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J. Biol. Chem., Vol. 280, Issue 10, 8831-8841, March 11, 2005

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Expression of Gastrointestinal Glutathione Peroxidase Is Inversely Correlated to the Presence of Hepatitis C Virus Subgenomic RNA in Human Liver Cells^*

Monika Morbitzer and Thomas Herget

From the AXXIMA Pharmaceuticals AG, Max-Lebsche-Platz 32, 81377 Munich, Germany

Received for publication, December 6, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
There is great medical need to develop novel therapies for treatment of human hepatitis C virus (HCV). By gene expression analysis of three HCV-subgenomic RNA replicon cell lines, we identified cellular proteins whose expression is affected by the presence of HCV and therefore may serve as drug targets. Data from cDNA array filter hybridization, as well as from Northern and Western blotting, revealed that the gastrointestinal-glutathione peroxidase (GI-GPx) was drastically down-regulated (up to 20-fold) in all replicon cell lines tested. Concomitantly, total cellular glutathione peroxidase activity was drastically reduced, which rendered these human liver cells more susceptible toward oxidative stress. Interferon caused down-regulation of the HCV-replicon followed by recovery of GI-GPx expression to nearly normal levels. Furthermore, expression of GI-GPx in replicon cells by gene transduction caused down-regulation of HCV RNA in a dose-dependent manner. Moreover, activating the endogenous gene coding for GI-GPx by all-trans-retinoic acid (RA) was sufficient to cause down-regulation of the HCV replicon. A small interfering RNA duplex abrogated GI-GPx up-regulation by RA and concomitantly suppression of HCV. The RA effect was dependent on the presence of sodium selenite, was reversible, and was independent of RNA-activated protein kinase. Taken together, these results show that HCV inhibits the expression of GI-GPx in replicon cells to promote its intracellular propagation. Modulation of GI-GPx activity may open new avenues of treatment for HCV patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The hepatitis C virus (HCV)¹ infects 2% of the population worldwide (1), and more than 80% of these patients become chronically infected causing recurring hepatitis, liver cirrhosis, and cancer. HCV-related end-stage liver disease is now the leading reason for liver transplantation. Interferon (IFN) monotherapy is effective in only 10% of HCV patients (2–4), and the combination of pegylated interferon and ribavirin can eradicate the virus in about 50% of patients (reviewed in Ref. 5). The mechanism of action of ribavirin has not been fully determined, but incorporation of ribavirin triphosphate into the HCV genome seems to induce error-prone replication to a nontolerable mutation rate (6). Binding of IFN/ leads to the dimerization of the respective receptors and activation of the Jak/STAT pathway, which turns on cellular genes known to be involved in antiviral responses (7). Among these are the Mx proteins, major histocompatibility antigens, 2',5'-oligoadenylate synthase, and the double-stranded RNA-specific protein kinase (PKR). The precise mechanisms by which HCV can resist the IFN response is not fully understood, but viral proteins E2 and NS5a seem to play a role in evasion of the cellular defense system by blocking PKR (reviewed in Ref. 8).

Molecular studies on HCV-host cell interaction have been hampered by the lack of small animal model systems (reviewed in Ref. 9). To overcome these limitations, a human hepatoma cell line (HuH7) was transfected with subgenomic HCV RNAs, called replicons (Fig. 1A) (10). Stable cell lines containing high levels of replicon RNA and viral proteins proved useful to study HCV replication as well as translation and processing of HCV proteins present in the system (10–12).

View larger version (50K): [in this window] [in a new window]   FIG. 1. Replicon cells express less GI-GPx mRNA and protein than control HuH7 cells. A, HCV is a single-stranded RNA virus of about 9.6 kb. The 5'- and 3'-UTRs flank a single open reading frame encoding at least 10 structural and nonstructural proteins (56). Viral proteins are generated as a polyprotein of about 3000 amino acids that is translated via the IRES located in the 5'-UTR (57). In the subgenomic replicon, the region that encodes the core proteins was replaced by the neomycin phosphotransferase gene (neo) and the IRES of the encephalomyocarditis virus. This IRES drives the translation of the polypeptide for the nonstructural proteins, whereas the selectable marker neo is expressed under the control of the original HCV IRES of the 5'-untranslated region (11, 12). B, HuH7 control cells (pcDNA3) and the HuH replicon cell lines 5-15, 11-7, and 9-13 were plated in 10-cm culture dishes (5 x 105 cells) and harvested after 3 days when cells were actively progressing through the cell cycle. Total RNA was isolated and 10 µg separated in a 1.2% agarose gel and used for Northern blot analysis. Blots were hybridized with radioactively labeled oligonucleotides (Probe 1 and Probe 2, upper panels) complementary to coding region and 3'-UTR, respectively, of the human GI-GPx mRNA. Blots were stripped and re-hybridized with two oligonucleotides (Probe 1 and Probe 2, lower panels) recognizing the cellular glutathione peroxidase (cGPx) mRNA. Membranes were exposed to Kodak x-ray films for 1 day (GI-GPx) and 2 days (cGPx), respectively, at –80 °C with intensifier screens. The blots shown are representatives of three independent experiments. C, replicon cells were grown for 3 days, when extracts were prepared and 20 µg of protein was separated by 12.5% PAGE for Western blotting using a GI-GPx-specific rabbit antiserum (upper panel) and an MnSOD antibody (middle panel). For loading control, membranes were re-probed with an -tubulin-specific antibody (lower panel). Bound antibody was detected by enhanced chemiluminescence (Amersham Biosciences). Positions of protein size marker are indicated on the left.

GI-GPx is one of four selenium-containing glutathione peroxidases, and its expression in rodents appears to be restricted to the epithelium of the gastrointestinal tract (13), whereas in humans it is also found in liver (14). Glutathione peroxidases are functioning primarily to counteract oxidative attack but also play roles in signaling (reviewed in Refs. 15 and 16). Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, are produced during aerobic metabolism. Glutathione peroxidases, along with superoxide dismutases and catalases, are considered the main antioxidant enzymes to encounter oxidative stress, which can cause severe cell damage (17). Selenoproteins like GPx incorporate selenocysteine (Sec) co-translationally into the polypeptide chain by a complex mechanism (reviewed in Refs. 18 and 19). Sec has its own code word, UGA, that serves a dual function of dictating Sec or the cessation of protein synthesis. The feature that distinguishes UGA as a Sec codon is the presence of a stem-loop structure called a Sec insertion sequence (SECIS) element within the 3'-UTR (18–21). About 25 selenoproteins with mostly unknown functions have been identified so far in humans and mice (22).

