Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

Abstract

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells.

METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography (HPLC).

RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild typeCYP2C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 ± 0.109 μmol•min^-1·g^-1 S9 protein or 8.62 ± 2.02 mol•min^-1·mol^-1 CYP, but was undetectable in parental CHL cell.

CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL-CYP2C9, efficiently expressing the protein of CYP2C9, was established.

Key Words: $[Keywords]

INTRODUCTION

Cytochrome P-450 (CYP) is a heme-containing enzyme widely distributed from bacteria to mammals, which catalyzes oxidative or reductive metabolism of a wide variety of substances including endogenous as well as exogenous compounds. Mammalian CYP present in liver microsomes is one of the key enzymatic mechanisms for the matabolism of drugs, pesticides, environmental pollutants, and carcinogens[1]. Mammals possess at least 17 distinct CYP gene families that together code for an estimated 50-60 individual CYP genes in any given species[2]. The human CYP2C subfamily comprises four members, CYP2C8, CYP2C9, CYP2C18 and CYP2C19[3], accounting for 20% of the total CYP in human liver. CYP2C9 is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs such as S-warfarin, phenytoin, tolbutamide, torsemide, losartan, fluoxetine, dapsone[4], cyclooxygenase-2 inhibitor: celecoxib[5,6], nonpeptide angiotensin II receptor antagonist: irbesartan[7] and numerous nonsteroidal anti-inflammatory drugs. It ranks among the most important drug metabolizing enzymes in humans[8].

The combination of gene technology and cell culture technology has provided new opportunities for studying proteins because any gene from any species encoding an protein may be cloned and expressed in bacterial, yeast, or mammalian cells in a defined way[9-18]. This approach to drug metabolism is of particular importance because some of the enzymes are difficult to purify and prepare in sufficient quantities, or expression levels are low, expression is organ-specificity, or the enzyme-product organs are very scarce. These restrictions apply especially for human enzymes. The heterologous expression of the cDNA allows to bypass these restrictions[19]. Human CYP2C9 previously has been expressed in E. coli[20], Salmonella typhimurium[21], yeast[22] COS-1[3], human liver epithelial cell THLE[23], and human hepatic cell line HepG2[24]. Several cell lines stably expressing human CYP1A1[25], CYP2B6[25], CYP2A6[26], CYP3A4[27], CYP2C18 (in press) and a phase II metabolism enzyme UDP-glucuronosyltransferase, UGT1A9[28] have been established in our laboratory. In this study human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and a transgenic cell line stably expressingCYP2C9 was established.

MATERIALS AND METHODS

Materials

Restriction endonucleases, Moloney murine leukemia virus (M-MuLV) reverse transcriptase were purchased from MBI Fermentas AB, Lithuania. Taq plus I DNA polymerase, dNTPs, PCR primers, DNA sequence primers and random hexamer primer were supplied or synthesized by Shanghai Sangon Biotechnology Corp. DNA sequencing kit was supplied by Perkin-Elmer Corp. The TRIzol reagent, G418, minimum essential media (MEM) and newborn bovine calf sera from Gibco. NADPH from Roche Molecular Biochemicals. Diethyl pyrocarbonate (DEPC), tolbutamide and hydroxytolbutamide were obtaineded from Sigma Chemical Company. T4 DNA ligase and pGEM-T vector system were supplied by Promega. Other chemical reagents used were all of analytical purity from commercial sources.

