[CANCER RESEARCH 50. 6949-6954. November I. 1990]

Stable Expression of Human Aromatase Complementary DNA in MammalianCells: A Useful System for Aromatase Inhibitor Screening1

Dujin Zhou, Denis Pompon, and Shiuan Chen2

Division of Immunology, Beckman Research Institute of the City of Hope. Duarte, California VIOIO ¡D.Z., S. C.], and Ceñirétie GénétiqueMoléculairetilt Centre\ational tie la Recherche Scientifique, Laboratoire Propre Associe a l'UniversitéPierre et Marie Curie. 91190 (¡if-sur-Yvette. France /l). P./

ABSTRACT

A mammalian cell expression plasmid, pH ,>'-\ro. containing the

human placenta aromatase complementary DNA was constructed. Theprepared plasmid was used to transfect breast cancer cells (MCF-7),noncancerous breast cells ( I lit] 1(10),and Chinese hamster ovary cellsby a stable expression method. While the maximum velocities for aromatase expressed in three types of cells »eredifferent (10-201 pmol of|'HjO| formed/h/mg) using |l/9,2|8--'H|androst-4-ene-3,17-dione as the

substrate, the apparent Michaelis-Menten constants »erefound to besimilar (39.9-57.8 PM) and »erewithin the range determined for theenzyme existing in human placenta. The expressed activities were inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione andaminoglutethimide, at concentrations that normally inhibit the humanplacenta! aromatase. However, it was found that the inhibition profileswere different for aromatase expressed in different types of cells, suggesting that other factors, such as the uptake of the inhibitor, may alsoplay a role in determining the inhibition efficiency. These constructedaromatase expressing mammalian cell lines will be very useful tools foraromatase inhibitor screening.

INTRODUCTION

Estrogens play an important role in breast cancer development, and aromatase cytochrome P-450 is a microsomal enzyme catalyzing the formation of estrogens from androgens.An abnormal expression of aromatase has been found in asignificant number of breast tumors (1-4). Although the significance of aromatase expression in breast tumors and the originof estrogen in these tissues are not yet completely understood,aromatase inhibitors are thought to be of value in treatingestrogen-dependent breast cancer by inhibition of estrogen production, especially in postmenopausal patients. Other investigators have reported that estrogens in postmenopausal womenare mostly produced at peripheral adipose tissues, and it hasbeen shown that the peripheral aromatase is not under gonad-otropin regulation (5). Therefore, complications due to a feedback regulatory mechanism, which increases LH' and follicle-

stimulating hormone after aromatase inhibitor treatment, willnot result in postmenopausal patients. In premenopausalwomen, LH and follicle-stimulating hormone stimulate thesynthesis of aromatase in ovaries and may counteract the effectsof some aromatase inhibitors, as reported for aminoglutethimide (6). Complete or partial tumor regression has been reported for postmenopausal patients treated with aromataseinhibitors, such as aminoglutethimide or 4-hydroxyandrostenedione (2, 7).

Aminoglutethimide was the first aromatase inhibitor used asa therapeutic agent for breast cancer. A number of more potent

Received 7/25/90; accepted 8/1/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 LJ.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH Grants CA 44735 and CA 33572.2To whom requests for reprints should be addressed.5The abbreviations used are: LH. leutinizing hormone; cDNA. complementary

DNA; CHO, Chinese hamster ovary: PCR. polymerase chain reactions; Km,Michaelis-Mcnten constants; Vm.K.maximum velocity.

and selective aromatase inhibitors have been synthesized duringthe last several years. Among them, 4-hydroxyandrostenedioneand CGS 16949A are now in clinical trials (7, 8). Since aromatase inhibitors are potentially useful drugs for treating breastcancer, the synthesis and screening of new aromatase inhibitorsremains an active area of research in many laboratories andpharmaceutical companies.

