Analytical Biochemistry 328 (2004) 147–154 www.elsevier.com/locate/yabio
Sesquiterpene lactones inhibit luciferase but not -galactosidase activity in vitro and ex vivo Maja T. Lindenmeyer,a Alfonso J. García-Piñeres,a Victor Castro,b and Irmgard Merforta,¤ a
Institut für Pharmazeutische Wissenschaften, Lehrstuhl für Pharmazeutische Biologie, Albert-Ludwigs-Universität Freiburg, Stefan-Meier-Str. 19, 79104 Freiburg, Germany b Escuela de Quimica, CIPRONA, Universidad de Costa Rica, San Jose, Costa Rica Received 29 October 2003
Abstract Reporter enzymes such as WreXy luciferase or -galactosidase of Escherichia coli are frequently used to study transcriptional activity of genes and to investigate the eVects of novel compounds on gene or transcription factor activity. It is generally assumed that the activity of these enzymes is unaVected by the treatment conditions. Therefore, this factor is not considered when interpreting the data obtained. Biologically active compounds such as sesquiterpene lactones (SLs) have also been tested in reporter gene assays for their inXuence on gene expression. Here we show in in vitro and ex vivo experiments that SLs inhibit WreXy luciferase activity probably by direct targeting of the enzyme while -galactosidase remains almost completely unaVected. The loss of luciferase activity after SL treatment could be an eVect of their sulfhydryl-modifying potency and the subsequent alteration of the enzyme’s tertiary structure. These results demonstrate that the eVect of the test substance on the reporter enzyme used should be taken into consideration when the transcriptional eVect of novel compounds is investigated. 2004 Elsevier Inc. All rights reserved. Keywords: Reporter gene assay; Luciferase; -Galactosidase; Sesquiterpene lactones; Parthenolide
Chronic inXammatory diseases, such as rheumathoid arthritis, asthma, and inXammatory and autoimmune diseases, are characterized by an increased expression of inXammatory genes. These diseases result from an interplay of genetic and environmental factors. One of these factors is the ubiquitous transcription factor NF-B which plays a crucial role in immune and inXammatory responses and cell growth by regulating the expression of speciWc cellular genes [1,2]. NF-B is a dimeric transcription factor formed by the hetero- or homodimerization of proteins of the rel family [3]. It has been shown that activation of NF-B that is consistent with IB degradation, NF-B nuclear translocation, or binding to the DNA does not imply transcriptional activation. Depending on the activating stimulus, NF-B itself is subject to posttranslational modiWcations that can ¤
Corresponding author. Fax: + 49-761-203-8383. E-mail address:
[email protected] (I. Merfort). 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.021
enhance transcriptional activation of NF-B-dependent genes [2,4]. As NF-B plays a pivotal role in inXammation, antagonizing NF-B activity is a potential therapeutic target to treat immune and inXammatory diseases. It has been shown that numerous synthetic and natural compounds block NF-B activation through multiple mechanisms by interfering with various steps of the NF-B activation cascade [1,5,6]. Thus, there are diVerent methods for screening the ability of a compound to block NF-B activation. To examine the eVect of inhibitors on NF-B nuclear translocation and DNA binding, electrophoretic mobility shift assays (EMSA)1 are used and reporter gene assays are performed to investigate eVects on B-dependent transcription [7]. 1 Abbreviations used: EMSA, electrophoretic mobility shift assay; SLS, sesquiterpene lactones; CMV, cytomegalovirus; HSV-TK, Herpes simplex virus thymidine kinase; PBS, phosphate-buVered saline; BSA, bovine serum albumin.
