[CANCER RESEARCH 46, 4933-4937, October 1986]

Stimulation of Cell Proliferation and Estrogenic Response by AdrenalCi9-A5-Steroids in the ZR-75-1 Human Breast Cancer Cell Line1

Richard Poulin and Fernand Labrie2

MRC Group in Molecular Endocrinology, Laval university Medical Center, Quebec GìV 4G2, Canada

ABSTRACT

We have examined the effect of androst-5-ene-3/3,170-diol (A5-diol)

and its precursors, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 30-sulfate (DHEAS), on the growth of the estrogen-sensitivehuman breast cancer cell line, ZR-75-1. While the cell number wasincreased up to 4-fold by maximal concentrations of estradici, A5-diolmaximally stimulated cell proliferation by approximately 3-fold. Sincethe half-maximal stimulation achieved by A5-diol is observed at 2.5 n\i

and the normal range of plasma concentrations of this steroid in womenis 1 to 3 UM,it is most likely that the stimulatory effect of A5-diol has

physiological significance. DHEA and DHEAS were much less effectivethan A'-diol in stimulating the proliferation of ZR-75-1 cells, the maximal

effect on cell number being 75% at the maximal dose used, namely 10U.M.The mitogenic effects of estradici and As-diol were competitively

inhibited by the antiestrogen LY156758 (keoxifene), while the effects ofDHEA and DHEAS were completely abolished by the antiestrogen. Theeffects of DHEA and A'-diol on cell proliferation are not likely to bemediated via their conversion to estrone or estradili!, since androstene-dione had no effect, while testosterone and dihydrotestosterone decreasedcell number by about 20%. The number of specific progesterone bindingsites was increased 3.7-, 3.2-, and 2.0-fold by A'-diol, DHEA, andDHEAS, respectively. The relative potency of the <VA'-steroids toincrease the number of progesterone-specific binding sites was comparable to their ability to stimulate cell proliferation. Direct competitionexperiments performed with intact cells in monolayer culture showedthat, under conditions of minimal metabolism, only A5-diol could significantly compete with estradiol for cellular estrogen-specific binding siteswith an apparent dissociation constant of 11 n\i. thus suggesting thatphysiological concentrations of Ci9-A5-steroids of adrenal origin could

exert an estrogenic stimulation of breast tumor growth without involvement of the aromatase pathway. The present data suggest not only thatestrone derived from androstenedione could play a role in estrogen-sensitive breast cancer in women but that A'-diol could well be the most

important estrogen in breast cancer in women.

INTRODUCTIONThe role of the "classical" estrogens (estradiol, estrone, and

estriol) in promoting the growth of estrogen-sensitive breastcancer is well recognized (2-5). In recent years, however, severalobservations have led to the suggestion that d9-A5-steroids ofadrenal origin, especially A5-diol,3 could include estrogenic

effects in target tissues (see Refs. 6 and 7 for review). Forinstance, plasma levels of A5-diol typical of those found in the

blood of Western women (1 to 3 HM) cause an uterotropicresponse in the sexually immature rat uterus (8). Moreover, inthe MCF-7 human breast cancer cell line, physiological concentrations of A5-diol and DHEAS stimulate the secretion of a M,

Received 3/21/86; revised 6/18/86; accepted 7/15/86.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.

1Supported by grants from the Medical Research Council of Canada. Parts ofthis work have been presented at the Annual Meeting of the Canadian Societyfor Clinical Investigation (1).

1To whom requests for reprints should be addressed.3The abbreviations used are: A'-diol. androst-5-ene-30,17/3-diol; DCC-treated

FBS, dextran-coated charcoal-treated fetal bovine serum; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone 3/9-sulfate; LY156758, [6-hydroxy-2 - (4 - hydroxyphenyl)benzo[A]thien - 3 - yl], |4 - [2 - (1 - piperidinyl)ethoxy]phenyl|-methanone hydrochloride; R5020, 17a,21-dimethyl-19-nor-pregna-5,9-diene-3,20-dione; ER, estrogen receptor.

