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Construction of Several Human-derived Stable Cell Lines Displaying Distinct Profiles of CYP3A4 Induction
Drug Metab. Pharmacokinet. 21 (2): 99–108 (2006).
Regular Article Construction of Several Human-derived Stable Cell Lines Displaying Distinct Proˆles of CYP3A4 Induction Wachiraporn NORACHARTTIYAPOT1, Yoko NAGAI1, Tsutomu MATSUBARA1,2, Masaaki MIYATA1, Miki SHIMADA1, Kiyoshi NAGATA1,* and Yasushi YAMAZOE1,2 1Division
of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2Comprehensive Research and Education Center for Planning of Drug Development and Clinical Education, Tohoku University 21st Century Center of Excellence Program. Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Cell lines which stably express reporter proteins through CYP3A4 gene activation have been developed for use in predicting CYP3A4 induction. Twelve clones showing distinct proˆles on chemicalinduced response were isolated. Among them, two clones showing high response for CYP3A4 inducers, namely clone 3-1-10 and 3-1-20, were further evaluated for their sensitivities, reproducibilities and applicabilities to predict CYP3A4 induction in human. Clone 3-1-10 showed higher response to rifampicin than to clotrimazole, whereas clone 3-1-20 had rather higher response to clotrimazole. Optimal plating density and highly reproducible response were observed at the range of 1.65–5.0×104 cell W cm2. Clear induction responses of more than ten chemicals were observed in both cell lines. The reporter activity was further dramatically increased after an introduction of human PXR. Induction with rifampicin was, however, not much altered between the absence and presence of hPXR. The luciferase activity remained unaltered and showed little ‰uctuation during the culture for more than 6 months. Due to the strikingly high sensitivity and reproducibility of this system, as compared to previously published systems, these HepG2-derived cell lines showing distinct response proˆles as developed in the present study will oŠer high advantages for chemical screening of CYP3A4 inducibility.
Key words: CYP3A4; induction; PXR; reporter assay; stable cell lines chemicals have been required. Currently, in vitro and in vivo assay systems are available. A large diŠerence, however, exists in the xenobiotic-induced response among experimental animal species and humans.7) Recently, a ligand-activated transcriptional factor, pregnane X receptor (PXR), has been shown to mediate the eŠects of xenobiotics on ligand-mediated expression of CYP3A genes in humans as well as in other experimental animals.8–10) PXR is also reported to be one of the major factors for the species diŠerences in the induction response.10,11) Human hepatocytes or liver slices have been used as in vitro assay systems. These human-derived cell line systems provide information on human-speciˆc induction responses and thus have clinical importance. The
Introduction Most of the exogenous lipophilic compounds including drugs and environmental pollutants are biotransformed by cytochrome P450 (CYP)-dependent reactions in the body.1,2) Human CYP3A4 is predominantly expressed in liver and intestine. The expression levels share 30 to 70z of the total cytochrome P450 content in these tissues3–5) and thus may account for the oxidative metabolism of more than 60z of clinically drugs.6) Due to being induced by a number of structurally unrelated compounds causing drug-drug interaction, we need to verify whether drug induces CYP3A4, particularly at drug development stage. Therefore, assay systems that estimate e‹ciently the CYP3A4 inducibility of test
Received; November 10, 2005, Accepted; December 29, 2005 *To whom correspondence should be addressed : Kiyoshi NAGATA, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. E-mail: nagataki＠mail.tains.tohoku.ac.jp Abbreviations used are: Ad; adenovirus, CYP; cytochrome P450, DMEM; Dulbecco's modiˆed Eagle's medium, DMSO; dimethyl sulfoxide, dNR; distal nuclear receptor-binding element, DR-3; direct repeat separated by 3 nucleotides, ER-6; everted-repeat separated by 6 nucleotides, hPXR; human pregnane X receptor, TCID50; tissue culture infection dose 50
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supply of the liver is, however, limited and expensive. Variation of CYP3A4 induction among individuals was another di‹culty. Large diŠerences in CYP3A4 induction proˆles, even though by treatment with the prototypical inducers of CYP3A4 such as rifampicin or clotrimazole, have been reported. In primary human hepatocyte cultures, CYP3A4 mRNA expression was induced strongly about 6.8- and 18.3-fold of control by treatment with clotrimazole and rifampicin.12) Roymans et al.13) and Hariparsad et al.14) reported that rifampicin induced about 6.2-fold of CYP3A4 activity in human hepatocytes. Madan et al.,15) however, reported that the eŠect of rifampicin on the CYP3A4 activity was variable, ranging from no increase to 145fold (average 10-fold, n＝61). It was also reported that clotrimazole and rifampicin enhanced CYP3A4 protein level to about 177 and 550z of control, respectively.16) Inconsistencies due to individual variability of liver samples are inevitable. Large diŠerences in induction proˆle may also arise during extended culture periods.17) To precisely predict CYP3A4 induction in humans, cell lines which are susceptible to the detection of chemicalinduced response are required. A system containing a human-derived gene reporter is one of the promising tools for induction assessment due to its stability and homogeneity.18) A number of systems for measurement of CYP induction have been developed by use of the reporter gene. Most systems require transfection of the plasmid gene construct prior to each experiment.17) Furthermore, the data are often varied among the experiments because of di‹culty in controlling transfection e‹ciency. Thus highly sensitive and reproducible methods of the reporter gene assay system are necessary. Although the mechanism of CYP3A4 transcriptional activation is not fully understood, CYP3A4 is considered to undergo activation through its interaction with PXR in the proximal promoter regions including ER-6 (－172 to －149) and distal xenobiotic-responsive enhancer module containing dNR1 (－7736 to －7716; DR-3), dNR2 (－7696 to －7669; ER-6) and dNR3 (－7290 to －7270; DR-3).19–26) Several of in vitro cell line systems which stably express reporter proteins have been reported.27,28) These systems consist of reporter gene constructs containing multimerized copies of either DR-3 or ER-6 response elements. As one example, Raucy et al.27) have established a cell line for determining CYP3A4 induction. A maximal induction at about 5.6-fold was observed at a 10 mM rifampicin treatment. Recently, a stable hepatoma cell line that was introduced hPXR gene as well as CYP3A4 enhancer and proximal promoter regions plus the luciferase reporter gene (p3A4-luciferase reporter construct) at a ratio of 10:1 was developed by Lemaire et al.28) These cell lines showed about a 14-fold increase of luciferase activity
