(CANCER RESEARCH 49. 4765-4769. September I. 1989]

Differential Ability of Antiestrogens to Stimulate Breast Cancer Cell (MCF-7)Growth in Vivo and in Vitro1

Marco M. Gottardis,2 Randall J. Wagner, Ernest C. Borden, and V. Craig Jordan3

Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792

ABSTRACT

We have previously described an MCF-7 breast cancer cell variant,MCF-7TAM, which is stimulated to grow in athymic mice by tamoxifen(TAM) (M. M. Gottardis and V. C. Jordan, Cancer Res., 48:5183-5187,1988). Earlier experiments have shown that TAM exhibits some profound estrogen-like effects in mice whereas TAM is less estrogenic inthe rat. The aim in these studies was to compare the ability of MCF-7TAM to grow in different host environments and to determine whetherthe TAM-stimulated phenotype could be maintained in vitro. Ovariec-tomized athymic mice and rats were implanted with 1-nini' pieces of

MCF-7TAM tumor and treated with estradiol, TAM, or control silasticcapsules. After 9 weeks of growth in either species, TAM or estradiol-treated groups both had sustained growth of MCF-7TAM compared withthe control groups. To determine the effects of estradiol and TAM onimmune function in athymic mice, splenocytes from treated or controlathymic mice, challenged with poly(I:C), were assayed for natural killer(NK) cell activity against 51Cr-labeled YACÕtarget cells. Both estradiol

and TAM abolished lytic activity by 12 weeks of treatment. To evaluatethe role of a decrease in NK-cell activity in the host on growth of MCF-7TAM xenografts we compared the growth effects in athymic and NK-cell deficient, ovariectomized beige mice. TAM stimulated MCF-7TAMin both beige and athymic mice; however, the tumor grew more rapidlyin control beige mice than in control athymic mice. This study demonstrated that TAM-stimulated growth could occur in vivo. However, TAMor 4-hydroxytamoxifen did not cause a stimulation of MCF-7TAMcompared with wild-type MCF-7 cells when experiments were conductedin vitro. These studies demonstrate that a suppression immune functioncan facilitate the growth of MCF-7TAM in athymic animals. However,additional components of the host environment contribute to TAM-stimulated growth in vivo.

INTRODUCTION

TAM,4 a nonsteroidal antiestrogen, is the antihormonal ther

apy of choice for the treatment of breast cancer (1, 2). Thetreatment regimens for adjuvant TAM therapy after mastectomy are becoming extended to 5 years or more (3-5) becauselaboratory evidence suggests that the antiestrogen is a tumor-istatic agent (6-8). However, it is important to build upon thesuccessful clinical application of this relatively (compared tochemotherapy) nontoxic drug, and to maintain the patients foras long as possible. Regretably, prolonged treatment regimenswith TAM may predispose the tumor to develop therapeuticresistance. We have previously described a number of potentialmechanisms for tumors to become resistant to antiestrogentherapy in the laboratory (9-14). It therefore becomes essentialto describe and to investigate the development of resistance to

Received 1/9/89; revised 5/19/89; accepted 6/7/89.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.

' Supported by USPHS Grant CA20432 from the National Cancer Institute.; Supported by Human Cancer Biology Training Grant NCI T32-CA09471.' To whom requests for reprints should be addressed, at Department of Human

Oncology, University of Wisconsin Clinical Cancer Center, 600 Highland Avenue,Madison, WI 53792.

4The abbreviations are: TAM. tamoxifen; ER, estrogen receptor; HT, 4-hydroxytamoxifen; NK, natural killer; LU, lytic units; poly(I:C), polyinosinic-polycytidylic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid;MEM. minimal essential media; HBSS, Hanks' balanced saline solution; IC50,effective concentration to produce 50% inhibition; HPLC, high-performanceliquid chromatography.

TAM therapy as the drug is now available to treat all stages ofbreast cancer.

TAM is not a pure antiestrogen. The drug appears to have aspecies-specific pharmacology in short-term tests: TAM is predominantly estrogenic in mice but is less estrogenic and predominantly antiestrogenic in rats and humans (1). Interestinglyenough, TAM and its metabolites inhibit the estradiol-stimu-lated growth of MCF-7 breast cancer cells transplanted intoathymic mice (8, 15). Nevertheless, long-term therapy withTAM results in the outgrowth of breast cancer cells that areresponsive to TAM for growth (14).