52,000 glycoprotein which is under specific estrogenic controlin these cells (9). Moreover, high (100 HM) concentrations ofA5-diol have been shown to stimulate thymidine kinase activity

and to increase cellular levels of progesterone receptors inMCF-7 cells (10). On the other hand, DHEA, the precursor ofA5-diol, stimulated the secretion of the M, 52,000 glycoproteinonly at concentrations 10- to 200-fold higher than the plasmalevels normally found in women (9).

In these studies, attempts to detect the formation of estradiolfrom Cii-A'-steroids were always negative (8-10). Consideringthe high affinity measured for the binding of A'-diol to the

estrogen receptor (Ad = 4.5 to 10 HM)from different tissues (7,10, 11), the effects observed in the above studies were likelymediated through direct interaction of As-diol with the estrogen

receptor and were reflecting direct estrogenic activity of thissteroid of adrenal origin.

Since the previous studies did not measure the possible effectof adrenal Ci9-A5-steroids on cancer cell growth, we have usedthe estrogen-sensitive human mammary cancer cell line ZR-75-1 to investigate a possible effect of the main d9-A5-steroids

originating from the human adrenal cortex, namely DHEAS,DHEA, and A5-diol, on the growth of these cells in vitro. These

cells have been shown to possess specific receptors for estrogens, progestins, androgens, and glucocorticoids (12) and toconsistently respond to physiological concentrations of estradiol in serum-containing (12, 13) as well as serum-free, hormone-supplemented medium (14, 15). Furthermore, the abilityof Ci9-A5-steroids to promote cell growth is also compared withtheir effect on the number of progesterone-specific binding sites.Finally, we document the estrogenic nature of the observedresponses to C]9-AS-steroids using an antiestrogen having pure

antagonistic activity in this system, namely LY156758 (keoxifene).

MATERIALS AND METHODS

Chemicals. 17/3-[2,4,6,7-3H]Estradiol (specific activity, 105 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). 17a-[/m>fA>'/-3H]R5020(specific activity, 87.0 Ci/mmol) and unlabeled

R5020 were obtained from New England Nuclear (Lachine, Quebec,Canada). Unlabeled 17/3-estradiol, As-diol, diethylstilbestrol, and dex-

amethasone were from Sigma. DHEA and DHEAS were obtained fromSteraloids (Pawling, NY). LY 156758 was kindly supplied by Dr. J. A.Clemens (Lilly Research Laboratories).

Maintenance of Stock Cell Cultures. The ZR-75-1 cell line (82ndpassage) was obtained from the American Type Culture Collection(Rockville, MD) and routinely cultured in RPMI 1640 supplementedwith 2 HIML-glutamine, 1 niM sodium pyruvate, 2 ¿igof bovine insulinper ml, 100 IU of penicillin per ml, 100 m¡of streptomycin per ml, and10% (v/v) fetal bovine serum (HyClone, Logan, UT) under a humidifiedatmosphere of 95% air:5% CO2 at 37'C. AH media and medium

supplements were purchased from Sigma, except for triiodothyronineand bovine insulin which were obtained from Collaborative Research(Waltham, MA). Cells were passaged weekly by treatment with 0.05%trypsin:0.02% EDTA (w/v) (Grand Island Biological Company, GrandIsland, NY). The cell cultures used for the experiments herein describedwere between passages 87 and 96.

Cell Growth Experiments. Cells in their logarithmic growth phasewere trypsinized, briefly centrifuged, and resuspended in RPMI 1640

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MITOGENIC EFFECTS OF C,,-A5-STEROIDS IN BREAST CANCER

supplemented as described above, except that the insulin concentrationwas 0.5 jig per ml, and that medium contained 5% (v/v) DCC-treatedFBS. We found that lowering the insulin concentration was necessaryin order to observe steroid effects, since higher concentrations of insulintended to mask the mitogenic effects of estrogens.4 Cells were then

placed in triplicate into Linbro 24 u ell plastic culture plates (Flow) ata density of 2 to 4 x IO4cells per ml.