Methods

Cloning of human CYP2C9 cDNA from a Chinese human liver The total RNA was extracted from a surgical specimen of human liver with TRIzol reagent according to the manufacture's instructions, and then the first strand of cDNA was reverse transcripted from mRNA. Procedure: 5 μg of the total RNA and 2 μg of random hexamer primer in deionized DEPC-treated water were denatured at 65 °C for 15 min, then 4 μL 5 × reverse transcription buffer, 3 μL 10 mmol•L⁻¹ dNTP, 1 μL M-MuLV reverse transcriptase (200 U) and essential deionized DEPC treated water was added to a total volume of 20 μL. The reaction was performed at 25 °C for 10 min, then at 42 °C for 1 h, and finally at 70 °C for 10 min to inactivate reverse transcriptase. The reactant then was stored at 4 °C. To amplify the human CYP2C9 cDNA by PCR, 2 μL of the reactant were mixed with 2 μL of 10 mmol•L⁻¹ each of dNTPs, 20 pmol of PCR primers and 4 u of Taq plus I DNA polymerase in 1 × PCR buffer containing 1.5 mmol•L⁻¹ MgCl₂. A total volume of 100 μL was reached by adding deionized water. Two specific 32 mer and 28 mer oligonucleotide PCR primers were designed according to the cDNA sequence of CYP2C9 clone 25 reported by Romkes et al[3] (GenBank accession no. M61855, J05326). The sense primer corresponding to base position 1 to 32 was 5'-GAGAAGGTACCAATGGATTCTCTTGTGGTCCT-3', with a restriction site of Kpn I, and the anti-sense one, corresponding to the base position from 1513 to 1540, was 5'- AGAGGAAAGAGAGCTCGAGGGACTGCAC-3' with a restriction site of Xho I. The PCR was performed at 94 °C 2 min, then 35 cycles of 94 °C 60 s, 60 °C 60 s, 72 °C 2 min, and lastly 72 °C 10 min. The product was stored at 4 °C. An aliquot of 10 μL from the PCR was subjected to electrophoresis in a 10 g•L⁻¹agarose gel stained with ethidium bromide.

Construction of recombinant pGEM-CYP2C9 and sequencing of CYP2C9 cDNA[29]

The PCR product of about 1.5 kb in length, recovered and purified by electroelution into dialysis bag was ligated with a clone vector, pGEM-T (Promega), by T4 DNA ligase. E. coli DH5α was transformed with the resulted recombinant pGEM-CYP2C9 and the replicated plasmid was harvested from the bacteria screened by ampicillin resistant and blue-white selection with X-gal and IPTG. The cDNA of YP2C9 cloned in pGEM-T was sequenced on both strands by dideoxy chain-termination method marked with BigDye with primers of T7 and SP6 promoters and two specific primers of 5'-TGCCTTGTGGAGTTGAGA-3' (463-483), and 5'-ACAGAGACGACAAGCACAAC-3' (907-926). The termination products were resolved and detected using an automated DNA sequencer (Perkin-Elmer-ABI Prism 310).

Construction of the pREP9 based expression plasmid for CYP2C9

The Kpn I/Xho I fragment of the human CYP2C9 cDNA cleaved from the recombinant pGEM-CYP2C9 recovered and purificated by electroelution into dialysis bag was cloned directly into a unique site Kpn I/Xho I within the multiple cloning sites of the mammalian expression vector pREP9 (Invitrogen) with T4 DNA ligase. The recombinant was transformed to E. coli Top 10, and screened by ampicillin resistant. The recombinant was identified by restriction mapping.

Transfection and selection[29]

Chinese hamster lung (CHL) cells were transfected with the resultant recombinant, pREP9-CYP2C9, using a modified calcium phosphate method. After 24 h incubation in MEM containing 10% newborn bovine calf sera at 37 °C, the culture was rinsed and re-fed with fresh growth medium. After 72 h post-transfection, the culture were split and then selected in the culture medium containing the neomycin analogue G418 (400 mg•L⁻¹). The selective medium was changed every 3-4 d to remove dead cells and to allow the growth of resistant colonies. After 1 mo, surviving colonies (termed CHL-CYP2C9) were harvested as a pool and propagated in medium containing G418.

Preparation of S9 of CHL-CYP2C9

CHL-CYP2C9 cells grown in the culture medium containing G418 (400 mg•L⁻¹) were rinsed with phosphate balanced solution (PBS), scraped and collected from the bottle with 11.5 g•L⁻¹ KCl aqua solution and then sonicated in 200 W, 5 s for 10 times with 10 s of interval break. The resulted homogenate was centrifuged at 9000 g at 4 °C for 20 min and the postmitochondrial supernatant (S9) was transferred carefully to a clean tube for assay or storage under -70 °C. The protein in S9 was determined by the method described by Lowry et al, with bovine serum albumin as standard. CYP was measured spectrally using the method of Johannesen et al[30].

