Proc. Nail. Acad. Sci. USAVol. 83, pp. 6387-6391, September 1986Biochemistry

Isolation and characterization of a complementary DNA specific forhuman aromatase-system cytochrome P-450 mRNA

(estrogen biosynthesis/regulation)

CLAUDIA T. EVANS*, DIANA B. LEDESMA*, TANYA Z. SCHULZt, EVAN R. SIMPSON*1,AND CAROLE R. MENDELSON**Cecil H. and Ida Green Center for Reproductive Biology Sciences, and Departments of Biochemistry and Obstetrics and Gynecology, The University ofTexas Health Science Center, Dallas, TX 75235; and tDepartment of Cell Biology, Baylor College of Medicine, Houston, TX 77030

Communicated by Jean D. Wilson, May 19, 1986

ABSTRACT A cloned complementary DNA sequence hasbeen isolated from a human placental cDNA library in thebacteriophage expression vector Agtll after screening withpolyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-45Or.^). A single recombinantclone, XhAROM1, was characterized by its ability to generatea 3-galactosidase fusion protein that reacted independentlywith polyclonal antibodies raised against .-galactosidase andcytochrome P-450Arm and with monoclonal antibodies specificfor cytochrome P-45OAro. The cDNA insert, which was foundto be 1.8 kilobases in length, was radiolabeled and used toanalyze poly(A)+ RNA isolated from human placenta and totalRNA isolated from human adipose stromal cells cultured in theabsence or presence of regulatory factors. The radiolabeledcDNA hybridized to several size species of mRNA in bothplacental and adipose stromal cell RNA fractions. Changes inthe levels of adipose stromal cell RNA that hybridized to thecDNA insert were associated with comparable changes in thelevels of translatable cytochrome P-450,om mRNA andaromatase system activity. These rmdings are indicative thatXhAROM1 contains DNA sequences complementary to humancytochrome P-450Aom mRNA and are suggestive that regula-tory factors affect aromatase activity by altering the transcrip-tional activity of the cytochrome P-450,,.,m gene.

The synthesis of estrogens from androgens is catalyzed by anenzyme complex termed the "aromatase" system, which iscomprised of a specific form of cytochrome P-450, aroma-tase-system cytochrome P450 (cytochrome P-450,o,9), anda flavoprotein, NADPH-cytochrome P-450 reductase(E.C.1.6.2.4). Cytochrome P450Arom belongs to a family offunctionally related proteins that comprise the cytochromeP-450 multigene family. The aromatase-system reactionsoccur in the endoplasmic reticulum and are of the mixed-function oxidase type. For each molecule of androgen con-verted to estrogen, three molecules of molecular oxygen andthree molecules of NADPH are required. Reducing equiva-lents from NADPH are transferred via the flavoprotein to thecytochrome P-450MAom, which binds the androgen substrateand inserts oxygen into the molecule, resulting in threesequential hydroxylations-two at the C-19 angular methylgroup and one at the C-2 position of the A ring. Thisapparently results in the loss ofthe C-19 angular methyl groupand the spontaneous aromatization ofthe A ring to a phenolicring structure (1-5).The aromatase system occupies a key position in the

steroid biosynthetic pathway and is subject to multifactorialregulation in the tissues in which it occurs. Estrogen forma-tion is a property of several tissues including granulosa cells

of the ovary (6), the placenta (1), adipose tissue (7, 8), Sertoli(9), and Leydig (10, 11) cells of the testis, and multiple sitesin the brain (12-14). In men of all ages and in postmenopausalwomen, the adipose tissue is the principal site of estrogenformation (15). Although the factors that regulate estrogenformation in ovarian granulosa cells have been well docu-mented, until recently little or nothing was known of thefactors that regulate aromatase-system activity in adiposetissue. Using human adipose stromal cells in culture as amodel system, it was found that glucocorticoids and ana-logues of cAMP markedly stimulate the aromatase-systemactivity of these cells (16, 17). Furthermore, phorbol esterswere observed to potentiate and epidermal growth factor(EGF) to inhibit this cAMP-mediated stimulation of aroma-tase-system activity (18). The cellular mechanisms wherebystimulatory and inhibitory factors regulate aromatase-systemactivity of adipose stromal cells and of ovarian granulosacells have not been determined.To investigate the molecular mechanisms that mediate the

regulation of aromatase-system activity, we have preparedpolyclonal and monoclonal antibodies against cytochromeP450Arom (19). In the present study, these antibodies nowhave been used to identify a cDNA clone specific for humancytochrome P-450 m from a phage Xgtll human placentalcDNA library. This clone has been characterized by differ-ential hybridization to RNA fractions containing differentamounts of translatable mRNA encoding cytochrome P-450,,vom. By use of this P-450Arom cDNA insert, it has beenshown that multiple species ofmRNA encoding cytochromeP-4SOArom occur in human cells expressing aromatase-systemactivity and that the levels of these species of mRNA inhuman adipose stromal cells incubated in the absence orpresence of regulatory factors are correlated with the levelsof translatable mRNA encoding cytochrome P-450ASom andwith aromatase-system activity.

