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Expression of hepatitis C virus core protein impairs DNA repair in human hepatoma cells
Cancer Letters 209 (2004) 197–205 www.elsevier.com/locate/canlet
Expression of hepatitis C virus core protein impairs DNA repair in human hepatoma cells Jos F. van Pelta,*, Tamara Severia, Tina Crabbe´a, Annemie Van Eetveldta, Chris Verslypea, Tania Roskamsb, Johan Feverya a
Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Herestraat 49, Leuven B 3000, Belgium b Department of Morphology and Molecular Pathology, University Hospital Gasthuisberg, Leuven, Belgium Received 28 September 2003; received in revised form 26 November 2003; accepted 28 November 2003
Abstract Several studies have documented the important association between hepatitis C virus (HCV) infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCVcore protein on DNA repair after UV-induced DNA damage. Therefore, we developed and characterized stably transfected HepG2 cell lines that express HCV-core protein as demonstrated by immunohistochemistry. These cells were significantly less capable to repair the DNA damage than control cells. This suppression of DNA repair by HCV-core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Interestingly, expression of the full-length HCV core did increase the cell doubling time in one of the cell lines we had developed that could not be attributed to an increase in apoptosis or change in telomerase activity in these cells. q 2003 Elsevier Ltd. All rights reserved. Keywords: Hepatitis C virus; Hepatocellular carcinoma; Liver; Cancer; Tumour
1. Introduction Since the discovery of the hepatitis C virus (HCV) in 1989, several studies have shown an important epidemiological association between long-standing HCV infection and hepatocellular carcinoma (HCC). Worldwide 250 000 – 400 000 new cases of HCC are * Corresponding author. Tel.: þ32-16-345835; fax: þ 32-16345846. E-mail address: [email protected] (J.F. van Pelt). 0304-3835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.canlet.2003.11.035
diagnosed annually [1]. In general, HCV infection precedes the development of HCC by 20– 30 years. A strong rise in the number of cases of HCC related to HCV is to be expected over the forthcoming years [2]. The mechanisms involved in the development of HCV-related HCC are still unknown. Several factors might interact in this process as outlined hereby. The oncogenesis may be related to the HCV-induced chronic inflammation. Due to this enhanced cell turnover, the repair of damaged DNA may be compromised rendering cells more susceptible to spontaneous or mutagen-induced alterations [3].
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In addition, the emergence of HCC in livers without cirrhosis or in nearly normal livers of HCV carriers also suggests a direct involvement of HCV in the hepatocarcinogenesis [4]. The absence of integration of the HCV genome in the host cell DNA and the lack of reverse transcriptase activities suggest a role for viral proteins of which HCV core is one of the candidates. The deduced amino acid sequence of the HCVcore protein indicates that it has a putative DNA binding motif and three potential nuclear localisation signals [5]. In the liver of infected patients, HCV-core protein is predominantly present in the cytoplasm [6]. HCV-core protein may modulate gene expression and apoptosis [7 – 9] and the transcription of cellular and viral promotors [10]. In patients, also truncated forms of HCV core might be expressed during HCC development but their clinical relevance needs further study [11]. Some similarities can be expected between the role of HCV-core protein and hepatitis B virus X protein (HBx) in the development of HCC based on their structural resemblance [12]. For HBx protein, it has already been reported that it can suppress DNA repair [13]. In the present study, we investigated whether HCV-core protein can modulate cellular mechanisms (DNA repair, telomerase activity) that in the normal situation protect the cell against the introduction of mutations. We developed human hepatoma cell lines that express HCV-core protein. In addition, we developed as one of the controls a cell line in which we have transfected HCV-core gene lacking the two hydrophobic domains. Furthermore, we will address the use of cell lines for the study of virus-induced HCC development.
2. Materials and methods 2.1. Cloning of viral genes HCV core and truncated HCV core gene were cloned by RT-PCR from RNA isolated from serum of a patient infected with HCV (genotype 1b). Briefly, RNA was extracted from 200 ml serum using Trizol LS (InVitrogen, Merelbeke, Belgium) according to the manufacturer’s instructions. cDNA synthesis was performed using 200 U M-MLV reverse transcriptase
(InVitrogen), 30 U RNase inhibitor (Amersham Pharmacia Biotech, Roosendaal, the Netherlands), 80 pmol random hexamer primers (Roche, Vilvoorde, Belgium), 10 mM dithiotreitol, 0.5 mM of each deoxynucleotide triphosphate (dNTP, Promega, Leiden, the Netherlands) in a total reaction volume of 20 ml. cDNA synthesis was carried out at 37 8C for 1 h. Specific primer sets were developed for the amplification of HCV core (cCORE-F: 50 -GCA.TCA.TGA.GCA.CAA.ATC.CTA.AAC.CYC.AAA.GAA.AAA.CCA.AAM.GWA.A-30 and cCORE-R: 50 -AAG.CGG.AAG.CTG.GGA.TGG.TCA.AAC.ARG.ACA.GCA.AAG. CYA.AGA.G-30 abbreviations: M, [A,C]; R, [A,G]; W, [A,T]; Y, [C,T]) and for truncated HCV core (P2: 50 -TAC.TGC.CTG.ATA.GGG.TGC.TTG.CG-30 and M7stop: 50 -CTT.AAC.CCA.ART.TRC.GCG.ACC.TAC.GCC-30 ). The amplified products were cloned into the pCR3 vector and of selected clones the sequence was determined (Eurogentec, Seraing, Belgium). 