Tolbutamide hydroxylase assay[22,31]

The CYP2C9 tolbutamide hydroxylase activity of S9 fraction was determined by high performance liquid chromatography (HPLC). The assay was performed in a total volume of 500 μL containing final concentrations of 5 mmol•L⁻¹ HEPES (pH7.4), 1.5 mmol•L⁻¹ MgCl₂, 0.1 mmol•L⁻¹ EDTA, 0.25 mg S9 and 1 mmol•L⁻¹ sodium tolbutamide. The reaction was initiated with 0.5 mmol•L⁻¹ NADPH and terminated after 60 min at 37 °C by the addition of 50 μL of 4 mmol•L⁻¹ HCl. Reaction product was extracted by vortex-mixing of 3 mL of water-saturated ethyl acetate with the mixture for 2 min. The organic layer was collected after centrifugation in a table top centrifuge at 1000 g for 5 min. After most of the ethyl acetate extract had air-dried, the rest was removed in a heating block at 75 °C. The residue was resolubilized in 200 μL of methanol, and reaction product, hydroxytolbutamide was then assayed using HPLC by injecting 20 μL of the solubilized extract on to a reversed phase column (Shim-pack CLC-ODS 15 cm ± 0.6 cm id, 10 μm particle size), using 0.5 g•L⁻¹ phosphoric acid, pH2.6, acetonitrile (6:4/V:V) as the mobile phase with a flow rate of 1 mL•min^-1. The column elution was monitored at 230 nm, and rates of product formation were determined from standard curves prepared by adding varying amounts of hydroxytolbutamide to incubations conducted without NADPH.

RESULTS

Construction of recombinants

The recombinant of pGEM-CYP2C9 (Figure 1) was constructed with the human CYP2C9 cDNA inserted into the cloning site of vector pGEM-T between the promoters of T7 and SP6. Selection and identification of the recombinant was carried out by Kpn I/Xho I endonuclease digestion and agarose gel electorphoresis (Figure 1). The cloned cDNA segment was sequenced. In comparison with the CYP2C9 clone 65 cDNA sequence reported by Romkes et al[3] (GenBank accession no. M61857, J05326), our preparation showed two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, i.e. L7, P382 respectively.

Figure 1 Scheme and electrophoresis identification of recombinant pGEM-CYP2C9. A: Scheme of recombinat of pGEM-CYP2C9; B: Electrophoresis identification of recombinant pGEM-CYP2C9; 1: Marker (λ/EcoR I and Hind III); 2: PCR product ofCYP2C9 (1.54 kb); 3: Recombinant of pGEM-CYP2C9 digested by Kpn I and Xho I; 4: pGEM-T vector

The Kpn I/Xho I fragment (1.5 kb) containing the complete CYP2C9 cDNA was subcloned into the Kpn I/Xho I site of mammalian expression vector pREP9 (Figure 2). Selection and identification of the recombinants were carried out by Kpn I/Xho I endonuclease digestion and agarose gel electrophoresis (Figure 2). The resulting plasmid was designated as pREP9-CYP2C9 and contained the entire coding region, along with 2 bp of the 5'end and 41 bp of the 3'end untranslated region of the CYP2C9 cDNA, respectively.

Figure 2 Scheme and electrophoresis identification of recombinant pREP9-CYP2C9. A: Scheme of pREP9-CYP2C9; B: Electrophoresis identification of recombinant pREP9-CYP2C9; 1: Marker (λ/EcoR I and Hind III); 2: PCR product ofCYP2C9 (1.54 kb); 3: Recombinant of pREP9-CYP2C9 digested by Kpn I and Xho I; 4: pREP9 vector

