Xin Li^{1, 2}, Ying-Nian Yu¹, Ge-Jian Zhu¹, Yu-Li Qian^1
1Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, China
²College of Pharmcy, Zhejiang University, Hangzhou, China
Supported by the National Natural Science Foundation of China (C39370805), Zhejiang Provincial Natural Science Foundation (300487) and the Excellent Youth Scientist Fund of Zhejiang Province
Correspondence to: Prof. Ying-Nian Yu, Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, China. zjhsin@yahoo.com
Telephone: +86-571-87217203, Fax: +86-571-87217149
Received 2001-06-19 Accepted 2001-10-20

Abstract
AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9.

METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E .Coli DH5α. The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recom binants, pREP9-UGT1A9, and selected by G418 (400 mg·L^-1) for one mo nth. The surv iving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in mediu m con taining G418 to obtain samples for UGT1A9 assays. The enzyme activity of CHL -UGT1A9 towards propranolol in S9 protein of the cell was determined by HPL C.

RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was id entical to that released by Gene Bank (GenBank accession number: AF056188) in co ding region. The recombinant constructed, pREP9-UGT1A9, contains the entire codi ng region, along with 18 bp of the 5’ and 55 bp of the 3’ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the pro tein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was fo und to be 101± 24 pmol·min^-1·mg^-1 protein (n=3), but was not detectab le in parental CHL cells.

CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chines e human liver and transfected into CHL cells. The CHL-UGT1A9 ce ll lines established efficiently expressed the protein of UGT1A9 for the fur ther enzyme study of drug glucuronidation.

Subject headings: UGT1A9; cloning; glucuronidation; cell li nes

Li X, Yu YN, Zhu GJ, Qian YL. Cloning of UGT1A9 cDNA from liver tissue s and its expression in CHL cells. World J Gastroenterol, 2001;7(6):841- 845

INTRODUCTION
Most organisms are exposed to a range of lipophilic compounds an d converted them into excretable hydrophilic compounds. This metabolism of forei gn compounds (xenobiotics) can be divided into two phases. For phase I metabolis m, a reactive group is mostly introduced into the xenobiotic molecule. These rea ctions are mainly catalyzed by the cytochrome P450 monooxygenase system which co nsists of cytochrome P450s (CYPs) and cytochrome P450 reductase (CPR). For phase II metabolism, the reactive metabolite is conjugated to small, hydrophilic endo genous molecules such as glucuronic acid. The conjugation of this cofactor to xe nobiotics is catalyzed by UDP-glucuronosyltransferases (UGTs). Since xenobiotic   metabolizing enzymes have to catalyze the metabolism of structurally very divers e substrates, the various enzyme systems (e.g. CYPs and UGTs) comprise several i sozymes that differ in their catalytic properties. The members of a given enzyme system have been grouped into families and subfamilies based on sequence homolo gies. In UGTs, two enzyme families termed UGT1 and UGT2 have been described.
      The UGT1 locus is highly conserved between species^[1]. UGT1A is a subfam ily of U GT1 gene complex that is located at chromosome 2q37. UGT1A subfamily is encoded by tandem individual promoters and their first exons are linked by differential splicing to four common exons. As one of the isoforms, UGT1A9, is mainly exp ress ed in liver. UGT1A9 can be induced by polycyclic aromatic hydrocarbons (PAHs ), a nd therefore the drug glucuronidation catalyzed by UGT1A9 will be increased in cigarette smokers who inhale PAHs^[2].
      Human hepatic UDP-glucuronosyltransferase s (UGT) is a family of microsomal enzymes that catalyze the glucuronidation of m any important drugs, xenobiotics and endogenous compounds. Attempts to character ize the microsomal enzymes by conventional purification technique are often frus trated due to its instability. UGT isoenzyme expressed by cells is a useful tool for characterizing UGT’s function. The cDNA cloning of UGTs from various sourc es (rabbit, rat, monkey, human beings, etc.) and their expression in cell line s were widely used for the gene characterization and function study of UGT isofor ms^[3-9]. In order to study tie drug metabolisms by UGTs, the cDNA encod ing UGT1A9 was cloned from human liver and expressed in Chinese hamster lung (CHL) cell line in this study. The enzyme expressed was extracted and its activity was assa y ed with a substrate of propranolol which is a nonselective β-adrenergic blocki ng agent and can be used widely clinically^[10].