2.2. Development of cell lines Plasmid DNA was purified from overnight (o/n) cultures of E. coli using a plasmid purification column (Nucleobond PC100, Macherey-Nagel, Du¨ ren, Germany). DNA was mixed with Lipofectin Reagent (InVitrogen) and added to HepG2 cells (HB-8065, ATCC, Rockville, MD, USA) using 1 mg of purified plasmid DNA/100 000 cells. Transfection was performed o/n in Optimem medium (InVitrogen) at 37 8C in a 5% CO2 incubator. The next day, Williams Medium E (InVitrogen) supplemented with 10% fetal calf serum, 2 mM L -glutamine, 20 mU/ml insulin, 50 nM dexamethasone, 100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml fungizone, 50 mg/ml gentamycin, 100 mg/ml vancomycin (WEM-C) was added. At day 3 after transfection, the medium was refreshed with WEM-C supplemented with Neomycine/G418 (InVitrogen) at a concentration of 800 mg/ml for the selection of plasmid-containing cells. After 14– 18 days, a number of G418-resistant groups of cells could be detected in the culture plates. Cells from separate wells were further subcloned in 96-well culture plates by limiting dilution in G418containing medium. Colonies that had developed in wells that initially contained a single transfectedHepG2 cell were expanded and evaluated for
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the presence of the desired DNA and the expression of RNA and protein. DNA and RNA were extracted from cells using Trizol according to the manufacturer’s instructions (InVitrogen). The presence of DNA and the expression of gene specific mRNA was evaluated by PCR or RT-PCR, respectively. 2.3. Detection of HCV core by immunohistochemistry To evaluate the protein expression of the transgene, cell lines were seeded in 4-well LabTex culture slides (Nunc, InVitrogen) and cultured in WEM-C until 80% confluency. Cultures were rinsed with phosphate buffer and fixed with acetone. HCV core and truncated HCV-core protein were detected by a standard twostep immunohistochemical detection method [14], using a mouse monoclonal antibody against HCV core (clone MA1-080, ABR, Golden, CO, USA) (1:10) as first antibody and a peroxidase-conjugated rabbit anti-mouse IgG (1:800) (Dako, Glostrup, Denmark) as the secondary antibody. Colour reaction was performed as described previously. 2.4. DNA repair in transfected cell lines For each condition, cells were seeded in a concentration series (25 000– 10 000 –5000 –2500– 1000 –0 cells/well) in a volume of 200 ml in a 96-well culture plate. The next day, the cells were UV radiated (lamp with a wave length 254 nm, 4 W). The lamp was placed at a distance of 30 cm that equals the energy of 14 J/cm2. Three series of cells were irradiated for 20 s and four series (controls) were not irradiated. Cells were cultured for three additional days after which 50 ml of XTT-solution (sodium 30 -[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) was added per well (Roche). Four hours after addition of the XTT, the absorbance was determined in a plate reader (Biorad Model 3550, measurement 490 nm, reference 655 nm, Hercules, CA, USA). For the calculations, we compared the conditions at which 5000 cells where seeded per well with or without UV exposure. 2.5. Growth characteristics of transfected cell lines Cells were grown in culture flasks to nearconfluency (90 –100%) as described above. Cells
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were trypsinized and the number of viable cells was determined using the Trypan blue exclusion test. For each cell line, a calibration curve was determined. Cells were plated in a 24-well plate in duplicate (range 5000– 250 000 cells per well in 500 ml); 2 h after cell seeding, 50 ml MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide) solution (Roche) was added to asses metabolic activity. This was followed 4 h later by 500 ml solubilization solution. The incubations were carried out at 37 8C under 5% CO2. After incubation o/n, the lysed cells were collected and the samples stored at 2 80 8C for spectophotometric measurement. To measure cell growth, 10 000 cells were added per well to 12 of a 24-well plate. At regular intervals, MTT-assay was performed for 2 wells starting at 2 h after cell seeding until 12 days and the samples stored at 2 80 8C. For the analysis, samples were quickly defrozen in a water bath at 37 8C and mixed by vortex; 200 ml of each sample was placed in a 96 well ELISA plate in triplicate. Absorbance was measured in a dual-wave spectophotometer (Biorad Model 3550, measurement 595 nm, reference 655 nm, Hercules, CA, USA). The number of cells in each well at different days was calculated from the calibration curve. 2.6. Apoptosis in transfected cell lines Cells were cultured in a 24 well plate at a density of 120 000 cells/well. Twenty-four hours after seeding, the cells in the control wells were exposed to 200 J/cm2 UV (wave length 254 nm). After 72 h, cells from exposed and non-exposed wells were harvested and apoptotic cell death was measured by quantitative sandwich-enzyme-immunoassay (Roche, Cell Death Detection ELISAplus) according to the manufacturer’s instructions. All determinations were done in triplicate. The degree of apoptosis for untreated cell was expressed relative to the positive control (UV-exposed) for the individual cell lines. 2.7. Determination of telomerase enzyme activity Telomerase activity in cultured cell lines was determined using the TeloTAGGG Telomerase PCR ELISAplus (Roche) according to the manufacturer’s instructions. Briefly, 200 000 cells were lysed and the supernatant collected. A dilution series was made
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(equivalent to 5000 – 500 – 100 – 25 – 1 cell/reaction) and PCR reaction mix and internal standard were added to the samples in duplicate. In the first step, telomerase added telomeric repeats to the 30 -end of the biotin-labelled synthetic primer. These elongation products and the internal control were amplified using PCR. The PCR products were divided into two; denatured and hybridized separately using probes to the telomeric repeats and to the internal standard. Resulting products were detected in a streptavidin microtiter plate using anti-digoxigenin conjugated with horseradish peroxidase and TMB (3,30 ,5,50 Tetramethylbenzidine) as substrate.
