[CANCERRESEARCH33.3149-3133,July I. I99.i|

Correlation of in Vitro and in VivoGrowth Suppression of MCF-7 Human BreastCancer by 2,3,7,8-Tetrachlorodibenzo-p-dioxin1

John F. Gierthy,2 James A. Bennett, Laurie M. Bradley, and Darvi S. Cutler

Watlsworth Center far Laboratories anil Reseatrh. »Ve»-York Slate Department of Health. Albany. 1220I-U5M ¡J.F. G.. D. S. C.¡.School of Public Health Science. University tit

Albany, Slale University of New York. Albany. 12201 ¡J.F. G.. L M. B. I. ami Department of Surgery. Albany Medical College. Albany. New York I220X ¡J.A. B.¡

ABSTRACT

The purpose of this study was to compare the effect of 2.3.7,8-tetra-chlorodibcnzo-p-dioxin (TCDD) on the in vitro and in vivo 170-estradiol(Ejl-dependent growth of MCF-7 human breast cancer cells. In culture, amajor component of postconfluent growth of MCF-7 cells is I^ dependent./« rir«.MCF-7 cells fail to grow as xenografts without exogenous E2support. Thus the effect of TCDD on postconduent MCF-7 growth inculture was compared with its effect on MCF-7 xenograft growth inimmunosuppressed mice. A concentration of 10 '' M I,? was optimal for

supporting postconfluent growth of MCF-7 cells in culture into multicel-lular aggregates (foci) on a monolayer background. The 5(1'. inhibitorydose of TCDD under these conditions was 3 *<10"'" \i, while K>-dependentfocus development was completely inhibited by 10~* MTCDD. Weekly i.p.

administration of TCDD (5 ug/kg) to mice bearing MCF-7 tumor xe

nografts resulted in inhibition of the tumor growth rate for the first 2weeks, followed by recovery to the control growth rate during the thirdweek. These recovered tumors were found to retain estrogen-dependent

growth as shown by second generation transplantation studies. The p.o.route of TCDD administration yielded a similar 2-week transient suppres

sion of grow th with a concentration of 8 ug TCDD/kg body weight but onlya 1-week growth rate latency with a 2-ug/kg body weight dose. A single5-ug/kg dose given 1 day after implantation was virtually noninhibitory.These results indicate that TCDD suppression of estrogen-dependentMCF-7 human breast cancer cell growth in vitro was predicative of asimilar growth suppression of MCF-7 solid tumor xenografts m vìvo.However, additional host-related factors must be involved in vivo, since

suppression of tumor growth is transient. These studies provide a basis forfuture in \-ivu investigations of TCDD endocrine toxicity by using the

M( 1 -7 tumor as a surrogate estrogen-responsive human organ and toexamine the efficacy of TCDD and related Ah receptor-mediated compounds in the management of human estrogen-dependent breast cancer.

INTRODUCTION

The environmental pollutant. TCDD,' exhibits a broad spectrum of

adverse responses which are tissue and species specific. In animals,this includes hyperkeratosis; edema; hyperplasia of the epithelium ofthe stomach, intestines, and urinary bladder: hepatocellulur damage;thymic involution: embryotoxicity and/or teratogenicity: and a prolonged wasting syndrome prior to death ( 1). The ultimate cause ofdeath in animals exposed to TCDD is not known. In animal studies.TCDD has been shown to be a potent carcinogen and tumor promotor(2). However. Kociba et al. (3). and subsequently others (4), haveshown that, although TCDD is a hepatocarcinogen in rodents, exposure to subtoxic doses results in a reduction of spontaneous, age-related tumors of the breast, uterus, and pituitary, indicating a tumor-

suppressive effect associated with hormonally responsive tissue.Interference with hormonally responsive tissue was further demonstrated when it was shown that TCDD-treated mice and rats do not

Received 11/23/92; accepted 4/23/93.The costs of publication of this article were defrayed in pan by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by National Institute of Environmental Health Sciences

Gram ES0356I.- To whom requests for reprints should be addressed.'The abbreviations used are: TCDD. 2.3.7.S-tetrachlorodiben?-O-/i-dioxin: E;. 17ß-

estradiol; DMSO. dimethyl sulfoxide: ID«,,.50r/i inhibitory dose.

display the uterotrophic response of increased uterine wet weight inresponse to 17ß-estradiol(5, 6). It has been suggested that many of thetoxic effects of TCDD in animals, such as depressed fertility, fetotox-

icity. teratogenicity. immunosuppression. and the wasting syndromecould be linked to its alteration of estrogenic activity (7).