We have previously shown (16) that the growth of the TAM-stimulated variant of MCF-7 cells, MCF-7TAM (14), can beinhibited by less estrogenic antiestrogens. However, the factthat the TAM-stimulated tumor is growing in a mouse host,where the drug exhibits estrogenic properties (1), raises questions about the role of the host in the growth of the tumor.

The aim of this investigation was to compare and contrastthe growth of MCF-7TAM in different environments. As previously stated, TAM is less estrogenic in the rat than the mouse(1); therefore, an initial study was undertaken to determinewhether MCF-7TAM tumors would grow during TAM treatment in athymic rats. However, one important aspect ofathymic animals is that they retain NK cell activity (17, 18).Estrogens lower NK cell activity in mice (19, 20); therefore, webelieved it was possible that TAM (an estrogen?) could lowerNK cell activity to promote MCF-7TAM growth indirectly.Beige mice have selectively impaired NK cell function (21), sothat if TAM-stimulated growth of MCF-7TAM was only dependent upon a modulation of NK cells in athymic mice, noadditional TAM-stimulated growth would occur compared withcontrols. Finally, the direct effects of estradiol, TAM, or itsmetabolite, 4-hydroxytamoxifen, were determined on MCF-7TAM cells in vitro.

MATERIALS AND METHODS

The TAM-stimulated breast cancer tumor, MCF-7TAM, was developed in athymic mice from an MCF-7 tumor failing TAM therapy (14).This tumor was subsequently maintained by passaging in ovariectomized athymic mice implanted s.c. with 5-mg TAM pellets (custom-made by Innovative Research of America, Toledo, OH). All tumorsused in these experiments were derived from in vivo passage 6.

Ovariectomized BALB/c 4-5-week-old athymic mice, 4-5-week-oldbeige Hsd:NlHS-hg/nu/xid mice or Hsdrathymic nude rat (rnu) (Spra-gue-Dawley, Indianapolis, Indiana) were implanted s.c. in the axillarymammary fat pads with 1-mnr' pieces of tumor as previously described

(14).Hormone Treatments. Silastic capsules used in the experiments were

made from Silastic tubing (Dow Corning, Midlands, MI) 0.078" insidediameter by 0.125" outside diameter out to a 2.0-cm size and filledwith TAM-free base (ICI Americas, Wilmington, DE) and ends pluggedwith Silastic Cement (Dow Corning). Estradiol capsules were cut to a1.0-cm size and filled with a matrix consisting by weight of three partsSilastic Medical Grade Elastomer (Dow Corning) and one part estradiol(Sigma Chemical, St. Louis, MO). All capsules were sterilized by 7-irradiation.

ER and TAM Determinations. Tumors excised from animals werefreshly homogenized in high salt (0.4 M KC1) buffer as previously

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described (14). MCF-7 cytosols were incubated with various concentrations of HT or TAM at 4°Cfor 2 h, then assayed following the protocol

outlined in a monoclonal estrogen receptor assay kit (Abbott Laboratories, North Chicago, IL).

Tumor cytosols were assayed for TAM and metabolites by extracting0.5 ml in 2.5 ml of hexane:amyl alcohol (98:2), drying down samplesunder nitrogen and redissolving in HPLC mobile phase. Samples wereassayed using an HPLC method previously described (22).

Tumor Measurements. Tumor measurements were performed weeklyusing calipers. Cross-sectional tumor area was calculated using theformula

/ H-

Preparation of Mouse Spleen Cells for Cytotoxicity Assay. Ovariec-tomized athymic mice (4-5 weeks old) were treated with TAM 2.0 cm,estradiol 1.0 cm, or placebo silastic capsules and sacrificed at 1,4, 6,or 12 weeks. Twenty-four h prior to sacrifice, mice were treated withan injection of 100 iig/0.1 ml i.p. of poly(I:C) (Miles Laboratories,Napaville, IL) dissolved in isotonic saline which was reannealed byheating at 70°C,then cooled.

After sacrifice, spleens and uteri were excised from animals anduterine wet weight was noted. Spleens were placed in RPM1 mediacontaining 25 mivi HEPES, 2 mM glutamine, 50 mg/liter gentamicin,and 10% fetal bovine serum (Hyclone, Logan, UT). Spleen cells weredisaggregated mechanically from spleen capsules and treated for 5 minwith Gey's hemolytic solution (23). Cells were washed three times withRPMI and incubated on a scrubbed nylon wool column for 1 h at 37°C

as described by Julius et al. (24). Nonadherent splenocytes were collected and quantitated using hemocytometer counting.