3. Results 3.1. Development of HCV core and truncated HCV-core expressing cell lines HepG2 cells were transfected with plasmids that contained HCV-core construct (T67), truncated HCVcore construct (T70) or empty vector (T68). For each construct, on an average 6 – 10 cell lines were developed. Using RT-PCR, cell lines that expressed the transgene the highest were selected (T67.2 for HCV core and T70.3 for truncated HCV core). The expression and localization of HCV-core protein in
Fig. 1. Anti-HCV core immunohistochemistry in developed cell lines. (A) T68.14 control cells transfected with the empty plasmid were stained for HCV core using a monoclonal antibody as described in materials and methods, no reactivity was observed. (B) and (C) T70.3 cells transfected with the truncated HCV core showed faint cytoplasmic reactivity (dotted arrows) and staining close to the chromosomes in cells that appeared to be in the process of cell division (double lined arrows). (D) and (E) A strong cytoplasmic staining was observed in T67.2 cells containing the full-length HCV core (thick arrows). (Original magnification 400 £ ).
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the cells were determined by immunohistochemistry. In cells transfected with a plasmid containing the fulllength HCV core the protein could be detected in the cytoplasm (Fig. 1D – 1E). Immunohistochemical staining was performed several times over a period of more than one year. Although the number of T67.2 cells that reacted could vary between 5– 40% (varying between different dates), cell line T67.2 always showed a strong positive reaction in at least 5% of the cells and was therefore selected for further analysis. In addition we studied cell line T67.12 that also expresses full-length HCV core protein but at a lower extent than T67.2 (1 – 2% on average). Expression of truncated HCV-core protein in T70.3 cells was in a diffuse pattern with grains of intense brown colour (Fig. 1C). In addition, T70.3 cells that appeared to undergo cell division showed a strong and distinct staining around condensed chromosomes (Fig. 1B). Cells of line T68.14 (containing the empty vector as confirmed by PCR on the DNA) (Fig. 1A) or non-transfected HepG2 cells did not stain using the same technique. When the first antibody was omitted, no staining could be observed in any of the cell lines (data not shown).
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the cell line with truncated HCV core (T70.3 cells) and intermediate between the extensively expressed HCV core (T67.2) and the empty vector (T68.14) cell lines.
3.2. DNA repair in transfected cell lines Using the XTT assay, the metabolic activity of HepG2 and transfected cells is linearly correlated with the number of cells within the range 2500 – 25.000 cells/well and under the conditions applied (data not shown). We decided to test at 5000 cells/well as was also applied by Jia et al. [13]. The metabolic activity of the cells was measured 72 h after the exposure to UV radiation. The reduction in cell number reflects the suppression of DNA repair and was calculated as the ratio of (ODnon-exposed 2 ODUV exposed)/(ODnon-exposed) £ 100%. All cell lines showed some reduction in metabolic activity after exposure to UV, but this reduction was only statistically significant for cell line T67.2 (Fig. 2A). Second, we compared the change induced by radiation between the different cell lines. The reduction in metabolic activity in UV-exposed T67.2 cells was significantly higher than observed in UV-exposed HepG2 cells or T68.14 cells. The reduction of the DNA repair in cell line T67.12, in which HCV core was expressed at a reduced level compared to T67.2, was similar to
Fig. 2. Reduction of DNA repair after UV exposure in transfected cell lines. (A) Cells were seeded in a concentration series in a volume of 200 ml in a 96-well culture plate. The next day, three series of cells were irradiated for 20 s and four series (controls) were not irradiated. Cells were cultured for an additional 3 days after which 50 ml of XTT-solution was added per well. Four hours later, the optical density was determined and the percentage change compared to non-exposed cells. Data were analysed using One-way ANOVA test and pairwise multiple comparison procedures (Tukey test). Number of separate determinations: HepG2 ðn ¼ 6Þ; T68.14 ðn ¼ 8Þ; T67.2 (high expression HCV core, n ¼ 7), T67.12 (low expression HCV core, n ¼ 3) and T70.3 ðn ¼ 6Þ: (B) The impact of UV-exposure on the DNA repair in T68.14 cells versus T67.2 cells was assayed in seven separate experiments. In each experiment, a more pronounced reduction of the metabolic activity per number of cells was observed for T67.2 cells versus T68.14. The mean difference in reduction (þ /2SD) was 11.5 þ /25.2%.
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When we compared the impact of UV-exposure on T68.14 cells versus T67.2 cells, in each separate experiment, we observed a more pronounced reduction of the metabolic activity for T67.2 cells versus T68.14 cells (Fig. 2B) (mean difference in reduction þ /2 SD:11.5% þ /2 5.2%). 3.3. Growth characteristics of developed cell lines For the separate cell lines, calibration curves were determined that correlate the metabolic activity with the number of cells (Fig. 3A). When cells were placed in a 24-well culture plate and the number of cells determined at consecutive days, we observed an increase in cell number (Fig. 3B). From this, we calculated the doubling time, for HepG2, T70.3 and T68.14; the doubling times were 1.1, 1.3 and 1.4 days, respectively, whereas for cell line T67.2, the calculated doubling time was 2.2 days. To investigate whether over-expression of HCVcore protein as in T67.2 cells stimulated cell death, we measured the apoptosis. The level of apoptosis in these cells in log phase (exponential increase of cell number) did not show significant differences (T67.2 cells: 7.1 þ /2 1.1% as compared to T68.14 cells: 8.2 þ /2 0.6%). 3.4. Determination of telomerase enzyme activity Telomerase activity was determined for T67.2 and T68.14 cells. We wanted to compare the cell number that corresponds with a 50% reduction of the maximal telomerase activity and therefore, a dilution series of cells was made. For each cell line, telomerase activity was determined 4-fold. We did not observe any difference in telomerase activity between T67.2 and T68.14 cells (Fig. 4).
4. Discussion We have developed HepG2 cell lines that stably express HCV-core protein (T67.2 and 67.12) or a variant with truncation of the two C-terminal hydrophobic domains (T70.3). We studied the subcellular localization of HCV core and the influence on DNA repair, cell proliferation and telomerase activity.