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Two of the commonly used reporter enzymes are WreXy luciferase and -galactosidase from Escherichia coli. FireXy luciferase is a 62-kDa molecular weight oxygenase with known crystal structure [8] that generates a bioluminescent signal by catalyzing the oxidation of its substrate luciferin in a two-step process [7,8]. -Galactosidase from E. coli is a glycosidase which catalyzes the hydrolysis and transgalactosylation of -D-galactosides. It is a well-characterized tetrameric enzyme which consists of identical subunits with molecular masses of 116 kDa and possesses four binding sites for galactosides [9–11]. In most cases, it is assumed that the activity of these reporter enzymes is unaVected by the treatment conditions, and interference of the speciWc treatment with the reporter enzyme is normally not considered when interpreting the data obtained from these assays. But both enzymes, WreXy luciferase and -galactosidase, can be inactivated under certain conditions such as heat shock [12] or in the presence of proteasome inhibitors [13], alkylating substances [14,15], substrate analogues [9,16], or other compounds [17,18]. As mentioned above, NF-B has become an important target in drug development as inhibitors of this transcription factor could prove to be eYcacious antiinXammatory agents. Among the NF-B-targeting natural compounds, sesquiterpene lactones (SLs) deserve special interest. They are the active constituents of many medicinal plants from the Asteraceae family, whose preparations are used for the treatment of inXammation in traditional medicine. It has been shown in various assays that plant extracts and the puriWed SLs possess antiinXammatory properties [19]. The antiinXammatory activity of SLs is most often chemically mediated by , -unsaturated carbonyl structures, such as an -methylene--lactone or an ,-unsubstituted cyclopentenone. These functional groups are known to react with nucleophiles, especially with cysteine sulfhydryl groups, in a Michael-type addition [19]. We have recently demonstrated that SLs inhibit activation of NF-B by alkylating its p65 subunit at Cys38, which participates in DNA binding [20,21]. As not only inhibition of DNA binding by SLs is of interest, several studies have been carried out to examine their eVect on NF-B-driven gene expression by using reporter gene assays, especially with luciferase as a reporter [21–25]. However, in all these studies it was not assessed whether the WreXy luciferase itself is targeted by SLs, which cannot be excluded because of their alkylating properties. For this reason we investigated the eVects of two diVerent SLs on WreXy luciferase ex vivo and in vitro. Our investigations show that WreXy luciferase is inhibited by SLs and loses its activity in a concentrationdependent manner, whereas -galactosidase, which was alternatively studied, remains almost completely unaVected. Thus, use of SLs in combination with the luciferase reporter enzyme may lead to a reduction of
enzyme activity and to a misinterpretation of quantitative data.
Materials and methods Test compounds Parthenolide was purchased from Sigma. 4,15Epoxy-miller-9E-enolide was isolated from leaves of Milleria quinqueXora [26]. Plasmids and constructs pNFB-Luc (Clontech) contains four tandem copies of the NF-B consensus sequence fused to a TATA-like region from Herpes simplex virus thymidine kinase (HSV-TK) promoter in front of a luciferase gene (luc). pgal-Basic (Clontech) is a mammalian reporter vector that lacks eukaryotic promoter and enhancer sequences and serves as a promoter-cloning vehicle for strong promoters. pRC/CMV, a mammalian cloning and expression vector containing a cytomegalovirus promoter, was purchased from Invitrogen. pM1--Gal expression vector (Roche Diagnostics) contains E. coli -galactosidase under the control of the human cytomegalovirus immediate-early promoter/enhancer. pCL encodes WreXy luciferase under the control of a cytomegalovirus promoter. It was constructed by using pNF-B-Luc (Clontech) as a template for PCR ampliWcation of the luciferase gene (luc) with the forward primer 50-CAG AAG CTT GGC ATT CCG GTA C-30 (HindIII site) and reverse primer 50-GCG ACT CTA GAA TTA CAC GGC GAT C-30 (XbaI site). The forward and reverse primers contain a recognition site for HindIII and XbaI, respectively, which are underlined. The PCR product was digested with HindIII and XbaI and ligated into pRc/CMV (Invitrogen). pNG encodes -galactosidase from E. coli