YAC-1 mouse lymphoma target cells (ATCC; Rockville, MD) werelabeled with 50 n\ of 5 mCi/ml 5lCr (New England Nuclear; Boston,

MA) for 90 min. The addition of 2000 target cells per well resulted ina maximum release value of between 5,000 and 10,000 cpm in 3%Triton X-100 (Sigma, St. Louis, MO) detergent-treated microwells.Different effector cell (splenocytes) to target cell (YAC-1) (E:T) ratios(100:1, 50:1, 25:1, 12.5:1, 6.25:1, and 3.125:1) were completed inquadruplicate in 96-well microtiter round-bottom wells (Costar, Cambridge, MA). Effector spleen and YAC-1 target cells were incubated for4 h at 37°Cand harvested using a Titertek supernatant collection

system (Skatron, Lier, Norway). Wells were included to determine thespontaneous release of 51Crfrom labeled YAC-1 cells. Filter plugs were

collected and the radioactivity determined with a gamma counter. Lyticunits/IO7 cells at 20% Cytotoxicity were calculated from the equations

developed by Pross (25).

^cpm = —¿�—(maximum release —¿�spontaneous release)1UU

(1 —¿�e~At)+ spontaneous release

where x is the E:T ratio, K is a rate constant, and A is the maximumattainable percentage lysis. The parameters A and A'are estimated using

nonlinear least squares analysis after transferring both sides of theequation to the log scale. LU are not defined when A is estimated to beless than 20% lysis. In these situations, an appropriate LU value wascalculated by fixing A at 21 % lysis. The formula for measuring LU in20% Cytotoxicity in IO7cells was

LU IO7IO7 effector cells -'»*('-?) Targets per well

Thus, one LU is defined as the number of effector cells required tocause 20% specific 5'Cr release.

Tissue Culture Experiments. MCF-7 cells were obtained from ATCC(Rockville, MD) and were used in these experiments at passage 145.MCF-7 wild-type cells were maintained in MEM media with 100 unitspenicillin/streptomycin/ml, 6 ng insulin/ml, 25 mM HEPES, and 10%calf serum (GIBCO) at 37°Cunder 5% CO2 conditions as previously

described (12). MCF-7TAM cells were obtained by mechanically dispersing MCF-7TAM tumors under sterile conditions. The cells were

maintained in MEM (without phenol red indicator; Sigma) with 10%charcoal-stripped serum and HT 10~' M (ICI Americas). Cells wereplated at 2.5 x IO4per well in 24-well dishes for all experiments. Before

plating cells, HT was removed by maintaining cells in MEM (withoutphenol red indicator) media and 10% charcoal-stripped serum for 7days with daily media changes. Compounds estradiol (Sigma), TAM orHT were dissolved in 100% cilia no I and added to media in a volume of0.1% ethanol. Ethanol alone was added to control. Media were changeddaily in all experiments. At the end of experiments, cells were disruptedin 0.1 x HBSS using sonication.

DNA determinations were made using a Hoechst 33258 (Calbi-ochem-Behring; LaJolla, CA) fluorescent dye assay as previously described (26) and DNA levels reported as ng DNA/ml.

Statistical Analysis. Differences in mean tumor area, DNA content,uterine wet weight, or LU between groups were measured using analysisof variance followed by unpaired Student's t test.

RESULTS

TAM-stimulated Growth of MCF-7TAM Tumors in AthymicMice and Athymic Rats. In the first set of experiments, wedetermined whether MCF-7TAM tumors could be stimulatedto grow in the athymic rat as well as the athymic mouse. Parallelsets of athymic rats and mice were transplanted with the sameMCF-7TAM tumor. After 9 weeks of treatment, 2.0-cm TAMcapsules produced a sustained tumor growth to a mean areasize (±SEM)of 0.75 ±0.14 cnr (n = 12) in the athymic mouse(Fig. IA), but only minimal growth was observed in untreatedcontrol animals. Estradiol stimulation with a 1.0-cm capsuleproduced similar growth during the 9 weeks of the experiment.The mean tumor area (±SEM) after 9 weeks of estradiol was0.5 ±0.04 cm2.