We report that, compared with control HuH7 cells, HCV-replicon cells expressed less GI-GPx and were more susceptible toward ROS-producing agents. Ectopic expression of GI-GPx in replicon cells resulted in a decrease of the HCV RNA and protein NS5a. We show that up-regulation of GI-GPx by retinoic acid and selenite caused drastic down-regulation of HCV in the replicon system. Therefore, retinoids, which exert their action by influencing gene networks, may be useful for treating HCV patients.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Cultures of the human hepatoma cell line HuH7 (23) and the HuH7 cell clones 5-15, 9-13, and 11-7, which contain the HCV replicons I₃₈₉/NS3–3', I₃₇₇/NS3–3', and I₃₇₇/NS2–3' (Fig. 1A), respectively, were propagated as described (10, 11). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated, fetal bovine serum (Invitrogen) and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. G418 (geneticin) (Invitrogen) was added to a final concentration of 750 µg/ml to medium used for culturing cell lines carrying HCV replicons. Cells were routinely passaged every 2–3 days at a dilution of 1:2 to 1:4, depending on density and cell line. Human interferon was purchased from Sigma. HEK 293 cells were transfected by the Ca²⁺-phosphate DNA co-precipitation method.

Screening of Arrays—Atlas^TM (Clontech) "apoptosis" and "stress" cDNA expression arrays include 205 and 234 human cDNAs, respectively, spotted in duplicate dots (10 ng of cDNA per dot) on positively charged nylon membranes. Probes were generated in a volume of 15 µl by reverse-transcribing 5 µg of total RNA in the presence of 50 µCi of [-³³P]dATP, dNTPs, Moloney murine leukemia virus reverse transcriptase, and a cDNA synthesis primer mix, which is a combination of primers specific for each type of Atlas^TM array. After removal of the non-incorporated nucleotides on a Sepharose spin column, efficiency of labeling was controlled by scintillation counting. Filters were prehybridized for 1 h and then hybridized with the radioactive probe (2 x 10⁶ cpm/ml) for 15 h at 68 °C. After washing two times with 2x SSC, 1% SDS and four times with 0.1x SSC, 0.5% SDS at 68 °C, membranes were exposed to x-ray films or to a PhosphorImaging screen (0.5–2 days). Data were utilized when the nine housekeeping genes of the filters generated similar signals for all four HuH7 cell lines. Only those signals, which were at least three times above background of filters, were considered as specific.

Northern Blot Analysis—Cultures were washed twice with ice-cold phosphate-buffered saline (PBS), and cellular RNA was extracted using the RNeasy Total RNA kit (Qiagen, Germany). 5 µg of total RNA per lane was separated in 1.2% agarose, 2.2 M formaldehyde gels and transferred onto Hybond^TM-N nylon membrane (Amersham Biosciences) by capillary blot with 10x SSC. Pre-hybridization was performed in 6x SSC containing 5x Denhardt's solution, 0.5% SDS, and 250 µg/ml denatured salmon sperm DNA at 65 °C for 4 h. For detection of mRNAs, specific antisense oligonucleotides (10 ng) were radioactively labeled with terminal transferase and 50 µCi of [-³²P]dATP according to the manufacturer's protocol (Roche Applied Science).

The sequences of probes used are as follows: PKR probe (coding region, 1293–1332; GenBank^TM accession number XM_002661.1), 5'-CGT TAT TAT ATT TAA CAC GTT TAA TAA CGT AAG TCT TTC C-3'; neo-probe (coding region of Tn-5), 5'-CCA GAT CAT CCT GAT CGA CAA GAC CGG CTT CC-3'; GI-GPx probe 1 (coding region, bp 526–560; GenBank^TM accession number XM_007364.1), 5'-TGG TTG GGA AGG TGC GGC TGT AGC GTC GGA AGG GC-3'; GI-GPx probe 2 (3'-UTR, bp 841–880; GenBank^TM accession number XM_007364.1), 5'-CCT CTC AGA CAC CAC CCA TGA GGG TTT AGG AAG GTG CCA T-3'; cGPx-probe 1 (bp 304–343; GenBank^TM accession number Y00433 [GenBank] ), 5'-GCC GCT AGC CGA GCA GCA CAC ATG GCG CAA TTG TCC AAG A-3'; and cGPx-probe 2 (bp 988–1027; GenBank^TM accession number Y00433 [GenBank] ), 5'-GTT CCT CCC TCG TAG GTT TAG AGG AAA CAC CCT CAT AGA T-3'.

The ³²P-labeled probes were added to freshly prepared pre-hybridization solution at 10⁶ cpm/ml and incubated with the membranes overnight at 65 °C. Filters were washed three times with 2x SSC, 0.2% SDS at 65 °C for 15 min each, followed by three washes with 0.2x SSC, 0.2% SDS at 60 °C for 15 min each. The blots were exposed overnight at –80 °C to Kodak X-Omat AR films with intensifier screens.

Preparation of Cell Lysates and Western Blot Analysis—Western blot analyses were performed as described previously (24). HuH7 cells were washed with ice-cold PBS, scraped with a rubber policeman, collected by centrifugation (10 min at 2000 x g), and resuspended in 200 µl of lysis buffer (50 mM Hepes, pH 7.5, 0.2 M NaCl, 1% Triton X-100, 0.4 mM EDTA, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 100 µg/ml leupeptin, 100 µg/ml aprotinin, 10 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 20 mM -glycerophosphate, and 0.1 mM sodium orthovanadate). The lysed cells were centrifuged for 10 min with 20,000 x g at 4 °C. Protein concentrations of the supernatants were determined by using the BCA reagents (Pierce).

