(CANCER RESEARCH 52, 290-294, January 15. I992|

17/3-Hydroxysteroid Dehydrogenase Gene Expression in Human Breast CancerCells: Regulation of Expression by a Progestin1

Matti Poutanen,2 Bruno Moncharmont, and Reijo Vihko

Biocenter and Department of Clinical Chemistry,* University ofOulu, SF-90220 Oulu, Finland

ABSTRACT

The expression of the 17/3-hydroxysteroid dehydrogenase (17-HSD)

gene in a series of human breast cancer cell lines was studied by Northernblot hybridization with a cDNA probe and by a time-resolved immuno-fluorometric assay using polyclonal antibodies against the enzyme protein. The 17-HSD enzyme protein concentration was measured in the800 x g cell extract. A high concentration was measured in the BT-20cell line, corresponding to one-fourth of the average concentration inplacenta! tissue. Western blot analysis indicated that the antigen corresponded to a single M, 35,000 band. In 2 other cell lines (MDA-MB-361 and T-47D), the 17-HSD protein concentration was much lower, butstill measurable, whereas in the remaining 5 cell lines (HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and ZR-75-1) it was below the detection limit of the assay. Treatment of the cells for 5 days with the syntheticprogestin, ORG2058, resulted in an increase of the 17-HSD proteinconcentration only in the T-47D cell line. By Northern blot analysis, alow level of 2.3-kilobase inKNA transcripts was detected in all 8 celllines. In addition, a 1.3-kilobase 17-HSD mRNA was present in thesamples from the 3 cell lines containing measurable amounts of 17-HSDprotein in the cell extract, and the band intensities were proportional tothe amount of protein measured with the immunofluorometric assay.Only in the 1-471) cell line did progestin treatment correspond to anincreased amount of the 17-HSD 1.3-kilobase mRNA. These resultssuggest that the 1.3-kilobase mRNA for 17-HSD is the form most closely-

associated with protein expression and is also the only form respondingto the progestin induction of the 17-HSD gene.

INTRODUCTION

Progesterone and estradici play an important role in thecontrol of growth, differentiation, and function of mammaryepithelial cells. In addition, a predominant role of estradiol inbreast cancer progression is generally accepted (1,2).

The reversible interconversion of 17/3-estradiol to the lessactive estrogen, estrone, is mediated by 17-HSD4 (EC 1.1.1.62).The presence of 17-HSD activity in normal (3-5) and malignant(3, 6, 7) human breast tissue is likely to modulate the localestrogen effect in these tissues. While in most tissues, such asthe endometrium, the oxidative reaction is predominant, thereis some evidence for an increased reductive activity of 17-HSDin breast cancer cells (8). This reaction would lead to thereported increase of estradiol in cancer tissue compared tonormal breast tissue. Therefore, 17-HSD may have a majorrole in the promotion and/or the progression of breast cancer.

Only limited knowledge is available concerning the mecha-

Received 7/10/91; accepted 10/28/91.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 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Research Council for Medicine of theAcademy of Finland and by the Foundation of the Finnish Cancer Institute.

2To whom requests for reprints should be addressed, at: Biocenter and Department of Clinical Chemistry, University of Oulu. SF-90220 Oulu, Finland.

3The Department of Clinical Chemistry is a WHO Collaborating Centre forResearch in Human Reproduction supported by the Ministries of Education, ofHealth and Social Affairs, and of Foreign Affairs, Finland.

4The abbreviations used are: 17-HSD. 17/i-hydroxysteroid dehydrogenase:ORG2058, 16<«-ethyl-21-hydroxy-19-nor-4-pregnene-3.20-dione; FCS. fetal calfscrum; PBS, phosphate-buffered saline; DCC. dextran-coated charcoal; BSA,bovine serum albumin; SDS, sodium dodecyl sulfate; TBS, Iris-buffered saline;SSC, standard saline-citrate.

nisms regulating 17-HSD in different tissues. The induction ofthe enzyme activity by progestins in the endometrium (9, 10)and in breast tissue (4, 5, 11) have been reported. Our recentdata indicate that the progestin-induced increase of the 17-HSD activity in these tissues is caused by enzyme proteinaccumulation (12, 13), thus strongly suggesting that progestinsare able to modulate estrogen metabolism by increasing 17-HSD gene expression.