A similar growth pattern for MCF-7TAM tumors was observed in the athymic rat (Fig. \B). Tumor size increased to0.74 ±0.06 cm2 with TAM while there was again no sustained

growth in control animals. Estradiol also stimulated MCF-7TAM tumors to grow to a mean area size (±SEM)of 0.54 ±0.05 cm2 during the 9-week experiment. Thus, despite theknown estrogen-like pharmacology of TAM in the mouse, the

E

o

1.00

0.75

0.50

0.25

0.000 2 4 6 810

Weeks

1.00

0.75

0-50

0.25

0.004 6

Weeks10

Fig. I. Growth of MCF-7TAM tumors (mean ±SE) (n = 12) in athymie mice(A) or athymic rats (B). O, animals treated with placebo capsules; D, animalstreated with estradiol 1.0-cm capsules; •¿�,animals treated with TAM 2.0-cmcapsules.

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MCF-7TAM tumor could be stimulated to grow with TAM ina host (the rat) where TAM is known to be less estrogenic.

Measurement of ER and TAM Content from MCF-7TAMTumors. We have previously noted that MCF-7TAM tumorswere ER positive (14). We believed it was important to confirmthat MCF-7TAM was still ER positive in the current passage(determined at Week 10 of the previous experiment; Fig. IA).Athymic mice, treated with estradici or TAM, were sacrificedand tumor cytosols prepared (see "Materials and Methods").

Tumor cytosols from TAM-treated animals which were extracted and analyzed by HPLC contained <1 ng/ml (3 HM) ofTAM. ER levels of MCF-7TAM tumors treated with TAMwere 201 ±20 fmol/mg of protein (mean ±SE; n = 8), whileER levels of animals treated with estradiol were significantlylower (P < 0.001), 35 ±3 fmol/mg of protein (mean ±SE; n= 8), respectively.

A recent report (27) has demonstrated that antiestrogens canaffect the binding of monoclonal antibodies to ER. We thereforedetermined the level of TAM and HT which would increase ERlevels as measured by the monoclonal antibody kit. MCF-7wild-type cytosols were incubated with various concentrationsof TAM and HT. Concentrations of 10~6 M of HT and TAM

increased ER levels in the monoclonal antibody assay by 60and 20%, respectively (Fig. 2). However, these concentrationswere considerably more than those analyzed in the TAM-treated tumor cytosols (<1 ng/ml, approximately 3 HM). Thusthe increased ER concentration cannot be attributed to TAMeffect on the ER-EIA alone.

Effect of TAM and Estradiol on NK Cell Activity in theAthymic Mouse. The long-term effects of sustained-releasepreparations of TAM and estradiol were determined in theovariectomized athymic mouse. Uterine wet weights at thevarious time points demonstrated the estrogen-like activity ofTAM, but the increases observed were not as large as those forestradiol treatment (Table 1). We have previously noted (28) asteady decrease in the uterine wet weights of ovariectomizedmice during the long-term TAM treatment using these silasticcapsules. The uteri are also refractory to estrogen challenge.The uterine weights of TAM-treated mice also declined in thisexperiment (Table 1).

Mice were challenged with an injection of poly(I:C) 24 hbefore sacrifice to increase NK cell activity. The splenic lymphocytes were isolated to determine NK cell activity for eachtreatment and compared with animals in the control groups(Table 2). Estradiol treatment affected the NK cell activity bythe first week of treatment, and the activity subsequentlydropped to approximately 6 LU/107 cells. TAM had less of an

effect than estradiol, but at 12 weeks, TAM caused a significant(P< 0.001) suppression of NK cell activity compared to control

Table 1 Uterine wet weight of athymic mice treated with estradiol (EJ or TAM

80

||TOg-sS ael«.

II,31 so

-11 -10 -9 -8 -7 -6LOG M Concentrations of Antiestrogens

Fig. 2. Effect of incubation of MCF-7 cytosols with antiestrogens on quanti-tation of estrogen receptors with Abbott Laboratories monoclonal antibody kit(each point mean ±SE; n = 3). O, tumor cytosol treated without antiestrogens;A, cytosols treated with HT; •¿�,cytosols treated with TAM.