Fig. 3. Growth characteristics of HepG2 cells and transfected cell lines. (A) A calibration curve was determined for each cell line investigated. Cells were plated in a 24-well plate in duplicate. Two hours after cell seeding, 50 ml MTT solution was added, followed 4 h later by 500 ml solubilization solution and by overnight incubation. The absorbance was measured in triplicate in a dual-wave spectophotometer (595 nm and reference wavelength 655 nm). (B) To determine growth rate 10 000 cells were added per well to 12 of a 24well plate. At regular time intervals an MTT-assay was performed, each sample in triplicate. The number of cells in each well at different days was calculated from the corresponding calibration curve. Each cell line was studied in three separate experiments.
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Fig. 4. Determination of telomerase enzyme activity. Telomerase activity in T68.14 and T67.2 cell lines was determined using the Telo TAGGG Telomerase PCR ELISAplus (Roche). After an elongation step, samples with internal control were amplified using PCR. The PCR products were divided in two, denatured and hybridised separately using probes to the telomeric repeats and to the internal standard. Resulting products were detected in a streptavidin microtiter plate using anti-digoxigenin conjugated with horseradish peroxidase and TMB as substrate. For each cell line, telomerase activity was determined 4-fold. No difference in telomerase activity was observed between T67.2 and T68.14 cells.
We could confirm that the full-length HCV core was localized exclusively in the cytoplasm of T67.2 cells (Fig. 1D – E) [6,15 – 18]. Although, all of the T67.2 cells contain the gene for HCV core, positive immunoreactivity could be demonstrated in only a fraction of the cells. The level of protein expression might be influenced by several factors such as the time period in culture before fixation, stage of cell cycle, cell – cell contact, etc. From in vitro studies, we know that truncated core is mainly localised in a (peri)nuclear area [18 –20]. However, it should be noted that some of these studies were performed using cell types other than human hepatocytes or hepatoma cells. We observed in T70.3 cells a faint reactivity with the antibody for HCV-core protein in the cytoplasm (Fig. 1C). Interestingly, the staining was stronger and more pronounced around remnants of the nucleus in T70.3 cells, which apparently were undergoing mitosis (Fig. 1B). This (peri)-nuclear staining was never seen in cells transfected with the full-length
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HCV core (T67.2 or T67.12) or in control cell lines, excluding false-positivity. Real-time PCR indicated that truncated HCV core is expressed at mRNA level comparable or slightly higher in T70.3 cells than HCV core is in T67.2 cells (data not shown). Despite this, the immunohistochemical detection of truncated core was much lower, this suggests that although truncated HCV core must be synthesized, the majority of the protein might have diffused or be transported out of the cells. Another explanation could be that truncated HCV-core protein is more rapidly degraded in the cells, which we cannot be excluded from our data. In patients with HCV, HCC develops in general over a period of 20 –30 years and involves phases with altered gene expression (up- and down-regulation). This development from normal cells to HCC is considered to involve several steps that lead to the accumulation of mutations, eventually transforming the cell to a malignant status. In the present study, we found indications that one such factor could be the suppression of DNA repair by HCV-core protein. It has been reported that DNA repair can be inhibited by HBx [13,21,22]. No such observations have been reported for HCV-core protein although some structural and functional resemblance between these two viral proteins exists [12]. In the present study, we could document that expression of HCV-core protein in HepG2 cells reduced the repair of UV-induced DNA damage (Fig. 2A). Within each of our seven separate experiments, the cells that contained the HCV core had a statistically significant reduction in DNA repair when compared to control T68.14 cells (Fig. 2B). This suppression of DNA repair was also less prominent in the cells expressing HCV core at a lower level (T67.12). In cells expressing truncated HCV core (T70.3), the reduction of the metabolic activity after UV exposure was not significantly different from that in the empty vector transfected cells T68.14 (Fig. 2A). We further studied the effect of HCV-core expression on cell proliferation in these cell lines. Data in the literature on the effect of HCV-core protein on the cell cycle are somewhat contradictory. Several groups describe the induction of cell proliferation by down-regulation of p21/waf [23,24] and pRb [8] or upregulation of cyclin E expression [25]. It has also been reported that HCV core could induce apoptosis [26,27] and upregulates p53 [28].
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Other studies show no [29] or reduced sensitivity towards apoptosis in cells expressing HCV core [30,31]. Part of the controversy can likely be attributed to either differences in the cells studied (different species, cell type and presence or absence of wild type p53) and to the localisation of the core protein depending on its size and processing (native, mature, truncated or fusion protein). In the present study, we observed in stably HCV-core transfected HepG2 cells (T67.2) an increase in cell doubling time (Fig. 3B) while the HCV core did not change the mitochondrial activity of the cells (Fig. 3A). In contrast, no significant effect on cell doubling time was observed for cells transfected with the empty plasmid (T68.14) as compared to HepG2 cells. An increase of cell doubling time was also previously reported for human osteosarcoma cells transfected with HCV core [32]. We further investigated whether HCV-core protein could induce apoptosis or suppress the telomerase activity (constitutively present in HepG2 cells) as a mechanism to reduce the overall cell doubling time in T67.2 cells, but no significant changes were observed (Fig. 4). With our model, we can study the subcellular distribution of HCV-core protein and its function within the liver cell. However, it is important to note that that there can always be some aspects that could be obscured by whatever model one chooses to use (here HepG2). There are several advantages in the use of HepG2 cells: (1) HepG2 cells (and Hep3B, Huh7) are derived from human liver, the target organ for hepatitis B and C virus, (2) the HepG2 cells resemble human hepatocytes closely with regard to protein expression and enzyme activity, (3) but in contrast to other frequently studied human hepatocyte cell lines such as Hep3B and Huh7, in HepG2 cells p53 protein is present and expressed as the wild-type. Using a stably transfected cell line has additionally practical advances. In the present study, we show that HCVcore protein can inhibit the DNA repair in vitro, this renders the cells more sensitive to acquire mutations that might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Further studies are required to determine for which phase of the developmental process of HCV-induced HCC our cells can be used as model.
Acknowledgements The authors wish to thank Mrs P. Aertsen for the immunohistochemical staining and Mrs L. Lan for performing real-time PCR quantitation of HCV core in the cells.