After 48 h, medium was replaced with fresh medium of identicalcomposition containing the indicated concentrations of steroids, in thepresence or absence of 300 nM LY156758. The final concentration ofethanol (0.1%, v/v) did not interfere with cell growth. Control culturesreceived the ethanol vehicle only. Cells were allowed to grow for an 8-day period with medium changes at Days 3, 5, and 7. Cultures werethen harvested by trypsinization, and cell numbers were measured witha Coulter Counter (Model ZM; Coulter Electronics, Hialeah, FL).Growth kinetics studies were performed essentially in the same manner,except that cells were harvested at the indicated times, and that mediumwas changed on Day 3, and then on every other day following theaddition of steroids.

Growth experiments were also performed using a serum-free, definedmedium modified from Calvo et ai (16). This medium contains (perml) 10 Mgof human transferrin, 0.5 Mgof bovine insulin, 100 ng ofhuman fibronectin, 100 ng of prostaglandin I-.....10 pmol of triiodothy-n >nme. and 10 pmol of dexamethasone in Dulbecco's modified Eagle'smedium:Ham's F-12 (1:1) supplemented with 15 DIM 4-(2-hydroxy-ethyl)-l-piperazineethanesulfonic acid, 2 HIM L-glutamine, and antibiotics. Cells were harvested in the usual fashion and plated in triplicatein Linbro 6-well plastic culture plates at a density of 6 x IO4cells perwell in RPMI 1640 supplemented with 2 mM L-glutamine, 1 HIMsodium pynivate, antibiotics, 0.5 *ig of bovine insulin per ml, and 5%DCC-treated FBS. Medium was changed daily for 3 days, after whichthe medium was replaced by the serum-free medium described above,following 3 washes of the cell cultures with 2 ml of Hanks' balanced

salt solution. Steroids were added at zero time at the indicated concentrations, and medium was changed on Days 3, 5, and 7 following theaddition of the steroids.Cells were harvested and counted after a growthperiod of 9 days in the serum-free medium.

Determination of Progesterone-specific Binding Sites. Exponentiallygrowing ZR-75-1 cells were harvested, resuspended in RPMI 1640supplemented with 5% DCC-treated FBS and 0.5 ng,of insulin per ml,and seeded in 24-well culture plates at 1 x Ml"cells/well in triplicate.

After 48 h, medium was replaced with medium of the same compositionsupplemented with increasing concentrations of steroids. Cells werethen grown for 4 days with one medium change 48 h after addition ofthe steroids. The number of specific progesterone binding sites wasthen determined using the whole-cell specific uptake of [3H]R5020 as

described (17). Briefly, following removal of medium, 0.5 ml of RPMI1640 supplemented with 0.1% bovine serum albumin, 6 nM [3H]R5020,

and 500 nM dexamethasone were added to cell monolayers. For eachpoint, 600 nM unlabeled R5020 was also included in triplicate wells inorder to determine nonspecific binding. The conditions used for incubation and extraction of radioactivity were exactly as described (17).Using this method, results obtained for the maximal cellular contentin progesterone binding sites in ZR-75-1 cells grown in RPMI 1640medium supplemented with 10% FBS and 10 nM estradici (174 ±21fmol/106 cells, n = 6) are similar to published values (12, 18).

Whole-Cell Specific Uptake of |3H|EstradioI. Cells in their logarithmic growth phase were seeded in triplicate into 24-well culture platesas described in the preceding section, except that 5% DCC-treated FBSin RPMI 1640 was kept free of steroids. Medium was changed everyother day following plating. When cultures reached near confluence(usually on Day 5), the medium was replaced with 0.5 ml of RPMI1640 supplemented with 0.1% bovine serum albumin, 5 nM |'II|-

estradiol plus or minus 500 nM diethylstilbestrol, and increasing concentrations of A5-diol, DHEA, or DHEAS. After 60 min, the radioactivity was extracted, and ER-specific labeling was determined as described (17). The duration of incubation with [3H]estradiol was found

to yield maximal values for spécifiebinding of the ligand (results notshown). Scatchard analysis (19) was performed using increasing con

centrations of 17|8-[3H]estradiol in the presence or absence of 500 nM

diethylstilbestrol.