To investigate the steroidal regulation of 17-HSD in breasttissue and the possible role of this enzyme in the pathogenesisof breast cancer, we have analyzed the expression and progestinregulation of 17-HSD in several human breast cancer cell lines.

MATERIALS AND METHODS

Chemicals and Reagents. [«-"P]dCTP (3000 Ci/mmol) was pur

chased from Amersham (Little Chalfont, United Kingdom). ORG 2058was a gift from Organon (Oss, The Netherlands). All cell culturematerials were obtained from Gibco (Grand Island, NY). Guanidineisothiocyanate was from Fluka (Buchs, Switzerland), cesium chlorideand oligo-dT cellulose were from Boehringer (Mannheim, Germany),agarose was from FCM BioProducts (Rockland, ME), and RNA molecular weight markers (RNA Ladder) were from BRL (Gaithersburg,MD). Other reagents not mentioned in the text were either from SigmaChemical Co. (St. Louis, MO) or Merck A.G. (Darmstadt, Germany)and were of the highest purity grade available.

Cell Culture. Human breast cancer cell lines (BT-20, HBL-100,MCF-7, MDA-MB-231, MDA-MB-361, MDA-MB-468, T-47D, andZR-75-1) were obtained from the American Type Culture Collection(Rockville, MD). Stock cultures of the cells were grown in RPMI 1640containing insulin (7.5 ng/ml). The medium was supplemented with10% PCS, penicillin (100 Mg/ml), and streptomycin (100 ¿ig/ml)andcontained phenol red as a pH indicator. Stock cultures were maintainedin almost continuous exponential growth by weekly passage of theappropriate number of cells following treatment with 0.05% trypsin-0.02% EDTA in Hanks' balanced salt solution without Mg2+and Ca2*.

For progestin treatments of the cells, the stock cultures were washedwith PBS, and the cells were harvested by trypsinization. After washingwith PBS, the cells were suspended in medium and seeded at about 5x 104/cm2 (a 1:3 split). The cells were allowed to attach overnight, after

which the medium was replaced with the indicated medium. Experiments were performed in the growth medium except that PCS waspretreated with DCC twice for 30 min at 45°C.The DCC treatment

used was found to remove over 99% of the estradiol from the fetalserum. The medium was changed on the third day and the cells wereharvested on the fifth day. The hormones were added to the mediumdirectly from lO.OOOxstock solutions in ethanol to achieve the indicated concentrations. An equal volume of pure ethanol was added tothe medium of the control cell culture.

Sample Preparation for Time-resolved Immunofluorometric Assay for17/J-Hydroxysteroid Dehydrogenase. After the plates were washed withPBS, the cells were harvested by scraping, washed with PBS, andhomogenized (5 x 107/ml) with a Douncer homogenizer at 4°Cin

buffer A (10 mM Na2HPO4, pH 7.5, 1 mivi EDTA, 0.5 HIM phenyl-methylsulfonyl fluoride, 0.02% NaN3, 20% glycerol). The homogenateswere centrifuged at 800 x g for IO min, and the supernatant was usedin the time-resolved immunofluorometric assay for 17-HSD.

Time-resolved Immunofluorometric Assay for 17-HSD. The time-resolved immunofluorometric assay was performed as described elsewhere (14) using a polyclonal antiserum against highly purified placen-

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BREAST 17(i-HYDROXYSTEROlD DEHYDROGENASE

tal 17-HSD (12). The only modification was that the 800 x g supernatant of the cell homogenates was used in the assay.