MATERIALS AND METHODS
Isolation of RNA from human liver tissue
Human liver tissue was obtained from a surgical sp ecimen of Chinese and stored at -80℃ until use. The total RNA was isolated wit h TRIzol reagent (Gibco Corp, USA)

UGT1A9 cDNA transcription
cDNA was transcr ipted from mRNA by revere transcriptase polymerase chain reaction (RT-PCR). Fiv e μg of the total RNA and 2 μg of random primer (SANGON, Shanghai) in deionized wate r containing DEPC (1g·L^-1) were denatured at 65℃ for 15 min, then 4μL 5 ×rever se transcriptase buffer, 3 μL10 mmol·L^-1 dNTP, 1 μL M-MuLV reverse tra nscriptase (200 U) (Fermentas) and essential deionized water containing DEPC (1g·L^-1 ) were added to have the total volume of 20 μL. The reaction was performed at 25℃ fo r 10 min, then 42℃ for 1 hour, and 70℃ for 10 min to inactivate the reverse tra nscr iptase. The product was finally held at 4℃. Two μL of the reactant was mixed w ith 2μL of 10 mmol·L^-1 dNTP, 30 pmol of PCR primers and 3.5 U of DNA ploym erase (Pe rkin-Elmer Corp). The total volume of 100 μL was reached by adding deionized w at er. Two 26 mer oligonucleotides as PCR primers were designed according to the DN A sequence of UGT1A9 (GenBank accession no. AF056188). The sense oligonucleo tide s corresponding to base positions 1 to 26 was 5’-CTAAGCTTCAGTTCTCTGATGGCT TG-3 ‘ w ith a restriction site of Hind III, and the anti-sense one, corresponding t o the bases position from 1641 to 1666, was 5’-GTTGGAAATGCCTAGGGAATGGTTC-3’. The poly merase chain reaction (PCR) was performed at 94℃ 2 min, then 94℃ 15 s, 60.1℃   30 s and 72℃ 2 min for 31 cycles, and 72℃ for 10 min. The product was finally held at 4℃. An agrose gel electrophoresis was carried out with 10 μL of the P CR solution to check the 1666bp DNA amplified.

Construction of recombinant pGEM-UGT1A9 and sequencing of UGT1A9
The PCR product of about 1.5 kb was isolated and ligated with pGEM-T (Promega) vector by T4 DNA ligase (Fermentas). E.Coli DH5a was transformed with the resulted recombinants pGEM-UGT1A9^{[11 ]} and t he posit ive bacteria colonies were screened by ampicillin resistant and blue-white scre e ning with X-gal and IPTG. The cDNA of UGT1A9 subcloned in pGEM-T was seque nced o n both strands by dideoxy chain-termination method marked with BigDye with prim e rs of T7 and SP6 promoters and a specific primer of 5’-CAAGTATCGTGTTGTTCGC-3 ‘. T he termination products were resolved and detected using an automated DNA sequen cer (Perkin-Elmer-ABI Prism 310, Foster City, CA).

Construction of the pREP9 based expression plasmid for UGT1A9
The Hind III-Not I fragment of the human UGT1A9 cDNA cleaved from t he selected and amplified recombinant pGEM-UGT1A9 by Hind III and Not I digestion was purified by agarose electrophoresis and cloned di rectly into a unique Hind III-Not I site within the multicloning site o f the mammalian expression vector pREP9 (Invitrogen, San Diego, CA) with T4 ligase.

Transfection and selection
Chinese hamster lung (CHL) cells were transfected wi th the resultant recombinants, pREP9-UGT1A9, using a calcium phosphate meth od^{[12 ]}. After 24 h incubation at 37℃, the culture was rinsed and re-fed with fre sh g rowth medium. Seventy-two h after transfection, the culture was split and then selected in the culture medium containing the neomycin analogue G418 (Gibco BRL, MD) (400 mg ·L^-1). The selective medium was changed every 3-4 d to remove dead ce lls and allow the growth of resistant colonies. After 1 mon, surviving clonies (termed C HL-UGT1A9) were harvested as a pool and propagated in medium containing G41 8.