for treatment with 10 mM rifampicin, while treatment with clotrimazole was less eŠective. In the present study, HepG2-derived cell lines containing tandemly ligated DNAs of the promoter and enhancer regions of the CYP3A4 gene, which exhibit highly reliable and stably express reporter protein have been established for chemical screening of CYP3A4 induction. Materials and Methods Materials: Rifampicin, clotrimazole, dexamethasone, griseofulvin, phenytoin, nifedipine, carbamazepine, and corticosterone were obtained from Sigma Chemical (St. Louis, USA). Omeprazole and troglitazone were kindly provided by Astra and Sankyo Co. LTD (Tokyo, JAPAN), respectively. The chemicals used for the in vitro study were dissolved in dimethylsulfoxide (DMSO). Restriction endonucleases and DNA modiˆcation enzymes were purchased from TaKaRa Shuzo (Kyoto, JAPAN). Dulbecco's modiˆed Eagle's medium (DMEM) and fetal calf serum were obtained from Gibco (Rockville, USA) and Roche Diagnostics (Indianapolis, USA), respectively. Cell culture and cell extract: Cells were cultivated under 5z CO2 at 379C in DMEM supplemented with 10z fetal calf serum, 100 units W mL of penicillin, 100 mg W mL of streptomycin, 0.25 mg W mL of amphotericin liter of glucose. The cells were seeded in B, and 3.5 g W 24-well tissue culture plates at 1×105 cells per well in 1 mL of DMEM. In the next day, the cell medium was changed with the medium containing drugs (0.1z DMSO) and cultured for 2 days. In the case of infection of the adenovirus vector containing hPXR (Ad hPXR), the day after seeding, cells were infected with 0.05 mL of Ad hPXR stock solution (5×104, 1.7×105, 1×106 TCID50 W well) for 1 h, and then the medium containing the test drug was added to the wells and cultured for 2 days. After incubation, the cells were washed with phosphate buŠer saline and suspended in 0.1 mL of Reporter Lysis BuŠer from Promega (Madison, USA) in a microcentrifuge tube. The cell suspension was C, and the centrifuged at 12,000×g for 5 minutes at 49 cell extract was used for the luciferase assay and determination of the protein concentration. Construction of adenovirus vector: Construction of Ad hPXR was performed according to the COS-TPC method with the aid of an Adenovirus Expression Vector Kit from TaKaRa Shuzo and also reported previously.29) Construction of pGL3-CYP3A4 and selection of clones responsive for inducers: The luciferase reporter plasmid, pGL3-Basic vector, lacking eukaryotic promoter and enhancer sequences, and pQBI including the neomycin resistant gene, were purchased from
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Fig. 1. Construction of pGL3–3A4–326–7.7k plasmid and selection of cloned cells stably expressing luciferase. The luciferase reporter plasmid, pGL3-basic vector, was inserted with the promoter region (－362 to ＋11 nt) and the distal nuclear receptor binding element (－7836 to －7208) of the CYP3A4 gene into its Hind III and Mlu I sites. The constructed pGL3–3A4–326–7.7k plasmid was also linearized with BamH I and pQB I was linearized with Bgl II and then these were ligated tandemly at a ratio of 5:1. HepG2 cells were seeded at 1.8×106 cells in 100 mm dish and the next-day ligated pGL3–3A4–326–7.7k was transfected and selected positive colonies with geneticin (900–1000 mg W mL) for 2 weeks.
Promega and from Wako Chemicals (Tokyo, JAPAN), respectively. The promoter region (－362 to ＋11nt) of the human CYP3A4 gene was ligated with the distal nuclear receptor binding element (－7836 to －7208) and then inserted into Hind III and Mlu I sites of the pGL3-Basic vector. The pGL3-CYP3A4–362–7.7k and pQBI linearized with BamH I and with Bgl II, respectively, were ligated tandemly. HepG2 cells were seeded at 1.8×106 cells in a 100 mm dish, and then ligated pGL3-CYP3A4–362–7.7k and pQBI at a ratio of 5:1 in molecular number was transfected to them the next day. After 2 weeks the positive colonies were selected with geneticin (900¿1000 mg W mL). The positive colonies were continued to culture furthermore and the induction response was measured by the luciferase assay method. Luciferase Assay: Luciferase assay was performed according to the manufacturer's instructions for Promega using the Luciferase Assay System and Turner 20 Luminometer (Promega, USA). The Designs TD-20 W activity was expressed as relative light units (RLU) W mg protein. Other Assays: Protein concentration was determined according to the procedure of Bradford with bovine serum albumin as the standard.30) Results Selection of cloned cells stably expressing luciferase:
Table 1. Response of cloned cells transfected with pGLCYP3A4– 362–7.7-DNA for inducers Cell number
Rifampicin (z of control)
Clotrimazole (z of control)
2-1-2 2-1-12 2-2-4 2-2-12 2-2-13 3-1-9 3-1-10 3-1-20 3-2-5 3-2-24 4-1-2 4-1-19
310 223 110 264 320 1677 5961 1576 884 465 71 191
107 100 260 36 236 1196 1911 9036 132 168 307 207
HepG2 cells were transfected with tandem-ligated pGLCYP3A4– 362–7.7 and selected resistant colonies with geneticin. Cloned cells were treated with rifampicin (30 mM) or clotrimazole (10 mM), for 48 hrs. Luciferase activity was normalized by protein concentration and the value was expressed as the percentage of 4 experiments.
pGL3-CYP3A4–362–7.7k constructed as described in MATERIALS AND METHODS contains the necessary elements as reported to highly respond to rifampicin.24) HepG2 cells were transfected with tandemly ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI or with non-ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI. Cells were cultivated in the presence of geneticin
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Fig. 2. In‰uence of cell density on reporter activity. Clone 3–1–10 and 3–1–20 were cultivated at 0.1, 0.33, 1, 3.3, 10 and 33×104 cells W well in 24-well tissue culture plates with 1 mL of DMEM, respectively. After 1 day, the medium was removed and the cells were treated with 10 mM of rifampicin or clotrimazole for 2 days and the lysates were harvested. The columns represent the means of 4 experiments. A. Response of clone 3–1–10 for 10 mM rifampicin. B. Response of clone 3–1–10 for 10 mM clotrimazole. C. Response of clone 3–1–20 for 10 mM rifampicin. D. Response of clone 3–1–20 for 10 mM clotrimazole.
(Fig. 1). Colonies were selected with their luciferase activities in the absence of chemical inducer. Finally, several clones with clear luciferase activities were selected from 144 colonies. No clear diŠerence was observed on the basal level of luciferase activities among the clones obtained by transfection with either tandemly ligated or non-ligated DNA. Selected clones expressing luciferase were further tested with their typical inducers, rifampicin and clotrimazole. The clones isolated from cells that transfected the non-ligated plasmid DNA showed low levels of luciferase activity. However, twelve clones from the cells that transfected the tandemly ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI showed strikingly high levels of reporter activity (Table 1). In particular, clone number 3–1–10 and 3–1–20 showed the highest response levels after treatment with drugs. In clone
3–1–10, the reporter activities were higher with rifampicin than with clotrimazole. The response of clone 3–1–20 was rather higher with clotrimazole than with rifampicin. In‰uence of cell density on reporter activity: Cloned cells were seeded at a series of 0.1, 0.33, 1, 3.3, 10 and 33×104 cells W well (0.5×103–1.65×105 cells W cm2 ) in 24well tissue culture plate in 1 mL of DMEM. The cells, after one-day culture, were treated with 10 mM rifampicin or 10 mM clotrimazole and kept for 2 days. The highest response was observed under condition of 3.3× 104 cells W well which was about 80z to 85z con‰uent as shown in Fig. 2A-D for both of the clone cells. In‰uence of concentration and cultured period on reporter activity: To verify the properties of clone 3–1–10 and 3–1–20, these cells were exposed at various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) and
Cell Responding Stably to CYP3A4 Induction
Fig. 3. In‰uence of drug-concentration and cultured period on reporter activity in clone 3–1–10. Clone 3–1–10 was seeded at 1×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM. After 1 day, the medium was removed and the cell were treated with various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) or clotrimazole (0.1, 1, 5, 10 mM) for 1, 2, and 3 days. Columns represent the means of 4 experiments and the error bar is a standard deviation. A. rifampicin. B. clotrimazole.