EXPERIMENTAL PROCEDURES

Poly(A)4 RNA Isolation and cDNA Synthesis. Total cellularRNA was extracted from human term placenta by theguanidine thiocyanate method (20). Poly(A)+ RNA wasisolated by two cycles of oligo(dT)-cellulose chromatography(21). Double-stranded cDNA synthesis, blunt-end EcoRIlinker addition, chimeric bacteriophage formation withEcoRI-cut Xgtll, in vitro recombinant bacteriophage pack-

Abbreviations: cytochrome P-450AOm, aromatase-system cyto-chrome P-450; Bt2cAMP, dibutyryl cAMP; EGF, epidermal growthfactor; kb, kilobase(s).tTo whom correspondence should be addressed at: Cecil H. and IdaGreen Center for Reproductive Biology Sciences, The University ofTexas Health Science Center at Dallas, 5323 Harry Hines Boule-vard, Dallas, TX 75235.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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aging, and amplification of the resultant cDNA library fol-lowed methods as described (22-24).

Screening of Bacteriophage Agtll cDNA Expression Li-brary. Approximately 1 x 106 phage (30,000 per plate) werescreened for cytochrome P-45OArom cDNA sequences by amodification (25) of the antibody screening procedure ofYoung and Davis (26). Nitrocellulose filters containing pro-tein released from lysed bacterial colonies were washed andtested for the presence of p-galactosidase-cytochrome P-450Arom fusion proteins as described (23, 24) by using rabbitpolyclonal antibodies (IgG no. 870) raised against humanplacental cytochrome P-450Arom, followed by incubation with1251-labeled goat anti-rabbit IgG and autoradiography. Filterslifted from replica plates were screened as above but weretreated with either preimmune rabbit IgG or without anti-body. Other controls also included filters lifted from replicaplates containing (i) bacteria infected with nonrecombinantXgtll, (ii) uninfected bacteria, and (iii) a blank agaroseoverlay. With this method, 45 positive clones were identifiedin the initial screen of the cDNA library. The 12 strongest-reacting clones were rescreened, and 6 of these that remainedpositive were plaque-purified and amplified by the plate-lysate method (27).The amplified phage had a titer of -1 x 1012 plaque-

forming units per ml and produced clear plaques on test platescontaining 5-bromo-4-choro-3-indolyl f-D-galactopyranoside(X-Gal), thereby indicating that the amplified clones re-mained recombinant. In addition, each individually amplifiedclone was rescreened and reacted positively with the rabbitpolyclonal IgG against cytochrome P-450Arom.

Size of the Cloned Cytochrome P-450AOm cDNA. DNA wasprepared from the phage produced from plate lysates of theplaque-purified clones (27). DNA (1 ,ug) from each clone wascut with EcoRI, and the inserts from the restriction enzymedigestion mixture were end-labeled with [32P]dATP by usingthe Klenow fragment of DNA polymerase I and were run ona 2% agarose gel. A HindIII digest of X phage DNA was usedfor molecular weight markers. The size of the largest cDNAinsert was estimated to be 1.8 kilobases (kb) and wasdesignated XhAROM1. The size of the cDNA insert from asecond positive recombinant Agtll clone was 1.5 kb and wasdesignated XhAROM2.

Construction of Lysogens, Preparation of /8-GalactosidaseFusion Proteins, and Immunoblot Analysis. Lysogens ofXhAROM1 and XhAROM2 were generated in the hflA Esch-erichia coli strain BNN Y1089. A logarithmic-phase culturewas infected at a multiplicity of 5 plaque-forming units percell at 32°C for 15 min, serially diluted, plated, and incubatedovernight at 30°C. Random colonies were picked, and indi-vidual lysogens were selected based upon their ability togrow at 30°C, to lyse after induction at 42°C, and to produceplaques at 420C when replica-plated on a BNN Y1088 lawn.