Preparation of S9 of CHL-UGT1A9
CHL-UGT1A9 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 11.5g·L^-1 KCl in aqua soluti on and t hen sonicated 3 s for 5 times with 5 s of interval break. The resulted homogenat e was centrifuged at 9000×g for 20 min and the supernatant (S9) was transfe rred carefully to a clean tube for assay or storage under-70℃. The protein in S9 wa s determined by the same method that was used in our previous paper^[13].

UGT assay
The UGT1A9 activities of S9 fraction were determined by the gl ucuronidation of propranolol. The assay was performed in a total volume of 100 μl containing final concentrations of 0.2 mmol·L^-1 propranolol, 1 mmol · L^-1 UDPGA, 1g ·L^-1 Triton X-100, 50 μg of S9 protein in 50 mmol· L^-1 Tris-HCl, 10 mmol·L^-1 MgCl₂ buffer, pH 7.8 at 37℃. The mixtures were pre-incubated and the glucur o nidation was started by the addition of UDPGA and stopped after 2 h by the addit io n of 100 μL of methanol. The mixtures were stirred thoroughly and centrifuged a t 10000 r·min^-1 for 10 min. Un-reacted propranolol in the layer of reacta nt was d etermined by HPLC and the enzyme activity was calculated according to the amount of propranolol declined after incubation.

HPLC analysis of propranolol metabolized by S9 of CHL-UGT1A9
The concentration of propranolol metabolized by S9 of CHL-UGT1A9 was assaye d by the HPLC procedure^[13]with modification to the mob ile phase. Twenty mL of the sample was applied to a reversed phase col umn ( Shim-pack CLC-ODS15cm×0.6cm id, 10 μm particle size). Propranolol was mon itored with a UV detector at 290nm. The mobile phase is made up with ammonium ac etate buffer (4.0 g ammonium acetate, 10 mL acetate acid and de-ionized water in 1 L)-methanol-acetonitrile (2:1:1), and to 500 mL mobile phase add 0.7 mL triethylamine as the elution modifier. The flow rate is 1.0mL·min^-1.

RESULTS
Construction of recombinants
The recombinant of pGEM-UGT1A9 (Figure 1) was const ructed with the human UGT1A9 inserted into the cloning site of vector pGEM- T bet ween the promoters of T7 and SP6. Selection and identification of the recombinan t was carried out by Hind III / Not I endonuclease digestion and agarose elector phoresis (Figure 3). The cloned DNA segments in selected recombinants were seque nced completely. According to the results of DNA sequencing, the cDNA in a selec ted recombinant was identical to the DNA sequence of UGT1A9 reported by Ciot ti-M et al (GenBank accession no. AF056188) in the reading frame. The restrictio n si tes of Hind III and Not I in the recombinant were used for the subclonin g of insertion fragment into an expression vector.

Figure 1(PDF) Scheme of pGEM-UGT1A9.

The Hind III/Not I fragment (1.5 kb) containing the complete UGT1A9 cDNA was s ubcloned into the Hind III/Not I site of mammalian expression vector pRE P9 (Figu re 2). Selection and identification of the recombinants were carried out by Hi nd III/Not I endonuclease digestion and agarose electrophoresis (Figure 3) . The re sulting plasmid was designated as pREP9-UGT1A9 which contained the entire c oding re gion, along with 18 bp of the 5’ and 55 bp of the 3’ untranslated region of th e UGT1A9 cDNA, respectively. In addition, the neo gene of the plasmid conf ers the G418 resistant phenotype to CHL cells for the selection of transfected cells.

Establishment of recombinant cell lines with UGT1A9 enzyme activi ty
CHL cells were transfected with pREP9-UGT1A9, and selected with G418 (400 mg·L^-1). The surviving clone was propagated a nd the cell line termed CHL-UGT1A9 was established. The preparation S9 was prepare d from CHL-UGT1A9 cells harvested for UGT1A9 activity assay by HPLC. Figure 4 shows t he typical elution of propranolol in incubation solution. The UGT enzyme act ivit y towards propranolol in S9 protein was found to be 101±24 pmol·min^-1·m g^-1 (n=3), but was not detectable in parental CHL cells.

