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METHODS IN CELL PHYSIOLOGY
1Department of Pharmacology and Therapeutics, University of Liverpool, United Kingdom; 2Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; and 3Department of Human Anatomy and Cell Biology, University of Liverpool, and 4Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom
Submitted 21 March 2005 ; accepted in final form 26 August 2005
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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B]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable. liver cells; proteins; CYP2E1; NRF2; GSTP1
The most promising and widely used technique for regulating heterologous gene expression has been the tetracycline-regulated gene expression system (16). The technique consists of consecutive transfections of the target cells with two transgenes, the first of which encodes a tetracycline-controlled transactivator protein (tTA) and the second of which is the modified gene of interest, with a tetracycline response element (TRE) in its 5' regulatory sequence. In the presence of tetracycline, or typically its more stable derivative doxycycline (dox), tTA is prevented from binding the TRE of its target gene and the system is inactive (tet-off system; Ref. 16). In an alternative version of this system, a mutated tTA, i.e., reverse tTA (rtTA), is used, whereby amino acid substitutions in the tTA protein yield a transcriptional activator in the presence of dox (17, 20, 32). This tet-on system has a number of inherent advantages over the tet-off approach: dox does not need to be permanently present in the cell environment to maintain the repressed state, as is the case with the tet-off cells. In addition, the need to remove all of the dox to activate genes has caused problems of variability in gene induction experiments using the tet-off system.
Although the tet-on approach circumvents these difficulties, the use of the first-generation tet-on system has been hampered by the phenomenon of "leakiness," which was sometimes observed to cause high background expression of inducible genes in the absence of dox (11, 12), and also the unsuitability of the tet-on approach in certain cell types, such as hepatocytes (13, 38). To address these issues, various modifications of the tet-on system have been tested, e.g., use of a silencer protein to reduce noninduced expression (12) and synthesis of a novel mutant rtTA protein (41). This second-generation rtTA, termed rtTA2S-M2, has enhanced trans-activation potential and a reduced affinity for the target gene promoter in the absence of dox (41). It has been tested in transgenic animals (26, 44) but has still not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. We have therefore compared the applicability of this novel system relative to the first-generation tet-on approach in two human and one mouse hepatoma cell lines and found that the new transactivator enables the generation of hepatocyte-derived cell lines in which expression of genes is fully controllable.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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The rtTA2S-M2 plasmid was described previously (10). The protein expressed from this plasmid is activated by dox to form a transcriptional activator capable of triggering transcription of genes with a series of tet operator (tetO) sequences aligned to form a TRE coupled to a cytomegalovirus promoter upstream of their transcription start site. Sequences corresponding to the full-length coding sequences of the genes investigated were obtained as follows: human glutathione S-transferase (GST) P1 (GSTP1) was amplified from a human lymphocyte cDNA library; mouse nuclear factor erythroid 2 p45-related factor (NRF)2 was amplified from a mouse NRF2 expression vector (kind gift of Ken Itoh and Masayuki Yamamoto, University of Tsukuba, Japan); human NFKB1 subunit of NF-B and human NRF1 were amplified from IMAGE clones obtained from the Medical Research Council (Cambridge, UK). PCR amplicons of each of these genes were obtained with MluI and NotI restriction tags to enable directional cloning into corresponding sites in the multiple cloning sequence of the dox-responsive pTRE2-hyg vector (Clontech, Oxford, UK). The identity and orientation of each of the coding sequences within each of the recombinant plasmids were checked by DNA sequencing of each of the strands. The rabbit cytochrome P-450 2E1 (CYP2E1) coding sequence within the dox-responsive vector pUHD10–3 was a kind gift of Lisa Bleyle and Dennis Koop (University of Oregon, Portland, OR) and was described previously (21).
Cell Transfection
One day before transfection, cells were seeded at a density of 0.5 x 106 cells/well in six-well culture plates. Stable transfection of the rtTA2S-M2 plasmid into the human hepatoma cell lines HuH7 and HepG2 and the mouse hepatoma cell line Hepa1c1c7 was carried out with Genejuice reagent (Novagen, Nottingham, UK) essentially as described by the manufacturer [1 µg/well at a ratio of 1:3 of DNA to Genejuice (wt/vol)]. Two days after transfection, cells were selected for rtTA2S-M2 integration with G418 antibiotic treatment for 3–4 wk (500 µg/ml for each cell line). Clones were selected for low basal activity and high absolute levels of induction in the luciferase assay, expanded, and cryopreserved. Transient transfections were carried out with Genejuice. Cells were lysed 16, 24, or 48 h after transfection and monitored for expression of the genes of interest. Stable transfections with the genes of interest were carried out as for the rtTA2S-M2 plasmid transfections, using hygromycin for selection (250 µg/ml for each of the clones tested).