Equal amounts of protein were separated by 12.5% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Immobilon P, Millipore, Bedford, MA) by semi-dry blotting. The membranes were blocked with PBS/Triton (0.1% Triton X-100 in PBS, pH 7.2), supplemented with 5% low fat milk powder (w/v) for 1 h. The primary antibodies were diluted in PBS, 0.1% Triton X-100 containing 1% low fat milk powder (w/v) and then incubated overnight at 4 °C. We used an NS5 antibody (Biogenesis, Cambridge, UK) (1:1000), a GI-GPx-specific polyclonal antibody, kindly obtained from R. Brigelius-Flohé (University of Potsdam, Germany) (1:500), MnSOD and PKR antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) (1:100), an -tubulin and a hemagglutinin tag-specific antibody, both diluted 1:2000 (DAKO, Hamburg, Germany). After washing, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit antibody (1:2000 in PBS, 0.1% Triton X-100 with 1% low fat milk powder; w/v) (Dako, Hamburg, Germany) for 1 h at room temperature to detect bound antibodies. Membranes were washed with PBS/Triton, and secondary antibodies were visualized by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) using medical x-ray films (Fuji).

Glutathione Peroxidase Assay—For measuring cellular glutathione peroxidase activity, we followed the manufacturer's instructions (Calbiochem). Briefly, cells were washed with ice-cold PBS, harvested with a rubber policeman in 5 mM EDTA, 1 mM dithiothreitol, and 50 mM Tris-HCl, pH 7.5, and lysed by three cycles of freezing and thawing. After spinning for 15 min at 10,000 x g (4 °C), protein concentration of the supernatant was determined with the BCA reagents. 180 µg of protein were applied per assay.

tert-Butyl hydroperoxide was used as substrate, and GPx activity was estimated indirectly. Oxidized glutathione, produced upon reduction of the peroxide by GPx, is recycled to its reduced state by glutathione reductase by oxidation of NADPH + H⁺ to NADP⁺. The oxidation of NADPH⁺ is accompanied by a decrease in absorbance at 340 nm, which provides a spectrophotometric means of monitoring GPx activity. The rate of decrease in the A₃₄₀ is directly proportional to the GPx activity in the sample. This assay does not discriminate between the activities of individual isozymes of the glutathione peroxidase gene family.

Cell Viability Assay—For quantification of the degree of cell death in cell culture, we employed the viability assay based on the reduction of tetrazolium salt to formazan by mitochondrial dehydrogenase activity. The assay was performed in 96-well microtiter plates (Greiner, Frickenhausen, Germany) as described previously (24), but Alamar Blue (Roche Applied Science) was used instead of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Eight wells per sample point were analyzed, and each experiment was repeated independently at least three times.

Transfection of HuH7 Cells—A 2.0-kb promoter fragment of the gpx2 gene was amplified by PCR utilizing genomic DNA isolated from 9-13 replicon cells and HEK 293 cells. The upstream primer (5'-GGG GAT TAA TTC CTA CAA ACA ATT CTC ATT TGC-3') contained an AsnI restriction site, and the downstream primer (5'-GGG GCT CGA GGC CTG AGC CCA CTT ATG-3') contained an XhoI restriction site. This fragment was fused with the luciferase gene, which was cloned into the pcDNA3 vector (Invitrogen) via the restriction sites PstI and XbaI, resulting in the pGPx-Luc vector. Sequence analysis revealed that the gpx2 promoter amplified from 9-13 replicon and HE293 cells were identical with published sequences (25). The pGPx-Luc plasmid was transfected into HuH7 cells and co-transfected with the vector pBabe6 (26) in 9-13 replicon cells, and stable transfectants were selected for 3 weeks with 1 mg/ml geneticin (G-418, Calbiochem) and 10 µg/ml puromycin (Sigma). Pools of drug-resistant cells were used for experiments.

siRNA Preparation and Transfection—We tested siRNAs targeting two positions of the human GPx2 mRNA (GenBank^TM accession number X68314 [GenBank] ). The sense sequence of duplex 1 siRNA was r(CCC UCU GGU UGG UGA UUC A)dTdT and of duplex 2 was r(GGA UGA UGG CAC CUU CCU A)dTdT. The control, nonsilencing siRNA was r(UUC UCC GAA CGU GUC ACG U)dTdT. The GPx2 and control siRNAs were designed by Qiagen-Xeragon (Germantown, MD). One control siRNA, r(GAC ACU GAG ACA CCA AUU GAC)dTdT targeting the NS5b coding sequence, was shown to suppress HCV effectively (27), although the 5'-UTR siRNA r(UUC UCC GAA CGU GUC ACG U) dTdT had little effect on HCV (28). All siRNAs were synthesized by Qiagen-Xeragon.

For annealing, a 20 µM siRNA solution was prepared, heated up to 90 °C for 1 min, and incubated at 37 °C for 60 min. The day before transfections, 5 x 10⁵ HuH7 5-15 cells were plated per well of a 6-well plate. Transfections were carried out with Lipofectamine 2000 Reagent (Invitrogen) in serum-free medium. The final siRNA concentration was 50 nM. After 3 days, transfections were repeated, and cells were incubated for another 3 days in the presence or absence of 1 µM all-trans-retinoic acid and 50 nM sodium selenite. Cultures were rinsed, harvested, and lysed, and 20 µg of protein was analyzed by Western blotting.