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Expression of hepatitis C virus core protein impairs DNA repair in human hepatoma cells Jos F. van Pelta,*, Tamara Severia, Tina Crabbe´a, Annemie Van Eetveldta, Chris Verslypea, Tania Roskamsb, Johan Feverya a
Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Herestraat 49, Leuven B 3000, Belgium b Department of Morphology and Molecular Pathology, University Hospital Gasthuisberg, Leuven, Belgium Received 28 September 2003; received in revised form 26 November 2003; accepted 28 November 2003
Abstract Several studies have documented the important association between hepatitis C virus (HCV) infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCVcore protein on DNA repair after UV-induced DNA damage. Therefore, we developed and characterized stably transfected HepG2 cell lines that express HCV-core protein as demonstrated by immunohistochemistry. These cells were significantly less capable to repair the DNA damage than control cells. This suppression of DNA repair by HCV-core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Interestingly, expression of the full-length HCV core did increase the cell doubling time in one of the cell lines we had developed that could not be attributed to an increase in apoptosis or change in telomerase activity in these cells. q 2003 Elsevier Ltd. All rights reserved. Keywords: Hepatitis C virus; Hepatocellular carcinoma; Liver; Cancer; Tumour
1. Introduction Since the discovery of the hepatitis C virus (HCV) in 1989, several studies have shown an important epidemiological association between long-standing HCV infection and hepatocellular carcinoma (HCC). Worldwide 250 000 – 400 000 new cases of HCC are * Corresponding author. Tel.: þ32-16-345835; fax: þ 32-16345846. E-mail address: [email protected] (J.F. van Pelt). 0304-3835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.canlet.2003.11.035
diagnosed annually [1]. In general, HCV infection precedes the development of HCC by 20– 30 years. A strong rise in the number of cases of HCC related to HCV is to be expected over the forthcoming years [2]. The mechanisms involved in the development of HCV-related HCC are still unknown. Several factors might interact in this process as outlined hereby. The oncogenesis may be related to the HCV-induced chronic inflammation. Due to this enhanced cell turnover, the repair of damaged DNA may be compromised rendering cells more susceptible to spontaneous or mutagen-induced alterations [3].
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In addition, the emergence of HCC in livers without cirrhosis or in nearly normal livers of HCV carriers also suggests a direct involvement of HCV in the hepatocarcinogenesis [4]. The absence of integration of the HCV genome in the host cell DNA and the lack of reverse transcriptase activities suggest a role for viral proteins of which HCV core is one of the candidates. The deduced amino acid sequence of the HCVcore protein indicates that it has a putative DNA binding motif and three potential nuclear localisation signals [5]. In the liver of infected patients, HCV-core protein is predominantly present in the cytoplasm [6]. HCV-core protein may modulate gene expression and apoptosis [7 – 9] and the transcription of cellular and viral promotors [10]. In patients, also truncated forms of HCV core might be expressed during HCC development but their clinical relevance needs further study [11]. Some similarities can be expected between the role of HCV-core protein and hepatitis B virus X protein (HBx) in the development of HCC based on their structural resemblance [12]. For HBx protein, it has already been reported that it can suppress DNA repair [13]. In the present study, we investigated whether HCV-core protein can modulate cellular mechanisms (DNA repair, telomerase activity) that in the normal situation protect the cell against the introduction of mutations. We developed human hepatoma cell lines that express HCV-core protein. In addition, we developed as one of the controls a cell line in which we have transfected HCV-core gene lacking the two hydrophobic domains. Furthermore, we will address the use of cell lines for the study of virus-induced HCC development.
2. Materials and methods 2.1. Cloning of viral genes HCV core and truncated HCV core gene were cloned by RT-PCR from RNA isolated from serum of a patient infected with HCV (genotype 1b). Briefly, RNA was extracted from 200 ml serum using Trizol LS (InVitrogen, Merelbeke, Belgium) according to the manufacturer’s instructions. cDNA synthesis was performed using 200 U M-MLV reverse transcriptase
(InVitrogen), 30 U RNase inhibitor (Amersham Pharmacia Biotech, Roosendaal, the Netherlands), 80 pmol random hexamer primers (Roche, Vilvoorde, Belgium), 10 mM dithiotreitol, 0.5 mM of each deoxynucleotide triphosphate (dNTP, Promega, Leiden, the Netherlands) in a total reaction volume of 20 ml. cDNA synthesis was carried out at 37 8C for 1 h. Specific primer sets were developed for the amplification of HCV core (cCORE-F: 50 -GCA.TCA.TGA.GCA.CAA.ATC.CTA.AAC.CYC.AAA.GAA.AAA.CCA.AAM.GWA.A-30 and cCORE-R: 50 -AAG.CGG.AAG.CTG.GGA.TGG.TCA.AAC.ARG.ACA.GCA.AAG. CYA.AGA.G-30 abbreviations: M, [A,C]; R, [A,G]; W, [A,T]; Y, [C,T]) and for truncated HCV core (P2: 50 -TAC.TGC.CTG.ATA.GGG.TGC.TTG.CG-30 and M7stop: 50 -CTT.AAC.CCA.ART.TRC.GCG.ACC.TAC.GCC-30 ). The amplified products were cloned into the pCR3 vector and of selected clones the sequence was determined (Eurogentec, Seraing, Belgium). 2.2. Development of cell lines Plasmid DNA was purified from overnight (o/n) cultures of E. coli using a plasmid purification column (Nucleobond PC100, Macherey-Nagel, Du¨ ren, Germany). DNA was mixed with Lipofectin Reagent (InVitrogen) and added to HepG2 cells (HB-8065, ATCC, Rockville, MD, USA) using 1 mg of purified plasmid DNA/100 000 cells. Transfection was performed o/n in Optimem medium (InVitrogen) at 37 8C in a 5% CO2 incubator. The next day, Williams Medium E (InVitrogen) supplemented with 10% fetal calf serum, 2 mM L -glutamine, 20 mU/ml insulin, 50 nM dexamethasone, 100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml fungizone, 50 mg/ml gentamycin, 100 mg/ml vancomycin (WEM-C) was added. At day 3 after transfection, the medium was refreshed with WEM-C supplemented with Neomycine/G418 (InVitrogen) at a concentration of 800 mg/ml for the selection of plasmid-containing cells. After 14– 18 days, a number of G418-resistant groups of cells could be detected in the culture plates. Cells from separate wells were further subcloned in 96-well culture plates by limiting dilution in G418containing medium. Colonies that had developed in wells that initially contained a single transfectedHepG2 cell were expanded and evaluated for