Luciferase Assay
Clones were tested for dox inducibility by transient transfection of 0.5 x 105 cells of each clone seeded in flat-bottomed 96-well plates, with 50 ng of pTRE2-luc dox-dependent luciferase reporter plasmid (Clontech). Four hours after transfection, clones were treated or not with 1 µg/ml dox in growth medium for at least 16 h. Cells in each well were then lysed with cell lysis buffer (Promega, Southampton, UK), BrightGlo luciferase substrate buffer (Promega) was added, and the chemiluminescence was measured in a plate reader (PerkinElmer, Pangbourne, UK) and recorded as arbitrary light units.
Western Blotting
Cell lysates were obtained from cells transiently or stably transfected with genes of interest by lysis in a Triton X-100 cell lysis buffer, followed by centrifugation at 1,500 g to pellet and remove the nuclei. Protein concentrations of the enucleated lysates were estimated by the method of Bradford (3). Equal amounts of protein (typically 10 µg) were separated by denaturing electrophoresis and detected and quantified by standard procedures.
Immunofluorescence Detection
A stable rtTA2S-M2-HuH7 clone transiently transfected with the CYP2E1-inducible plasmid was grown in a LabTek II chamber slide system and treated or not with 1 µg/ml dox for 16 h. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 10% fetal bovine serum in PBS, they were incubated with the anti-CYP2E1 antiserum (diluted 1:200 in 2% fetal bovine serum in PBS) at 37°C for 1 h, washed with PBS, stained with fluorescein isothiocyanate-conjugated anti-rabbit antibody (diluted 1:200 in 2% fetal bovine serum in PBS), and then washed with PBS. Immunofluorescence microscopy analysis was performed with a Nikon E600. For detection of stably transfected NRF2, the HepG2-NRF2-WT19 cells were grown in a LabTek II and treated or not with 1 µg/ml dox for 16 h. Cells were then fixed, permeabilized, and stained with anti-NRF2 antiserum. Hoechst 33258 stain was included in the final washes of the cells to visualize the nuclei. Immunofluorescence confocal microscopy analysis was performed with a Leica SP2 AOBS.
Analysis of CYP2E1 Activity
For the measurement of p-nitrophenol hydroxylation by the method of Reinke and Moyer (35), cells were incubated at 37°C for 2 h in 100 µl of Hanks' buffered saline containing 200 µM p-nitrophenol. To this was added 50 µl of 0.06 M perchloric acid. After vortexing and centrifugation, 100 µl of the supernatant was added to a 96-well plate with 50 µl of 10 M sodium hydroxide. The plate was read at 550 nm and quantified with p-nitrocatechol standards. For the measurement of chlorzoxazone hydroxylation, a method adapted from that of Peter et al. (34) was used. Briefly, cells were suspended in 6 ml of hepatocyte incubation buffer containing chlorzoxazone (200 µM) and incubated at 37°C, with gentle shaking, for 3 h. The reactions were terminated by the addition of 6 ml of acetonitrile and internal standard (2-benzoxazolinone) and analyzed by reverse-phase HPLC.
Analysis of GST Activity
Aliquots of lysates were placed in a 96-well plate with 320 µl of potassium phosphate buffer (0.1 M; pH 6.5) containing 1.5 mM 2,4-dinitrochlorobenzene (DNCB) and 1.5 mM GSH. The formation of the DNCB-glutathione conjugate was determined from the increase in absorbance at 340 nm, according to Habig and Jakoby (19).
Isotope-Coded Affinity Tag Analysis
A cell extract enriched for microsomal proteins was prepared from a CYP2E1-expressing clone by differential centrifugation (23). An aliquot of 100 µg was denatured, reduced, labeled with heavy isotope-coded affinity tag reagent, and digested with trypsin per the manufacturer's instructions (Applied Biosystems) (18). After cation exchange and avidin affinity chromatography, the sample was subjected to mass spectrometry (MS/MS) analysis.
Two-Dimensional Gel Electrophoresis
Cells were incubated under the indicated conditions, washed twice with ice-cold PBS, harvested in PBS by gentle scraping, and then washed several times in PBS. The final cell pellet was lysed in 70 µl of isoelectric focusing sample buffer [7 M urea, 2 M thiourea, 4% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris, 1% (wt/vol) DTT], subjected to a freeze-thaw cycle at –70°C, and centrifuged at 17,000 g and 4°C for 30 min. The supernatant was assayed for protein content and separated by two-dimensional (2D)-PAGE, following the method of Gorg et al. (15). The gels were stained with colloidal Coomassie blue and scanned with a GS-710 imaging densitometer (Bio-Rad, Hemel Hempstead, UK).