Steroid Receptor Assay. The determinations of progestin and estrogen receptors were performed in triplicate in cells from the stockcultures. For the assay of the cytosolic receptors, aliquots of cytosol(100 ii\) were incubated overnight at 4°Cwith 5 nM [3H]17/i-estradiol,

in the presence of 0.5 ^M testosterone (for determination of estrogenreceptor), or with 10 nM ['H]ORG2058 (for determination of proges

terone receptor), in a final volume of 200 n\. The nonspecific bindingwas measured in a parallel incubation by adding a 200-fold excess ofthe corresponding unlabeled steroid to the reaction medium. The unbound ligand was then removed by addition of 1 ml of DCC suspension,followed by a 15-min incubation at 4°C.The sample was centrifuged

and the radioactivity of an 800-iil aliquot was counted.The protein concentration was measured with the Bio-Rad protein

assay (Richmond, CA), using BSA as a standard.Polyacrylamide Gel Electrophoresis and Immunoblotting. SDS-poly-

acrylamide gel electrophoresis was performed using 10% polyacryl-amide gels ( 15). After electrophoresis, the proteins were electrophor-etically transferred to nitrocellulose membranes (16). The membranewas incubated with 20% PCS in 50 mM Tris-HCl, pH 8.0, containing150 mM NaCl (TBS-buffer) at room temperature for 30 min, and thenfor 2 h with polyclonal anti-17-HSD antibodies (12) diluted 1:200 inTBS-buffer, containing 5% FCS. The membrane was then washed 3times for 5 min with TBS-buffer and incubated for 2 h with protein A-peroxidase (20 ng/ml) in TBS, containing 5% FCS. The membrane wasthen sequentially washed with TBS-buffer, TBS containing 0.1% Non-idet P-40, and TBS-buffer, for 5 min each. The immunoreactive proteinswere visualized using 4-chloro-l-naphtol as substrate (0.6 mg/ml in90% TBS, 10% methanol).

Isolation of DNA and Southern Blot Hybridization. Genomic DNAwas isolated from the T-47D and BT-20 human breast cancer cell linesand from human leukocytes using a published procedure (17). GenomicDNA (10 Mg)was cleaved to completion with restriction enzymes (Seal,Hindlll, and EcoRl) and resolved by electrophoresis on a 0.8% agarosegel for 27 h at 25 V. The DNA was then blotted onto a nitrocellulosemembrane by capillarity, fixed to the membrane at 80°Cfor 2 h, andprehybridized for 2 h at 65°Cin 5x SSC (Ix SSC: 150 mM NaCl-15

mM sodium citrate, pH 7.0), containing 0.1% BSA, 0.1% Ficoll, 0.1%polyvinyl pyrrolidone, 0.1% SDS, and 50 Mg/ml salmon sperm DNA.A 1.0-kilobase EcoRl-Sac\ fragment of 17-HSD cDNA ( 18) was labeledby nick translation (Promega, Madison, WI) with [«-'2P]dCTP,and the

hybridization was then carried out overnight in the indicated solutionat 65°C.The membrane was then washed twice in 2x SSC, containing0.1% SDS, for 15 min each at 65°C,and twice further in Ix SSC,containing 0.1% SDS, for 15 min each at 65°C.The same membranewas probed with a [«-"PjdCTP-labeled 1.1-kilobase fcoRI fragmentcovering the entire coding region of the catecol-O-methyltransferasegene (19). The hybridization was carried out overnight, as indicatedabove, at 42°C.After hybridization, the membrane was rinsed with 1x

SSC, containing 0.1% SDS at room temperature and 3 times morewith 1x SSC, containing 0.1 % SDS, for 15 min each at 60°C.After

hybridization, the membrane was exposed to Kodak XAR film (Rochester, NY) for 3 days.

Isolation of RNA and Northern Hybridization. Cell culture disheswere washed with PBS, and the cells were harvested by scraping, washedwith PBS, and collected by centrifugation. Total RNA was prepared byhomogenizing the cells (1 g/16 ml) in 4 M guanidine thiocyanate,followed by centrifugation (21 h, at 179,000 x g) of the homogenatethrough a cushion of 5.7 M CsCl, and ethanol precipitation (20).Poly(A)+ RNA was isolated by one cycle of oligo-dT cellulose chro-matography (21). For each sample, 10 n% of poly(A)* RNA was subjected to electrophoresis in a 1% (w/v) agarose-formaldehyde gel (21)except in the case of the samples from the BT-20 cell line and placenta,in which 20 /xg of total RNA were used. The RNAs were transferredovernight to a Hybond N nylon membrane (Amersham), according tothe manufacturer's instructions.