Figure 2(PDF) Scheme of pREP9-UGT1A9.
Figure 3(PDF) Electrophoresis identification of recombinants constr ucted.
Lanes 1: Marker (λ/EcoR I and Hind III); 2: PCR produ ct of UGT1A9 from Chinese human liver; 3: recombinant of pGEM-UGT1A9; 4 : recombi nant of pGEM-UGT1A9 cleaved by Hind III and Not I; 5: recombinant o f pREP9-UGT1A9; 6: recombinant of pREP9-UGT1A9 cleaved by Hind III and Not I; 7:pREP9 expression vector; 8:Marker (λ/Hind III).
Figure 4(PDF) Chromatogram o f propranolol after incubation with S9 prepared from CHL-UGT1A9 cell. A Shi m-pac k CLC-ODS column (15cm×0.6cm i.d.) was used. The mobile phase was constituted with ammonium acetate buffer-methanol-acetonitrile (2:2:1) and 1.4mL·L ^-1 triethy lamine with the flow rate at 1.0mL·min^-1. Propranolol was monitored at 290nm. propranolol: t_R=9.065min.

DISCUSSION
UGTs are involved in the conjugation of UDP-glucuronic acid (UDPGA) to a variety of chemicals, drugs, and endogenous compounds. The elimination of hydrophobic chemicals from cells is aided by their conversion to water-soluble glucuronides. UGTs are closely relatied to the system of cytochrome P450 monooxygenase, and involved in the transportation of c a rrier and the passage of drugs through cell phospholipid bilayer. In most cases, the lipophilic compounds are converted by phase I metabolism to the substrate f o r glucuronidation by obtaining an essential function (such as carbon, nitrogen, sulfur and oxygen), but in many cases xenobiotics and endogenous substances canalso be glucuronidated by UGTs without the phase I metabolism. The xenobiotic me tabolizing cytochrome P450 monooxygenase system and the UGTs reside mainly in th e endoplasmic reticulum. However, CYPs and the CPR are localized on the cytosolic side of the endoplasmic reticulum, which the UGTs are localized on its lumin al side^[14]. UGTs are latent enzymes, needing activation (in general by detergents) to express its maximal activity.
      Numerous reports revealed that glucuronidation is a major pathway involved in the metabolism of drugs, exogeno us, and numerous endogenous compounds such as bile acids and steroid hormones. E ach UGTs family or subfamily has its own substrates but the substrate spectrum a re partly overlapped. UGT1 has substrates such as thyroid hormone^[15], SN-38^[16], bilirubin^[17,18], opioids, bile acids, fatty acids, retinoids, c iprofibrate , furosemide, dilunisa, catechol estrogens, coumarins, flavonoids, anthraquinone s, EM-652 (an active antiestrogen)^[19] and phenolic compounds^{[20 ]}. UGT2 cataly zes substrates such as estrogens, androgens, morphine, AZT, and retinoic acid, e pirubicin^{[16, 21,22, 23]}, etc. UGT1A9 is a member of UGT1A subfamily . The endoge nous substrates for UGT1A9 are estrone, 4-hydroxyestrone, ethinylestradiol, retinoic acids, etc, and exogenous substrates include propofol, propranolol, paracet amol, S-naproxen, ketoprofen, ibuprofen, entacapone, some mutagenic arylamine s, etc^{[2, 24-26]}. UGT1A9 was found to have regioselectivity on the g lucuronidation of hydroxyl group of carbohydrate-containing drugs^[27].
      UGTs are expres sed extensively in organs and tissues, and they may play a key role in the regul ation of the level and action of steroid hormones in steroid target tissues. Org ans that express UGTs include liver, kidney, gastrointestinal tract^{[28-29 ]}, olfactory ^[30], jejunum, ileum^[31], prostate^[32-33], colon^[34]. UGT1A9 is mainly expressed in liver, and also expressed in steroid targets^[35] and colon^[34].
      UGTs are inducible enzymes. In most cases this induction is due to increased transcription of the corresponding genes but sometimes it is also due to an improved stability of proteins. The pattern of enzymes affected is dependent on the inducing agent. Usually, phenobarbital induces mainly enzymes within UGT2 famil y, and methylcholanthrene induces enzymes belonging to the UGT1 family^{[28 ]}. Oth er chemicals that induce UGTs include aryl hydrocarbon receptor ligands or oltip raz^[36], flavonoid chrysin^[37], and t-butylhydroquinone and 2,3 ,7,8-tetrachloro dibenzo-p-dioxin^[38], etc. UGT1A9 can be induced by polycyclic aro matic hydro carbons (PAHs)^[39]. On the other hand, UGTs can also be inhibited, for example by uridine diphosphate^[40], and N-glycosylation is involved in the func tional properties of UDP-glucuronosyltransferase enzymes^[41].