P-Galactosidase fusion proteins were prepared by growinglogarithmic-phase cultures of the individual lysogen coloniesat 30°C, inducing at 42°C for 15 min, adding isopropyl,B-D-thiogalactopyranoside (IPTG) to a final concentration of10 mM, and continuing incubation at 37°C for 30 min. Thecells were harvested by centrifugation at room temperature,resuspended in cold 50 mM Tris HCl, pH 7.5/150 mM NaCl,subjected to freeze/thaw cycle, and briefly sonicated at 4°C.Cellular debris was removed by centrifugation, and theprotein concentration of the supernatant was assayed by theBradford method (28). Crude bacterial proteins (50 ,ug) fromeach induced lysogen were separated on three identicalNaDodSO4/polyacrylamide gels (7.5%) (29) and electroblot-ted onto nitrocellulose (30). The protein blots were washed,blocked, and treated with (i) rabbit anti-,f-galactosidase IgGand 1251-labeled goat anti-rabbit IgG; (ii) rabbit polyclonalanti-cytochrome P-45OArom IgG no. 870 and 1251I-labeled goatanti-rabbit IgG; and (iii) mouse monoclonal anti-P-45OArom

IgG no. 58F10/6 and 125I-labeled rabbit anti-mouse IgG. Themethods used for washing, blocking, and treating with IgGwere the same as those described for the antibody screeningof the Xgtll cDNA expression library (26).

Subcloning of the KhAROMl Fragment into pBR322. TheEcoRI fragment of the P-450Aom clone, XhAROM1, wassubcloned into the corresponding restriction site of pBR322by ligating the fragment to the complementary ends ofphosphatase-treated pBR322 under standard conditions andwas used to transform E. coli RRI by described methods (27).Clones containing pBR322 recombinants bearing the 1.8-kbinsert from XhAROM1 were designated phAROM1.

Differential Hybridization of RNA Isolated from CulturedHuman Adipose Stromal Cells. Primary monolayer cultures ofhuman adipose stromal cells were prepared as described (18).At confluence, the cells were placed in serum-free mediumfor 24 hr, and then were maintained for 48 hr in the absence(control) or presence of 1 mM dibutyryl cAMP (Bt2cAMP),1 mM Bt2cAMP with 100 nM phorbol 12,13-diacetate, or 1mM Bt2cAMP with EGF at 20 ng/ml. Aromatase-systemactivity was assayed in triplicate cultures as described (31)and was expressed as pmol of [3H]androstenedione metab-olized x (mg of protein)f-12 hr-1. Total RNA (20 ,ug) isolatedfrom the cells by a modification of the guanidine thiocyanatemethod (32) was analyzed by RNA blotting (27) with the1.8-kb EcoRI insert of phAROM1 radiolabeled with phos-phorus-32 by nick-translation (27).

RESULTSIdentification of Phage Containing cDNA Sequences Com-

plementary to the mRNA Encoding Human P-45OArm. Afterthe Xgtll human placental cDNA library was screened, sixrecombinant clones were selected that expressed proteinsthat reacted with the polyclonal IgG raised against humanplacental P-450Arom. The six recombinants contained cDNAinserts that ranged in size from 0.5 to 1.8 kb. Lysogens werethen constructed from two of the positive clones, designatedXhAROM1 (1.8 kb) and XhAROM2 (1.5 kb), and the inducedfusion proteins were characterized by their capacity to reactwith polyclonal IgGs against ,B-galactosidase (Fig. LA) andcytochrome P-450Arom (Fig. 1B) and with a monoclonal IgGagainst cytochrome P-450Arom (Fig. 1C). The findings areindicative that a single fusion protein produced by cellslysogenized with XhAROM1 is recognized by all three anti-bodies (Fig. 1 A-C, lanes 4 and 5). Therefore, XhAROM1contained a cDNA insert in frame with the lacZ gene, whichcoded for the sites recognized by the polyclonal and mono-clonal anti-cytochrome P-45OArom IgGs. Consequently,AhAROM1 was tentatively identified as a clone that con-tained cDNA sequences encoding human cytochrome P-45OArom. The fusion protein encoded by XhAROM1 (=150kDa) was larger in size than authentic 13-galactosidase (116kDa), and this molecular mass difference (z35 kt)a) wassuggestive that the clone, XhAROM1, contained codingsequences for at least 60% of the mature P-450Aom protein (55kDa). The other clone, XhAROM2, produced a hybrid proteinthat reacted with the polyclonal IgGs against ,-galactosidaseand cytochrome P-450Arom but failed to react with themonoclonal anti-cytochrome P-450Arom IgG.