Adenovirus Construction, Purification, and Infection—The adenovirus used here were all E1- and E3-defective derivatives of adenovirus type 5 (reviewed in Ref. 29). The coding region for GI-GPx (0.7 kb) was amplified by PCR by using an upstream primer containing a HindIII recognition site (5'-GCG CAA GCT TAT GGC TTT CAT TGC CAA GTC CTT C-3', start codon underlined) and a downstream primer containing an XbaI site (5'-GTT CAT CTA GAT ATG GCA ACT TTA AGG AGG CGC TTG-3'). The 3'-UTR (0.3 kb) of the GI-GPx mRNA, containing a SECIS element, was amplified using the upstream primer 5'-GCC CTC GAG ATG TGA ACT GCT CAA CAC ACA G-3' with an XhoI recognition site and the downstream primer 5'-CCA CGC GGC CGC TTT ATT GGT CTC TTC TAG CAG AGT GGC-3' covering the polyadenylation site (AAUAAA) and containing a NotI recognition site for cloning. RNA isolated from HuH7 cells was reverse-transcribed by random priming and used as template for PCR. The cDNA coding for human GI-GPx was cloned with and without 3'-UTR into the transfer plasmid (pPM7) between the cytomegalovirus immediate early promoter/enhancer and the rabbit -globin intron/polyadenylation signal. This expression cassette was inserted into a bacterial plasmid-borne adenovirus genome using recombination in bacteria (30, 31). A cloned version of the novel genome was identified by DNA analysis, and the viral genome was released from the plasmid by restriction enzyme digestion, and virus replication was initiated by transfecting the genome into HEK 293 cells. Virus was amplified in modified HEK 293 cells and purified from cell lysates using CsCl density gradient centrifugation as described (31). Virus was quantified by protein content using the conversion factor 1 mg/ml pure virion protein = 3.4 x 10¹² viral particles/ml (31). The control viruses AdJ5 and AdLuc were described previously (32).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GI-GPx Is Down-regulated in Replicon Cells—Interference of HCV with cellular signaling events can be reflected in altered gene expression. Possible influences and dependences of HCV RNA replication and HCV proteins on host cell transcription are accessible to analysis with cDNA arrays. Three replicon cell lines (HuH 9-13, HuH 5-15, and HuH 11-7) (10, 11) were analyzed for alterations in cellular RNA expression patterns. HuH7-pcDNA3 cells are HuH7 cells resistant to G-418 by integration of a neo gene-carrying plasmid (pcDNA3, Invitrogen) and served as control. Radioactively labeled complex cDNA probes were derived from the RNA of these four cell lines and were hybridized with the cDNA array membranes (stress and apoptosis arrays, Atlas^TM, Clontech). Results from this novel signal tranduction microarray analysis revealed significant down-regulation (10–20-fold) of GI-GPx message in the three replicon cell lines (data not shown).

To verify the results obtained by cDNA array screening, we performed Northern blot analyses utilizing 10 µg of total RNA of each cell line and two different radioactively labeled DNA oligonucleotides as probes. The sequences were deduced from the coding region (GI-GPx; probe 1) and from the 3'-UTR (GI-GPx; probe 2) (Fig. 1B). Densitometric scanning of autoradiograms of three independent experiments established that the mRNA coding for GI-GPx is about 10- (HuH 5-15) and 20-fold (HuH 11-7 and HuH 9-13) down-regulated in replicon cell lines compared with control cell line HuH7 pcDNA3 (Fig. 1B). We performed Northern blot analyses with RNA isolated from four naive HuH7 cell lines with various numbers of passages (passage 15, 41, 80, and 136) to rule out that due to clonal selection the HuH7 pcDNA3 reference cell line overexpressed the GI-GPx mRNA. These cell cultures expressed similar levels of GI-GPx mRNA as the HuH7 pcDNA3 reference cell line (data not shown). Thus, the low amount of GI-GPx mRNA in the replicon cell lines was linked to the presence of HCV-RNA and/or HCV-proteins.

Furthermore, we wondered whether the cytoplasmic member of the glutathione peroxidase family expressed in hepatocytes, the cellular glutathione peroxidase (cGPx), compensates for the deficiency of the GI-GPx message. Stripping the membranes (Fig. 1B) and hybridizing them with radioactively labeled DNA oligonucleotides specific for human cGPx revealed similar cGPx mRNA levels in control cells and replicon cells (Fig. 1B, lower panels). These data demonstrate that the GI-GPx gene is specifically affected by the HCV replicon.

The level of GI-GPx mRNA (Fig. 1B) was also reflected on the protein level. GI-GPx protein was readily detectable in pcDNA3 cells, was hardly detectable in 5-15, and undetectable in 9-13 and 11-7 replicons (Fig. 1C, upper panel). Furthermore, we studied the expression of MnSOD, another enzyme involved in detoxification of cellular radicals. Western blotting demonstrated that levels of MnSOD protein were similar in the three replicon cell lines and in pcDNA3 control cells (Fig. 1C, middle panel).

Replicon Cells Are Susceptible to Oxidative Stress—We addressed physiological consequences of the transcriptional down-regulation of the GI-GPx gene. Therefore, cellular extracts of pcDNA3 control cells and the three replicon cell lines (HuH 5-15, 11-7, and 9-13) were prepared to estimate activity of the cellular glutathione peroxidase. The data show that indeed the three replicon cell lines exhibited only about half of the GPx activity of the pcDNA3 control cells (Fig. 2A). These findings prompted us to investigate whether the replicon cells were more susceptible toward treatment with radical and oxidative stress-producing compounds. Replicon cell lines and control cells (HuH7 pcDNA3) were incubated for 24 h with various concentrations of paraquat (methyl viologen), and the viability of the cultures was measured using the Alamar Blue assay. Paraquat, a bipyridol herbicide that uses molecular oxygen to produce superoxide anion radical and subsequently hydrogen peroxide (33), impaired viability of replicon cells more severely than of HuH7 pcDNA3 control cells (Fig. 2B). The estimated LD₅₀ values for paraquat calculated from three independent experiments were 260 ± 50 µM for HuH 9-13, 270 ± 75 µM for HuH 5-15, 310 ± 65 µM for HuH 11-7, and 3 mM±120 µM for HuH pcDNA3. Thus, the HCV replicon cells were about 10-fold more susceptible toward this radical-producing compound. Similar results were obtained with the ROS-generating agent 2,3-dimethoxy-1,4-naphthoquinone (data not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 2. A, glutathione peroxidase activity is reduced in HuH7 replicon cells. HuH7 replicon cells and pcDNA3 control cells were plated and harvested (described in legend of Fig. 1B), and 180 µg of protein of cytoplasmic extract was used for estimation of glutathione peroxidase activity as described under "Experimental Procedures." The mean change (±S.E.) of extinction at 340 nm reflecting glutathione peroxidase activity for each cell line is illustrated. The replicon cell lines HuH 5-15, 11-7, and 9-13 show lower glutathione peroxidase activity than the control cell line pcDNA3. B, replicon cells are susceptible toward oxidative stress. HuH7 cultures were plated in 96-well microtiter plates (5 x 103 cells/well) and, 3 days after seeding, treated for 24 h with the concentration of paraquat indicated (up to 5 x 103 µM). The cell viability was then determined in triplicates with the help of an Alamar Blue assay and compared with untreated cells (=100% viability). Although pcDNA3 control cells (circle) showed an LD50 of about 3 x 103 µM paraquat, the replicon cell lines 5-15 (square), 11-7 (triangle), and 9-13 (rhombus) had LD50 values between 260 and 310 µM. A typical result of three experiments is depicted.