clotrimazole (0.1, 1, 5, 10 mM) for 1, 2 and 3 days. The enhanced reporter activities in clone 3–1–10 and 3–1–20 were observed at the concentration of more than 1 mM. In clone 3–1–10, 20-, 50- and 52-fold increases were observed at 10 mM and also 73-, 340- and 950-fold increases were also observed at 100 mM rifampicin for 1, 2 and 3 days of treatments, respectively (Fig. 3A). In the presence of 10 mM clotrimazole, about 3-, 18- and 40-fold increases were observed after 1, 2 and 3 days of treatment, respectively (Fig. 3B). In the case of clone 3–1–20, 4-, 5- and 6-fold increases were observed at 10 mM rifampicin. Addition of 100 mM rifampicin resulted in about 6-, 50- and 140-fold increases for 1, 2 and 3 days of treatment, respectively (Fig. 4A). In similar experiment, the luciferase activity
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Fig. 4. In‰uence of drug-concentration and cultured period on reporter activity in clone 3–1–20. well in 24-well tissue Clone 3–1–20 was seeded at 1×105 cells W culture plates with 1 mL of DMEM. After 1 day, the medium was removed and the cell was treated with various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) or clotrimazole (0.1, 1, 5, 10 mM) for 1, 2, and 3 days. Columns represent the means of 4 experiments and the error bar is a standard deviation. A. rifampicin. B. clotrimazole.
was increased about 7-, 26- and 82-fold at 10 mM clotrimazole for 1, 2 and 3 days of treatment, respectively (Fig. 4B). In‰uence of human PXR on reporter activity: In‰uence of human PXR on CYP3A4-reporter activity was examined by infection with adenovirus including human PXR (Ad hPXR) expression vector. Clone 3–1–10 and 3–1–20 were infected with Ad hPXR at the well to validate titer from 5×104 to 1×106 TCID50 W hPXR eŠect on the reporter activity. Luciferase activity was enhanced by the introduction of Ad hPXR, and dependent on the amount of the infected Ad hPXR as shown in Fig. 5A and 5B. These activities increased in
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Fig. 5. In‰uence of human PXR on reporter activity. well tissue Clone 3–1–10 and 3–1–20 were seeded at 1×105 cells W culture plates with 1 mL of DMEM. After 1 day, the cells were infected with Ad hPXR and Ad Lac Z at the titer of 5×104 to 1×106 TCID50 W well, the medium was removed and the cells were treated with 10 mM rifampicin for 2 days. The value was expressed as normalized RLU. Columns represent the means of 6 experiments. A. clone 3–1–10. B. clone 3–1–20.
the presence of rifampicin in both cloned cells gradually decreased with an increase amount of the infective Ad hPXR. The peaks at about 50- and 7.5-fold increases were observed under the condition of the 5×104 TCID50 W well for clone 3–1–10 and 3–1–20, respectively (Fig. 6A and 6B). Surprisingly, in the clone 3–1–10, the Ad hPXR-activated luciferase activity with rifampicin was slightly lower than the luciferase activity observed in the Ad LacZ-activated luciferase activity (Fig. 6A). In‰uence of various drugs on CYP3A4 induction: Various drugs were exposed to clone 3–1–10 and 3–1–20 at 10 mM for 2 and 3 days to verify the response for CYP3A4 induction. All the chemicals examined, except for phenytoin, increased the reporter activity. In clone 3–1–10, about 2.5- and 5.4-fold increases for
Fig. 6. In‰uence of human PXR on the induction in clones. Luciferase activity of Ad hPXR-infected cells which were measured in Fig. 5. was expressed as induction fold compared with those value of Ad Lac Z. Columns represent the means of 6 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with hPXR-control. A. clone 3–1–10. B. clone 3–1–20.
griseofulvin, about 3.3- and 4.1-fold increases for nifedipine, 1.9- and 3.3-fold increases for omeprazole and about 1.8- and 2.6-fold increases for troglitazone were observed in 2 and 3 days of treatment, respectively (Fig. 7). In clone 3–1–20, about 1.9- and 5.4-fold increases for griseofulvin, about 3- and 4.5-fold increases for nifedipine, about 2.5- and 3.5-fold increases for omeprazole were observed at 2 and 3 days of treatment, respectively. Addition of troglitazone also resulted in about 1.9- and 2.1-fold increases at 2 and 3 days of treatment, respectively (Fig. 8). In‰uence of cell passage on reporter activity: To validate the in‰uence of cell passage on reporter activity, clone 3–1–20 was continually cultured for more than 6 months. The reporter activity showed a high responsive level about 9-fold of control in the presence of 10 mM rifampicin for 2 days of treatment and was
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Fig. 7. In‰uence of various drugs on reporter activity in clone 3–1–10. Clone 3–1–10 was seeded at 1.5×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM 1 day before drugs treatment. The cells were treated with various drugs at 10 mM for 2 and 3 days and the lysate was harvested. Columns represent the means of 4 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with control (DMEM).
Fig. 8. In‰uence of various drugs on reporter activity in clone 3–1–20. Clone 3–1–20 was seeded at 1.5×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM 1 day before drugs treatment. The cells were treated with various drugs at 10 mM for 2 and 3 days and the lysate was harvested. Columns represent the means of 4 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with control (DMEM).
sustained for at least 65 passages as shown in Fig. 9. Discussion In the present study, we established several cell lines for screening of CYP3A4 induction. These cells stably express reporter protein mediated through the CYP3A4 gene. Among them, the clone 3–1–10 and 3–1–20 show clearly distinct responses on drug-induced CYP3A4
gene transactivation. To assess the response proˆle, reproducibility and stability of these cell lines, we further studied the eŠect of inducers in diŠerent concentration and culture periods as well as the eŠect of cell number per well of tissue culture plate. A stable hepatoma cell line expressing hPXR and the luciferase reporter through CYP3A4 distal and proximal promoter has been established to assess the
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Fig. 9. In‰uence of cultured period on luciferase activity in clone 3–1–20. Clone 3–1–20 continually cultured for indicated months was plated in 24-well tissue culture plates at the density of 1.5×105 cells W well the day before drug treatment. The cells were treated with 10 mM rifampicin for 2 days and the lysate was harvested. These values represent 4 experiments and the error bar is a standard deviation.
Fig. 10. Comparison of luciferase activity between 24- and 96-well tissue culture plates in clone 3–1–20. Clone 3–1–20 was plated in 24-well tissue culture plates at the density of 1.5×105 cells W well and in 96-well tissue culture plates at the density of 0.5×105 cells W well the day before drug treatment. The cell was treated with 10 mM rifampicin for 2 days and the lysate was harvested. The column of 24-well tissue culture plate represents the means of 4 experiments and the column of 96-well tissue culture plate represents the means of 6 experiments and the error bar is a standard deviation.