RNA was fixed to the membrane by UV irradiation. The membranewas then prehybridized for 3 h at 65"C in 5 x SSPE (Ix SSPE = 0.15

M NaCl, 10 mM sodium phosphate buffer, 0.1 mM EDTA, pH 7.4),containing 50% formamide, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.5% SDS, and 20 Mg/ml salmon sperm DNA. The hybridization with a ["P]-labeled 1.0-kilobase cDNA fragment of 17-HSD(18) was then carried out overnight at 65°C.The membrane was thenwashed with 2x SSPE, containing 0.1% SDS, for 15 min at 65°C,andtwice with Ix SSPE, containing 0.1% SDS, for 15 min each at 65°C.

To quantitate the amount of mRNA applied to the gel, the membraneswere also hybridized with a human -y-actin cDNA-probe (American

Type Culture Collection, Rockville, MD). The membranes were exposed to Kodak XAR film (Rochester, NY).

RESULTS

The concentration of the 17-HSD enzyme protein was measured in the 800 x g supernatant from 8 different breast cancercell lines. Cells were grown in the presence of charcoal-strippedserum, or treated for 5 days with 10 n\i ORG2058, a syntheticprogestin. The results of the time-resolved immunofluorometricassay of 17-HSD are presented in Table 1. The results of theestrogen and progesterone receptor ligand-binding assays givenin Table 1 are from the cytosol fractions of cells from the stockcultures. The 17-HSD enzyme concentration showed a greatvariation among the cell lines. In 5 of them, it was below thedetection limit of our assay. In 3 cell lines (BT-20, MDA-MB-361, and T-47D), the basal level was measurable, but only inone of them (T-47D) was progestin treatment able to increasethe enzyme concentration 2-fold (P = 0.015, t test), as reportedpreviously (13). In this cell line, a high concentration of theprogesterone receptor was detected, as expected (22).

The highest concentration of 17-HSD enzyme protein wasobserved in the cell extract of the BT-20 cells, and it corresponded to about one-fourth of the concentration measured in

the cytosol from term placenta. In this cell line, the concentrations of estrogen and progestin receptors were low (Table 1).Western blot analysis of the BT-20 cell and placenta! extracts

Table 1 / 7-HSD in human breast cancer cell linesThe cells were grown in the presence or absence of 10 nM progestin. ORG 2058. The concentrations shown for 17-HSD are the mean ±SD of triplicate samples,

analyzed with the time-resolved immunofluorometric assay, from 800 x g fractions of the cell homogenates. The concentrations of estrogen and progestin receptorswere measured in triplicate in the cytosol fractions of cells from the stock cultures using ligand-binding assays.

17-HSD (ng/mgprotein)Cell

lineBT-20T-47DMDA-MB-361ZR-75-1MCF-7MDA-MB-231MDA

MB-468HBL-100Control1630

±1212.4±0.461.9

±0.17«0.2«0.2«0.2«0.2«0.2ORG

20581540±

1434.2±0.61°2.0

±0.38«0.2«0.2«0.2«0.2«0.2Estrogen

receptor(fmol/mgprotein)424101643442Progestin

receptor(fmol/mgprotein)201176201102115995

' P = 0.015.

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BREAST 17/i-HVDROXYSTEROID DEM'S DROGENASE

indicated that, in both, the antigen of the polyclonal antibodiesto 17-HSD migrated as a single band with the expected M,35,000 (Fig. 1). Aliquots of genomic DNA from the BT-20 andT-47D cell lines, digested with different restriction enzymes,were analyzed by Southern blot, using a cDNA probe containinga fragment of the 17-HSD gene sequence, and compared to thesame amount of genomic DNA from human leukocytes analyzed in parallel. The result, presented in Fig. 2, indicated thatgene amplification was not responsible for the high level of 17-HSD gene expression detected in the BT-20 cell extract. Acontrol hybridization of the filter with cDNA for catecol-O-

methyltransferase gene, located on chromosome 22 (23),showed that small differences in the hybridization figure visible

kDa

98-69-

46-

30-

1 2Fig. I. Western blot analysis of the cytosol fractions from human placenta

(¡Mne/) and from the BT-20 human breast cancer cell line (Lane 2). The totalamount of protein applied to the lanes was 50 jig.