RESULTS

As illustrated in Fig. 1.i, an 8-day incubation with maximalconcentrations of estradici increased ZR-75-1 cell number byabout 3.5-fold. The concentration of estradici required to induce half-maximal stimulation of cell proliferation was approximately 5 pM. It can be seen in Fig. IB that A5-diol has a strongmitogenic effect on ZR-75-1 cells, this steroid leading to amaximal increase in cell number 2.8-fold above control values,and the half-maximal effect being observed at approximately2.5 nM. It should be mentioned that this concentration lieswithin the range of normal serum levels of As-diol in women (1

to 3 nM) (20, 21).On the other hand, DHEA and DHEAS had a much less

potent action on cell proliferation (Fig. 1, C and />), bothsteroids increasing cell number up to about 75% above controlat the maximal concentration used (10 /JM). DHEA was morepotent than its 3/8-sulfate ester, a significant increase in cellnumber being observed at concentrations greater than 100 nM,while DHEAS did not have a significant effect on the growthof ZR-75-1 cells at concentrations lower than 3000 HM.

O-O CONTROL•-•LY156758 (300 nM)

j (LOG M) A5 - DKX (LOG M)

JTo

Z

8

-13 -12 -11 -10 -9 -8 -7 -13 -12 -11 -10 -9 -8 -7 -6 -5

4 Unpublished observations.

OHEA (LOG M) DHEAS (LOG M)

Fig. 1. Effect of increasing concentrations of estradici and Cn-A'-steroids onthe proliferation of ZR-71-1 cells in culture and its inhibition by the antiestrogenLY 156758. Cells were plated at an initial density of 2.0 x IO4cells/well in 24-well culture plates in RPMI 1640-5% DCC-treated FBS. After 48 h, estradici( l ). A5-diol (/O. DHEA (O. or DHEAS (/>) was added with fresh medium atthe indicated concentrations in the presence (•)or absence (O) of 300 nMLY 156758. Cell numbers were measured after 8 days in the presence of thesteroids. Points, means of triplicate determinations from a representative experiment; bars, SE.

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MITOGENIC EFFECTS OF CVA'-STEROIDS IN BREAST CANCER

The potent antiestrogen LY156758, a benzothiophene derivative having minimal agonistic activity in vivo (22) and behavingas a pure estrogen antagonist in rat pituitary gonadotrophs (23),was next used to assess the estrogenic nature of C|9-A5-steroid

action. It can be seen in Fig. 1 that the antiestrogen aloneexerted a 50% inhibition of cell growth, this inhibitory effectremaining maximal up to 0.3 UMestradici and 0.3 MMA5-diol.

That the effect of LY156758 was not cytotoxic is indicated bythe finding that 100 nM estradiol could completely reverse theeffect of the antiestrogen. The inhibitory effect of the antiestrogen was thus of a competitive nature for both estradiol and A5-

diol, the calculated (24) KA value of LY156758 action being0.54 HM. Growth stimulation by the less potent DHEA andDHEAS was completely abolished by LY156758 at all steroidconcentrations used (Fig. 1, C and D).

Almost identical results were obtained when the cells wereincubated with estradiol and A5-diol in a serum-free, hormone-supplemented medium (Fig. 2). Under these conditions, a 9-day incubation period in the presence of maximal concentrations of estradiol led to a 4-fold increase in cell number, a half-maximal effect being observed at about 5 psi estradiol. Likewise, cell proliferation was stimulated up to 3-fold followingincubation of ZR-75-1 cells with A5-diol, a half-maximal effectbeing observed at about 1.5 nM A5-diol.

The expression of the progesterone receptor is known to bespecifically under estrogenic control in human breast cancercells (25). We have thus measured the level of progesterone-specific binding sites after incubation of the ZR-75-1 cells for4 days in the presence of estradiol and of the CI9-A5-steroids.As illustrated in Fig. 3, the specific uptake of 3H-labeled R5020was increased up to 5-fold in ZR-75-1 cells incubated withestradiol. When present up to 10 pM, A5-diol, DHEA, andDHEAS maximally increased the number of high-affinityR5020 binding sites by 3.7-, 3.2-, and 2.0-fold, respectively. Ahalf-maximal stimulatory effect was obtained at 0.025, 13, and200 nM with estradiol, A5-diol, and DHEA, respectively. Therefore, the relative potency of the C|9-A5-steroids in increasing

the number of specific progesterone binding sites is parallel totheir respective ability to stimulate cell proliferation.