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the presence of the desired DNA and the expression of RNA and protein. DNA and RNA were extracted from cells using Trizol according to the manufacturer’s instructions (InVitrogen). The presence of DNA and the expression of gene specific mRNA was evaluated by PCR or RT-PCR, respectively. 2.3. Detection of HCV core by immunohistochemistry To evaluate the protein expression of the transgene, cell lines were seeded in 4-well LabTex culture slides (Nunc, InVitrogen) and cultured in WEM-C until 80% confluency. Cultures were rinsed with phosphate buffer and fixed with acetone. HCV core and truncated HCV-core protein were detected by a standard twostep immunohistochemical detection method [14], using a mouse monoclonal antibody against HCV core (clone MA1-080, ABR, Golden, CO, USA) (1:10) as first antibody and a peroxidase-conjugated rabbit anti-mouse IgG (1:800) (Dako, Glostrup, Denmark) as the secondary antibody. Colour reaction was performed as described previously. 2.4. DNA repair in transfected cell lines For each condition, cells were seeded in a concentration series (25 000– 10 000 –5000 –2500– 1000 –0 cells/well) in a volume of 200 ml in a 96-well culture plate. The next day, the cells were UV radiated (lamp with a wave length 254 nm, 4 W). The lamp was placed at a distance of 30 cm that equals the energy of 14 J/cm2. Three series of cells were irradiated for 20 s and four series (controls) were not irradiated. Cells were cultured for three additional days after which 50 ml of XTT-solution (sodium 30 -[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) was added per well (Roche). Four hours after addition of the XTT, the absorbance was determined in a plate reader (Biorad Model 3550, measurement 490 nm, reference 655 nm, Hercules, CA, USA). For the calculations, we compared the conditions at which 5000 cells where seeded per well with or without UV exposure. 2.5. Growth characteristics of transfected cell lines Cells were grown in culture flasks to nearconfluency (90 –100%) as described above. Cells
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were trypsinized and the number of viable cells was determined using the Trypan blue exclusion test. For each cell line, a calibration curve was determined. Cells were plated in a 24-well plate in duplicate (range 5000– 250 000 cells per well in 500 ml); 2 h after cell seeding, 50 ml MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide) solution (Roche) was added to asses metabolic activity. This was followed 4 h later by 500 ml solubilization solution. The incubations were carried out at 37 8C under 5% CO2. After incubation o/n, the lysed cells were collected and the samples stored at 2 80 8C for spectophotometric measurement. To measure cell growth, 10 000 cells were added per well to 12 of a 24-well plate. At regular intervals, MTT-assay was performed for 2 wells starting at 2 h after cell seeding until 12 days and the samples stored at 2 80 8C. For the analysis, samples were quickly defrozen in a water bath at 37 8C and mixed by vortex; 200 ml of each sample was placed in a 96 well ELISA plate in triplicate. Absorbance was measured in a dual-wave spectophotometer (Biorad Model 3550, measurement 595 nm, reference 655 nm, Hercules, CA, USA). The number of cells in each well at different days was calculated from the calibration curve. 2.6. Apoptosis in transfected cell lines Cells were cultured in a 24 well plate at a density of 120 000 cells/well. Twenty-four hours after seeding, the cells in the control wells were exposed to 200 J/cm2 UV (wave length 254 nm). After 72 h, cells from exposed and non-exposed wells were harvested and apoptotic cell death was measured by quantitative sandwich-enzyme-immunoassay (Roche, Cell Death Detection ELISAplus) according to the manufacturer’s instructions. All determinations were done in triplicate. The degree of apoptosis for untreated cell was expressed relative to the positive control (UV-exposed) for the individual cell lines. 2.7. Determination of telomerase enzyme activity Telomerase activity in cultured cell lines was determined using the TeloTAGGG Telomerase PCR ELISAplus (Roche) according to the manufacturer’s instructions. Briefly, 200 000 cells were lysed and the supernatant collected. A dilution series was made
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(equivalent to 5000 – 500 – 100 – 25 – 1 cell/reaction) and PCR reaction mix and internal standard were added to the samples in duplicate. In the first step, telomerase added telomeric repeats to the 30 -end of the biotin-labelled synthetic primer. These elongation products and the internal control were amplified using PCR. The PCR products were divided into two; denatured and hybridized separately using probes to the telomeric repeats and to the internal standard. Resulting products were detected in a streptavidin microtiter plate using anti-digoxigenin conjugated with horseradish peroxidase and TMB (3,30 ,5,50 Tetramethylbenzidine) as substrate.
3. Results 3.1. Development of HCV core and truncated HCV-core expressing cell lines HepG2 cells were transfected with plasmids that contained HCV-core construct (T67), truncated HCVcore construct (T70) or empty vector (T68). For each construct, on an average 6 – 10 cell lines were developed. Using RT-PCR, cell lines that expressed the transgene the highest were selected (T67.2 for HCV core and T70.3 for truncated HCV core). The expression and localization of HCV-core protein in
Fig. 1. Anti-HCV core immunohistochemistry in developed cell lines. (A) T68.14 control cells transfected with the empty plasmid were stained for HCV core using a monoclonal antibody as described in materials and methods, no reactivity was observed. (B) and (C) T70.3 cells transfected with the truncated HCV core showed faint cytoplasmic reactivity (dotted arrows) and staining close to the chromosomes in cells that appeared to be in the process of cell division (double lined arrows). (D) and (E) A strong cytoplasmic staining was observed in T67.2 cells containing the full-length HCV core (thick arrows). (Original magnification 400 £ ).