View larger version (65K): [in this window] [in a new window]   FIG. 3. Interferon causes down-regulation of the replicon followed by up-regulation of GI-GPx. The HuH7 pcDNA3 control cells and the replicon cell lines 5-15, 11-7, and 9-13 were plated as described (see legend of Fig. 1B), and after 3 days the cultures were treated for 2 and 4 days with 1000 units/ml IFN. Cells were then harvested, and RNA was prepared. 10 µg of total RNA was used for Northern blot analysis. For detection of GI-GPx mRNA, probe 1 was used (see Fig. 1B). Although IFN caused rapid depletion of HCV RNA within 2 days, a subsequent up-regulation of GI-GPx mRNA was observed within 4 days. As control for IFN response, PKR mRNA was analyzed (lower panels). Exposure time for all blots was 2 days at –80 °C with intensifier screen. The positions of the 18 S rRNA and 28 S rRNA are shown on the left.

Effect of Overexpression of GI-GPx on HCV Replicon—To gain more direct proof that GI-GPx prevents the replication of genomic HCV RNA, we overexpressed the human GI-GPx cDNA in replicon cells. Therefore, the GI-GPx coding region was cloned into expression vector pPM7. To allow efficient readthrough of the stop codon coding for selenocysteine, we fused the 3'-UTR of the GI-GPx mRNA containing the SECIS element to the GI-GPx coding region. To control the effectiveness of these constructs, HEK 293 cells were transiently transfected for 1 day with plasmids containing the GI-GPx coding region with and without the 3'-UTR of GI-GPx. Only the construct with 3'-UTR of GI-GPx produced evident amounts of GI-GPx protein (Fig. 4A). Measuring activities of the transfected cultures disclosed a 50% increase in glutathione peroxidase activity when the GI-GPx +3'-UTR construct was used proving the functioning of the constructs (Fig. 4A).

View larger version (40K): [in this window] [in a new window]   FIG. 4. A, transient expression of GI-GPx requires 3'-UTR. The pPM7 plasmid without additional insert (mock) or with the human hemagglutinin-tagged GI-GPx coding region with 3'-UTR (+3UTR) of the GI-GPx gene including the SECIS or without 3'-UTR (–3UTR) was transfected into HEK 293 cells, which were seeded in a 6-well plate (4 x 105 cells per well) the day before transfection. After 1 day, cultures were harvested, and 20 µg of cellular protein was analyzed by Western blotting utilizing a monoclonal hemagglutinin antibody. Pronounced expression of GI-GPx (arrow) was only observed when the SECIS element of the 3'-UTR was present. In parallel, glutathione peroxidase activity assays were performed by using 50 µg of cellular protein of the transfected cells. The values for the relative activities are presented (mock control = 100%). The HEK 293 culture transfected with the GI-GPx +3'-UTR construct showed significant higher levels (1.5-fold) of glutathione peroxidase activity than the mock-transfected culture or cultures transfected without the SECIS element. B, expression of GI-GPx causes down-regulation of replicon. The replicon cell lines 5-15, 11-7, and 9-13 were plated at a density of 105 cells per well of a 6-well plate and infected with 103 adenoviral particles/cell, containing either the green fluorescent protein (GFP) as negative control, the GI-GPx cDNA without the 3'-UTR (–3'-UTR), and the GI-GPx cDNA with the 3'-UTR containing the SECIS (+3'-UTR), as indicated. After 4 days post-infection cells were harvested, lysed, and 10 µg of protein separated on a 12.5% polyacrylamide gel for Western blot analysis. Monitoring expression of the transduced GI-GPx cDNA without 3'-UTR (–3'-UTR) revealed no (cell line 5-15 and 9-13) or only weak expression (cell line 11-7). However, drastic GI-GPx expression was achieved with the GI-GPx +3'-UTR construct. Western blot analyses show a strong down-regulation of the HCV protein NS5a in all replicon cell lines infected with the GI-GPx+3'-UTR construct. Thus, expression of NS5a was inversely correlated to levels of GI-GPx. Loading efficacy and integrity of proteins were controlled by re-blotting with an -tubulin antibody. The figures depicted exemplify results from three independent experiments.

Seven days after transduction, expression of GI-GPx was still high and NS5a remained down-regulated in these cells (data not shown). These data demonstrate again the inverse correlation between presence of GI-GPx and HCV replicon.

Effect of Retinoic Acid on Replicon—In order to utilize a mechanism to activate the endogenous gpx2 gene, coding for GI-GPx, we examined the promoter of the human gene (GenBank^TM accession number AF199441 [GenBank] ). We identified several putative retinoic acid-response elements in the promoter and in the first intron. Indeed, treatment of the breast tumor cell line MCF-7 with all-trans-retinoic acid (RA), a classical morphogen that can induce differentiation and apoptosis (24, 53), increased both GI-GPx mRNA level and enzymatic activity (34). The induction by RA was specific for the expression of the gpx2 gene, but not of the gpx1 gene coding for the cellular cGPx (34).