kb

23-

9.4-

6.6-

4.4-

T-47D

123417-HSD

BT-20

2 3

leukocytes

1234

COMT23-

Fig. 2. Southern blot analysis of DNA from BT-20 and T-47D human breastcancer cells and human leukocytes. Ten ng of undigested genomic DNA (1). orDNA digested with Seal (2), ////»/III (3). or £coRI(4). Top. hybridization obtainedwith 17-HSD cDNA; bottom, hybridization obtained with catechol-O-methyl-transferase (COMT) cDNA.

kb

17HSDT-47D MDA-MB-361 ZR-75-1

CO COCO

MCf-7

CO

"•M

M«1.3-

0.9-

Qctin

B 17HSDP BT-20

c O

MDA-MB-231 MDA-MB-468 HBl-100 T-47D

C O C O C O C O

kb

2.3-

Fig. 3. Northern blot analysis of RNA of human breast cancer cell lines grownin the absence(c) or presenceof 10 nM ORG2058 (O) for 5 days. Ten fig of poly-(A)* RNA (T-47D. MDA-MB-361. ZR-75-1, MCF-7. MDA-MB-231, MDA-MB-468, HBL-100), 20 ^g of total RNA [placenta (P), BT-20 cells]. Top,hybridization obtained with 17-HSD cDNA. A, exposure time 4 days; B, exposuretime 8 days (MDA-MB-231, MDA-MB-468, HBL-100), or 2 days (P, BT-20, T-47D). Bottom, hybridization obtained with actin cDNA (exposure time 1 day).

in the lanes of the BT-20 were due to variations in the amountof DNA applied to the gel. The 2 genes for 17-HSD arelocalized in tandem on chromosome 17 (24-26), and bothhybridize with the probe used in the experiment. Two Hindlllsites are localized between the 2 genes, and no other Hindl\lsites are present in the genes.5 Thus, the digestion pattern

obtained with Hind\\\ enzyme (Fig. 2, Lane 4), containing 2fragments, is due to the presence of both of the 17-HSD genesin the genomic DNA of the BT-20 cells.

The Northern blot analysis of the RNA purified from thesame panel of cell lines is presented in Fig. 3. Three differentmRNA species of 0.9, 1.3, and 2.3 kilobases were hybridizedwith a 17-HSD probe. The signal of the 2.3-kilobase band wasvisible in all of the samples. In the samples from the cell lineswhere the enzyme protein concentration was below the assaydetection limit, this was the only 17-HSD mRNA species

5 H. Peltoketo, persona] communication.

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BREAST 170-HYDROXYSTEROlD DEHYDROGENASE

present, with the exception of the ZR-75-1, which also expressed a mRNA species of 0.9 kilobase. In MCF-7 cells, thesignal was almost exclusively restricted to the 2.3-kilobase band,with a very faint signal at about 0.6 kilobase. An intense 1.3-kilobase band was the prominent band in the BT-20 cell extractand in a mRNA sample from human placenta. A less intense1.3-kilobase band was also present in the samples from the celllines in which the enzyme protein was present in low, butmeasurable concentrations (MDA-MB-361 and T-47D). In theT-47D samples, a 0.9-kilobase band was also observed. Proges-

tin treatment of the cultured cells resulted in an increase in theintensity only of the 1.3-kilobase band in the T-47D sample.Quantitative densitometric scanning of the autoradiographsfrom 2 separate experiments indicated a 2-fold induction of themRNA (data not shown).

DISCUSSION

It has been suggested that multiple forms of 17-HSD exist(27, 28) and that the placental enzyme is different from that ofthe endometrium (29). However, using a polyclonal antibodyraised against placental 17-HSD, we have previously demonstrated the expression and progestin induction of a placental-like 17-HSD in the human endometrium (12) and in the T-47Dhuman breast cancer cell line (13).