The most common method for aromatase inhibitor screeninginvolves an in vitro enzyme assay using human placenta! micro-somes that express a significant level of aromatase. This assaymethod is usually performed in the presence of a NADPH-cytochrome P-450 reducíasepreparation, and a NADPH-re-generating system. This is obviously an artificial system, andthe assay can be affected by all the components included. Thereare also methods using animal tissues that express aromatase.For example. Hausler et al. (9) recently reported the use of LH-treated adult female hamster ovarian tissues for aromataseinhibitor screening. The method using animal tissues cannoteliminate the issue of tissue heterogeneity, which may create aproblem when one wants to compare results from experimentto experiment. In addition, the assay requires tissues from alarge number of animals. Furthermore, although the methodreported by Hausler et al. (9) takes into consideration theinteraction of inhibitors with different steroidogenic enzymes,it does not address the problem that not much is known aboutaromatase or other steroidogenic enzymes in hamster ovary. Itis not unusual to find that an enzyme, when expressed indifferent tissues of the same species or in the same tissue butfrom different species, responds differently to different inhibitors. For example. Wing et al. (10) have demonstrated that 4-hydroxyandrostenedione is a more potent inhibitor for humanplacenta! aromatase than the rat ovary enzyme, while aminoglutethimide is a better inhibitor for the rat ovary enzyme.

Aromatase inhibitor screening using cultured human cellsthat express aromatase would be a good choice (e.g., Refs. 11-13). The assay could be performed using intact human cells,resembling more of the in vivo situation. However, since cultured cells express aromatase at a low level, the enzyme assaywould still require a long incubation time (normally, 3 to 6 h).In addition, cultured cells often have to be treated with properhormones to induce the expression of aromatase to a level thatcould be measured. In consideration of the problems encountered for currently available screening methods, we have recently developed a new system for aromatase inhibitor screening. We have transfected mammalian cells with an expressionplasmid containing full-length aromatase cDNA and have gen

erated mammalian cell lines expressing high levels of humanaromatase. Aromatase is stably expressed in these cells, and thehigh level of expressed enzyme allows the enzyme assay to becarried out with 30-min incubation.

This article describes the construction of the aromataseexpression plasmid and the transfection experiments. We performed kinetic analysis to show that the expressed aromatasehas properties very similar to those of the enzyme present in

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human placenta. We also demonstrate that the expressed aro-matase is inhibited by 4-hydroxyandrostenedione and amino-glutethimide. Human aromatase cDNA has been previouslyexpressed in COS cells by a transient expression method (14),and recently expressed in yeast by a stable expression method(15).

MATERIALS AND METHODS

Chemicals. T4 kinase, T4 DNA ligase, Klenow fragment, and restriction endonucleases were obtained from Boehringer Mannheim Biochemical (Indianapolis, IN) and Bethesda Research Laboratories(Gaithersburg, MD). Radiolabeled nucleotides were from New EnglandNuclear (Boston, MA), and DNA sequencing kits were from UnitedStates Biochemicals (Cleveland, OH). The mammalian cell expressionvector, pH ßApr-1-neo, was obtained from Gunning et al. (16).

Aromatase Expression in Mammalian Cells. The full-length humanaromatase cDNA was cloned in this laboratory as described previously(15). The procedure for the construction of the aromatase expressionplasmid, pH /i-Aro, will be described in detail in "Results and Discussion." Aromatase was expressed in human breast cancer cells (MCF-

7), noncancerous human breast cells (HBL-100), and CHO cells bytransfection using the expression plasmid pH ß-Aro.The transfectionexperiments were done using Lipofectin'" following the manufacturer's

protocol (Bethesda Research Laboratories). The cells were incubatedat 37°Cwith 5% CO2 for 24 h in Ham's F12 media, after which media

containing 10% fetal calf serum were added. After an additional 48 h,the cells were transferred to selective media (Ham's F12) containing

G418 (600 Mg/ml for MCF-7 and CHO cells, and 300 Mg/ml for HBL-100 cells). After 2 weeks of selection, the cells were screened foraromatase expression by Southern blot analysis. Northern blot analysis,the polymerase chain reaction, and enzyme activity.