In order to assess potent activators of the gpx2 promoter, we stably transfected naive HuH7 and 9-13 replicon cells with a construct consisting of the human gpx2 promoter (2 kb) upstream of the luciferase reporter gene. Incubating stable pools of transfectants with several retinoids (9-cis-RA, all-trans-RA, 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid, 13-cis-RA, methoprene acid, AM-580, and 4-hydroxyphenylretinamide; 1 µM each) for 3 days revealed that all-trans-RA was the most potent inductor of the gpx2 promoter (2.5-fold), followed by 9-cis-RA, 13-cis-RA, and 4-hydroxyphenylretinamide (Fig. 5A). The pattern was similar in HuH7 and 9-13 replicon cells (data not shown) and implies that RA receptors, but not retinoid X receptors, were involved in gpx2 promoter regulation.

View larger version (63K): [in this window] [in a new window]   FIG. 5. A, HuH7 transfectants treatment with retinoids. HuH7 cells were transfected with a reporter construct consisting of the gpx2 promoter and the luciferase reporter gene. Pools of stably transfected cultures were generated and left either untreated (Co) or incubated with the following retinoids (final concentration 1 µM each): 9-cis-retinoic acid (9-cis-RA), all-trans-retinoic acid (All-trans-RA), TTNPB (4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid), 13-cis-retinoic acid (13-cis-RA), methoprene acid (Methopr. Acid), AM-580, 4-hydroxyphenylretinamide (hydroxyphenyl.) or with the solvent Me2SO (DMSO) (0.1%) for 3 days. The cells were then extracted, and 50 µg of protein was used to determine luciferase activity, depicted as relative light units (RLU). The average (±S.E.) of three independent experiments is shown. Similar results were observed with stable pools of replicon cell line 9-13 (data not shown). B, retinoids cause down-regulation of NS5a expression. One day after plating, cultures of replicon cell line 9-13 were incubated for 3 days with retinoids (10 µM each) as indicated or left untreated (Co). Cells were harvested, and 10 µg of protein was analyzed for expression of NS5a by Western blotting (upper panel). A strong effect was observed with all-trans-RA, 4-hydroxyphenylretinamide, 9-cis-, and 13-cis-RA. The blot was re-probed with an -tubulin antibody to demonstrate equal loading of the 10% polyacrylamide gel. C, all-trans-retinoic acid caused up-regulation of GI-GPx and down-regulation of HCV replicon in a dose-dependent manner. Replicon cells 9-13 remained untreated (lane 1) or were treated with 50 nM sodium selenite (Se) (lane 2), 10 µM all-trans-retinoic acid (RA) (lane 3), or with increasing amounts of RA as indicated (lanes 4–9) in the presence of 50 nM sodium selenite. Levels of GI-GPx, NS5a, and -tubulin as loading control were determined by Western blotting. In the presence of selenite, all-trans-RA induced GI-GPx expression in a dose-dependent manner, and NS5a expression was inversely correlated to the levels of GI-GPx. D, effect of Gpx2 siRNA on NS5a and GI-GPx protein levels after treatment of HCV replicon cells with retinoic acid. HuH7 replicon cells were transfected with 50 nM siRNA as depicted. Cells were then incubated with 1 µM all-trans-retinoic acid and 50 nM sodium selenite (+RA/Se) or left untreated (–RA/Se) for 3 days. Cultures were analyzed for expression of NS5a, GI-GPx, and -tubulin. Control (Contr.) siRNA had a nonsilencing sequence, and the siRNAs 5'-UTR and NS5b recognized the respective sequences of HCV (see Fig. 1A).

In order to elucidate the effect of retinoids on HCV in more detail, HuH7 replicon cells were incubated with increasing concentrations (from 10^–4 to 10 µM) of all-trans-retinoic acid in the presence of 50 nM sodium selenite for 1 week. The GI-GPx mRNA (data not shown) and protein levels increased in a dose-dependent manner (Fig. 5C, lanes 4–9). The half-maximal induction was at 1 µM RA, which is in good agreement with effects mediated by retinoic acid receptors (24). In parallel, we studied the effect of activating GI-GPx expression via RA treatment on the HCV replicon by monitoring NS5a levels. In fact, 1 and 10 µM RA (Fig. 5C, lanes 8 and 9) caused significant down-regulation of the NS5a protein. At this concentration we did not observe any cytotoxic effects of RA either on naive HuH7 cells or on replicon cells. Furthermore, growth curves of all cell lines over 1 week in the presence or absence of 1 µM RA (±50 nM sodium selenite) were indistinguishable from control cultures (data not shown).

Notably, RA (10 µM) without sodium selenite hardly induced GI-GPx protein expression (Fig. 5C, lane 3). This finding implies that selenite has to be present in the culture medium for efficient translation of selenocysteine proteins as already described before for hepatocytes (35). Fig. 5C also shows that selenite itself caused a slight increase of GI-GPx protein levels which, however, was not sufficient to affect NS5a levels (lane 2). Even cultivating the replicon cell lines for several passages (3 weeks) in the presence of 50 nM sodium selenite did not cause down-regulation of HCV (data not shown). The finding that RA caused NS5a down-regulation only in the presence of selenite argues that the effect was indeed mediated by the selenocysteine protein GI-GPx.

To strengthen this theory siRNA experiments were performed. Therefore, we ordered two sets of Gpx2 siRNA (Qiagen) and found that only duplex 1 was efficient in down-regulating the GI-GPx expression (see Fig. 5D, middle panel, lane 7). Replicon 5-15 cultures were transfected with GI-GPx siRNAs and again after 3 days. After incubation for an additional 3 days in the presence or absence of all-trans-retinoic acid (1 µM) and sodium selenite (50 nM), expression of NS5a, GI-GPx, and PKR was analyzed. Although cultures transfected with a non-silencing control siRNA responded to the RA/sodium selenite treatment by down-regulation of NS5a (Fig. 5D, lanes 3 and 4) as the control cells (transfection procedure only) did (lanes 1 and 2), this down-regulation was severely reduced by Gpx2 siRNA (compare lane 8 with lanes 2 and 4). This inhibitory effect was not complete, which may be because not all cells took up the siRNA in sufficient amounts or because minor effects of RA on HCV were independent of GI-GPx. The siRNAs targeting the 5'-UTR and the NS5b coding sequence of HCV served as controls. Although the 5'-UTR siRNA had minor consequences on NS5a levels (Fig. 5D, lane 5) as described before (27), the NS5b siRNA effectively suppressed NS5a expression (lane 6), as shown previously (28). The nonsilencing control siRNA and the 5'-UTR siRNA demonstrate that transfection of the siRNAs shown here did not trigger the IFN response (36). Furthermore, neither RA nor transfection of siRNAs caused induction of PKR expression (data not shown).