Characterization of Protein Spots
The putative GSTP1 was excised from the 2D gel and subjected to in-gel tryptic digestion by the method of Courchesne and Patterson (6) before MS. Confirmation of identity of the protein was achieved by nano-liquid chromatography (LC)-electron spray ionization (ESI)-MS/MS analysis.
3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl-2H-Tetrazolium Bromide Assay for Cell Viability
Cells were seeded at a density of 1 x 104/well in a 96-well plate and treated with dox for 24 h. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was then added to each well (to a final concentration of 1 µg/ml) and incubated at 37°C for 4 h. An equal volume of lysis buffer was then added to the wells and incubated at 37°C for 4 h. Absorbance in each well was then measured at 570 nm.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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We successfully established dox-inducible hepatoma cells with the second-generation tet-on activator in two human cell lines, HepG2 and HuH7, and a mouse hepatoma cell line, Hepa1c1c7. As found previously by other groups, we were unable to develop tet-on inducible hepatocyte-derived cells with the conventional first-generation rtTA tet-on activator and/or the silencer protein, tTSE (data not shown). It is known that overexpression of the VP16 domain in the rtTA protein can titrate out components of the normal transcription machinery, resulting in "squelching" of components of general transcription, e.g., TFIIB (28), TFIID (39), and TFIIH (43). We therefore tested the applicability of a recently described transcriptional modulator, rtTA2S-M2, in which the full-length VP16 domain has been substituted with a multimerized acidic peptide derived from VP16 itself and the sequence has been optimized to improve expression (41). Stable clones were selected and tested for dox inducibility by transient transfection with a luciferase expression plasmid under the control of a TRE. Representative screening experiments are shown in Fig. 1. These data indicate that rtTA2S-M2 is suitable for use as a regulator within the hepatocyte-derived cells tested. The selected clones from each of the cell types have much higher levels of expression of the luciferase reporter, as well as greater differences in the fold induction, compared with our data from the conventional rtTA activator alone or in combination with the tTSE silencer. From low basal activity, the induction of clones that were considered to be potentially useful ranged from >10-fold up to >1,000-fold.
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Transient expression.
We next tested some of the selected human clones for inducibility of transiently transfected heterologous genes known to be involved in a number of different pharmacologically relevant processes in the liver, namely phase I metabolism, i.e., CYP2E1, and phase II metabolism, i.e., GSTP1 and transcription factors responsible for sensing chemical stress signals, i.e., NRF2, NRF1, and the NFKB1 subunit of NF-B. Expression of a rabbit CYP2E1 sequence was successfully achieved in our HepG2 and Huh7 clones (Fig. 2A); parental HepG2 cells are known not to express CYP2E1 (7); nothing has been published regarding expression of CYP2E1 in HuH7 cells. We did not detect CYP2E1 in either cell type before transfection, as shown by Western blotting and immunofluorescence staining (Fig. 2A). On the basis of enzyme kinetic parameters, few differences exist among mammalian species with respect to activity of the CYP2E1 enzyme (2); therefore, the rabbit CYP2E1 was deemed a useful model of human CYP2E1 activity. Inducible expression was also achieved for human GSTP1 in our HepG2 clones (see Fig. 2B), the parental form of which does not express detectable GSTP1 (30). Induction over 48 h did not increase expression above that observed after 24 h. Transient transfection of mouse NRF2 also yielded dox-inducible expression of this transcription factor (Fig. 2C). A mouse NRF2 sequence was used here because these cells express endogenous NRF2 (this is barely detectable where cells have not been exposed to any chemical stress because the protein is constitutively degraded before activation; for reviews see Refs. 31 and 25). Levels of expression within our cells appeared comparable with those achieved with a positive control tet-on rtTA2S-M2-transfected HeLa clone, HR2sM2, as previously described (Ref. 24; compare NRF2 induced in the HuH7 clone with that in the HeLa clone in Fig. 2C). Transient transfection of genes encoding human NRF1 and NFKB1 also yielded dox-inducible expression of these transcription factors (Fig. 2, D and E), indicating the utility of these cells for a variety of genes.
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16-h exposure to 1 µg/ml dox, although GSTP1 can be detected after just 4-h exposure (Fig. 6A). Maximal GSTP1 expression was observed at 1 µg/ml dox (Fig. 6B), although expression can be seen to be increased above baseline at 10 ng/ml dox. In both time course and dose-response experiments, induction of GSTP1 correlated with its GST activity, measured by DNCB conjugation with glutathione, indicating that the expressed GSTP1 was functionally active.