capability of several xenobiotic agents to induce CYP3A4 and CYP2B6.28) In those study, about 15-, 7and º1.7-fold increases were observed in rifampicin, clotrimazole and others drugs, respectively, after treatment with 10 mM of each compound. As shown in Fig. 3 and Fig. 4, newly developed cell lines shown in the present study exhibit much higher extents of induction response. The high response seems to result in multiple pGL3-CYP3A4–362–7.7k DNAs inserted into chromosome by transfection of the tandemly ligated DNA. The induction response was augmented with the increase of chemical concentration and culture period examined. The maximal level of induction was observed after 3 days of treatment. In the presence of rifampicin, about 20-, 50- and 52-fold increases were shown at 10 mM and reached their peaks about 73-, 340- and 950fold increases at 100 mM after 1, 2 and 3 days of treat-
ment in clone 3–1–10, respectively. In the presence of clotrimazole, about 7-, 26- and 82-fold increases were observed at 10 mM after 1, 2 and 3 days of treatment, respectively, in clone 3–1–20. Interestingly, both cells have also shown diŠerences in cell character and cell proliferation. The cause of these diŠerences is still obscure, but random integration to chromosome in HepG2 may produce a subtle change of the CYP3A4 gene structure leading to a change in interaction at the gene transcription with proteins and nuclear receptors or alter an expression level of nuclear proteins mediating CYP3A4 induction. Several recent studies have demonstrated that PXR is a key regulator of the CYP3A4 induction in a range of species.8–10) This receptor interacts with certain xenobiotics and binds to speciˆc DNA elements in the CYP3A4 gene9,10,24,26) resulting in an increased gene transactivation. To validate the eŠect of hPXR on reporter activity and induction, adenovirus vector was used for transient transfection of hPXR. Expression of hPXR stimulated luciferase activity, which depended on the increase of the infection (Fig. 5A and 5B). Surprisingly, the induced luciferase activity after normalized by protein amount in clone 3–1–20 was increased by the expression of hPXR, whereas those observed in clone 3–1–10 was decreased. We do not know the reason exactly, but possibly the expression level of the other nuclear receptor that formed a heterodimer with hPXR involved in the induction may not be su‹cient in the cloned cells that overexpressed hPXR by adenovirus. By the way, these data suggest that overexpression of hPXR is not essential for enhancement of CYP3A4 induction in our systems. A large amount of evidence suggested that cell density and cell proliferation also aŠect CYP3A4 regulation.31) At the low density of cell per cm2, hepatocytes become progressively more spread and ‰attened, and such changes in cell morphology are often accompanied with phenotypic changes.18) Excess levels of densities rather interfere with cell attachment to cause loss of optimal mono-layer formation31) as do the interaction of cell surface to chemicals. As shown in Fig. 2, a highly reproducible response was observed at plating density of 3.3×104–1.0×105 cells W well (1.65–5.0×104 cells W cm2 ). No increase was observed at low cell densities used. High density at cell plating also resulted in a low level of induction. Therefore, the abundance of nutrients in medium, pH-change, cell con‰uence, cell condition (including cell proliferation rate) may cause the inconsistencies of such extents of induction. In our studies, maximum induction was obtained at 3.3×104–1×105 cells W well, of which density is close to previous reports19) (size of 24-well tissue culture plate is about 2 cm2 W well for BD Biosciences product), and consequently we chose to seed at this range of cell density. Maximal
Cell Responding Stably to CYP3A4 Induction
growth was also observed at about 13 passages per month and 11 passages per month for clone 3–1–10 and 3–1–20, respectively. Recent papers17) have showed that in continuous culture, at the early stage of cell passage, luciferase induction dropped during increased culture period of passages. After 30 or 40 passages, the responsiveness in MDA-kb2 stably expressing an androgen-responsive reporter is stable and stabilized for more than 80 passages.17) To access whether our cloned cells are available for high through-put screening at the early stage of drug development, we have examined the eŠect of tissue culture plate size on reporter activity and the stability of clone 3–1–20 by culture for several months. No clear diŠerent response was detected on reporter activity among diŠerent tissue culture plates (Fig. 10) and was kept stable at a high responsive level during long culture periods of at least 6 months (about 65 passages) as shown in Fig. 9. Since the cells were derived from a single stable clone, inter- and intra-assay variability would be reduced. In general, there is a large individual diŠerence in the proˆle of CYP3A4 induction in human livers.15) The diŠerent induction proˆle of human liver primary cells responsive to clotrimazole and rifampicin was also observed.12) In our developed cell lines, most of the test drugs enhanced the luciferase activity and the signiˆcance of the induction levels observed were diŠerent, especially for clotrimazole- and rifampicinactivated CYP3A4 induction. At present, in 9 facilities, the usefulness of these cell lines for chemical screening of CYP3A4 inducibility is being evaluated. The high response for a number of drugs and high reproducibility were conˆrmed in these facilities. In addition, these cell lines are available to assess CYP3A4 inducibility by exploring pesticides. Therefore the induction study using both cell lines is very useful for validation of CYP3A4 induction not only by drugs but also environmental pollutants. In conclusion, we have established two cell lines showing a diŠerent property in the reporter activity induced by drugs through the CYP3A4 gene. These cloned cells also responded at a signiˆcant level of CYP3A4 induction to treatment with various drugs. Judging from their high responses, stabilities and reproducibilities, our established cloned cells are very useful for the screening CYP3A4 induction at the early stage of drug development. Acknowledgements: This work was supported by a Grant-in-Aid from the Ministry of Education, Sciences and Culture (Ministry of Education, Culture, Sports, Sciences and Technology), the Ministry of Health and Welfare (Ministry of Health, Labour, and Welfare) of Japan and Comprehensive Research and Education Center for Planning of Drug Development and Clinical
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Education, Tohoku University 21st Century Center of Excellence Program. References 1)
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Nebert, D. W. and Russell, D. W.: Clinical importance of the cytochromes P450. Lancet, 360: 1155–1162 (2002). Guengerich, F. P.: Human cytochrome P450 enzymes. In Ortiz de Montellano, P. R. (ed): Cytochrome P450; Structure, Mechanism, and Biochemistry, New York, Kluwer Academic W Plenum Publisher, 2005, pp. 377–530. Kawano, S., Kamataki, T., Yasumori, T., Yamazoe, Y. and Kato, R.: Puriˆcation of human liver cytochrome P-450 catalyzing testosterone 6 beta-hydroxylation. J. Biochem. (Tokyo), 102: 493–501 (1987). Kolars, J. C., Lown, K. S., Schmiedlin-Ren, P., Ghosh, M., Fang, C., Wrighton, S. A., Merion, R. M. and Watkins, P. B.: CYP3A gene expression in human gut epithelium. Pharmacogenetics, 4: 247–259 (1994). Shimada, T., Yamazaki, H., Mimura, M., Wakamiya, N., Ueng, Y. F., Guengerich, F. P. and Inui, Y.: Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal liver and adult lungs. Drug Metab. Dispos., 24: 515–522 (1996). Cholerton, S., Daly, A. K. and Idle, J. R.: The role of individual human cytochromes P450 in drug metabolism and clinical response. Trends Pharmacol. Sci., 13: 434–439 (1992). Lu, C. and Li, A. P.: Species comparison in P450 induction: eŠects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague-Dawley rat, minipig, and beagle dog. Chem. Biol. Interact., 134: 271–281 (2001). Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T. and Kliewer, S. A.: The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest., 102: 1016–1023 (1998). Handschin, C. and Meyer, U. A.: Induction of drug metabolism: the role of nuclear receptors. Pharmacol. Rev., 55: 649–673 (2003). Williams, S. N., Dunham, E. and Bradˆld, C. A.: Induction of cytochrome P450 enzymes. In Ortiz de Montellano, P. R. (ed): Cytochrome P450; Structure, Mechanism, and Biochemistry, New York, Kluwer Academic W Plenum Publisher, 2005, pp. 323–346. Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G. B., Hamilton, G. A., McKee, D. D., Tomkinson, N. C., LeCluyse, E. L., Lambert, M. H., Willson, T. M., Kliewer, S. A. and Moore, J. T.: The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol., 14: 27–39 (2000). Luo, G., Cunningham, M., Kim, S., Burn, T., Lin, J., Sinz, M., Hamilton, G., Rizzo, C., Jolley, S., Gilbert, D., Downey, A., Mudra, D., Graham, R., Carroll, K.,
