDS–PAGE followed by silver staining. (A) SDS–PAGE in the absence of HeLa nuclear extracts; (B) SDS–PAGE after the addition of HeLa nuclear extracts. The experiments have been independently conducted three times; shown is one representative experiment.

in terms of colony forming ability (7,19). At the end of these treatments, cell extracts were prepared as described in Materials and methods. As shown in Figure 4, a diminished protein binding is observed at 50 µM NiCl2 and higher, resulting in 62% residual binding at 50 µM and 14% at 600 µM; lower concentrations of 5 and 25 µM showed no effect (data not shown). One possible mechanism of binding inhibition consists in the competition of nickel ions with divalent magnesium ions, and the inhibition of the incision frequency after UVirradiation has been shown to be partly reversible by the addition of excess magnesium(II) (7). Therefore, we investigated whether magnesium(II) is able to reverse the inhibitory effect of nickel in our test system. When the concentration of magnesium(II) in the gel-shift buffer was enhanced from 5 mM (standard reaction) to 10 mM, the protein binding of nuclear extracts from nickel-treated cells is restored almost completely; only at 600 µM nickel(II) a reduction of the binding capacity to 27% of the control is still observed. In the case of cadmium(II), a dose-dependent reduction of DNA– protein interactions is seen at all concentrations applied; at 0.5 µM, the protein-binding to the UV-irradiated oligonucleotide is reduced to 58% and at 5 µM to 11% compared to untreated control cells (Figure 5). In contrast to the results obtained with nickel(II), an enhanced magnesium-concentration in the gel-shift buffer had no impact on the observed inhibition (data not shown); however, the addition of 100 µM zinc(II) to the binding reaction led to a largely restored binding behaviour at all cadmium-concentrations applied.

Fig. 4. Effect of nickel(II) on the specific binding of proteins to UVirradiated DNA and protective interaction of magnesium(II). HeLa cells were incubated for 24 h with the respective concentrations of NiCl2 before the preparation of the nuclear protein extracts and the conduction of the gelmobility-shift assay. The binding reaction was carried out in the presence of poly(dI-dC) and a 5-fold excess of unlabeled, unirradiated oligonucleotide of the same sequence (specific competitor). (A) Gel-shift reaction in the presence of 5 mM MgCl2; (B) gel-shift reaction in the presence of 10 mM MgCl2; (C) quantification of the data shown in (A) and (B) after densitometrical evaluation; shown are mean values of three determinations 6 SD.

Discussion In the present study we demonstrated that nickel(II) and cadmium(II) disturb DNA–protein interactions involved in DNA damage recognition during nucleotide excision repair. Two proteins were identified which bind with high affinity to UV-irradiated DNA; by applying a magnetic separation procedure, the molecular weights of the proteins were determined to be ~34 kDa and ~40 kDa, respectively. The identity of the proteins is not known at present. Regarding the 40 kDa protein, it could correspond to the DNA damage recognition protein XPA, which appears at the 40 kDa position in SDS gel electrophoresis and which is absolutely required for the initiation of repair events (20). However, the addition of an antibody specific for the XPA protein did not yield a ‘supershift’ in the gel mobility shift assay (data not shown), indicating that the binding factor derived in the present study is distinct from XPA. The existence of additional proteins involved in the cellular response to UV-induced DNA damage beyond known DNA repair enzymes has also been described by Wakasugi et al. (12). They identified novel and not yet characterized proteins from HeLa nuclear extracts which exert high affinities to UV-irradiated DNA, including one 40 kDa protein. Nevertheless, further studies are required to character619

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Fig. 5. Effect of cadmium(II) on the specific binding of proteins to UVirradiated DNA and protective interaction of zinc(II). HeLa cells were incubated for 2 h with the respective concentrations of CdCl2 before the preparation of the nuclear protein extracts and the conduction of the gelmobility-shift assay. The binding reaction was carried out in the presence of poly(dI-dC) and a 5-fold excess of unlabeled, unirradiated oligonucleotide of the same sequence (specific competitor). (A) Gel-shift reaction without ZnCl2; (B) gel-shift reaction in the presence of 100 µM ZnCl2; (C) quantification of the data shown in (A) and (B) after densitometrical evaluation; shown are mean values of three determinations 6 SD.

ize the UV-damaged-DNA binding proteins obtained in the present study. Concerning the effects of nickel(II) and cadmium(II) on the interactions between nuclear proteins and damaged DNA, the results provide new insights into the mechanisms leading to the inhibition of nucleotide excision repair. They demonstrate that compounds of both metals act on the very first step of the repair process, namely the recognition of the DNA damage induced by UVC-irradiation. These DNA–protein interactions are disturbed after treatment of the HeLa cells with low, noncytotoxic concentrations of the respective metals. In the case of nickel(II), 50 µM decrease the protein binding to 62% compared to the control; this is ~15-fold below the concentration, where the colony forming ability starts to decline under these treatment conditions (7). Similarily, the incubation of the cells with as little as 0.5 µM cadmium(II) reduces the protein binding to 58%, while the colony forming ability is affected only at concentrations higher than 20 µM (19). In principle, several reasons could account for the observed disturbance of DNA–protein interactions, including a reduced transcription and/or translation of the respective proteins, the 620

distortion of DNA structures or the inactivation of the proteins due to metal binding. Our results support the last alternative. Since the nuclear protein fractions are derived from metaltreated cells, changes in DNA structures in these cells are not expected to have any impact on the outcome of the experiments. Furthermore, the nearly complete restoration of the DNA– protein interactions after the addition of excess magnesium(II) or zinc(II), respectively, to the nuclear extracts excludes a reduction in the level of the specific proteins in the metaltreated cells. Therefore, the experiments presented in this study point towards a protein inactivation by nickel(II) and cadmium(II), where the competition with essential metal ions seems to play a predominant role. With respect to cadmium, the observed inhibition was almost completely reversible by the addition of excess zinc(II) to the gel-shift reaction. This supports results described by Nocentini (8), who showed that the cadmium-induced repair inhibition after UV-irradiation was diminished when the cells were simultaneously incubated with cadmium(II) and zinc(II). One possible explanation for the inhibitory effects of cadmium(II) in our study could be the replacement of zinc ions by cadmium(II) in zinc finger structures. Several DNA repair enzymes have been shown to contain zinc finger motifs in their DNA binding domain, including the DNA damage recognition proteins XPA (20) and RPA (21). In this context, cadmium(II) has been shown in several studies to displace zinc(II) in transcription factors with similar DNA binding motifs (e.g., 22–24). In the case of nickel(II), the displacement of magnesium ions from DNA damage recognition proteins appears to be relevant; this is in agreement with our previous study, where the inhibition of the incision step in nucleotide excision repair by nickel(II) was reversible by the addition of excess magnesium(II) (7). In summary, our results supply further evidence that the inhibition of nucleotide excision repair provides a relevant mechanism in nickel- and cadmium-induced genotoxicity. Most evident from the high cancer-proneness of xeroderma pigmentosum patients, the recognition of DNA damage and the initiation of DNA repair events is essential for the prevention of mutations and cancer. Since the disturbance of DNA damage recognition by nickel(II) and cadmium(II) is observed at low and therefore relevant concentrations of the metal compounds, it might well account for their carcinogenic and cocarcinogenic properties. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, grant no. Ha 2372/1–1, and by the University of Bremen, Graduiertenkolleg Gesundheitswissenschaften.

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