Restriction Endonuclease Mapping. The EcoRI fragment ofthe P-450Arom clone, XhAROM1, was subcloned into theEcoRI site of pBR322. Restriction endonuclease analysis ofphAROM1 was performed, and the locations of the corre-sponding restriction sites are depicted in Fig. 2. The cDNAinsert contained single restriction sites at 0.4, 0.6, and 1.2 kbthat were sensitive to digestion by Cla, Xho, and Sca,respectively, while two Kpn-sensitive sites mapped at 0.45kband 1.4 kb.

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A

1 2 3 4 5kDa

200-

_U116- --92- i

B

1 2 3 4 5

., 4. _ ._ _. o ..A.4i"O _w ..

C

12 3 4 5

li

66-

45-

FIG. 1. Construction oflysogens, preparation of ,-galactosidase fusion proteins, and identification ofthe cytochrome P-450om cDNA clone.E. coli strain Y1089 was lysogenized with the positive Xgtll clones and the ,-galactosidase fusion proteins were prepared as described. Celllysate protein (50 ,ug) from each lysogen was separated on 7.5% NaDodSO4/polyacrylamide gels. Three identical gels were run, electroblottedonto nitrocellulose, and treated with rabbit anti--Bgalactosidase IgG and 15I-labeled goat anti-rabbit IgG (A), rabbit polyclonal anti-cytochromeP-450A,,,1 IgG and '25I-labeled goat anti-rabbit IgG (B), and mouse monoclonal anti-cytochrome P-450A,. IgG and '25I-labeled rabbit anti-mouseIgG (C). Lanes 1-5 are the autoradiograms of the immunoblots of the proteins from Y1089 lysogenized with: nonrecombinant Xgtll (lanes 1);a recombinant clone that failed to accumulate fusion protein (lanes 2); an immunopositive recombinant, XhAROM2 (lanes 3); and fusion proteinsisolated from two independent Y1089 colonies lysogenized with XhAROM1 (lanes 4 and 5).

Size ofmRNA Species Complementary to XhAROMl Insert.Cytochrome P-450Arom is a protein of 55 kDa and must beencoded by mRNAs at least 1.7 kb in size. To determinewhether the cloned cDNA was complementary to mRNA ofsufficient size to code for full-length cytochrome P450,&om,hybridization was performed with human placental mRNA.Since the abundance ofcytochrome P-450ArommRNA is quitelow (about 0.02% of human placental mRNA), the mRNAencoding P-450Arm was enriched by size fractionation ofhuman placental poly(A)+ RNA (100 ,ug) on a nondenaturingsucrose density gradient (Fig. 3). Duplicate aliquots of eachfraction were analyzed by RNA blotting and by in vitrotranslation and immunoprecipitation (Fig. 3). The fractions(15-20) containing mRNA that encoded immunoprecipitablecytochrome P-450krom, a 55-kDa protein, were found tosediment between the 18S and 28S regions of the gradient.These individual fractions were then separated by denaturingagarose electrophoresis and were hybridized with the 32P-labeled 1.8-kb cDNA insert from XhAROM1 (Fig. 3). Eachsucrose density gradient fraction that contained translatablemRNA encoding mature cytochrome P45OArom protein hy-bridized with the cDNA insert from XhAROM1. Further-more, from the broad range of mRNA fractions encodingfull-length cytochrome P-450Arom and from the hybridizationprofile, it appears that multiple sizes of mRNA encodeP450OAom. Further evidence that the phage, XhAROM1,

phAROM1EcoRI Cla Xho

Kpn

Sca

Kpn1.8 kb

FIG. 2. Restriction endonuclease map of the cDNA insert eA-coding human cytochrome P450,m. Restriction sites were deter-mined by a combination of single, partial, and double digests ofphAROMl.

carried a cDNA insert hybridizing to multiple sizes of RNAwas obtained by examining the hybridization of the cDNA tototal poly(A)+ RNA isolated from human placenta. Thehybridization of the 1.8-kb insert from XhAROM1 as afunction of increasing concentrations of human placentalmRNA is shown in Fig. 4. At least three distinct sizes ofmRNA from human placenta (3.0 kb, 2.7 kb, and 2.4 kb)

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1 2 3 4

28S

18S

0.56-

FIG. 4. Electroblot analysis of human placental poly(A)+ RNA.Lanes: 1-4, poly(A)+ RNA from human placenta at 1 ,ug (lane 1), 2.5,g (lane 2), 5.0 ,ug (lane 3), and 10 ,ug (lane 4); 5, poly(A)- RNA fromhuman placenta; 6, 3 jg of poly(A)+ RNA from rabbit lung. mRNAwas separated on a 1.25% denaturing agarose gel, electroblotted ontoZeta-Probe, and hybridized with 32P-labeled 1.8-kb cDNA insertfrom phAROM1.

hybridized to the cloned cDNA insert, and these mRNAscorresponded to the range of sizes of mRNA from whichcytochrome P-450Arom was immunoisolated, as shown in Fig.3.