The MCF-7 cell line, which was isolated from a pleural metastasis

of a human breast adenocurcinoma. has been used as a model to studyestrogen-dependent tumors (8). When transplanted to immunosuppressed mice. MCF-7 cells form tumors only with concurrent administration of estrogen. This is inhibited by cotreatment with antiestro-gens. In vitro, estrogen stimulates MCF-7 cell proliferation in

preconfluent and especially postconfluent cultures (9). Postconfluemcell growth in vitro is a characteristic of cancer cells ( 10). The induction of postconfluent cell proliferation in MCF-7 cultures by I7ß-estradiol results in the development of three-dimensional cellular aggregates or foci. This E^-dependent focus development is inhibited bythe estrogen-receptor hlockers tamoxifen and LY 156758 (9).

The antiestrogenicity of TCDD has been demonstrated in MCF-7cultures by the suppression of estrogen-enhanced secretion of tissueplasminogen activator (11) and also by reduction of other estrogen-

mediated effects including cathepsin D, and Mr 34.000 and M,160.000 proteins (12). Of particular interest was the ability of lowlevels of TCDD to suppress E2-enhanced postconfluent cell proliferation and focus development in MCF-7 cultures (13. 14).

Having established the antiestrogenicity of TCDD in human MCF-7

breast cancer cells in vitro, the utility of these cells as a human modelfor studying the antiestrogenic effects of TCDD in vivo was investigated. MCF-7 cells grown as xenografts in immunosuppressed micewere used to determine the effect of TCDD on E2-dependent MCF-7

tumor growth in vivo. The results reported here demonstrate thatTCDD treatment suppresses E^-stimulated growth of human MCF-7

breast cancer xenografts. This extends the /;; vitm studies of TCDDantiestrogenicity in human cells to an in vivo environment. It providesthe foundation for an in vivo model that can be used to study theantitumor effects of TCDD and its analogues against estrogen-dependent breast cancer as well as providing a surrogate human "targetorgan" in which to examine the antiestrogenic effects of environmen

tal exposure to this class of halogenated hydrocarbons.

MATERIALS AND METHODS

Chemicals, Animals, and Cells. TCDD was obtained from CambridgeIsotope Laboratories. Woburn. MA. Its purity was determined by mass spec-troscopy to be >99%. 17ß-Estradiol was obtained from the Sigma Chemical

Co.. St. Louis. MO. and DMSO was from the Aldrich Chemical Co.. Milwaukee. Wl. Cyclosporine A (Sandimmune. IV) was purchased from Sandoz Inc.,

East Hanover. NJ, and C57BL/6 x DBA/2 (hereafter called B6D2F,) micewere obtained from The Jackson Laboratory. Bar Harbor. ME.

A strain of the human metastatic mammary adenocurcinomu cell lineMCF-7 was obtained from Dr. Alberto C. Baldi, .nstitute for Experimental

Biology and Medicine. Buenos Aires. Argentina. Stock cultures were maintained in plastic tissue culture flasks (Costar), using medium consisting ofDulhecco's modified Eagle's medium wilh penicillin ( 1(X)units/ml) and strep

tomycin ( 1(X)ng/ml) supplemented with 59Õ-calf serum (Hyclone. Logan, UT.

10 ng/ml insulin, i-glutamine (2 IÃŽIM).and nonessential amino acids. The

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HUMAN BRKAST CANCLR (¡ROWTH SUPPRESSION BY TCDD

complete medium was filler sterili/ed using 5(X)-ml 0.2-fim pore si/e plastic

Nalgene filter units from Nalgene. Rochester. NY.Focus Development. Stock MCF-7 cells were suspended in medium atier

treatment with trypsin (0.25% I. seeded into 24-well plastic tissue culture plates(2 cm-/well) at a density of IO5 cells/well in I ml of medium, and incubatedat 37°Cin a humidified atmosphere containing 5% CO:. The cultures were

refed at 24 h and every 4-5 days thereafter with 2 ml medium supplementedwith I X 10"**M 17ß-estradiol.a concentration which induces maximum focus

development (9). and indicated concentrations of TCDD in DMSO (<O.I9í

final concentration I or DMSO alone. After 14 days the cultures were fixed withformalin in pH 7.4 buffer (130 IHMNaCI. 2 imi KCI. 8.8 m.MNa,HPO4. 1 imiKH:PO4) and stained with I'/i rhodamine B. Foci were counted by using a

New Brunswick automated colony counter modified lo magnify the image ofthe microscopic multicellular foci (9). The foci retained the red rhodamine Bslain to a greater extent than did surrounding monolayer cells, affording appropriate contrast for enumeration. All experimental protocols used phenolred-free medium.