Southern and Northern Blot Analyses. The genomic DNA was isolated from cultured cells according to the method of Davis et al. (17),digested with restriction enzymes, electrophoresed in a 0.8% agarosegel, and then transferred to Zetaprobe membranes using the methodprovided by Bio-Rad. The bound DNA was hybridized using aromatasecDNA 2.4-kilobase fragment as the probe. The rest of the procedurefor Southern blot analysis was identical to that described previously(18).

The Northern blot analysis was performed according to the proceduredescribed previously by Pompon et al. (15). The probe was the aromatase cDNA 2.4-kilobase fragment.

Aromatase Expression Analyzed by Polymerase Chain Reactions.Total RNA was isolated from cultured cells by the method of Chirgwinet al. (19). PCR were performed according to the procedure of Saiki etal. (20) using the isolated RNA (100 ng/reaction) as templates. Thereactions were initiated with avian myeloblastosis virus reverse tran-scriptase (2 units), and followed by Tag polymerase (5 units) in thepresence of two primers (0.2 ^mol of each of two primers) withsequences derived from aromatase cDNA, 5'-ATCTCTGGAGAG-GAAACACTCATTA-3' and 5'-CTGACAGAGCTTTCATAAA-GAAGGG-3' (reverse primer). The reactions were carried out for 40

cycles. The DNA products (198 base pairs) were analyzed in a 1.8%agarose gel, and the bands were visualized by ethidium bromide staining. The DNA products were transferred to Zetaprobe membranes(Bio-Rad), followed by hybridization using a 12P-labeled probe derived

from the aromatase cDNA sequence in the region between the first twoprimers. The sequence of the latter oligonucleotide was 5'-ATTA-CAGCTCTCGATTCGGCAGCAA-3'. Hybridization using such a

third probe further ensured that the PCR products were those expected.The conditions for prehybridization and hybridization were accordingto the Bio-Rad instruction manual.

Aromatase Assay in Cells Transfected with pH 0-Aro Plasmid. Thetransfected cells expressed a high level of aromatase as indicated byactivity measurement. The enzyme assay was modified from a previousmethod (5) and performed directly on cultured cells without purificationof the enzyme. Cells were grown to confluence in six-well cell culture

plates, and were washed twice with serum-free cell culture mediumbefore assay. The substrate, androst-4-ene-3,17-dione[l/i,2fi-1H(A')]

(specific activity, 41.8 Ci/mmol; specific activity at \ii position, 18 Ci/mmol), was dissolved in serum-free cell culture medium, filter-sterilized, and then added into each well. The assay mixture also containedprogesterone (1 ^M), which inhibited the 5«-reductasein the cells. Thislatter enzyme utilized the same substrates as aromatase. After a 30-minincubation at 37°Cfollowed by a 5-min incubation on ice, 1 ml of

culture medium was withdrawn from each well. The culture mediumwas initially mixed with an equal volume of chloroform to extractunused substrate, and further treated with dextran-treated charcoal.Charcoal was removed by a brief centrifugation. and the supernatantcontaining the product, tritiated water, was counted. The protein concentration was determined after dissolving cells in 0.5 N NaOH by themethod of Bradford (21).

The ['HjHjO release assay for human aromatase expressed in these

cells was validated by a product isolation assay. The latter assay wasperformed using [7-'H]androstenedione (specific activity, 28 Ci/mmol)as the substrate. After a 30-min incubation at 37"C followed by a 5-min incubation on ice, [4-'4C)estrone (specific activity, 56.4 mCi/mmol)was added for quantitation of recovery'- One ml of culture medium was

withdrawn from each well, and the culture medium was mixed with anequal volume of chloroform to extract the steroids. The chloroformextract was concentrated, and the product, estrone (including [7-3HJ-estrone and (4-'4C]estrone), was isolated by reverse-phase high-pressure

liquid chromatography with a mobile phase of 55% acctonitrile usingan Ultracarb 5 ODS column (4.6 x 250 mm) from Phenomenex Co.(Torrance, CA) and quantitated by scintillation counting.