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the cells were determined by immunohistochemistry. In cells transfected with a plasmid containing the fulllength HCV core the protein could be detected in the cytoplasm (Fig. 1D – 1E). Immunohistochemical staining was performed several times over a period of more than one year. Although the number of T67.2 cells that reacted could vary between 5– 40% (varying between different dates), cell line T67.2 always showed a strong positive reaction in at least 5% of the cells and was therefore selected for further analysis. In addition we studied cell line T67.12 that also expresses full-length HCV core protein but at a lower extent than T67.2 (1 – 2% on average). Expression of truncated HCV-core protein in T70.3 cells was in a diffuse pattern with grains of intense brown colour (Fig. 1C). In addition, T70.3 cells that appeared to undergo cell division showed a strong and distinct staining around condensed chromosomes (Fig. 1B). Cells of line T68.14 (containing the empty vector as confirmed by PCR on the DNA) (Fig. 1A) or non-transfected HepG2 cells did not stain using the same technique. When the first antibody was omitted, no staining could be observed in any of the cell lines (data not shown).
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the cell line with truncated HCV core (T70.3 cells) and intermediate between the extensively expressed HCV core (T67.2) and the empty vector (T68.14) cell lines.
3.2. DNA repair in transfected cell lines Using the XTT assay, the metabolic activity of HepG2 and transfected cells is linearly correlated with the number of cells within the range 2500 – 25.000 cells/well and under the conditions applied (data not shown). We decided to test at 5000 cells/well as was also applied by Jia et al. [13]. The metabolic activity of the cells was measured 72 h after the exposure to UV radiation. The reduction in cell number reflects the suppression of DNA repair and was calculated as the ratio of (ODnon-exposed 2 ODUV exposed)/(ODnon-exposed) £ 100%. All cell lines showed some reduction in metabolic activity after exposure to UV, but this reduction was only statistically significant for cell line T67.2 (Fig. 2A). Second, we compared the change induced by radiation between the different cell lines. The reduction in metabolic activity in UV-exposed T67.2 cells was significantly higher than observed in UV-exposed HepG2 cells or T68.14 cells. The reduction of the DNA repair in cell line T67.12, in which HCV core was expressed at a reduced level compared to T67.2, was similar to
Fig. 2. Reduction of DNA repair after UV exposure in transfected cell lines. (A) Cells were seeded in a concentration series in a volume of 200 ml in a 96-well culture plate. The next day, three series of cells were irradiated for 20 s and four series (controls) were not irradiated. Cells were cultured for an additional 3 days after which 50 ml of XTT-solution was added per well. Four hours later, the optical density was determined and the percentage change compared to non-exposed cells. Data were analysed using One-way ANOVA test and pairwise multiple comparison procedures (Tukey test). Number of separate determinations: HepG2 ðn ¼ 6Þ; T68.14 ðn ¼ 8Þ; T67.2 (high expression HCV core, n ¼ 7), T67.12 (low expression HCV core, n ¼ 3) and T70.3 ðn ¼ 6Þ: (B) The impact of UV-exposure on the DNA repair in T68.14 cells versus T67.2 cells was assayed in seven separate experiments. In each experiment, a more pronounced reduction of the metabolic activity per number of cells was observed for T67.2 cells versus T68.14. The mean difference in reduction (þ /2SD) was 11.5 þ /25.2%.
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When we compared the impact of UV-exposure on T68.14 cells versus T67.2 cells, in each separate experiment, we observed a more pronounced reduction of the metabolic activity for T67.2 cells versus T68.14 cells (Fig. 2B) (mean difference in reduction þ /2 SD:11.5% þ /2 5.2%). 3.3. Growth characteristics of developed cell lines For the separate cell lines, calibration curves were determined that correlate the metabolic activity with the number of cells (Fig. 3A). When cells were placed in a 24-well culture plate and the number of cells determined at consecutive days, we observed an increase in cell number (Fig. 3B). From this, we calculated the doubling time, for HepG2, T70.3 and T68.14; the doubling times were 1.1, 1.3 and 1.4 days, respectively, whereas for cell line T67.2, the calculated doubling time was 2.2 days. To investigate whether over-expression of HCVcore protein as in T67.2 cells stimulated cell death, we measured the apoptosis. The level of apoptosis in these cells in log phase (exponential increase of cell number) did not show significant differences (T67.2 cells: 7.1 þ /2 1.1% as compared to T68.14 cells: 8.2 þ /2 0.6%). 3.4. Determination of telomerase enzyme activity Telomerase activity was determined for T67.2 and T68.14 cells. We wanted to compare the cell number that corresponds with a 50% reduction of the maximal telomerase activity and therefore, a dilution series of cells was made. For each cell line, telomerase activity was determined 4-fold. We did not observe any difference in telomerase activity between T67.2 and T68.14 cells (Fig. 4).
4. Discussion We have developed HepG2 cell lines that stably express HCV-core protein (T67.2 and 67.12) or a variant with truncation of the two C-terminal hydrophobic domains (T70.3). We studied the subcellular localization of HCV core and the influence on DNA repair, cell proliferation and telomerase activity.
Fig. 3. Growth characteristics of HepG2 cells and transfected cell lines. (A) A calibration curve was determined for each cell line investigated. Cells were plated in a 24-well plate in duplicate. Two hours after cell seeding, 50 ml MTT solution was added, followed 4 h later by 500 ml solubilization solution and by overnight incubation. The absorbance was measured in triplicate in a dual-wave spectophotometer (595 nm and reference wavelength 655 nm). (B) To determine growth rate 10 000 cells were added per well to 12 of a 24well plate. At regular time intervals an MTT-assay was performed, each sample in triplicate. The number of cells in each well at different days was calculated from the corresponding calibration curve. Each cell line was studied in three separate experiments.