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Xie, J., Madan, A., Parkinson, A., Christ, D., Selling, B., LeCluyse, E. and Gan, L. S.: CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metab. Dispos., 30: 795–804 (2002). Roymans, D., Van, Looveren, C., Leone, A., Parker, J. B., McMillian, M., Johnson, M. D., Koganti, A., Gilissen, R., Silber, P., Mannens, G. and Meuldermans, W.: Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes. Biochem. Pharmacol., 67: 427–437 (2004). Hariparsad, N., Nallani, S. C., Sane, R. S., Buckley, D. J., Buckley, A. R. and Desai, P. B.: Induction of CYP3A4 by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital. J. Clin. Pharmacol., 44: 1273–1281 (2004). Madan, A., Graham, R. A., Carroll, K. M., Mudra, D. R., Burton, L. A., Krueger, L. A., Downey, A. D., Czerwinski, M., Forster, J., Ribadeneira, M. D., Gan, L. S., LeCluyse, E. L., Zech, K., Robertson, P.-Jr., Koch, P., Antonian, L., Wagner, G., Yu, L. and Parkinson, A.: EŠects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab. Dispos., 31: 421–431 (2003). Raucy, J. L.: Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products. Drug Metab. Dispos., 31: 533–539 (2003). Wilson, V. S., Bobseine, K., Lambright, C. R. and Gray, L. E.-Jr.: A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicol. Sci., 66: 69–81 (2002). LeCluyse, E. L.: Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur. J. Pharm. Sci., 13: 343–368 (2001). Hashimoto, H., Toide, K., Kitamura, R., Fujita, M., Tagawa, S., Itoh, S. and Kamataki, T.: Gene structure of CYP3A4, an adult-speciˆc form of cytochrome P450 in human livers, and its transcriptional control. Eur. J. Biochem., 218: 585–595 (1993). Kocarek, T. A., Schuetz, E. G., Strom, S. C., Fisher, R. A. and Guzelian, P. S.: Comparative analysis of cytochrome P4503A induction in primary cultures of rat, rabbit, and human hepatocytes. Drug Metab. Dispos., 23: 415–421 (1995). Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P. and Berkenstam: A Identiˆcation of a human nuclear receptor deˆnes a new
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Regular Article Construction of Several Human-derived Stable Cell Lines Displaying Distinct Proˆles of CYP3A4 Induction Wachiraporn NORACHARTTIYAPOT1, Yoko NAGAI1, Tsutomu MATSUBARA1,2, Masaaki MIYATA1, Miki SHIMADA1, Kiyoshi NAGATA1,* and Yasushi YAMAZOE1,2 1Division
of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2Comprehensive Research and Education Center for Planning of Drug Development and Clinical Education, Tohoku University 21st Century Center of Excellence Program. Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: Cell lines which stably express reporter proteins through CYP3A4 gene activation have been developed for use in predicting CYP3A4 induction. Twelve clones showing distinct proˆles on chemicalinduced response were isolated. Among them, two clones showing high response for CYP3A4 inducers, namely clone 3-1-10 and 3-1-20, were further evaluated for their sensitivities, reproducibilities and applicabilities to predict CYP3A4 induction in human. Clone 3-1-10 showed higher response to rifampicin than to clotrimazole, whereas clone 3-1-20 had rather higher response to clotrimazole. Optimal plating density and highly reproducible response were observed at the range of 1.65–5.0×104 cell W cm2. Clear induction responses of more than ten chemicals were observed in both cell lines. The reporter activity was further dramatically increased after an introduction of human PXR. Induction with rifampicin was, however, not much altered between the absence and presence of hPXR. The luciferase activity remained unaltered and showed little ‰uctuation during the culture for more than 6 months. Due to the strikingly high sensitivity and reproducibility of this system, as compared to previously published systems, these HepG2-derived cell lines showing distinct response proˆles as developed in the present study will oŠer high advantages for chemical screening of CYP3A4 inducibility.
Key words: CYP3A4; induction; PXR; reporter assay; stable cell lines chemicals have been required. Currently, in vitro and in vivo assay systems are available. A large diŠerence, however, exists in the xenobiotic-induced response among experimental animal species and humans.7) Recently, a ligand-activated transcriptional factor, pregnane X receptor (PXR), has been shown to mediate the eŠects of xenobiotics on ligand-mediated expression of CYP3A genes in humans as well as in other experimental animals.8–10) PXR is also reported to be one of the major factors for the species diŠerences in the induction response.10,11) Human hepatocytes or liver slices have been used as in vitro assay systems. These human-derived cell line systems provide information on human-speciˆc induction responses and thus have clinical importance. The
Introduction Most of the exogenous lipophilic compounds including drugs and environmental pollutants are biotransformed by cytochrome P450 (CYP)-dependent reactions in the body.1,2) Human CYP3A4 is predominantly expressed in liver and intestine. The expression levels share 30 to 70z of the total cytochrome P450 content in these tissues3–5) and thus may account for the oxidative metabolism of more than 60z of clinically drugs.6) Due to being induced by a number of structurally unrelated compounds causing drug-drug interaction, we need to verify whether drug induces CYP3A4, particularly at drug development stage. Therefore, assay systems that estimate e‹ciently the CYP3A4 inducibility of test
Received; November 10, 2005, Accepted; December 29, 2005 *To whom correspondence should be addressed : Kiyoshi NAGATA, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. E-mail: nagataki＠mail.tains.tohoku.ac.jp Abbreviations used are: Ad; adenovirus, CYP; cytochrome P450, DMEM; Dulbecco's modiˆed Eagle's medium, DMSO; dimethyl sulfoxide, dNR; distal nuclear receptor-binding element, DR-3; direct repeat separated by 3 nucleotides, ER-6; everted-repeat separated by 6 nucleotides, hPXR; human pregnane X receptor, TCID50; tissue culture infection dose 50