In Viro Tumor Growth. Human MCF-7 breast cancer cells from culturewere solidified by a two-step process which included centrifugaron of 10

million cells into a cell pellet followed by exposure of the cell pellet to 15 filof fibrinogen (50 fig/ml) and 10 i¿\of thrombin (50 units/ml) for 30 minutesat 37°C.The second slep of this process congealed the cells in a fibrin clot.

Each clot was cut into four pieces of equal si/e. Each piece contained approximately 2.5 X 10'' cells. The pieces were implanted under the kidney capsule

of male B6D2F| mice immunosuppressed by daily treatment with cvclosporineA (60 mg/kg S.C.). We have previously shown that these mice are at least asgood and perhaps somewhat belter than nude mice in supporting tumor xe-

nograti growth (15). Male mice were used for betler control of E: dosage.Moreover, unsupplemented 10-week-old female mice could not support tumor

grow Ih (data not show n ). Two perpendicular diameters of the tumor xenograftswere measured in situ by using a dissecting microscope equipped with anocular micrometer. Tumor dimensions were measured during laparotomy immediately after implantation and at weekly intervals for 3 weeks thereafter.Tumor volumes were calculated atier each observation with the use of theformula:

V ?"'i. V :-<*T A. "fv-ÄiiSffT'

-

.?*••

which assumes the shape of the mass to be an ellipsoid of revolution around thelong diameter (/>l. 17ß-Estradiol was suspended in peanut oil. and 0.05 mg

was inoculated i.m. every 4 days beginning immediately after tumor implantation. This was found to be the optimal dose and schedule to support MCF-7tumor growth (see "Results"). TCDD was dissolved in corn oil and injected i.p.

or administered by p.o. gavage on days I. 8. and 15 after tumor implantation.All animal experimentation was done at Albany Medical College using Institutional Animal Care Use Committee approved protocols.

RESULTS

E2 treatment stimulates MCF-7 cell proliferation in vitro (9). The

majority of this stimulation is seen after the culture forms a confluentmonolayer and this E2-dependent postconfluent cell proliferation re

sults in the development of cellular aggregates superimposed on amonolayer background (Fig. I, A and B). These aggregates weredesignated foci, polarity-independent three-dimensional solid arrays

of cells (9). The foci first emerged from the monolayer about 3 daysafter the culture became confluent, and were thus a postconfluentevent. This estrogen-dependent focus development may be a consequence of the tumor origin of these cells. Induction of foci in contact-

inhibitable mouse fibroblast cultures by chemicals is routinely used inin vitro assays as an indicator of carcinogenesis (16). Development ofE;,-induced foci in MCF-7 cultures is inhibited by treatment withTCDD (Fig. 1C) ( 14). For a focus-inducing E2 concentration of 10~9M, the ID.,,, of TCDD is approximately 3 x IO'"' M(Fig. 2).

In order to determine whether TCDD would inhibit estrogen-dependent growth of MCF-7 cells in vivo, MCF-7 cells were grown assolid tumor xenografts under the kidney capsule of immunosup-

G

fig. 1. Inhibition by TCDD of foci induced with E2 in MCF-7 cultures. MCF-7 cellswere cultured untreated (A ). Irealed with I X I0~g M I7ß-estradiol (ß).or treated with 1X IO-" M I7ß-esiradioland 1 x IQ-" MTCDD (O for 14 days with medium replacement

every 4-5 days as described. Cultures were fixed in formalin and stained with rhodamineB. Note the absence of foci in A (untreated), foci development ¡nH wilh I7ß-eslradioltreatment, and inhibition of these loci in C with TCDD. Bar. I(X) jim.