The estrogen-like effects of A5-diol, DHEA, and DHEAS

could have been mediated through metabolism by an endogenous aromatase with the subsequent formation of the knownestrogenic compounds estrone and/or estradiol (26-28). This

0-0 E2•-•As-Öd.

-12 -11 -10 -9 -8 -7 -6

STEROID (LOOM)

Fig. 2. Effect of increasing concentrations of estradiol (E2) and A5-diol on theproliferation of ZR-75-1 cells in serum-free, hormone-supplemented medium (cf."Materials and Methods"). Cells were plated at an initial density of 6.0 x IO4cells/well in 6-well culture plates in RPMI 1640-5% DCC-treated FBS. After 96h. the medium was changed for serum-free medium containing the indicatedconcentrations of estradiol (O) or A5-diol (•).Cell numbers were measured after

10 days in the presence of the steroids. Points, mean of triplicate determinationsfrom a representative experiment; hur.\, SE.

~ 200-

3

100-

o-oE2

-13 -12 -11 -10 -9 -8 -7 -6 -5

STEROID (LOG M)

Fig. 3. Effect of estradiol and Ci»-A5-steroidson the specific uptake of[3H]R5020 by ZR-75-1 cells in monolayer cultures. Cells were plated at an initialdensity of 1.0 x 10* cells/well in 24-well culture plates in RPMI 1640-5% DCC-treated FBS. After 48 h, the indicated concentrations of estradiol (O) (E2), A9-diol (•),DHEA (II). or DHEAS (•)were added with fresh medium. After 4 days,medium was removed, and the whole-cell specific uptake of [JH]R5020 wasmeasured as described in "Materials and Methods." Points, mean of triplicatedeterminations; bars, SE. For each treatment, the nonspecific uptake of radioli-gand was determined by adding 500 nM unlabeled R5020 to parallel triplicatecultures.

¿p-ox•-^cetuCO2z_l

_J7-6-5-4

-3-2-O

CONTROL•6«DT•

OHTAA4-DIONEA

DHEAS0DHEA»

A5-DIOLtA

2 4 6 8 10

INCUBATION TIME (DAYS)

12

Fig. 4. Effect of estradiol, Ci»-A*-steroids,and androgens on the cell growthkinetics of ZR-75-1 cells. Cells were initially plated at 5.5 x IO4cells/well in 24-well culture plates in RPMI 1640-5% DCC-treated FBS. After 48 h, 300 nM ofthe indicated steroids were added with fresh medium of identical composition.Control cells (O) received the ethanol vehicle only (0.1%). Cells from each groupwere harvested and counted daily for a 12-day period in the presence of theindicated steroids. Points, mean of triplicate determinations; bars, SE. /:'.., 17/3-estradiol; 7",testosterone; DHT, 5a-dihydrotestosterone; &'-DIONE, androstene-

dione.

possibility was examined by comparing the effect of androstene-dione, testosterone, and 5o-dihydrotestosterone on cell growthkinetics with that observed with estradiol and with the C)9-A5-steroids. Fig. 4 shows that estradiol, As-diol, and DHEA (each

at 300 HM) decreased generation time by 50 ±1, 57 ±1, and63 ±2%, respectively, as compared with control cells having ageneration time of 120 h. The density level reached by cellsgrown in the presence of A5-diol was 84% of that observed with

estradiol. However, cells incubated with 300 nM DHEA hadapparently not reached a plateau even after 12 days of growth,suggesting a delayed action of DHEA at the dose used on theproliferation of ZR-75-1 cells.

DHEAS, on the other hand, had no significant effect on cellgrowth at the concentration used (300 nM), in accordance withthe results of dose-response experiments (cf. Fig. ID). Furthermore, androstenedione had no effect on cell number, whiletestosterone and 5a-dihydrotestosterone caused an approximately 20% inhibition of cell growth (Fig. 4).