The RA Effect on the Replicon Is Reversible—Because it is known that RA can have significant effects on cell cycle and differentiation, we investigated whether the replicon cell lines experienced permanent alterations upon RA treatment. The 5-15 cells were cultivated for four passages (each 4 days) in the presence (+) and absence of all-trans-RA (–) (Fig. 6). RA caused up-regulation of GI-GPx expression and concomitantly down-regulation of NS5a after 3 days (passage 1) (Fig. 6A, lane 2). The decline of NS5a expression to barely detectable levels was even more pronounced during the next three passages (Fig. 6A, lanes 4, 7, and 10). Removal of RA after one passage (Fig. 6A, +/–) caused reduced expression of GI-GPx and recovery of NS5a levels (Fig. 6A, compare lanes 4 and 5). The GI-GPx expression was completely lost after one more passage in the absence of RA (Fig. 6A, lane 8). Densitometric scanning of the Western blot analyses demonstrated first that the RA effect on up-regulation of GI-GPx and down-regulation of NS5a was reversible and second that the expression of NS5a and GI-GPx was strictly and inversely correlated (Fig. 6, B and C).

View larger version (33K): [in this window] [in a new window]   FIG. 6. A, wash-out of retinoic acid causes recovery of HCV. Replicon cells 9-13 were incubated for 4 days with (+RA) or without (–RA) retinoic acid (passage 1; P1). The cells continued to be passaged without (–RA; lane 3) or with RA (+RA; lane 4). In addition, the culture of passage 1 containing RA was subcultured without RA (+/–RA; lane 5). These three cultures (–RA, +RA, and +/–RA) were passaged two more times every 4 days (P3 and P4). Western blotting revealed that long term treatment with RA drastically reduced NS5a levels (lanes 7 and 10). Furthermore, removal of RA led to a reduction of GI-GPx to undetectable levels and a concomitant recovery of NS5a levels after two passages (lanes 8 and 11). B, the levels of NS5a were quantified by densitometric scanning of the autoradiograms (A). Whereas NS5a levels remained quite constant of control cultures (–RA, circles), the NS5a levels in RA-treated cultures decreased down to 5% of control (+RA, diamonds). Most interestingly, NS5a levels of cultures treated with RA recovered within two passages, when RA was left out (+/–RA; squares). C, the levels of GI-GPx protein were quantified as described for NS5a (B). Removal of RA (+/–RA; square) caused rapid decline of GI-GPx levels within two passages. One typical example out of three is depicted.

View larger version (29K): [in this window] [in a new window]   FIG. 7. PKR is not involved in retinoic acid-mediated but is involved in IFN-mediated HCV down-regulation. Replicon cells (9–13) were treated for 4 days with 50 nM sodium selenite (Se), 1 µM all-trans-retinoic acid (RA), or with 1 µM all-trans-retinoic acid together with 50 nM sodium selenite (RA/Se). RA, in combination with sodium selenite, induced a significant increase in GI-GPx level and down-regulation of HCV protein NS5a without induction of PKR. -Interferon (1000 units/ml, plus 50 nM sodium selenite) (IFN), in comparison, caused NS5a down-regulation by induction of PKR expression. Co, control.

These data are in favor of a novel PKR-independent pathway, by which RA leads to HCV down-regulation. Using subtherapeutic concentrations of IFN alone and in combination with all-trans-RA substantiated this hypothesis. Interferon caused a dose-dependent down-regulation of NS5a, which was further enhanced by addition of all-trans-RA (data not shown). The finding that RA acts independently of interferon implies the possibility to cure patients who do not respond to interferon standard therapy with RA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The present study demonstrates that human liver cells harboring a subgenomic HCV replicon RNA express drastically reduced levels of gastrointestinal glutathione peroxidase. The inverse correlation between HCV and GI-GPx was revealed by several lines of evidence (summarized in Fig. 8). 1) Cells housing the HCV replicon express 10–20-fold less GI-GPx than cells without replicon. 2) Treating replicon cells with interferon caused disappearance of HCV RNA and HCV protein NS5a, as described previously (11). HCV down-regulation was followed by a recovery of GI-GPx mRNA levels (this study). 3) Forced expression of GI-GPx in replicon cells by adenoviral gene transfer induced down-regulation of HCV RNA and NS5a. 4) Activating the promoter of the cellular gpx2 gene (coding for GI-GPx) by nuclear receptor ligand retinoic acid in a time- and dose-dependent manner caused down-regulation of HCV. Wash out experiments demonstrated that this effect of RA on GI-GPx expression and HCV replicon was reversible. The retinoic acid effect depends on selenium and is abrogated by Gpx2 siRNA.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Model of HCV and GI-GPx interplay. Naive HuH7 hepatic cells produce GI-GPx by the active gpx2 gene. 1, upon transfection with the HCV replicon, expression of the gpx2 gene is shut down. 2, treatment of replicon cells with IFN induces the innate immune reaction. With the elimination of the HCV replicon by the IFN response, the suppressive influence of HCV on the gpx2 gene disappears. 3, on the other hand, expression of GI-GPx in replicon cells by adenoviral gene transfer or by 4 activation of the cellular gpx2 promoter with retinoic acid (RA) cause down-regulation of the replicon.

Selenocysteine proteins do not only function to protect against oxidative stress but seem to have other crucial roles in maintaining a healthy physiology of the liver (40, 41). Selenium was proposed to have anticarcinogenic functions, and recently it was shown that in progressed stages of colorectal cancer expression of selenoprotein GI-GPx was decreased (42, 58). Selenium has also been implicated in enhancing immune functions and thereby slowing the progression of AIDS in human immunodeficiency virus-positive patients (reviewed in Ref. 40). Our data now point to a more direct antiviral function of the selenoprotein GI-GPx in HCV infection, and it remains to be seen if the same applies to HIV. Because deficiency in dietary selenium results in decreased levels of selenoproteins, thus compromising biological processes that are maintained by these proteins, it will be interesting to investigate whether selenium lack supports HCV spreading in patients.