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supply of the liver is, however, limited and expensive. Variation of CYP3A4 induction among individuals was another di‹culty. Large diŠerences in CYP3A4 induction proˆles, even though by treatment with the prototypical inducers of CYP3A4 such as rifampicin or clotrimazole, have been reported. In primary human hepatocyte cultures, CYP3A4 mRNA expression was induced strongly about 6.8- and 18.3-fold of control by treatment with clotrimazole and rifampicin.12) Roymans et al.13) and Hariparsad et al.14) reported that rifampicin induced about 6.2-fold of CYP3A4 activity in human hepatocytes. Madan et al.,15) however, reported that the eŠect of rifampicin on the CYP3A4 activity was variable, ranging from no increase to 145fold (average 10-fold, n＝61). It was also reported that clotrimazole and rifampicin enhanced CYP3A4 protein level to about 177 and 550z of control, respectively.16) Inconsistencies due to individual variability of liver samples are inevitable. Large diŠerences in induction proˆle may also arise during extended culture periods.17) To precisely predict CYP3A4 induction in humans, cell lines which are susceptible to the detection of chemicalinduced response are required. A system containing a human-derived gene reporter is one of the promising tools for induction assessment due to its stability and homogeneity.18) A number of systems for measurement of CYP induction have been developed by use of the reporter gene. Most systems require transfection of the plasmid gene construct prior to each experiment.17) Furthermore, the data are often varied among the experiments because of di‹culty in controlling transfection e‹ciency. Thus highly sensitive and reproducible methods of the reporter gene assay system are necessary. Although the mechanism of CYP3A4 transcriptional activation is not fully understood, CYP3A4 is considered to undergo activation through its interaction with PXR in the proximal promoter regions including ER-6 (－172 to －149) and distal xenobiotic-responsive enhancer module containing dNR1 (－7736 to －7716; DR-3), dNR2 (－7696 to －7669; ER-6) and dNR3 (－7290 to －7270; DR-3).19–26) Several of in vitro cell line systems which stably express reporter proteins have been reported.27,28) These systems consist of reporter gene constructs containing multimerized copies of either DR-3 or ER-6 response elements. As one example, Raucy et al.27) have established a cell line for determining CYP3A4 induction. A maximal induction at about 5.6-fold was observed at a 10 mM rifampicin treatment. Recently, a stable hepatoma cell line that was introduced hPXR gene as well as CYP3A4 enhancer and proximal promoter regions plus the luciferase reporter gene (p3A4-luciferase reporter construct) at a ratio of 10:1 was developed by Lemaire et al.28) These cell lines showed about a 14-fold increase of luciferase activity
for treatment with 10 mM rifampicin, while treatment with clotrimazole was less eŠective. In the present study, HepG2-derived cell lines containing tandemly ligated DNAs of the promoter and enhancer regions of the CYP3A4 gene, which exhibit highly reliable and stably express reporter protein have been established for chemical screening of CYP3A4 induction. Materials and Methods Materials: Rifampicin, clotrimazole, dexamethasone, griseofulvin, phenytoin, nifedipine, carbamazepine, and corticosterone were obtained from Sigma Chemical (St. Louis, USA). Omeprazole and troglitazone were kindly provided by Astra and Sankyo Co. LTD (Tokyo, JAPAN), respectively. The chemicals used for the in vitro study were dissolved in dimethylsulfoxide (DMSO). Restriction endonucleases and DNA modiˆcation enzymes were purchased from TaKaRa Shuzo (Kyoto, JAPAN). Dulbecco's modiˆed Eagle's medium (DMEM) and fetal calf serum were obtained from Gibco (Rockville, USA) and Roche Diagnostics (Indianapolis, USA), respectively. Cell culture and cell extract: Cells were cultivated under 5z CO2 at 379C in DMEM supplemented with 10z fetal calf serum, 100 units W mL of penicillin, 100 mg W mL of streptomycin, 0.25 mg W mL of amphotericin liter of glucose. The cells were seeded in B, and 3.5 g W 24-well tissue culture plates at 1×105 cells per well in 1 mL of DMEM. In the next day, the cell medium was changed with the medium containing drugs (0.1z DMSO) and cultured for 2 days. In the case of infection of the adenovirus vector containing hPXR (Ad hPXR), the day after seeding, cells were infected with 0.05 mL of Ad hPXR stock solution (5×104, 1.7×105, 1×106 TCID50 W well) for 1 h, and then the medium containing the test drug was added to the wells and cultured for 2 days. After incubation, the cells were washed with phosphate buŠer saline and suspended in 0.1 mL of Reporter Lysis BuŠer from Promega (Madison, USA) in a microcentrifuge tube. The cell suspension was C, and the centrifuged at 12,000×g for 5 minutes at 49 cell extract was used for the luciferase assay and determination of the protein concentration. Construction of adenovirus vector: Construction of Ad hPXR was performed according to the COS-TPC method with the aid of an Adenovirus Expression Vector Kit from TaKaRa Shuzo and also reported previously.29) Construction of pGL3-CYP3A4 and selection of clones responsive for inducers: The luciferase reporter plasmid, pGL3-Basic vector, lacking eukaryotic promoter and enhancer sequences, and pQBI including the neomycin resistant gene, were purchased from
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Fig. 1. Construction of pGL3–3A4–326–7.7k plasmid and selection of cloned cells stably expressing luciferase. The luciferase reporter plasmid, pGL3-basic vector, was inserted with the promoter region (－362 to ＋11 nt) and the distal nuclear receptor binding element (－7836 to －7208) of the CYP3A4 gene into its Hind III and Mlu I sites. The constructed pGL3–3A4–326–7.7k plasmid was also linearized with BamH I and pQB I was linearized with Bgl II and then these were ligated tandemly at a ratio of 5:1. HepG2 cells were seeded at 1.8×106 cells in 100 mm dish and the next-day ligated pGL3–3A4–326–7.7k was transfected and selected positive colonies with geneticin (900–1000 mg W mL) for 2 weeks.
Promega and from Wako Chemicals (Tokyo, JAPAN), respectively. The promoter region (－362 to ＋11nt) of the human CYP3A4 gene was ligated with the distal nuclear receptor binding element (－7836 to －7208) and then inserted into Hind III and Mlu I sites of the pGL3-Basic vector. The pGL3-CYP3A4–362–7.7k and pQBI linearized with BamH I and with Bgl II, respectively, were ligated tandemly. HepG2 cells were seeded at 1.8×106 cells in a 100 mm dish, and then ligated pGL3-CYP3A4–362–7.7k and pQBI at a ratio of 5:1 in molecular number was transfected to them the next day. After 2 weeks the positive colonies were selected with geneticin (900¿1000 mg W mL). The positive colonies were continued to culture furthermore and the induction response was measured by the luciferase assay method. Luciferase Assay: Luciferase assay was performed according to the manufacturer's instructions for Promega using the Luciferase Assay System and Turner 20 Luminometer (Promega, USA). The Designs TD-20 W activity was expressed as relative light units (RLU) W mg protein. Other Assays: Protein concentration was determined according to the procedure of Bradford with bovine serum albumin as the standard.30) Results Selection of cloned cells stably expressing luciferase:
Table 1. Response of cloned cells transfected with pGLCYP3A4– 362–7.7-DNA for inducers Cell number
Rifampicin (z of control)
Clotrimazole (z of control)
2-1-2 2-1-12 2-2-4 2-2-12 2-2-13 3-1-9 3-1-10 3-1-20 3-2-5 3-2-24 4-1-2 4-1-19
310 223 110 264 320 1677 5961 1576 884 465 71 191
107 100 260 36 236 1196 1911 9036 132 168 307 207
HepG2 cells were transfected with tandem-ligated pGLCYP3A4– 362–7.7 and selected resistant colonies with geneticin. Cloned cells were treated with rifampicin (30 mM) or clotrimazole (10 mM), for 48 hrs. Luciferase activity was normalized by protein concentration and the value was expressed as the percentage of 4 experiments.
pGL3-CYP3A4–362–7.7k constructed as described in MATERIALS AND METHODS contains the necessary elements as reported to highly respond to rifampicin.24) HepG2 cells were transfected with tandemly ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI or with non-ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI. Cells were cultivated in the presence of geneticin
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Fig. 2. In‰uence of cell density on reporter activity. Clone 3–1–10 and 3–1–20 were cultivated at 0.1, 0.33, 1, 3.3, 10 and 33×104 cells W well in 24-well tissue culture plates with 1 mL of DMEM, respectively. After 1 day, the medium was removed and the cells were treated with 10 mM of rifampicin or clotrimazole for 2 days and the lysates were harvested. The columns represent the means of 4 experiments. A. Response of clone 3–1–10 for 10 mM rifampicin. B. Response of clone 3–1–10 for 10 mM clotrimazole. C. Response of clone 3–1–20 for 10 mM rifampicin. D. Response of clone 3–1–20 for 10 mM clotrimazole.