under the control of four tandem copies of the NF-B consensus sequence fused to a TATA-like promotor region from HSV-TK promoter. It was constructed by using pNF-B-Luc (Clontech) as a template for PCR ampliWcation of the NF-B promotor region with the forward primer 50-GTA GGC TGT CTC GAG TGC AAG TG-30 (XhoI site) and the reverse primer 50CGT TAT GCC AAG CTT CTG CTT CAT C-30 (HindIII site). The forward and reverse primers contain a recognition site for XhoI and HindIII, respectively, which are underlined. The PCR product was digested with XhoI and HindIII and ligated into p-gal-Basic (Clontech). Cell culture 293 cells were maintained in Dulbecco’s modiWed Eagle’s medium (Gibco), supplemented with 10% fetal
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bovine serum (Sigma), 100 IU/ml penicillin, and 100 g/ ml streptomycin (Roche Diagnostics). TNF- was purchased from Roche Diagnostics. Transient transfection and luciferase assay 293 cells were plated 12–16 h prior to transfection at a density of 1 £ 105 cells per each well in a six-well tissue culture dish and transfected with the newly constructed vector pCL. Transfections were performed by using SuperFect (Qiagen) according to the manufaturer’s recommendations. Two days after transfection, cells were treated with the test compounds for 1 h prior to harvesting. Cells were then harvested in lysis buVer (supplemented with the Luciferase Reporter Gene System from Roche Diagnostics) and WreXy luciferase activity was measured using the Luciferase Reporter Gene Assay System from Roche Diagnostics. Luciferase activity was then normalized to total cellular protein using the Protein Assay ESL (Roche Diagnostics). Experiments were performed in triplicate and all experiments were repeated at least three times. Transient transfection and -galactosidase assay 293 cells were plated on a 12-well tissue culture dish 12–16 h prior to transfection at a density of 5 £ 104 cells per each well and transfected with pM1--Gal (Roche Diagnostics). Transfections were performed by using SuperFect (Qiagen) according to the manufacturer’s recommendations. Forty-eight hours after transfection cells were treated with the test compounds for 1 h prior to harvesting. Cells were then harvested in lysis buVer (supplemented with the -Gal Reporter Gene System from Roche Diagnostics) and -galactosidase activity was measured using the -Gal Reporter Gene System from Roche Diagnostics. -Galactosidase activity was then normalized to total cellular protein using the Protein Assay ESL (Roche Diagnostics). Data represent means § standard deviation from at least three independent measurements in triplicate. For monitoring the NF-B-dependent gene expression, 293 cells were plated on a 12-well tissue culture dish 12–16 h prior to transfection at a densitiy of 1 £ 105 cells per each well and transfected with the newly constructed vector pNG. Forty-eight hours after transfection cells were treated with the test compounds for 1 h and subsequently stimulated for 3 h with 5 ng/ml TNF-, after which lysates were prepared and measured for galactosidase activity as described above. All experiments were performed in triplicate and were repeated at least three times. Determination of the transfection rate by X-gal staining 293 cells were plated on a 12-well tissue culture dish 12–16 h prior to transfection at a density of 5 £ 104 cells
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per each well and transfected with pM1--Gal (Roche Diagnostics). Transfections were performed by using SuperFect (Qiagen) according to the manufacturer’s recommendations. Forty-eight hours after transfection cells were washed twice with PBS and then Wxed with 0.25% (v/v) glutaraldehyde solution for 10 min at room temperature. After washing three times with PBS cells were stained with X-gal reagent (100 mg/ml 5-bromo-4chloro-3-indolyl--D-galactoside in dimethyl-formamide), diluted 1:100 with X-gal solution (5 mM K3Fe (CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2), and incubated in the dark at 37 °C for 3 h. Analysis of X-gal staining demonstrated a transfection eYciency of 40–50%. In vitro luciferase assay To study the direct inhibition of the enzyme, recombinant WreXy luciferase (Promega) was diluted in Luciferase Reporter Gene Assay lysis buVer (Roche Diagnostics), supplemented with 1 mg/ml BSA (Sigma), to obtain a concentration of 1 ng/15 l. This luciferase solution was treated with the test compounds for 1 h at room temperature and then the activity