Tissue Specificity ofRNA Hybridizing to XhAROM1 Insert.When 32P-labeled XhAROM1 insert was hybridized to filterscontaining RNA from several human fetal tissues, stronghybridization was observed only to placental RNA. Weakerhybridization was seen to RNA from liver, a fetal tissueknown to contain aromatase-system activity, whereas heart,kidney, and lung RNAs all failed to hybridize to XhAROM1(data not shown). Human placental poly(A)- RNA andpoly(A)+ RNA from rabbit lung, which does not containdetectable aromatase-system activity, also failed to hybridizeto the 1.8-kb cDNA insert from XhAROM1 (Fig. 4).

Differential Hybridization of XhAROM1 to RNA fromHuman Adipose Stromal Cells Maintained in Culture in thePresence of Factors that Regulate Aromatase-System Activity.The sequence abundance of P-45OArom mRNA was analyzedin human adipose stromal cells cultured in the absence(control) or presence of Bt2cAMP, Bt2cAMP/phorbol 12,13-diacetate, or Bt2cAMP/EGF by RNA blotting and hybrid-ization to 32P-labeled insert from XhAROM1 (Fig. 5). Therewas no detectable hybridization of RNA to the radiolabeledcDNA in control samples, nor in those treated with phorbol12,13-diacetate or EGF alone (not shown), while hybridiza-tion of an -3-kb RNA band was observed in Bt2cAMP-treated cells (Fig. 5). This 3.0-kb band corresponded to thelargest RNA species that hybridized to XhAROM1 in blothybridizations ofhuman placental mRNA. A further increasein the hybridization of the 3.0-kb RNA band was observed incells treated with Bt2cAMP/phorbol 12,13-diacetate, whileno hybridization was observed in the RNA isolated from

FIG. 5. Electroblot analysis of total RNA isolated from culturedhuman adipose stromal cells. Total RNA was isolated from humanadipose stromal cells after 48 hr of incubation in the absence orpresence of regulatory factors. The RNA was separated on a 1.25%denaturing agarose gel, electroblotted onto Zeta-Probe, and hybrid-ized with 32P-labeled 1.8-kb cDNA insert from phAROM1. Lanes:1-4, 20 ,ug of total RNA isolated from cells treated with no additions(control) (lane 1), 1 mM Bt2cAMP (lane 2), 1 mM Bt2cAMP/100 nMphorbol 12,13 diacetate (lane 3), and 1 mM Bt2cAMP/20 ng of EGFper ml (lane 4).

Bt2cAMP/EGF-treated cells (Fig. 5). These increases anddecreases in hybridizable mRNA were paralleled by respec-tive increases or decreases in the levels of translatablecytochrome P-450,&om mRNA (data not shown) and inaromatase-system activity of these cells. The aromatase-system activity (x ± SEM) in triplicate cultures of cellstreated with the same four conditions as above was 0.8 ±0.05, 37 ± 2.6, 166± 19, and 1.8 ± 0.02 pmol'(mg protein)l-2hr-1 for control, Bt2cAMP-, Bt2cAMP/phorbol 12,13-diace-tate-, and Bt2cAMP/EGF-treated cultures, respectively.Bt2cAMP stimulated aromatase-system activity =45-fold ascompared to controls; the addition of phorbol ester togetherwith Bt2cAMP caused a potentiation of the stimulation ofactivity observed in the presence of Bt2cAMP alone (-200-fold over control cells), while EGF inhibited the Bt2cAMP-mediated stimulation of aromatase-system activity by =90%.Phorbol 12,13-diacetate and EGF, when added by them-selves, had little effect on aromatase-system activity.