RESULTS AND DISCUSSION

Design and Construction of Aromatase Expression Plasmid.We decided to develop mammalian cell lines expressing highlevels of aromatase for the purpose of screening aromataseinhibitors. A stable gene expression method was chosen becauseit offers advantages over transient expression in the stablemaintenance of transfected DNA in cells.

Aromotose cDNA

Klenow fragmentt- dNTP

Aromatose cDNA

Fig. 1. Construction of the mammalian expression vector for aromatase. pHfi-Aro. The detailed description of (he construction of this vector is in "Resultsand Discussion." Restriction sites: B, BamHl: E. EcoR¡:(i, BgH\; //, ///'/»/Ill;.9,Sail: T, Siu\. b, restriction sites were blunt-ended. Amp*, ampicillin-rcsistancegene; SV-Neo. neomycin-resistance gene.

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HUMAN AROMATASE EXPRESSION

Fig. 2. Southern blot analysis of genomicDNA isolated from untransfected and trans-fected cells. Genomic DNA (15 /jg) was extracted from the cells and digested with EcoRl.DNA samples were those isolated from: Lane1, untransfected HBL-100 cells; Lane 2. HBL-100 cells transfected with pH ji-Aro: Lane 3.untransfected MCF-7 cells: Lane 4. MCF-7cells transfected with the expression vector,pH lìApr-1-neo; Lane 5, MCF-7 cells transfected with pH pi-Aro; Lane 6, untransfectedCHO cells; Lane 7, CHO cells transfected withpH piApr-1-neo; Lane 8, CHO cells transfectedwith pH pi-Aro. The restricted DNA was elec-trophoresed in a 0.8cíagarose gel and subsequently transferred to Zetaprobe membranes.The bound DNA was hybridized with 32P-labeled 2.4-kilobase Aro 1 probe (18). Sincethe 8.0-kilobase band is not clearly shown inLane 5 of the first blot, results from a differentblot are also shown. Lanes 9, 10, and // areDNA samples isolated from HBL-100, MCF-7. and CHO cells transfected with pH pi-Aro,respectively.

12345678 9 10 11

11.0Kb-8.0 Kb-

48 Kb-

12345 67 1 234567 8

-6.0 Kb

-3.0 Kb

-2.0 Kb

Fig. 3. Northern blot analysis of RNA isolated from untransfected and transfected cells. RNA samples were those isolated from: Lane I, untransfected HBL-100 cells; Lane 2. HBL-100 cells transfected with pH fi-Aro; Lane 3. untransfectedMCF-7 cells; Lane 4, MCF-7 cells transfected with the expression vector, pH ßApr-1-neo; Lane 5. MCF-7 cells transfected with pH pi-Aro; Lane 6, untransfectedCHO cells; Lane 7. CHO cells transfected with pH pi-Aro. The RNA waselectrophoresed in a O.S^r agarose gel containing formaldehyde and transferredto Genetran 45 membrane. The membrane was hybridized with 32P-labeled 2.4-kilobase Aro 1 probe.

The expression vector was that reported by Gunning et al.(16), pH fi Apr-1-neo. This vector contained 3 kilobases of the/3-actin promoter region plus a 5' untranslated region andintervening sequence 1 linked at the 3' splice site to a short

DNA polylinker segment containing unique Sail, Himflll, andBamHl restriction endonuclease sites followed by a SV40 pol-yadenylation signal, an ampicillin-resistance gene, and a neo-mycin-resistance gene. Therefore, the expression of aromatase