was assayed with the Luciferase Reporter Gene Assay. Experiments were performed in triplicate and were repeated at least two times. In vitro -galactosidase assay To investigate the direct inhibition of the -galactosidase enzyme, recombinant -galactosidase from E. coli (Roche Diagnostics), diluted in -Gal Reporter Gene Assay lysis buVer (Roche Diagnostics), was used at a concentration of 0.05 ng/l and treated with the test compunds for 1 h at 37 °C; then -galactosidase activity was assayed with the -Gal Reporter Gene Assay. Experiments were performed in triplicate and were repeated at least two times. Statistical analysis Statistical analysis was performed using the Origin 7.0 software. Data are reported as means § SD and analyzed using an independent t test (two groups); p 0 0.05 is considered statistically signiWcant (*p 0 0.05, **p 0 0.005 versus positive control, op 0 0.05, oop 0 0.005 comparison between in vitro and ex vivo data.
Results Sesquiterpene lactones aVect luciferase activity To investigate whether SLs interfere with luciferase activity, 293 cells were transiently transfected with a luciferase reporter gene driven by the cytomegalovirus promoter. These transfected cells were left untreated or
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treated for 1 h at 37 °C with diVerent concentrations of two SLs, parthenolide or 4,15-epoxy-miller-9E-enolide (Fig. 1). Cells were subsequently harvested and luciferase activity was determined in total cell extracts and normalized to total protein concentration (Figs. 2A and B). Luciferase activity was inhibited by both SLs in a concentration-dependent manner. Compared with the untreated cells, which produce luciferase constitutively, treatment with 40 M parthenolide or 10 M 4,15epoxy-miller-9E-enolide led to a loss of »80% of luciferase activity. To exclude cytotoxic eVects of the SLs, cell viability after treatment with parthenolide or 4,15epoxy-miller-9E-enolide tested with trypan blue staining was more than 90% (data not shown). To conWrm that inactivation of luciferase activity was due to a direct inhibition of the enzyme, and not to any other phenomenon, recombinant WreXy luciferase was incubated for 1 h at room temperature with the same concentrations of the SLs and then its activity was assayed (Figs. 2A and B). Recombinant luciferase was also inhibited in a concentration-dependent manner. In contrast to the ex vivo data, the loss of luciferase activity in the in vitro experiments was lower at the same SL concentration. This could be due on the one hand to the lower incubation temperature (20 °C instead of 37 °C with all cell cultures), given that the enzyme loses its activity at higher temperatures [12]. On the other hand, SLs could bind to components of the reaction mixture such as BSA, which is added in the in vitro experiment at a 1 mg/ml concentration as a stabilizer for recombinant luciferase. Data obtained in our group show that SLs bind to BSA (S. Wagner, current thesis). This would lead to smaller amount of SLs available for reaction with luciferase. Interestingly, 4,15-epoxy-miller-9E-enolide shows bimodal behavior in the ex vivo data (Fig. 2B). To explore this phenomenon a time course using the two lowest concentrations was carried out. Transiently transfected cells were incubated with 1 or 2.5 M 4,15epoxy-miller-9E-enolide for various time intervals at 37 °C and subsequently harvested, and luciferase activity was determined in total cell extracts (Figs. 3A and B). The same procedure was performed with the recombinant enzyme at 20 °C. At a 1 M concentration, a
Fig. 2. Reduction of luciferase activity ex vivo ( ) and in vitro (䊏) by parthenolide (A) and 4,15-epoxy-miller-9E-enolide (B). For the ex vivo experiments, 293 cells were transiently transfected with pCL. Transfected cells were left untreated (+) or treated with the indicated concentrations of parthenolide [5–40 M] (A) or 4,15-epoxy-miller9E-enolide [1–10 M] (B) for 1 h. Subsequently, cells were harvested and WreXy luciferase activity was measured using the Luciferase Reporter Gene Assay System from Roche Diagnostics. Mean values from at least three independent experiments performed in triplicate are shown. In in vitro test, recombinant luciferase at a concentration of 1 ng/15 l was incubated with the same concentrations of parthenolide (A) or 4,15-epoxy-miller-9E-enolide (B) for 1 h at room temperature or left untreated (+). Mean values from at least two independent experiments performed in triplicate are shown.