DISCUSSIONPolyclonal and monoclonal antibodies directed against thecytochrome P-45OArom component of the human placentalaromatase enzyme complex were used to identify a clonedcDNA insert specific for human cytochrome P-450Aom.Approximately 1 x 106 recombinant phage from a Xgtllhuman placental cDNA library were screened initially withpolyclonal antibodies against cytochrome P-450Aom. Thej-galactosidase fusion proteins produced by these phagewere characterized by NaDodSO4/polyacrylamide gel elec-trophoresis and immunoblotting. A single hybrid proteinproduced by the cDNA clone XhAROM1 was shown toindependently react with a polyclonal IgG against ,B-galac-tosidase as well as with polyclonal and monoclonal IgGsspecific for human cytochrome P-45OArom. The finding that

1 23 4 5 6

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2 3.1-9.4-6.6-4.4-

2.3-2.0-

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the fusion protein produced by the constructed lysogens was-35 kDa greater in size than O-galactosidase is suggestivethat the clone XhAROM1 codes for at least 60%o of the matureP-45OArom protein. Upon restriction digestion with EcoRI,XhAROM1 was found to contain a 1.8-kb cDNA insert,sufficient in size to code for the P-450Aom protein.Further identification of XhAROM1 as a clone containing

cDNA sequences specific for human P-450,&Om was providedby sucrose density gradient fractionation of poly(A)+ RNAfrom human placenta, in vitro translation, and RNA blotting.Only the fractions that contained mRNAs encoding im-munoprecipitable cytochrome P-450,&,m hybridized to the1.8-kb cDNA insert from XhAROML. From the range ofmRNA fractions that both hybridized to hAROMi cDNA andencoded for mature P-450Aom protein, it appears thatcytochrome P-450Arom is encoded by mRNAs of differentsizes. This conclusion was verified by the finding that thehAROM1 cDNA hybridized to at least three distinct mRNAspecies in blots of human placental poly(A)+ RNA, whichwere in the same size range as those mRNAs that coded forthe cytochrome P-450,,om protein in the sucrose densitygradient fractions. The discrete mRNA species that hybrid-ized to hAROM1 cDNA may arise as a consequence ofdifferential processing. The presence of multiple mRNAs foranother steroidogenic cytochrome P-450, 11/-hydroxylasecytochrome P-450, has been reported (33).To characterize XhAROM1 in greater detail, the cDNA

insert was subcloned into the EcoRI site of pBR322 andanalyzed by restriction endonuclease digestion. Several re-striction fragments, 400-600 bp in length, were generatedthat contained compatible sequences in the multiple cloningregion of the phage M13 mpl8 vector. These fragments arebeing sequenced to determine the primary structure of thecytochrome P-450Aom protein. Preliminary sequence datahas led to the identification of an open reading framecontaining the following sequence:

5'... .TTCCACGTGAAG-PheHisValLys-

ACATTGCAAGGACAGTGTGTTGAGAGCATACAGAAG-ThrLeuGlnGlyGlnCysValGluSerIleGlnLys-ATACACGAC....3'IleHisAsp .

The 12-amino acid sequence in the center is identical to thecysteine-containing peptide T-16, reported by Chen et al. (34)to be present in their preparation of purified cytochromeP-45OArom. This is strong supportive evidence that XhAROM1does indeed contain a cDNA sequence specific for humancytochrome P450OArom.

This conclusion was further supported by the finding thatthe 32P-labeled hAROM1 cDNA hybridized differentially toblots of RNA isolated from adipose stromal cells cultured inthe absence or presence of factors that have been shown toregulate aromatase-system activity of these cells. In partic-ular, the activity is induced by Bt2cAMP/phorbol 12,13-diacetate, and this induction is antagonized by EGF. Thechanges in the relative amounts of hybridizable mRNAcorresponded not only to the changes in the aromatase-system activity of these cells but also to the relative levels oftranslatable mRNA encoding cytochrome P-450Arom (unpub-lished observations).These data are indicative that hAROMl contains DNA

sequences complementary to mRNA encoding humancytochrome P-45OArom and that the above effectors mayregulate aromatase-system activity ofhuman adipose stromalcells by altering the transcriptional activity of the humancytochrome P-450ASOm gene.

The authors gratefully acknowledge the helpful comments andsuggestions of Drs. Bert W. O'Malley and David Russell and thankMs. Selina Newsome and Ms. Rosemary Bell for expert editorialassistance. This research was supported, in part, by NationalInstitutes of Health Grants R01-AM31206 and 5-P01-AG00306.C.T.E. was supported, in part, by National Institutes ofHealth Grant5-T32-HD07190.

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Biochemistry: Evans et al.

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