In the present study we show that, in the BT-20 human breastcancer cell line, the molecular weight of the 17-HSD is thesame as that measured for the placental enzyme. The data thusshow that at least one of the enzymes having 17-HSD activityin the breast cancer cells is most probably identical to theplacental 17-HSD. This conclusion is further supported byidentical data obtained in primary human breast cancer tissueextracts.6

The BT-20 cell line, which contains the highest concentrationof 17-HSD protein, does not show the morphological characteristics of epithelial glandular cells when grown under standardconditions (30). In addition, this cell line does not contain thealkaline phosphatase isoenzyme typical of breast tissue, butexpresses the isoenzyme phenotype of term placenta and otherembryonic tissues (31). The relationship of these findings tothe high level of 17-HSD expression detected is unknown. Highlevels of 17-HSD gene expression are typical of placental tissue.The BT-20 cell line may, therefore, have some embryonic- orplacental-like properties that could be related to the highexpression of 17-HSD. However, we cannot exclude the possibility that an unknown autocrine effect by growth factor(s)could be responsible for the high expression of 17-HSD in thiscell line. Inasmuch as the high level of 17-HSD in BT-20 cellswas not caused by a progestin and the gene structure of 17-

HSD present in these cells was normal as judged by a Southernblot analysis, this cell line may be a good model to studyadditional factors regulating 17-HSD in breast cancer tissue.

The variation in the relative expression of 17-HSD mRNAsin the breast cancer cell lines reported in this article has alsobeen observed in some human tissues (24). While the primarystructure of the 2.3- and 1.3-kilobase mRNAs is well documented (18, 32), the structure and origin of the 0.9-kilobasemRNA remains unknown. This short mRNA, together withthe faint 0.6-kilobase band detected in the MCF-7 cells, mayrepresent specific degradation products of the native 17-HSDmRNAs (1.3 and 2.3 kilobases), because they are not long

' M. Poutanen, V. Isomaa, V-P. Lehto, and R. Vihko, submitted for publica-

enough to include the total coding region of the subunit of 17-HSD. In the 3 cell lines in which the expression of 17-HSDprotein was measurable, the 1.3-kilobase 17-HSD mRNA wasexpressed and the band intensities were proportional to theamount of protein measured with the immunofluorometricassay. The 1.3-kilobase mRNA is also the most representativeband in the placental tissue. Furthermore, the observed induction by progestin in the T-47D cells only affected the 1.3-kilobase mRNA. Recent findings have shown that the treatmentof cultured human granulosa cells with cAMP reduced theabundance of the 1.3-kilobase mRNA of 17-HSD, whereas the2.3-kilobase mRNA was not affected (25). These findings areconsistent with our results, suggesting that the transcription ofthe 1.3-kilobase mRNA is more efficiently regulated than thatof the 2.3-kilobase mRNA. The finding that the progestin-induced increase of the 17-HSD protein concentration andenzyme activity (13) was reflected by an increase in the expression of the 1.3-kilobase mRNA is also in line with previousresults, which showed that actinomycin D and cycloheximidereduced the progestin-induced increase of 17-HSD activity innormal breast epithelial cells in culture (4).

The translation efficiency of the 3 mRNA species may bedifferent. At the short 5'-end of the 1.3-kilobase mRNA, the

sequence containing the initiation codon corresponds to theoptimal sequence for initiation by eukaryotic ribosomes (33). Ithas been suggested that the additional ATG triplets upstreamof the one used for the initiation of translation of the 17-HSDprotein could modulate the efficiency of translation of the 2.3-kilobase mRNA (32). The detection of a 17-HSD-specific 2.3-kilobase band in the mRNA of all the cell lines in which the17-HSD protein was below the detection limit of our assayindicated that the transcription of the 17-HSD gene was nevercompletely turned off.

ACKNOWLEDGMENTS

We are grateful to Hellevi Peltoketo, M.Sc., for the analysis of thegene amplification. We thank Liisa Kaarela for her skillful technicalassistance.

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on July 2, 2015. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1992;52:290-294. Cancer Res   Matti Poutanen, Bruno Moncharmont and Reijo Vihko  Breast Cancer Cells: Regulation of Expression by a Progestin

-Hydroxysteroid Dehydrogenase Gene Expression in Humanβ17

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