decrease of luciferase activity was observed only for the ex vivo data. For the higher concentration of 2.5 M a slight activity loss with increasing time was observed in the in vitro experiments, while the ex vivo data showed bimodal behavior. -Galactosidase activity remains nearly unaVected by sesquiterpene lactones
Fig. 1. Structures of the investigated sesquiterpene lactones.
To Wnd an alternative reporter enzyme for monitoring NF-B-dependent gene expression we investigated whether SLs would interfere with -galactosidase activity. For the ex vivo experiments, 293 cells were
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Fig. 3. Time course of the reduction of luciferase activity ex vivo ( ) and in vitro (䊏) by 1 M (A) or 2.5 M (B) 4,15-epoxy-miller-9E-enolide. For the ex vivo experiments, 293 cells were transiently transfected with pCL. Transfected cells were left untreated (+) or treated with 1 M (A) or 2.5 M (B) of 4,15-epoxy-miller-9E-enolide for the indicated time (15–120 min). Subsequently, cells were harvested and WreXy luciferase activity was measured using the Luciferase Reporter Gene Assay System from Roche Diagnostics. Mean values from at least three independent experiments performed in triplicate are shown. In the in vitro test, recombinant luciferase at a concentration of 1 ng/l was incubated with 1 M (A) or 2.5 M (B) of 4,15-epoxy-miller-9Eenolide at room temperature for the same incubating times or left untreated (+). Mean values from at least two independent experiments performed in triplicate are shown.
transiently transfected with pM1--Gal plasmid, which contains E. coli -galactosidase under the control of the human cytomegalovirus immediate-early promoter/ enhancer. The transfected cells were left untreated or treated for 1 h at 37 °C with various concentrations of parthenolide or 4,15-epoxy-miller-9E-enolide. -Galactosidase activity was determined in the total cell extracts and normalized to total protein concentration (Figs. 4A and B). Both parthenolide and 4,15-epoxy-miller-9Eenolide showed negligible inhibiting eVects on -galactosidase. To corroborate these data recombinant E. coli -
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Fig. 4. InXuence of parthenolide (A) and 4,15-epoxy-miller-9E-enolide (B) on -galactosidase activity ex vivo ( ) and in vitro (䊏). For the ex vivo experiments, 293 cells were transiently transfected with pM1-Gal. Transfected cells were left untreated (+) or treated with the indicated concentrations of parthenolide [5–40 M] (A) or 4,15epoxy-miller-9E-enolide [1–10 M] (B) for 1 h. Subsequently, cells were harvested and -galactosidase activity was measured using the -Gal Reporter Gene System from Roche Diagnostics. Mean values from at least three independent experiments performed in triplicate are shown. In the in vitro test, recombinant -galactosidase at a concentration of 0.05 ng/l was incubated with the same concentrations of parthenolide (A) or 4,15-epoxy-miller-9E-enolide (B) for 1 h at room temperature or left untreated (+). Mean values from at least two independent experiments performed in triplicate are shown.