(Fig. 1). Colonies were selected with their luciferase activities in the absence of chemical inducer. Finally, several clones with clear luciferase activities were selected from 144 colonies. No clear diŠerence was observed on the basal level of luciferase activities among the clones obtained by transfection with either tandemly ligated or non-ligated DNA. Selected clones expressing luciferase were further tested with their typical inducers, rifampicin and clotrimazole. The clones isolated from cells that transfected the non-ligated plasmid DNA showed low levels of luciferase activity. However, twelve clones from the cells that transfected the tandemly ligated DNA of pGL3-CYP3A4–362–7.7k and pQBI showed strikingly high levels of reporter activity (Table 1). In particular, clone number 3–1–10 and 3–1–20 showed the highest response levels after treatment with drugs. In clone
3–1–10, the reporter activities were higher with rifampicin than with clotrimazole. The response of clone 3–1–20 was rather higher with clotrimazole than with rifampicin. In‰uence of cell density on reporter activity: Cloned cells were seeded at a series of 0.1, 0.33, 1, 3.3, 10 and 33×104 cells W well (0.5×103–1.65×105 cells W cm2 ) in 24well tissue culture plate in 1 mL of DMEM. The cells, after one-day culture, were treated with 10 mM rifampicin or 10 mM clotrimazole and kept for 2 days. The highest response was observed under condition of 3.3× 104 cells W well which was about 80z to 85z con‰uent as shown in Fig. 2A-D for both of the clone cells. In‰uence of concentration and cultured period on reporter activity: To verify the properties of clone 3–1–10 and 3–1–20, these cells were exposed at various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) and
Cell Responding Stably to CYP3A4 Induction
Fig. 3. In‰uence of drug-concentration and cultured period on reporter activity in clone 3–1–10. Clone 3–1–10 was seeded at 1×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM. After 1 day, the medium was removed and the cell were treated with various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) or clotrimazole (0.1, 1, 5, 10 mM) for 1, 2, and 3 days. Columns represent the means of 4 experiments and the error bar is a standard deviation. A. rifampicin. B. clotrimazole.
clotrimazole (0.1, 1, 5, 10 mM) for 1, 2 and 3 days. The enhanced reporter activities in clone 3–1–10 and 3–1–20 were observed at the concentration of more than 1 mM. In clone 3–1–10, 20-, 50- and 52-fold increases were observed at 10 mM and also 73-, 340- and 950-fold increases were also observed at 100 mM rifampicin for 1, 2 and 3 days of treatments, respectively (Fig. 3A). In the presence of 10 mM clotrimazole, about 3-, 18- and 40-fold increases were observed after 1, 2 and 3 days of treatment, respectively (Fig. 3B). In the case of clone 3–1–20, 4-, 5- and 6-fold increases were observed at 10 mM rifampicin. Addition of 100 mM rifampicin resulted in about 6-, 50- and 140-fold increases for 1, 2 and 3 days of treatment, respectively (Fig. 4A). In similar experiment, the luciferase activity
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Fig. 4. In‰uence of drug-concentration and cultured period on reporter activity in clone 3–1–20. well in 24-well tissue Clone 3–1–20 was seeded at 1×105 cells W culture plates with 1 mL of DMEM. After 1 day, the medium was removed and the cell was treated with various concentrations of rifampicin (0.1, 1, 5, 10, 100 mM) or clotrimazole (0.1, 1, 5, 10 mM) for 1, 2, and 3 days. Columns represent the means of 4 experiments and the error bar is a standard deviation. A. rifampicin. B. clotrimazole.
was increased about 7-, 26- and 82-fold at 10 mM clotrimazole for 1, 2 and 3 days of treatment, respectively (Fig. 4B). In‰uence of human PXR on reporter activity: In‰uence of human PXR on CYP3A4-reporter activity was examined by infection with adenovirus including human PXR (Ad hPXR) expression vector. Clone 3–1–10 and 3–1–20 were infected with Ad hPXR at the well to validate titer from 5×104 to 1×106 TCID50 W hPXR eŠect on the reporter activity. Luciferase activity was enhanced by the introduction of Ad hPXR, and dependent on the amount of the infected Ad hPXR as shown in Fig. 5A and 5B. These activities increased in
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Fig. 5. In‰uence of human PXR on reporter activity. well tissue Clone 3–1–10 and 3–1–20 were seeded at 1×105 cells W culture plates with 1 mL of DMEM. After 1 day, the cells were infected with Ad hPXR and Ad Lac Z at the titer of 5×104 to 1×106 TCID50 W well, the medium was removed and the cells were treated with 10 mM rifampicin for 2 days. The value was expressed as normalized RLU. Columns represent the means of 6 experiments. A. clone 3–1–10. B. clone 3–1–20.
the presence of rifampicin in both cloned cells gradually decreased with an increase amount of the infective Ad hPXR. The peaks at about 50- and 7.5-fold increases were observed under the condition of the 5×104 TCID50 W well for clone 3–1–10 and 3–1–20, respectively (Fig. 6A and 6B). Surprisingly, in the clone 3–1–10, the Ad hPXR-activated luciferase activity with rifampicin was slightly lower than the luciferase activity observed in the Ad LacZ-activated luciferase activity (Fig. 6A). In‰uence of various drugs on CYP3A4 induction: Various drugs were exposed to clone 3–1–10 and 3–1–20 at 10 mM for 2 and 3 days to verify the response for CYP3A4 induction. All the chemicals examined, except for phenytoin, increased the reporter activity. In clone 3–1–10, about 2.5- and 5.4-fold increases for
Fig. 6. In‰uence of human PXR on the induction in clones. Luciferase activity of Ad hPXR-infected cells which were measured in Fig. 5. was expressed as induction fold compared with those value of Ad Lac Z. Columns represent the means of 6 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with hPXR-control. A. clone 3–1–10. B. clone 3–1–20.
griseofulvin, about 3.3- and 4.1-fold increases for nifedipine, 1.9- and 3.3-fold increases for omeprazole and about 1.8- and 2.6-fold increases for troglitazone were observed in 2 and 3 days of treatment, respectively (Fig. 7). In clone 3–1–20, about 1.9- and 5.4-fold increases for griseofulvin, about 3- and 4.5-fold increases for nifedipine, about 2.5- and 3.5-fold increases for omeprazole were observed at 2 and 3 days of treatment, respectively. Addition of troglitazone also resulted in about 1.9- and 2.1-fold increases at 2 and 3 days of treatment, respectively (Fig. 8). In‰uence of cell passage on reporter activity: To validate the in‰uence of cell passage on reporter activity, clone 3–1–20 was continually cultured for more than 6 months. The reporter activity showed a high responsive level about 9-fold of control in the presence of 10 mM rifampicin for 2 days of treatment and was
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Fig. 7. In‰uence of various drugs on reporter activity in clone 3–1–10. Clone 3–1–10 was seeded at 1.5×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM 1 day before drugs treatment. The cells were treated with various drugs at 10 mM for 2 and 3 days and the lysate was harvested. Columns represent the means of 4 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with control (DMEM).