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Fig. 4. Determination of telomerase enzyme activity. Telomerase activity in T68.14 and T67.2 cell lines was determined using the Telo TAGGG Telomerase PCR ELISAplus (Roche). After an elongation step, samples with internal control were amplified using PCR. The PCR products were divided in two, denatured and hybridised separately using probes to the telomeric repeats and to the internal standard. Resulting products were detected in a streptavidin microtiter plate using anti-digoxigenin conjugated with horseradish peroxidase and TMB as substrate. For each cell line, telomerase activity was determined 4-fold. No difference in telomerase activity was observed between T67.2 and T68.14 cells.
We could confirm that the full-length HCV core was localized exclusively in the cytoplasm of T67.2 cells (Fig. 1D – E) [6,15 – 18]. Although, all of the T67.2 cells contain the gene for HCV core, positive immunoreactivity could be demonstrated in only a fraction of the cells. The level of protein expression might be influenced by several factors such as the time period in culture before fixation, stage of cell cycle, cell – cell contact, etc. From in vitro studies, we know that truncated core is mainly localised in a (peri)nuclear area [18 –20]. However, it should be noted that some of these studies were performed using cell types other than human hepatocytes or hepatoma cells. We observed in T70.3 cells a faint reactivity with the antibody for HCV-core protein in the cytoplasm (Fig. 1C). Interestingly, the staining was stronger and more pronounced around remnants of the nucleus in T70.3 cells, which apparently were undergoing mitosis (Fig. 1B). This (peri)-nuclear staining was never seen in cells transfected with the full-length
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HCV core (T67.2 or T67.12) or in control cell lines, excluding false-positivity. Real-time PCR indicated that truncated HCV core is expressed at mRNA level comparable or slightly higher in T70.3 cells than HCV core is in T67.2 cells (data not shown). Despite this, the immunohistochemical detection of truncated core was much lower, this suggests that although truncated HCV core must be synthesized, the majority of the protein might have diffused or be transported out of the cells. Another explanation could be that truncated HCV-core protein is more rapidly degraded in the cells, which we cannot be excluded from our data. In patients with HCV, HCC develops in general over a period of 20 –30 years and involves phases with altered gene expression (up- and down-regulation). This development from normal cells to HCC is considered to involve several steps that lead to the accumulation of mutations, eventually transforming the cell to a malignant status. In the present study, we found indications that one such factor could be the suppression of DNA repair by HCV-core protein. It has been reported that DNA repair can be inhibited by HBx [13,21,22]. No such observations have been reported for HCV-core protein although some structural and functional resemblance between these two viral proteins exists [12]. In the present study, we could document that expression of HCV-core protein in HepG2 cells reduced the repair of UV-induced DNA damage (Fig. 2A). Within each of our seven separate experiments, the cells that contained the HCV core had a statistically significant reduction in DNA repair when compared to control T68.14 cells (Fig. 2B). This suppression of DNA repair was also less prominent in the cells expressing HCV core at a lower level (T67.12). In cells expressing truncated HCV core (T70.3), the reduction of the metabolic activity after UV exposure was not significantly different from that in the empty vector transfected cells T68.14 (Fig. 2A). We further studied the effect of HCV-core expression on cell proliferation in these cell lines. Data in the literature on the effect of HCV-core protein on the cell cycle are somewhat contradictory. Several groups describe the induction of cell proliferation by down-regulation of p21/waf [23,24] and pRb [8] or upregulation of cyclin E expression [25]. It has also been reported that HCV core could induce apoptosis [26,27] and upregulates p53 [28].
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Other studies show no [29] or reduced sensitivity towards apoptosis in cells expressing HCV core [30,31]. Part of the controversy can likely be attributed to either differences in the cells studied (different species, cell type and presence or absence of wild type p53) and to the localisation of the core protein depending on its size and processing (native, mature, truncated or fusion protein). In the present study, we observed in stably HCV-core transfected HepG2 cells (T67.2) an increase in cell doubling time (Fig. 3B) while the HCV core did not change the mitochondrial activity of the cells (Fig. 3A). In contrast, no significant effect on cell doubling time was observed for cells transfected with the empty plasmid (T68.14) as compared to HepG2 cells. An increase of cell doubling time was also previously reported for human osteosarcoma cells transfected with HCV core [32]. We further investigated whether HCV-core protein could induce apoptosis or suppress the telomerase activity (constitutively present in HepG2 cells) as a mechanism to reduce the overall cell doubling time in T67.2 cells, but no significant changes were observed (Fig. 4). With our model, we can study the subcellular distribution of HCV-core protein and its function within the liver cell. However, it is important to note that that there can always be some aspects that could be obscured by whatever model one chooses to use (here HepG2). There are several advantages in the use of HepG2 cells: (1) HepG2 cells (and Hep3B, Huh7) are derived from human liver, the target organ for hepatitis B and C virus, (2) the HepG2 cells resemble human hepatocytes closely with regard to protein expression and enzyme activity, (3) but in contrast to other frequently studied human hepatocyte cell lines such as Hep3B and Huh7, in HepG2 cells p53 protein is present and expressed as the wild-type. Using a stably transfected cell line has additionally practical advances. In the present study, we show that HCVcore protein can inhibit the DNA repair in vitro, this renders the cells more sensitive to acquire mutations that might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Further studies are required to determine for which phase of the developmental process of HCV-induced HCC our cells can be used as model.
Acknowledgements The authors wish to thank Mrs P. Aertsen for the immunohistochemical staining and Mrs L. Lan for performing real-time PCR quantitation of HCV core in the cells.
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