galactosidase was treated with the two substances for 1 h at 37 °C. The same results as those in the ex vivo experiments were obtained. Sesquiterpene lactones inhibit NF-B-dependent gene expression Given that luciferase is directly attacked by SLs, the strength of inhibition of NF-B-driven gene expression after incubating with these substances can be misinterpreted. Luciferase is not the most appropiate enzyme in
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a reporter gene assay for alkylating compounds, such as SLs. As a consequence, previous analyses that used luciferase as a reporter gene to assign an inhibitory role to SLs on NF-B-dependent transcription must be reassessed [23,27]. -Galactosidase was not aVected by SLs. For this reason, we studied their inXuence on NF-Bdriven gene expression with a -galactosidase reporter system. We transiently transfected 293 cells with a galactosidase reporter gene under the control of four tandem copies of the NF-B consensus sequence fused to a TATA-like promotor region from Herpes simplex virus thymidine kinase promoter. These cells were incubated with various concentrations of parthenolide or 4,15-epoxy-miller-9E-enolide for 1 h and stimulated for 3 h with 5 ng/ml TNF-. Cells were subsequently harvested; -galactosidase activity was determined in total
Fig. 5. Inhibition of NF-B-dependent gene expression by parthenolide (A) or 4,15-epoxy-miller-9E-enolide (B). 293 cells were transiently transfected with pNG. Transfected cells were left untreated (¡) or stimulated with 5 ng/ml TNF- alone for 3 h (+) or pretreated for 1 h with the indicated concentrations of parthenolide (A) or 4,15-epoxymiller-9E-enolide (B) and subsequently stimulated with 5 ng/ml TNF- for 3 h. Cells were then harvested and -galactosidase activity was measured using the -Gal Reporter Gene System from Roche Diagnostics. Mean values from at least three independent experiments performed in triplicate are shown.
cell extracts and normalized to total protein concentration (Figs. 5A and B). The induction of the NF-B transcription by stimulating cells with TNF- was suppressed in a concentration-dependent manner. No cytotoxic eVects were observed. Cell viability was higher than 90% after pretreatment with SLs and stimulation with TNF- (data not shown). Furthermore, whether constitutively expressed -galactosidase produced from pM1--Gal remains unaVected after treatment with 20 M parthenolide or 10 M 4,15-epoxy-miller-9Eenolide was tested. Even after 4 h of treatment, -galactosidase was not aVected (data not shown).
Discussion Reporter enzymes such as chloramphenicol acetyltransferase, WreXy luciferase, and -galactosidase from E. coli are frequently used for studies of gene regulation, gene activity, and expression in eukaryotic cells [7]. In particular WreXy luciferase has been extensively applied in molecular and cell biology studies because of its negligible background and high sensitivity and the relative simplicity of the assay [28,29]. These reporter enzymes are used with diVerent cell types and treatment conditions and, in general, the interference of the experimental treatment on the reporter enzyme activity is not considered. The biologically active SLs belong to those natural products that have also been tested in reporter gene assays for their inXuence on gene expression [21,22,24, 25,30]. Although their alkylating potency is known, until now studies on the eVect of SL treatment on the activity of reporter enzymes such as luciferase or -galactosidase have not been conducted. Here we show in in vitro and ex vivo experiments that two diVerent SLs, parthenolide and 4,15-epoxy-miller-9E-enolide, inhibit WreXy luciferase activity by directly targeting the enzyme, while galactosidase from E. coli remains almost completely unaVected (Figs. 2A, B and 4A, B). Because of the short half-life of luciferase (t1/2 D 3–4 h in mammalian cells) [31] an incubation time of 1 h was used. A higher inhibition will probably be observed with longer incubation times. FireXy luciferase possesses four cysteine residues (Cys81, Cys216, Cys285, Cys391). One cysteine residue (Cys391) is located close to the active site, while the others are further away [8]. Even though mutagenesis studies could show that cysteine residues are not absolutely required for bioluminescence activity [15], signiWcant changes in luciferase activity were observed after treatment with the alkylant N-tosyl-L-phenylalanine chloromethyl ketone or cysteine mutagenesis [15], suggesting that an alteration in the tertiary structure of the enzyme could aVect the active site. Consequently, the loss of luciferase activity after SL treatment could also be an eVect of their sulfhydryl-modifying potency and