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The inducible CYP2E1-, GSTP1-, and NRF2-expressing clones have been cryopreserved and regrown for as many as 25 passages without obvious loss of induction, confirming the robustness of the rtTA2S-M2 activator approach in these cells (data not shown). rtTA2S-M2-expressing clones of each cell type were also checked to ensure that the levels of dox used for induction (normally 
1 µg/ml) did not cause cytotoxicity. In a MTT assay, no toxicity was observed up to 3 µg/ml dox treatment (3 times that generally required for maximum induction) after 24 h of exposure (data not shown).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Our approach yields robust and highly inducible hepatocyte-derived cells, which appear to be excellent host cells for the genes that were tested. We did not find it necessary to use a virally mediated gene transfer technique, as was recently shown for establishment of an alternative tet-on regulation system in HepG2 cells (27). Our method is therefore amenable to development in most laboratories, without the need for specialized vector transfection facilities. Each cell line that had been selected for further study exhibited low uninduced and high absolute levels of expression, providing ample scope for precisely controlling gene expression levels, because in each case the extent of dox induction was related directly to dose. In addition, because induction does not rely upon the removal of an inhibitor, long-term culture in the presence of inhibitor is not required, avoiding possible compensatory changes in the cells. To determine the utility of these clones, they were tested with five genes of relevance to liver biology, which were all found to be expressed at the transient level. Stable clones of cells with three of these genes, i.e., CYP2E1, GSTP1, and NRF2, were created, and these exhibited temporal and dose-dependent regulation of gene expression. The gene products were expressed to high levels and were shown to be functionally active. With global protein analysis, it was possible to detect many protein changes as a consequence of expression of one of these genes, GSTP1. These changes were related to the duration and/or extent of expression of the GSTP1. In fact, GSTP1 is well recognized as a multifunctional protein, capable not only of conjugation of GSH with a variety of electrophilic compounds (22, 37) but also of modulating other biochemical pathways, such as intracellular signaling processes, including the jun kinase pathway (1, 8, 36), and activation of the essential antioxidant protein peroxiredoxin (29). We are therefore presently using this novel system to investigate the nature of the changes observed on GSTP1 induction.
The maximal rate of reaction of our CYP2E1-expressing cells, i.e., 399 pmol 6-hydroxyclorzoxazone formed·min–1·mg total cell protein–1, is higher than that reported for the only other known inducible CYP2E1 cell line, i.e., human HeLa tetHeLa2E1–12 cells (21), which exhibit an activity of 158 pmol·min–1·mg total cell protein–1. Although Chen and Cederbaum (5) previously developed a HepG2 cell line expressing CYP2E1 at approximately 10-fold higher levels than those seen in the HeLa inducible cells, expression of CYP2E1 in these cells caused significant growth retardation effects compared with parental cells. Our system therefore potentially enables the setting of a more physiologically relevant level of expression with which to explore the role of CYP2E1 in drug metabolism and toxicity.
The development of a fully NRF2-inducible cell line may prove useful in further defining the role of this facilitator of cell defense. We (14) and others (4, 9) have shown the importance of NRF2 in the rapid response of the liver to chemical stress elicited by prototypic hepatotoxicants such as acetaminophen. We are presently exploring, with a proteomic approach, the effect of controlling the level of NRF2, to help us define the critical chemical changes in NRF2 and its interacting partners that are thought to be necessary for its activation and the triggering of the cellular defense response.
Importantly, the use of proteomic analysis is to our knowledge the first report of a study of dox induction on the cell proteome and indicates that the levels of dox necessary for protein synthesis do not appear, by visual inspection and semiautomated spot analysis with ImageMaster software of 2D gels, to have a statistically significant effect on the expression of other proteins.
An earlier report indicated toxicity of dox at levels as low as 0.2 µg/ml in PC-12 cells (10), although toxicity was not observed until 96 h after exposure of cells to dox. No cytotoxic effects of dox were observed on our rtTA2S-M2-expressing clones of any of the three cell lines tested, over a 24-h period, which is the typical duration of our experiments, at concentrations up to 3 µg/ml, i.e., generally above those found to be necessary for maximal heterologous gene expression.
The approach described here may provide a useful tool in drug discovery and development programs or for chemical toxicity studies. Idiosyncratic liver damage is a major problem in drug development because testing of inbred animals does not provide the variation in biochemical profile seen in the human population. Furthermore, toxicity test systems based on cell lines or even primary hepatocytes may not possess critical activation or defense pathways (33, 40). Moreover, the difficulty in understanding the mechanisms underlying idiosyncratic hepatotoxicity is not just that there are no good animal models but that most humans are not good models (42). Our approach is readily manipulable such that individuals at the extremes of the biochemical spectrum may be accurately modeled. Moreover, this system would be applicable to hepatotoxicity tests incorporating mutant forms of drug-activating or -metabolizing proteins, as well as highly variable levels of wild-type proteins.
In summary, we have produced hepatoma cell lines incorporating a dox-inducible gene regulation mechanism. These cells are amenable to the controlled expression of individual gene targets and should prove valuable physiological, pharmacological, and toxicological tools for defining the functional roles of proteins involved in liver cell biology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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