Here we show for the first time that the selenocysteine protein GI-GPx plays a crucial function in limiting the HCV replicon in human liver cells, and one might speculate that GI-GPx carries antiviral properties. Our novel findings support the hypothesis that GI-GPx, apart from being a barrier against hydroperoxide absorption, may also be involved in cell growth, differentiation, and ultimately in regulating signal transduction (reviewed in Refs. 16 and 58). In a recent study, it was shown that endogenously produced ROS did not lead to NF-B activation but instead lowered the magnitude of its activation (43). Therefore, it may be an advantage for HCV to abolish GI-GPx expression in order to prevent an NF-B-driven anti-viral host response. Although it is not known how the HCV replicon system interferes with the cellular signal transduction machinery to induce down-regulation of the gpx2 gene, it is conceivable that NS5a is involved. Transfection of NS5a itself seems to be sufficient to trigger the elevation of ROS leading to the activation of several cellular transcription factors like NF-B and STAT-3 (44). Several studies demonstrated that HCV-infected liver cells in patients (45) and in vitro (46, 47) are characterized by a high level of oxidative stress.

A major contribution to nucleic acid damage is caused by oxidative stress due to H₂O₂ and other endogenous reactive oxygen species (48). Therefore, it is conceivable that the down-regulation of GI-GPx expression is accountable for, first, the high rate of mutations in HCV and development of quasispecies and, second, the development of liver fibrosis and cancer (49). Over a long term period, an inappropriate composition of the cellular antioxidant defense system might facilitate DNA damage and contribute to the increased cancer risk in HCV patients. Not surprisingly, we identified in our array analysis several genes, which are involved in DNA repair, up-regulated in replicon cells. Among these were the growth arrest and DNA damage-inducible protein GADD153, the DNA repair protein RAD54, the DNA excision repair protein ERCC1, and the damage-specific DNA-binding protein p48 subunit.² Another consequence of the down-regulation of GI-GPx may be the promotion of synthesis of HCV proteins. Under conditions of cellular stress, the rate of translation for most cellular mRNAs is reduced. However, mRNAs containing an internal ribosomal entry site (IRES) are selectively translated under these conditions. In particular, translation mediated by the viral IRES is enhanced following prolonged exposure to anoxia (50). Consistent with this model is the observation that antioxidants block replication of HCV in the subgenomic replicon system (46).

As stated above, it is conceivable to cure HCV patients by treatment with ROS-producing agents in order to selectively kill the highly susceptible HCV-positive hepatocytes. Furthermore, our data obtained with the adenoviral gene transduction system or by activation of the endogenous promoter of the cellular GI-GPx gene by retinoic acid demonstrate that enhanced expression of GI-GPx supports the host cell in limiting HCV replication. As shown here in human replicon cells, RA can activate the putative retinoic acid-response element(s) of the gpx2 gene. Increasing the GI-GPx activity to reduce cellular radical levels may form the basis for valuable treatments of HCV patients. The differential regulation and the tissue-specific expression of GI-GPx may allow finding means to up-regulate the GI-GPx protein in HCV-infected liver cells.

It is worth mentioning that more than additive effects in inhibiting the replicon were obtained when RA was applied in the presence of ribavirin or subtherapeutic concentrations of interferon (data not shown), both belonging to the standard regimen for treatment of HCV patients and shown to be effective in the replicon system (11, 51). Because the inhibitory effect of RA on the replicon acts independently of an interferon response (Fig. 7), RA may be useful for treating patients who do not react on interferon treatment, the so-called "nonresponders." About 50% of chronic HCV patients are nonresponders facing cirrhosis, cancer, and, finally, liver transplant. Furthermore, the inhibitory effect of RA on HCV infection in vivo may be even stronger than in the subgenomic replicon system. In recent studies it was determined that the HCV core protein, which is absent in replicon cells (Fig. 1A), can increase expression of retinoic acid-response elements containing genes and RA-mediated apoptosis (52–54).

The effects of RA on replicon cells described here were recently confirmed by a study in humans. A patent application (59) describes examples for successful application of retinoic acid to HCV-positive patients. Within 3 weeks of treatment with all-trans-RA, the viral load dropped to undetectable levels. It is likely that the underlying mechanism is based on the up-regulation of GI-GPx as described here.

Our results present new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigating retinoic acid as a stand-alone therapy or in combination with interferon. Therefore, it will be crucial to elucidate and understand in more detail the virus/host cell interaction and the controlling elements regulating expression of GI-GPx.

	FOOTNOTES

 
^* 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.

To whom correspondence should be addressed: Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany. Tel.: 49-61-51-72-6494; Fax: 49-61-51-72-5039; E-mail: thomas.herget{at}merck.de.

¹ The abbreviations used are: HCV, hepatitis C virus; GPx, glutathione peroxidase; GI-GPx, gastrointestinal glutathione peroxidase; GPx2, gene coding for GI-GPx; RA, retinoic acid; ROS, reactive oxygen species; Sec, selenocysteine; SECIS, selenocysteine inserting sequence; UTR, untranslated region; IRES, internal ribosomal entry site; SOD, superoxide dismutase; PBS, phosphate-buffered saline; PKR, RNA-activated protein kinase; siRNA, small interfering RNA; IFN, interferon.

² M. Morbitzer and T. Herget, unpublished data.

	ACKNOWLEDGMENTS

 
We are grateful to Prof. Dr. R. Brigelius-Flohé (University Potsdam, Germany), Prof. Dr. R. Bartenschlager (University of Heidelberg, Germany), Prof. Dr. A. Ullrich (MPI Martinsried, Munich, Germany), Prof. Dr. P. Galle and Dr. D. Strand (University Mainz, Germany), Dr. M. Grell (Merck KGaA, Darmstadt, Germany), and Dr. B. Klebl, Dr. M. Cotten, and Dr. F. Krätzer (Axxima AG) for providing reagents and discussing results.

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