Establishment of transgenic cell lines with CYP2C9 enzyme activity

CHL cells were transfected with pREP9-CYP2C9, and selected with G418 (400 mg•L⁻¹). The surviving colonies were propagated and a cell line termed CHL-CYP2C9 was established. The tolbutamide hydroxylase activity of CYP2C9 in S9 fraction of CHL-CYP2C9 cells was assayed by HPLC. A typical elution profile of hydroxytolbutamide in extracts is shown (Figure 3).CYP2C9 enzyme activity with tolbutamide was found to be 0.465 ± 0.109 μmol•min^-1•g^-1 S9 protein or 8.62 ± 2.02 mol•min^-1•mol^-1 CYP (n = 3), but none was detectable in parental CHL cells. The CYP content was 57.7 nmol•g^-1 S9 protein from CHL-CYP2C9 but no detectable CYP was present in CHL cell.

Figure 3 Representative chromatogram of extractsA Shim-pack CLC-ODS column (15 cm ± 0. 6 cm i.d.) was used. The mobile phase was constituted with phosphoric acid (pH2.6), acetonitrile (6:4/V:V) with the flow rate at 1 mL·min^-1. Hydroxytolbutamide was monitored at 230 nm. A: hydroxytolbutamide; B: tolbutamide

DISCUSSION

Direct cloning of human CYP cDNAs from cDNA libraries generally has been successful using anti-rodent or anti-human CYP antibodies and rodent CYP cDNA probes. But these cloning procedures are applicable only for the most abundantly expressedCYP mRNAs. Using the RT-PCR to clone low abundance CYP cDNA is a simple and direct method. CYP2C9 mRNA was present in human liver[32], HepG2 cells[33], kidney, testes, adrenal gland, prostate, ovary, duodenum[34], and brain tumors[35]. The pGEM-T vector system possesing single 3'-T overhangs at the insertion site greatly improves the efficiency of ligation of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for PCR products generateed by Taq Plus I DNA polymerases.

The human CYP2C9 gene is located on chromosome 10q24. Up to date, 12 CYP2C9 alleles have been identified (see: Table 1 and CYP2C9 alleles nomenclature http://www.imm.ki.se/CYPalleles/cyp2c9.htm). CYP2C9*1 is the wild type of humanCYP2C9. CYP2C9*2 exhibit a base substitute 430C > T, resulting in a R144C substitution which has been suggested to affect the interaction between the CYP enzyme molecule and the cytochrome P450 reductase[36]; this may explain the slower metabolism of some CYP2C9 substrates such as S-warfarin and tolbutamide[8,37]. CYP2C9*3 has a base substitute 1075A > C, which leads to an I359L substitute. Takanashi et al[38] expressed the CYP2C9*1 and CYP2C9*3 cDNA in yeast and examined the kinetics of seven individual metabolic reactions by wild-type CYP2C9*1 and its CYP2C9*3 variant. Their results indicated that the I359L exchange significantly reduces the catalytic activity with all CYP2C9-mediated substrates studied, although the extent of the reduction in activity and kinetic parameters varied between different substrates. Interestingly Kidd et al[39] reported a male Caucasian, homozygous for CYP2C9*3, who poorly metabolized phenytoin and glipizide/tolbutamide. This study establishes that the I359L mutation is responsible for the poor metabolizer phenotype. The CYP2C9*2 and CYP2C9*3 are responsible for the poor metabolizing celecoxib[5], losartan[40], torsemide[41]. CYP2C9*4[42] has a base substitute of 1076T > C, leading to a I359T substitution. Ieiri et al[43] evaluated the catalytic activity of three variants (I, L, and T) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system. The specific catalytic activities were assessed by diclofenac-4'-hydroxylation. The in vitro study revealed that recombinant I359, L359, and T359 (2 batches) exhibited a mean Km of 2.0, 16.5 and (3.8 and 2.9) μmol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol•min^-1·nmol^-1CYP, respectively. TheCYP2C9*5 variant is derived from a 1080C > G transversion in exon 7 of CYP2C9 that leads to a D360E substitution in the encoded protein[44]. The CYP2C9*5 variant was found to be expressed in African-Americans with a frequency of approximately 3% in this population group. This variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. The in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates compared to individuals expressing the wild-type protein[44]. Kidd et al[45] reported a new CYP2C9 allele (CYP2C9*6) with the deletion of an adenine at base pair 818 of the cDNA. The clearance of phenytoin in this individual is estimated to be approximately 17% of that observed in normal patients. The frequency of this allele was 0.6% in 79 African-Americans and 0% in 172 Caucasians. Shintani et al[46] reported that mutations in the 5'-flanking region of the humanCYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity. Compared with CYP2C9*1 cDNA, there are two base differences in the CYP2C9 cDNA cloned by us, but the encoded amino acid sequence remains unchanged. This obviously is the molecular basis for full enzymatic activity.