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MITOGENIC EFFECTS OF C,,-A5-STEROIDS IN BREAST CANCER

Experiments were next designed to study the competition byA5-diol, DHEA, and DHEAS of the specific uptake of [3H]-

estradiol by intact cells in monolayer culture. Fig. 5 illustratesthe characteristics of | 'H lustraciio I specific uptake in intact ZR-

75-1 cells. Linear Scatchard analysis (19) showed that, underthe conditions used, estradici binds to 16.0 ±2.8 fmol ofspecific binding sites per 10* cells (9600 ±1700 sites per cell)

at an apparent dissociation constant of 0.60 ±0.09 nisi.In order to measure the relative affinity of the Ci9-A5-steroids

for the ER, cells were exposed to 5 nM [3H]estradiol for 60 minin the presence of increasing concentrations of A5-diol, DHEA,

or DHEAS. As shown on Fig. 6, among the steroids tested,only A5-diol had a marked ability to compete with estradici forhigh-affinity binding sites as assessed by the specific uptake ofthe radioligand. The apparent dissociation constant (A',) value

for As-diol, calculated according to Cheng and Prusoff (24),

was 11 nM. This value is in close agreement with those alreadyobtained by other methods on breast cancer specimens (11) as

0 5 10 15B (fmol / 10* CELLS)

Fig. 5. I. uptake of [3H]estradiol by ZR-7S-1 cells incubated for 60 min at37*C with increasing concentrations of radioligand in the presence or absence of

500 nM diethylstilbestrol in order to measure nonspecific binding. Other detailsare as described in "Materials and Methods." Total (O), nonspecific (D), andspecific (•)binding of ['Hjestradiol (/'////•:.)to whole cells represents the means

±SE of measurements from triplicate wells. For each treatment, the nonspecificuptake of radioligand was determined by adding 500 nM diethylstilbestrol toparallel triplicate cultures. B. Scatchard plot of the experiment shown in A. B/F,bound/free.

100i

80

60

40

20

0J-12 -11-10 -9 -8 -7 -6 -5

STEROID (LOG M)

Fig. 6. Specific uptake of [3H]estradiol by ZR-75-1 cells incubated for 60 minat 37'C in the presence of Cn-A*-steroids. Cells were plated and grown toconfluency in RPMI 1640-5% DCC-treated FBS in the absence of steroids, asdescribed in "Materials and Methods." The whole-cell specific uptake of radioligand was then measured in the presence of A5-diol (O), DHEA (•),or DHEAS

(•)at the indicated concentrations, in RPMI 1640 medium containing 5 nM!3H]estradiol. Points, mean of triplicate determinations; han. SE. For eachtreatment, the nonspecific uptake of radioligand was determined by adding 500nM diethylstilbestrol to parallel triplicate cultures.

well as in MCF-7 cells (10). Although DHEA and DHEAShave significant effects on the proliferation of the /R 75-1 cellsin the range of concentrations tested in the competitive bindingstudies summarized in Fig. 6, they do not exhibit a commensurate ability to inhibit the specific uptake of [3H]estradiol in

these cells.

DISCUSSION

The present results clearly demonstrate, for the first time,that C,9-As-steroids of adrenal origin stimulate growth of estrogen-sensitive breast cancer cells. The present demonstration ofthe effect of A5-diol, DHEA, and DHEAS on the proliferationof the ZR-75-1 human breast cancer cells extends the observations made by others on the estrogenic effects of these steroidson the secretion of specific proteins (9) and on the level ofprogesterone receptors in MCF-7 cells (10), another estrogen-responsive human breast cancer cell line.