—1.35 kb—1.08kb—0.87kb

-OfiOkb

/031 kb¿-0.27kb—0.23kb—0.19kb

-0.12 kb—0.07 kb

Fig. 4. PCR analysis of aromatase RNA transcript. Five % of the resultingPCR products were subjected to electrophoresis on a 2/r agarose gel and transferred to Zetaprobe membrane. The membrane was hybridized with a "P-labeledprobe as indicated in "Materials and Methods." The analysis was performedusing RNA isolated from: Lane 2, untransfected HBL-100 cells; Lane 3, HBL-100 cells transfected with pH fi-Aro; Lane 4, untransfected MCF-7 cells; Lane 5,MCF-7 cells transfected with pH fi-Aro; Lane 6, untransfected CHO cells; Lane7, CHO cells transfected with pH pi-Aro. Lane 1 contains the RNA markers, andLane 8 contains the PCR products generated using human placenta! RNA as thetemplate.

cDNA was under control of a strong /3-actin promoter. Cellscontaining the expression plasmid were selected by their resistance to neomycin.

Aromatase cDNA was prepared by digesting pAroXl?, apreviously constructed aromatase yeast expression plasmid(15), with restriction enzymes, Bgfll and Stul (see Fig. 1). The1.9-kilobase aromatase cDNA fragment was purified. As described in the previous publication (15), this 1.9-kilobase fragment was without the 5'-untranslated region of the cDNA andhad a very short 3'-flanking region. The end of the cDNAfragment created by Bglll digestion had a 3'-OH recessed end,

which was filled in using Klenow polymerase and the appropriate deoxynucleotides to form a blunt end. This filled-in aro-

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HUMAN AROMATASE EXPRESSION

2,000,000

I 1,500,000

J 1,000,000

500,000

234Incubation time (hr)

.08

-fe

r06

•=.04

.02

O .04 .08 .12 .16 .20

Fig. 5. Enzymatic activity of aromatase expressed in CHO cells transfectedby pH rf-Aro. A, time-dependency of [3H]H;O release. The counting efficiency for3H of the scintillation counter we used is 65%. B, double-reciprocal plot of the

activity of the expressed aromatase. The incubation time was 30 min.

Table l Michaelis parameters for the expressed aromatase in three types of cellsThe assay conditions are those described in "Materials and Methods." The

protein concentrations were total protein concentrations in these cells.

Cell lines Km(nM)

„„(pmol [3H] H2O

formed/h/mg)

CHOMCF7HBL-100

57.855.639.9

201.21026.7

100 2004-OHA cone. (nM)

10 20AG cone. (fj.M)

Fig. 6. Inhibition of the aromatase expressed in MCF-7 cells (O), HBL-100cells (•),and CHO cells (A) by 4-hydroxyandrostenedione (A; 4-OHA) andaminoglutethimide (B; AC). The concentration of the substrate, [It¡,2fi-'H|andro-stenedione. »as100 nM. The assay conditions are those described in "Materialsand Methods."

matase cDNA fragment was then ligated to a Sail and Hindlttrestricted and filled-in expression vector, pH ßApr-1-neo.Plasmids bearing the cDNA insert in the orientation in whichthe Bgl\\ site was flanking the ß-actinpromoter, pH 0-Aro,were selected and used for expression experiments (Fig. 1).

Expression of Human Aromatase in Mammalian Cells. Thesuccessful expression of aromatase cDNA in three types of cellswas initially demonstrated by the presence of the expressionplasmid DNA in transfected cells through Southern blot analysis (Fig. 2), and by analyzing RNA transcripts through Northernblot analysis (Fig. 3) and PCR analysis (Fig. 4). The Southernblot analysis indicated the successful transfection of the cellswith the pH /i-Aro plasmid. An 8.0-kilobase band, EcoRl restricted fragment of pH 0-Aro, was detected only in the cellstransfected with this plasmid (Fig. 2, Lanes 2, 5, and 8-11).We estimated the ratio of the copy numbers of pH ß-AroinHBL-100, MCF-7, and CHO cells to be 6:1:15 by comparingthe intensity of the 8.0-kilobase band (Fig. 2, Lanes 9, 10, and11, respectively). The reason for the differences in transfectionefficiencies for different cells is not currently understood. Interestingly, the transfection efficiency for the same type of cellsremains unchanged from experiment to experiment. Aromatasegene is amplified in MCF-7 cells'1; therefore, the hybridizing