Fig. 8. In‰uence of various drugs on reporter activity in clone 3–1–20. Clone 3–1–20 was seeded at 1.5×105 cells W well in 24-well tissue culture plates with 1 mL of DMEM 1 day before drugs treatment. The cells were treated with various drugs at 10 mM for 2 and 3 days and the lysate was harvested. Columns represent the means of 4 experiments and the error bar is a standard deviation, *Pº0.05 (Student's t-test) as compared with control (DMEM).
sustained for at least 65 passages as shown in Fig. 9. Discussion In the present study, we established several cell lines for screening of CYP3A4 induction. These cells stably express reporter protein mediated through the CYP3A4 gene. Among them, the clone 3–1–10 and 3–1–20 show clearly distinct responses on drug-induced CYP3A4
gene transactivation. To assess the response proˆle, reproducibility and stability of these cell lines, we further studied the eŠect of inducers in diŠerent concentration and culture periods as well as the eŠect of cell number per well of tissue culture plate. A stable hepatoma cell line expressing hPXR and the luciferase reporter through CYP3A4 distal and proximal promoter has been established to assess the
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Fig. 9. In‰uence of cultured period on luciferase activity in clone 3–1–20. Clone 3–1–20 continually cultured for indicated months was plated in 24-well tissue culture plates at the density of 1.5×105 cells W well the day before drug treatment. The cells were treated with 10 mM rifampicin for 2 days and the lysate was harvested. These values represent 4 experiments and the error bar is a standard deviation.
Fig. 10. Comparison of luciferase activity between 24- and 96-well tissue culture plates in clone 3–1–20. Clone 3–1–20 was plated in 24-well tissue culture plates at the density of 1.5×105 cells W well and in 96-well tissue culture plates at the density of 0.5×105 cells W well the day before drug treatment. The cell was treated with 10 mM rifampicin for 2 days and the lysate was harvested. The column of 24-well tissue culture plate represents the means of 4 experiments and the column of 96-well tissue culture plate represents the means of 6 experiments and the error bar is a standard deviation.
capability of several xenobiotic agents to induce CYP3A4 and CYP2B6.28) In those study, about 15-, 7and º1.7-fold increases were observed in rifampicin, clotrimazole and others drugs, respectively, after treatment with 10 mM of each compound. As shown in Fig. 3 and Fig. 4, newly developed cell lines shown in the present study exhibit much higher extents of induction response. The high response seems to result in multiple pGL3-CYP3A4–362–7.7k DNAs inserted into chromosome by transfection of the tandemly ligated DNA. The induction response was augmented with the increase of chemical concentration and culture period examined. The maximal level of induction was observed after 3 days of treatment. In the presence of rifampicin, about 20-, 50- and 52-fold increases were shown at 10 mM and reached their peaks about 73-, 340- and 950fold increases at 100 mM after 1, 2 and 3 days of treat-
ment in clone 3–1–10, respectively. In the presence of clotrimazole, about 7-, 26- and 82-fold increases were observed at 10 mM after 1, 2 and 3 days of treatment, respectively, in clone 3–1–20. Interestingly, both cells have also shown diŠerences in cell character and cell proliferation. The cause of these diŠerences is still obscure, but random integration to chromosome in HepG2 may produce a subtle change of the CYP3A4 gene structure leading to a change in interaction at the gene transcription with proteins and nuclear receptors or alter an expression level of nuclear proteins mediating CYP3A4 induction. Several recent studies have demonstrated that PXR is a key regulator of the CYP3A4 induction in a range of species.8–10) This receptor interacts with certain xenobiotics and binds to speciˆc DNA elements in the CYP3A4 gene9,10,24,26) resulting in an increased gene transactivation. To validate the eŠect of hPXR on reporter activity and induction, adenovirus vector was used for transient transfection of hPXR. Expression of hPXR stimulated luciferase activity, which depended on the increase of the infection (Fig. 5A and 5B). Surprisingly, the induced luciferase activity after normalized by protein amount in clone 3–1–20 was increased by the expression of hPXR, whereas those observed in clone 3–1–10 was decreased. We do not know the reason exactly, but possibly the expression level of the other nuclear receptor that formed a heterodimer with hPXR involved in the induction may not be su‹cient in the cloned cells that overexpressed hPXR by adenovirus. By the way, these data suggest that overexpression of hPXR is not essential for enhancement of CYP3A4 induction in our systems. A large amount of evidence suggested that cell density and cell proliferation also aŠect CYP3A4 regulation.31) At the low density of cell per cm2, hepatocytes become progressively more spread and ‰attened, and such changes in cell morphology are often accompanied with phenotypic changes.18) Excess levels of densities rather interfere with cell attachment to cause loss of optimal mono-layer formation31) as do the interaction of cell surface to chemicals. As shown in Fig. 2, a highly reproducible response was observed at plating density of 3.3×104–1.0×105 cells W well (1.65–5.0×104 cells W cm2 ). No increase was observed at low cell densities used. High density at cell plating also resulted in a low level of induction. Therefore, the abundance of nutrients in medium, pH-change, cell con‰uence, cell condition (including cell proliferation rate) may cause the inconsistencies of such extents of induction. In our studies, maximum induction was obtained at 3.3×104–1×105 cells W well, of which density is close to previous reports19) (size of 24-well tissue culture plate is about 2 cm2 W well for BD Biosciences product), and consequently we chose to seed at this range of cell density. Maximal
Cell Responding Stably to CYP3A4 Induction
growth was also observed at about 13 passages per month and 11 passages per month for clone 3–1–10 and 3–1–20, respectively. Recent papers17) have showed that in continuous culture, at the early stage of cell passage, luciferase induction dropped during increased culture period of passages. After 30 or 40 passages, the responsiveness in MDA-kb2 stably expressing an androgen-responsive reporter is stable and stabilized for more than 80 passages.17) To access whether our cloned cells are available for high through-put screening at the early stage of drug development, we have examined the eŠect of tissue culture plate size on reporter activity and the stability of clone 3–1–20 by culture for several months. No clear diŠerent response was detected on reporter activity among diŠerent tissue culture plates (Fig. 10) and was kept stable at a high responsive level during long culture periods of at least 6 months (about 65 passages) as shown in Fig. 9. Since the cells were derived from a single stable clone, inter- and intra-assay variability would be reduced. In general, there is a large individual diŠerence in the proˆle of CYP3A4 induction in human livers.15) The diŠerent induction proˆle of human liver primary cells responsive to clotrimazole and rifampicin was also observed.12) In our developed cell lines, most of the test drugs enhanced the luciferase activity and the signiˆcance of the induction levels observed were diŠerent, especially for clotrimazole- and rifampicinactivated CYP3A4 induction. At present, in 9 facilities, the usefulness of these cell lines for chemical screening of CYP3A4 inducibility is being evaluated. The high response for a number of drugs and high reproducibility were conˆrmed in these facilities. In addition, these cell lines are available to assess CYP3A4 inducibility by exploring pesticides. Therefore the induction study using both cell lines is very useful for validation of CYP3A4 induction not only by drugs but also environmental pollutants. In conclusion, we have established two cell lines showing a diŠerent property in the reporter activity induced by drugs through the CYP3A4 gene. These cloned cells also responded at a signiˆcant level of CYP3A4 induction to treatment with various drugs. Judging from their high responses, stabilities and reproducibilities, our established cloned cells are very useful for the screening CYP3A4 induction at the early stage of drug development. Acknowledgements: This work was supported by a Grant-in-Aid from the Ministry of Education, Sciences and Culture (Ministry of Education, Culture, Sports, Sciences and Technology), the Ministry of Health and Welfare (Ministry of Health, Labour, and Welfare) of Japan and Comprehensive Research and Education Center for Planning of Drug Development and Clinical
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