pressed mice. Tumor implantation in the subrenal capsule site waspreferred over a s.c. site because it consistently resulted in 100<7r

tumor takes and immediate tumor growth ( 17). In the s.c. sites in theregion of the mammary fat pads, tumor takes were less than 509r andtumor growth was delayed and slow (data not shown) in both male orfemale mice. Estrogen support was provided by estradiol contained inSilastic tubing implants, which were placed in the s.c. space of theflank region resulting in an E2 serum concentration of 100 pg/ml (17),or by i.m. injection of estradiol suspended in 0.1 ml of peanut oil. Asshown in Fig. 3, MCF-7 breast cancer cells were completely depen

dent for growth on an exogenous source of estrogen following implantation of this tumor under the kidney capsule of immunosuppressed male mice. Estradiol contained in Silastic tubing implantssupported consistent tumor growth over a 2-week period. In subse

quent experiments it was found that tumors continued to grow over a4-week period. Estradiol delivered by i.m. injection in peanut oilsupported consistent and immediate tumor growth over the 2-week

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MIMAN URLASI (ASÕ I.k (ÕRCAVIH SI I'I'KI SSION HY TCDI)

150

100

Oo

50

O -12 -11 -10 -9

Log M TCDD

-8 UT

Fig. 2. The dose-response effect of TCDD on focus development in MCF-7 cultures.MCF-7 cultures similar to those described in Fig. I were fixed after I4 days of incubationin the presence of the indicated concentrations of TCDD in DMSO (•)and I X IO"1*M

173-estradio!. Cultures were fixed in formalin and stained with rhodamine B. and f(X'i

were counted as described. Each point is an average of duplicate cultures of a representative experiment ±range (bars). UT (O), untreated.

300

14

DAYS AFTER IMPLANTATION

Fig. 3. Effect of I7ß-estradiol on growth of MCF-7 tumor xenograiis. MCF-7 tumorpieces were inserted beneath the renal capsule of male B6D2F, mice. Each mousereceived one tumor piece containing approximately 2.5 x IO* tumor cells. Implantationwas followed by I7ß-estradiol supplementation by silastic implant (•)(17), i.m. injections of 17ß-estradiol in peanut oil given every 4 days starting on the day of tumor

implantation: A. 0.02 mg. A. 0.05 mg; D, 0.10 mg: or •¿�.one i.m. injection of 0.10 mg17ß-estradiol in peanut oil; or O, peanut oil alone. Tumor si/e was measured at theindicated times after implantation and expressed as percent of si/.e at day 0 (4 mm1). Each

point is an average of 5 mice ±SE (bars).

observation period when a minimum of 0.05 mg of E2 was givenevery 4 days. Injection of 0.1 mg of E2 on this schedule also supportedtumor growth, but injection of 0.5 mg of E2 became lethal after thethird injection on this schedule (data not shown). A single injection of0.1 mg of E2 supported tumor growth for 7 days, but the growth ratebegan to regress by 14 days after tumor implantation. Injection of 0.05mg of E2 every 4 days appeared to be the minimum amount of E2needed to sustain immediate and consistent tumor growth. Thus thisregimen was chosen for subsequent experiments, since it was thoughtthat it would be most sensitive to changes wrought by exposure toTCDD.

In the next series of studies, mice with MCF-7 cells growing as a

solid tumor under the kidney capsule received i.p. injections of TCDDat weekly intervals beginning 1 day after tumor implantation. It wasfelt that the i.p. route of administration of TCDD would be the optimal

route for directly exposing the tumor to unmodified TCDD, whichwere the experimental conditions used in the tissue culture studies. Asshown in Fig. 4, weekly treatment with TCDD significantly inhibitedestrogen-stimulated growth of MCF-7 tumors. Inhibition was greatest

during the first 2 weeks of tumor growth. However, during the thirdweek, tumors became refractory to TCDD and grew at a rate whichparalleled that found in the group treated only with E2. This recoveryin growth rate resulted in similar differences in tumor size betweenTCDD-treated and E^-stimulated controls on days 21 and 14 after

tumor implantation. Injection i.p. of corn oil (the vehicle for TCDD)in estrogen-supported mice results in tumor growth similar to that

found in the group treated only with E2 (data not shown). Following

400

300 -

'Ss«—¿� 200 -LUIS)

C/3

rr

i100

20

DAYS AFTER IMPLANTATION

Fig. 4. Effect of i.p. TCDD treatment on I7ß-estradiol-supported MCF-7 tumor growthin male B6D2Fi mice. MCF-7 cells were implanted under the renal capsule. Micewere supplemented with 0.05 mg 17ß-estradiolas described in Fig. 3 (A, D) or unsup-plemented (O. n = 6) and exposed to 5 /ig TCDD/kg in body weight in corn oil(D, n = I7) or corn oil alone (A, O) by i.p. injection. Tumor volume was determinedat the indicated limes after implantation and is expressed as percentage of size at day 0(4 mm') ±SE (bars). *. P < 0.05 when compared with MCF-7 tumors in I7ß-estradiol-supplemenled mice not treated with TCDD (A, n = 9) using Student's / test.