signals for the endogenous aromatase gene fragments (11-,5.2-, 5.0-, and 4.8-kilobase bands) in these cells are stronger(see Fig. 2, Lanes 3-5 and 10). Southern blot analysis ofuntransfected CHO cells (Fig. 2, Lane 6) indicated that thestructure of the hamster aromatase gene was significantly different from that of the human aromatase gene because thepotential hybridizing bands for the endogenous aromatase genefragments are weaker and different from those found in theHBL-100 and MCF-7 cells. Northern blot analysis revealed

that the aromatase RNA transcripts are synthesized in thetransfected cells. The expected size of the aromatase RNAtranscript was 1.9 kilobases, and it was found in the transfectedHBL-100 and CHO cells (Fig. 3, Lanes 2 and 7). It is interestingto note that there were two sizes of aromatase transcript, whichwere much larger than the expected 1.9-kilobase transcript, inthe transfected MCF-7 cells (Fig. 3, Lane 5) hybridizing withthe aromatase cDNA probe. This latter finding is not currentlyunderstood, but it is suggested to be caused by improper RNAprocessing in this type of breast cancer cell. No aromatase RNAtranscript was detected in untransfected cells or in cells treatedwith the expression vector pH (i Apr-1-neo (Fig. 3, Lanes I, 3,4, and 6). Clear PCR products were found for all three types ofcells transfected with the aromatase expression plasmid (Fig.4, Lanes 3, 5, and 7) using the PCR condition described in"Materials and Methods" and carrying the amplification for 40

cycles. The results from the PCR analysis also indicated thatboth untransfected HBL-100 and MCF-7 cells (Fig. 4, Lanes 2and 4, respectively) expressed a very low level of the enzyme,because the cDNA product of the RNA transcript could bedetected after PCR amplification. Brueggemeier and Katlic (11)have shown that MCF-7 cells do contain aromatase but atrelatively low levels. In our laboratory, aromatase activity couldbe detected, at a measurable level, in the untransfected cellsonly after incubation for 6 h. A minor PCR product with a sizeof 0.27 kilobase was generated using placenta RNA as thetemplate (see Fig. 4, Lane 8) and was a result of mispriming.It could be seen only by hybridization and when the concentration of the RNA message for aromatase was high.

' D. Zhou. E. Chen, and S. Chen, manuscript in preparation.

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The definitive proof for the expression of active protein inthese cells came from enzyme activity measurement. Usingaromatase expressed in CHO cells as an example, the enzymereaction reached saturation after 2.5 h using assay conditionsdescribed in "Materials and Methods" (Fig. 5A). The aromatasewas expressed at such a level that 19 pmol of ['HJH.O (500,000

cpm; counting efficiency, 65%) were formed after incubating100 pmol of [l&Zß-'Hjandrostenedione for 30 min with 5 xIO5 CHO cells carrying aromatase expression plasmid, while0.12 pmol of ['H]H2O (3,000 cpm) was formed upon incubating