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the subsequent alteration of the enzyme’s tertiary structure. However, it cannot be excluded that additional reactions of SLs with other amino acids, such as lysine, may also contribute to the inhibitory eVect. This would be in accordance with recent studies, which showed that SLs do not solely react with the exposed sulfhydryl group in human serum albumin (S. Wagner, current thesis). Concerning the bimodal behavior of the SL 4,15epoxy-miller-9-E-enolide, further investigations are required to explain this unexpected result. Despite the fact that -galactosidase possesses 16 cysteine residues in the reduced form per subunit [10], a negligible eVect on its activity could be observed with the two SLs. This result shows that SLs diVer from the alkylating reagents such as iodoacetamide or bromoacetamide which reversibly alkylate the -galactosidase at a methionyl residue near the active site [32]. The X-ray structure of -galactosidase [10] showed that three cysteine residues (Cys402, Cys500, Cys536) are situated near the active site of the enzyme [9,10], while the remaining cysteine residues are distributed on the enzyme surface or in an inaccessible position or at some distance from the active site. Therefore, alkylation of residues located on the surface should not aVect the enzyme activity, as an alteration of the tertiary structure is not expected. Consequently, only the three cysteine residues near the active site could have eVects on the catalytical activity after modiWcations with an alkylating reagent. As no inhibition of the enzyme was observed, a modiWcation of these residues can be excluded or, at least, they should not aVect the enzyme’s activity. This result shows that, although SLs are unspeciWc alkylating reagents, they still possess some selectivity for their target residues. By comparing Figs. 2–4, it becomes evident that inhibitions in ex vivo experiments are nearly always stronger than those in in vitro. This might be explained by a negative eVect of SLs on the CMV promoter. The strong cytomegalovirus promoter is often used in expression plasmids for transient transfection experiments and in viral vectors for infection experiments. Its enhancer region contains many cis-acting elements and its activity is stimulated by several cellular transcription factors, such as NF-B/rel, AP-1, and CREB/ATF [33,34]. We have previously demonstrated that activation of the transcription factors NF-B and AP-1 is inhibited by SLs [20]. Therefore, an inhibition of these transcription factors could lead to a lower CMV promoter activity and subsequently to detection of lower reporter enzyme activities. As -galactosidase appeared to be a more reliable reporter than luciferase for use with SLs, we used this reporter gene assay and showed that both SLs prevent the induction of B-dependent gene expression in a concentration-dependent manner (Figs. 5A and B). The data correlated with those obtained in the NF-B EMSA, in
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which 4,15-epoxy-miller-9E-enolide also prevented NF-B DNA binding at a lower concentration than parthenolide [35]. In summary, we provided evidence that SLs directly interfere with luciferase while -galactosidase remains unaVected after treatment, suggesting that for alkylating reagents, such as SLs, -galactosidase rather than luciferase is the reporter enzyme of choice to investigate the inXuence on the transcriptional activity and to avoid misinterpretation of results. Data obtained with SLs in the luciferase reporter gene assay should carefully be considered and, if used, should be used only for qualitative and not quantitative analysis. Thus, IC50 values deduced from experiments with SLs in the luciferase reporter gene assay should be avoided [36]. Together with previous reports our studies underline the necessity to include a control that monitors the direct eVect of the experimental conditions on the reporter enzyme before using it in a reporter gene assay.
Acknowledgments We gratefully acknowledge the Wnancial support of the Volkswagen-Stiftung. MTL acknowledges the Landesgraduiertenförderung Baden-Württemberg for a scholarship. AGP thanks funding by the Deutscher Akademischer Austauschdienst and the Baden-Württemberg Stipendium.
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