      To clone and express UGTs in cells can help screen substrates that an isoenz yme is responsible. The prod uction of a UGT enzyme protein using transgenic cell lines is a practical manner to study its function^[42-43]. We report here the cloning of UGT1A9 cDNA and es tablishment of a CHL cell line expressing UGT1A9 from a Chinese human liver. The full-length cDNA, UGT1A9, that encodes for a human UDP-glucuronosyltransf erase protein, was isolated from a Chinese human liver total RNA. To achieve high expr ession levels of UGT1A9, the UGT1A9 cDNA was cloned into the eukaryotic expressi on vector pREP9, which we had previously used in this laboratory for the express ion of human CYP450 1A1, 2B6, 3A4, etc in CHL cells^[44-45]. The sali ent feature of this vector has an EBV origin of replication and nuclear antigen (EBNA-1) to   allow high-copy episomal replication in mammal cell lines. The Rous sarcoma vir us long terminal repeat (RSV LTR) early promoter controls the expression of the U GT1A9 cDNA. As noted under “Results", the isolated clone contains a 1592-nuc leoti de open reading frame flanked by 18 and 55 base pairs of 5’ and 3’ noncoding s eq uences, respectively. The DNA sequence in the reading code frame is identical to that reported (GenBank accession no. AF056188). The expression of a protein tha t catalyzed the glucuronidation of propranolol was proven in the Chinese hamster lung cells transfected with the recombinant plasmid pREP9-UGT1A9.
      Conjugatio n with glucuronic acid is an important biotransformation pathway for a large num ber of clinically used drugs. In human intestinal, UGTs play an important role i n the detoxification of xenobiotics compounds and, in some cases, may limit the bioavailability of therapeutic agents^[20]. The deficient of a UGTs isoe nzyme, m ay cause disease and clinical incident^[46-47], the typical example was serious adverse events associated with chloramphenicol toxicity in neonates. Human UGTs are regulated in cases of healthy condition and exposure of harmful environmenta l carcinogens^[48-50]. Moreover, UGT was identified as an antigenic tar get in a subgroup of liver- kidney microsomal auto-antibodies^[51]. Hence, it is very ne cessary to undertake the study of functions and characteristics of UGTs. Over th e last decade, some research papers were published about the usage of cloned and expressed human UGTs for the assessment of human drug conjugations and identification potential drug interactions^[6-8]. However, the information gap s till exi sts regarding the enzymatic aspects of UGTs to drugs elimination and its potenti al impact on therapy. More researches on the drug metabolism by UGTs are necessa ry for effective translation of scientific information into clinically applicabl e knowledge. As has been shown with the CYPs, coupling of basic and clinical sci ence is needed to continually improve our understanding of the UGTs. Man y factors are known to influence the activities of UGTs involved in drug metabol ism, hence plasma clearances of glucuronidated drugs. Such factors include ag e (especially neonatal period), cigarette smoking, diet, certain disease states, drug therapy, ethnicity, genetics and hormonal effects. Knowledge of the profil e, substrate specificities and regulation of human UGTs remains limited and cons equently it is still generally not possible to predict the effects of specific e nvironmental and genetic factors on the metabolism and pharmacokinetics of indiv idual glucuronidated drugs. Future investigations must define the substrate spec ificities of the various UGTs and investigate mechanisms by which the separate i sozymes are regulated. Only then will it become possible to rationalize (and pr edict) the alterations in pharmacokinetics and response to glucuronidated drugs in specific patient groups.

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