250 F

0 7 14

DAYS AFTER IMPLANTATION

Fig. 5. Characterization of 17/3-estradiol growth dependency of second-generationMCF-7 tumors from TCDD-treated mice. On day 21, tumors in the 17ß-esiradiolplusTCDD group described in Fig. 4 were resected and transplanted a second time under thekidney capsule in new, I7ß-estradiol-supported (A) or untreated (U) male B6D2F| mice.The results are compared with first-generation MCF-7 transplants with (A) and without<•)17ß-estradiolsupplementation. Tumor volume was determined at the indicated timesafter implantation and is expressed as percentage of size at day 0 (4 mm1). Each point is

an average of five mice ±SE (bars).

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HUMAN BREAST CANÅ’R GROWTH SUPPRESSION BY TCDD

10 15 20

DAYS AFTER IMPLANTATION

Fig. 6. Effect of p.o. TCDD treatment on I7ß-esiradiol-supporied MCF-7 tumorgrowth in male B6D2F, mice. MCF-7 cells were implanted under the renal capsule in

mice supplemented with 0.05 mg E; as described in Fig. 3 (A. G. •¿�)or unsupplemcnled(O. n = 4). and exposed to 2 /ig ever) 4 days (Zi, «= 8I or 8 ng {•.n = 7> of TCDD

in corn oil/kg body weight or corn oil alone (¿i,C) by oral gavage. Tumor volume wasdetermined at the indicated times utter implantation and is expressed as percentage of sizeal day 0 (5 mm') ±SE (burs}. *. P < 0.05 when compared to MCF-7 tumors in17/3-esiradiol-supplememed mice not treated with TCDD (¿Xn = 6) using Student's t test.

measurement of tumor size on day 21. tumors ¡nthe E? plus TCDDgroup were resected and transplanted under the kidney capsule in new.Ei-supported or untreated mice. In both cases this second transplantgeneration of tumors remained E2-dependent. although there was an

initial lag in the growth of tumors that had been exposed to TCDD inthe first generation (Fig. 5). These results indicated that, upon recovery of growth, the TCDD-treated MCF-7 tumor retained its characteristic E2-dependent growth properties.

The p.o. route of TCDD administration was evaluated, since it wasa more distant route from the site of the tumor and a route by whichTCDD from the environment could enter the body. A single p.o. doseof 8 /-ig/kg TCDD given I day after tumor implantation significantlyinhibited tumor growth for 14 days, followed by a recovery in tumorgrowth rate for the next 7 days (Fig. 6). A lower dose of TCDD givenmore frequently (2 /-ig/kg every 4 days) initially slowed tumor growthrate, but during the second and third weeks of treatment there was arebound effect in the tumors, which resulted in a similar growth ratecompared with tumors in estrogen-supported mice not treated with

TCDD. A single p.o. dose of 5 fig^? given 1 day after tumor implantation was virtually noninhibitory (data not shown). All of thesein vivo studies followed a pattern whereby treatment with TCDDcaused an initial inhibition of estrogen-stimulated MCF-7 breast can

cer growth followed by a recovery in the tumor, which made itrefractory to continued treatment with TCDD.

DISCUSSION

In this study we demonstrated that treatment of mice with TCDDresults in suppression of E2-dependent growth of xenotransplantedhuman MCF-7 breast cancer. This sensitivity of MCF-7 tumor xe-

nografts is in agreement with the cell culture results which demonstrated inhibition by TCDD of E2-dependent postconfluent growth ofMCF-7 cells into discrete foci ( 14). In culture. TCDD was quite potent(ID.,,, 3 X 10-'" M)relative to tamoxifen (ID5(, IfT6 M)and LY 156758(IDs,, IO"8 M) which had been previously tested in this system (9). In

vivo, TCDD significantly inhibited tumor growth for 2 weeks but thentumors resumed growth despite continued administration of TCDD. Incontrast, tamoxifen given p.o. to mice at a dose of 25 fig/mouse/day

produced a sustained growth inhibition of this tumor in vivo (18). Thusin discussing our results, consideration must be given to not only themechanism of antiestrogenicity of TCDD but also to the basis for thetransient nature of its inhibition of estrogen-dependent tumor growth