Table 1 Nomenclature and characteristics of CYP2C9 alleles.

Allele	Protein	Nucleotide changes	Effect	Enzyme activity		Ref.
				In vivo	in vitro
CYP2C9*1	CYP2C9.1	None		Normal	Normal	3
CYP2C9*2	CYP2C9.2	430C > T	R144C		Decr	36
CYP2C9*3	CYP2C9.3	1075A > C	I359L	Decr	Decr	37, 38, 39, 43
CYP2C9*4	CYP2C9.4	1076T > C	I359T			42, 43
CYP2C9*5	CYP2C9.5	1080C > G	D360E	Decr		44
CYP2C9*6		818delA	frame shift	Decr		45
CYP2C9*7-*12						In press

To express the functional activity of a CYP, a cell evidently must have adequate heme supply, either by intracellular biosynthesis or extracellular provision[47]. CYPs also require other enzymatic components for full activity, including the flavoprotein NADPH-P450 oxidoreductase (OR) and, in some cases, cytochrome b5. The OR must interact directly with the CYP to transfer the required two electrons from NADPH. Cytochrome b5 is necessary for increasing electron transfer for certain CYP forms and specific substrates. The CHL is the cell line originally derived from the lung of a newborn female Chinese hamster and has no or very limited activities of CYP enzymes, but has adequate OR and cytochrome b5 levels to support CYP activities.

To achieve high expression levels of CYP2C9, the CYP2C9 cDNA was cloned into the eukaryotic expression vector pREP9, which had previously been used in this laboratory for the expression of human CYP1A1, CYP2B6, CYP2A6, CYP3A4 and UGT1A9 in CHL cells[25-28]. The salient feature of this vector has an Epstein Barr Virus origin of replication and nuclear antigen (EBNA-1) to allow high-copy episomal replication in mammal cell lines. The Rous sarcoma virus long terminal repeat (RSV LTR) early promoter controls the expression of the CYP2C9 cDNA.

Tolbutamide (1-butyl-3-p-tolylsulfonylurea) is an oral hypoglycemic agent which is being used in the treatment of diabetes. In humans it undergoes CYP-catalyzed hydroxylation of the tolyl methyl group which is the initial and rate-limiting reaction followed by further oxidation by cytosolic dehydrogenases yielding carboxytolbutamide. Overall this pathway accounts for up to 85% of tolbutamide clearance in humans. Evidence that CYP2C9 is solely responsible for tolbutamide hydroxylation is convincing and tolbutamide is widely accepted as a prototypic substrate for the assessment of hepatic CYP2C9 activity, both in vitro and in vivo[8].

We used tolbutamide as a substrate for evaluating the expressing of human CYP2C9 activity in CHL-CYP2C9 cell. The tolbutamide hydroxylase activity was 0.465 ± 0.109 μmol•min^-1·g^-1 S9 protein or 8.62 ± 2.02 mol•min^-1•mol^-1CYP. These value, were somewhat higher than these obtained with recombinant CYP2C9 purified from E. coli: 4.67-4.96 nmol•min^-1•nmol^-1CYP[48] or human liver microsomes: 0.189 ± 0.0083 nmol•min^-1•mg^-1 microsome[49] and 2.29-4.33 nmol•min^-1•nmol^-1 CYP[48]. This clearly stated that CHL- CYP2C9 expressed the CYP2C9 efficiently and this may be a useful tool for further studies of its enzymatic function and mechanism.