It is likely that As-diol, at physiological concentrations, actsas a genuine estrogen on the ZR-75-1 cells through its directinteraction with the ER. The high affinity of the steroid withthe ER, as measured by direct competition with the short-termuptake of radiolabeled estradici by whole cells, supports thenotion that metabolic conversion is not required for the bindingof As-diol to high-affinity intracellular sites for estrogens. Infact, we have observed that A5-diol is not converted to "classical" estrogens such as estradici or estrone in metabolic studies

of ZR-75-1 cells grown under conditions identical to those usedin the experiments presented here.4 Furthermore, any conversion of A5-diol to estradici via the formation of androstenedione

and testosterone is unlikely to be significant, since the presentdata show that androstenedione has no significant effect whiletestosterone was inhibitory on the proliferation of ZR-75-1

cells.Since the half-maximal stimulation of cancer cell growth was

observed at 1.5 to 2.5 nM and the serum levels of A5-diol in

adult women are 1 to 3 nM (20, 21), the present data clearlyindicate the potential role of As-diol in breast cancer growth

and development in women. DHEA and DHEAS, on the otherhand, are much less potent. Nevertheless, in the case ofDHEAS, the concentrations needed to increase cell number liein the higher part of the physiological range of plasma levels,especially in premenopausal women (29). Only supraphysiolog-ical concentrations of DHEA, however, had a mitogenic effecton the ZR-75-1 cells. The competitive nature of the inhibitionof the growth-promoting action of A5-diol, DHEA, and DHEAS

observed when cells were incubated simultaneously with theantiestrogen LY156758 clearly suggests that the effect of thethree steroids is mediated by the ER.

On the other hand, DHEA and DHEAS, which are the mainprecursors of A5-diol in the human through conversion by

peripheral tissues (6), are likely to undergo metabolic transformation before exerting their estrogenic action. In fact, in short-term studies where metabolism was minimal, we could notdemonstrate any significant displacement of [3H]estradiol-spe-cific uptake by DHEA or DHEAS in intact ZR-75-1 cells (thispaper) or with the estrogen receptor prepared from humanbreast cancer biopsies or rat anterior pituitary gland.5 More

over, despite the fact that no difference was seen in the affinityof DHEA and DHEAS for the estrogen receptor, the noncon-jugated form DHEA is clearly more potent than its 30-sulfateand less than As-diol, its 17/8-hydroxyIated derivative, in increasing cell number and the amount of high-affinity progesterone binding sites in the ZR-75-1 cells. In addition, DHEA has

5J. Simard and F. Labrie, unpublished observations.

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MITOGENIC EFFECTS OF C,,-A5-STEROIDS IN BREAST CANCER

REFERENCES

19.

an action similar, albeit somewhat delayed, to A5-diol on cell

proliferation (cf. Fig. 4). Since DHEA can be converted intoA5-diol in ZR-75-1 cells4 as well as in MCF-7 cells (9), it is 13

likely that the weaker estrogenic activity of DHEA, as comparedwith that of A5-diol, found in the present study, is related to thetime delay of its metabolism into A5-diol.

The implications of the present findings for breast cancer are '

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sustained estrogenic stimulus in responsive breast tumor cells,the serum level of A5-diol being maintained by continuous n.

peripheral conversion from DHEAS and DHEA (30, 31).Moreover, the cytosolic concentrations of A5-diol and DHEAin breast tissue are 2- to 5-fold higher than in plasma (32-34), 18and breast cancer tissue homogenates are known to convertDHEA into A5-diol (35, 36).

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breast cancer cell growth indicates that estrogens of adrenalorigin, either in the form of A5-diol or of its precursors, should 20be taken into consideration for the efficient control of estrogen-sensitive breast cancer. Thus, medical or surgical oophorectomy 21offers no more than a partial blockade of estrogens in premen-opausal women (37). Neutralization by a pure antiestrogen of 22the continuous action of Ci9-A5-steroids should therefore con

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(40), plasma levels of the latter are 12 to 24 times higher thanthose of estrone (6). Unless estrone and estrone sulfate are very 29.efficiently converted into the more active estradiol by 170-hydroxysteroid dehydrogenase present in most breast tumors(33, 39), A5-diol could well constitute the most important 30.

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1986;46:4933-4937. Cancer Res   Richard Poulin and Fernand Labrie  Cell Line

-Steroids in the ZR-75-1 Human Breast Cancer5∆-19Adrenal CStimulation of Cell Proliferation and Estrogenic Response by

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