with either untransfected cells or cells transfected with theexpression vector only. These results demonstrate that thetransfected cells express a high level of aromatase, and definitiveresults can be obtained by performing the assay with an incubation time of 30 min. Figure SB serves as an example andshows that the aromatase expressed in CHO cells had activityfollowing normal Michaelis-Menten kinetics. Table I summarizes the Km and Vmaxfor aromatase expressed in three celllines. The tritiated water assay for aromatase expressed in thesecells was validated with a concurrent product extraction assay.The Km values for aromatase expressed in CHO cells determined by the simultaneous product extraction assay and thetritiated water assay were 54.9 and 68.7 n\i, respectively. TheVmaxvalue determined by the product extraction assay was 79%of that determined by the tritiated water release assay. Thedifferences in the level of the expression of aromatase in thesethree types of transfected cells (i.e., differences in Vmaxvalues)probably resulted from differences in transfection efficiency asindicated by Southern blot analysis. However, we cannot eliminate the possibility that the high VmaNvalue for aromataseexpressed in CHO cells indicates more active enzyme in CHOcells than in other cells. Although the levels of the expressionof aromatase in MCF-7 and HBL-100 cells were lower thanthat in CHO cells, they were much higher than the endogenousactivity. We could not detect a significant level of aromataseactivity after a 30-min incubation of untransfected cells. Whilethe levels of the expressed aromatase activity varied amongdifferent cells, the Km values were similar to those reported inthe literature (e.g., Ref. 22). The similarity in the Kn,values forthe enzyme expressed in different cells and that existing inhuman placenta suggested that the expressed aromatase enzymeprobably had the same conformation as the enzyme present inhuman placenta.

Figure 6 shows that the expressed aromatase was inhibitedby 4-hydroxyandrostenedione and aminoglutethimide. 4-Hy-droxyandrostenedione is a substrate analogue that was suggested to bind to the active site of the enzyme (23). The bindingof aminoglutethimide to aromatase was shown to induce acytochrome P-450 type II spectrum, suggesting that this compound binds to the heme prosthetic group of the enzyme (24).It was interesting to find that the inhibitory profiles weredifferent for aromatase expressed in different types of cells.Among three types of cells, HBL-100 cells were most sensitiveto 4-hydroxyandrostenedione, and CHO cells were most sensitive to aminoglutethimide. Since the same expressed enzyme inthree types of cells responded differently to two inhibitors, thisvariation in inhibition profiles would be most likely to resultfrom other differences among these cells in responding todifferent inhibitors. For example, cell membrane variations mayaffect the uptake rate of inhibitors in the three types of cells.This finding is interesting and stresses the importance of choosing a proper system for aromatase inhibitor screening. Withthis in mind, aromatase inhibitor screening using human pla-

cental microsomes or animal tissues as the assay systems maynot be relevant in searching for the most effective inhibitor fortreating breast cancer. For this reason we are now expressingaromatase in MCF-7 breast cancer cells and HBL-100 noncan-cerous breast cells. Although the inhibitory profiles for twoinhibitors are slightly different in three types of cells, the 50%inhibitory concentration values for the inhibition are within therange determined for human placental aromatase.

In conclusion, we have stably expressed a high level of humanaromatase in three types of cultured mammalian cells using aconstructed mammalian cell expression plasmid containing thehuman placenta aromatase cDNA, pH 0-Aro. The expressedaromatase has enzymatic properties similar to the placentalenzyme as demonstrated by normal kinetic analyses and byinhibition studies using two aromatase inhibitors at the expected concentrations. We believe that these cells should be auseful system for aromatase inhibitor screening. Certainly, theywill be useful models to determine the reasons for the differences in responding to aromatase inhibitors among differentcells.

ACKNOWLEDGMENTS

We would like to thank Dr. Kay Rutherfurd in helping us set up thehigh-pressure liquid chromatography condition for the product extraction assay for aromatase.

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13. Berkovitz. G. D.. Fujimoto. M.. Brown, T. R., Brodie, A. M., and Migeon.C. J. Aromatase activity in cultured human genital skin fibroblasts. J. Clin.Endocrinol. Metab.. 59: 665-671. 1984.

14. Crobin, C. J.. Graham-Lorence, S., McPhaul, M., Mason. J. I.. Mendelson,C. R.. and Simpson. E. R. Isolation of a full-length cDNA insert encodinghuman aromatase system cytochrome P-450 and its expression in nonster-oidogenic cells. Proc. Nati. Acad. Sci. USA, 85: 8948-8952. 1988.

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1990;50:6949-6954. Cancer Res   Dujin Zhou, Denis Pompon and Shiuan Chen  ScreeningMammalian Cells: A Useful System for Aromatase Inhibitor Stable Expression of Human Aromatase Complementary DNA in

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