HI vivo.The mechanism of TCDD antiestrogenicity is controversial. Much

of the activity associated with TCDD segregates with the Ah locus.The Ah locus comprises a group of genes which are modulated by thecytosolic Ah receptor when it is bound to the appropriate ligund. Theaffinity of polychlorinated dioxins for this receptor depends on theposition of chlorine substitutions, with TCDD demonstrating the mostavid binding and highest activity ( 1).

TCDD has been shown to reduce the number of occupied nuclearestrogen receptors ¡nMCF-7 cultures with a 50% effective concentration of about IO"'' M( 19). From these studies, it has been suggested

that the TCDD-mediated antiestrogenicity occurs through estrogenreceptor down-regulation in MCF-7 cells (20). However, previous

examination of whole cell estrogen receptor performed in our laboratory indicated no effect on total estrogen receptor levels by TCDDtreatment (11). This apparent inconsistency may be explained by analternative hypothesis of antiestrogenicity based on an Ah receptor-dependent induction by TCDD of cytochrome P-450 mixed-function

oxidases, which degrade E2 to less active metabolites as it transversesthe cytoplasm. TCDD induces the cytochrome P-45()s in MCF-7 cells,

resulting in the metabolism of E2 at the 2. 4. 6a, and I5a positions,with most of the activity being at positions 2 and 4(21). This activityis dose dependent, starting at a concentration of I0~" MTCDD. with

a 5()9c effective concentration of 0.5 IIMand maximal stimulation at 10n.MTCDD (22). The Vlmlvfor 2- and 4-E2 hydroxylation is about I JUM.

suggesting a high capacity for metabolism (22). The magnitude of thisE2 metabolism in TCDD-treated MCF-7 cells i;i vitro suggests that E2depletion could play a role in the antiestrogenicity of TCDD in MCF-7

cultures. It is possible that this putative in vivo metabolism is restricted to the intracellular environment of estrogen-responsive target

tissue and would not necessarily result in lower circulating E2 levels,which are under homeostatic control. Instead, the E2 could be metabolized during passage through P-450-containing cytoplasmic mem

branes. This would result in less E2 reaching the estrogen receptorlocated in the nucleus, a decrease in estrogen receptor binding, and thereduction of occupied nuclear receptor in response to TCDD. asdescribed by others ( 19), without affecting the total estrogen receptorin MCF-7 cells.

The basis for the transient nature of tumor suppression by TCDD invivo is unknown. Tissue dosimetry studies were not carried out: thuswe do not know whether a change in bioavailability of TCDD at thetumor site occurred over time. However, this transience does concurwith other recent studies of TCDD. TCDD lowers estrogen receptorlevels in the livers of rodents, but this depression is transient andestrogen receptor levels return to control levels within a few weeks inspite of a maintained body burden of TCDD (23). Another studydemonstrated that the effect of TCDD on thymic atrophy is transientwith a duration of 11-18 days (24). The possibility that the MCF-7

transplant in this study had become estrogen independent and thusrefractory to the antiestrogenic effects of TCDD was shown to beinvalid by second generation transplant experiments, which demonstrated the tumor to still be estrogen dependent.

The results described here indicate that TCDD treatment causes anantiestrogenic response to a human estrogen responsive tissue in vivo.This can be used as a model for Ah locus-mediated antitumor studiesas well as providing a surrogate for an estrogen-responsive human

tissue to examine the antiestrogen potential of TCDD in vivo. Thetransient nature of this suppressive effect may be the result of increasing E2 levels that override the TCDD induction of this effect, regard-

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HUMAN BREAST CANCER GROWTH SUPPRESSION BY TCDD

less of the actual mechanism, or it may be based on a more fundamental effect of TCDD, suggesting a compensatory mechanism of thewhole organism in response to xenobiotic exposure. Examination ofthese possibilities is in progress.

ACKNOWLEDGMENTS

We gratefully acknowledge the secretarial assistance of Jill Ranetta.

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-dioxinpHuman Breast Cancer by 2,3,7,8-Tetrachlorodibenzo- Growth Suppression of MCF-7in Vivo and in VitroCorrelation of

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