TreatmentgroupControl

capsulesEj 1.0-cm capsules'TAM 2.0-cm capsules'*112.4

+ 0.6°•¿�*

62.8 ±6.256.3 ±2.3Weeks

oftreatment461211.1

±0.9 14.1 ±0.8 11.4± 1.363.3 ±5.0 70.1 ±6.6 87.9 ±9.337.1 ±3.0 40.4 ±2.3 29.6 ±4.9

°n = 4 observations per treatment at each time point.* Values expressed as (mean ±SE) in mg.' All Entreated groups significant from control group (P < 0.001 ) by unpaired

Student's / test.rfAH TAM-treated groups significant from control groups (P < 0.01) by

unpaired Student's / test.

[19.8 ±3.7 LU/107 cells versus 173.4 ±28.0 LU/107 cells

(mean ±SE), respectively].In a separate experiment, the NK cell activity of splenic

lymphocytes from ovariectomized athymic mice was comparedwith beige athymic mice 24 h after a challenge with poly(I:C)(four mice in each group). No NK cell activity was detected insplenic lymphocytes from beige mice whereas the NK cellactivity of the athymic mice was 220 ±12.6 LU/107 cells.

TAM-stimulated Growth of MCF-7TAM Tumors in AthymicMice and Beige Mice. The differential growth of MCF-7TAMwas studied in beige and athymic mice. Parallel groups of micewere implanted with 1-mm' pieces of the same MCF-7TAM

tumor and TAM or control (placebo) capsules were implanted.After 9 weeks of therapy (Fig. 3), MCF-7TAM tumors were

stimulated to grow with TAM administration in both beige andathymic mice to a mean area size (±SEM)of 0.71 ±0.10 cm2and 0.68 ±0.11cm2, respectively. However, although there wasminimal growth of MCF-7TAM tumors in the athymic mousecontrol after 9 weeks, 0.12 ±0.01 cm2 (mean ±SEM), the

beige mouse control had over a twofold increase in tumor size,0.31 ±0.03 cm2. Thus, MCF-7TAM tumors can grow in beige

mice without TAM supplementation; however, this growth issignificantly lower than in TAM-treated beige mice.

Characteristics of MCF-7TAM Cells in Vitro. Tumors fromTAM-stimulated MCF-7TAM athymic mice were excised andcells grown in culture under conditions without phenol red andin 10% stripped calf serum. Experiments were done in parallelwith wild-type MCF-7 cells which had been maintained innonestrogenic conditions 7 days before the experiment. Theresults shown in Fig. 4 are representative of three experimentsto investigate the ability of estradiol and HT to stimulate MCF-7TAM cells in vitro. There was a significant increase in DNAcontent in cells treated with HT compared with controls (P <0.01), but there was no concentration-related increase in DNAcontent for the antiestrogen. (Data from MCF-7 cells treatedwith concentrations of 10~9-10"~6Mof HT were superimposable

so, for clarity, one line is shown in Fig. 4.) The results weresimilar when TAM was used in place of HT. In contrast,estradiol stimulated a 5-fold increase in DNA compared withcontrols. Wild-type MCF-7 cells showed a similar pattern ofantiestrogen and estrogen stimulation (data not shown).

Finally, we determined whether estradiol-stimulated growthof MCF-7TAM cells could be prevented by antiestrogens (Fig.5). Increasing concentrations of HT inhibited increases in DNAper well produced by estradiol (10~'°M); the IC50 of HT was 5x IO'8 M (Fig. 5).

DISCUSSION

These studies demonstrate that TAM-stimulated growth ofMCF-7-derived tumors occurs in vivo, in either athymic rats ormice, and is partly dependent on the level of NK cell activity inthe animals. The MCF-7TAM tumors did grow more rapidlyin NK cell deficient (beige) mice so that part of the growth of

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Table 2 NK cytoloxicily assay of nylon wool-treated alhymic mouse splenocytes after estrogen ana' antiestrogen treatment

TreatmentgroupControl

capsulesE2 1.0-cm capsulesTAM 2.0-cm capsulesWeeks

oftreatment1122.2

±21.7°'*

72.7 ±20.050.6 ±10.34180.0±

31.622.0 ±7.5'

165.9 ±24.86666.7

±138.46.9 ±1.88r

115.0 + 32.0''12173.4±

28.06.3 ±1.6°

19.9 ±3.74''

" n = 4 mice per group at each time point.* Values expressed as (mean ±SE) in LU/107 cells.' Significantly different from controls (P < 0.001 ) by unpaired Student's / test.J Significantly different from controls (P < 0.001 ) by unpaired Student's t test.

1.00

0.75

0.50

0.25

0.004 6

Weeks8 10

Fig. 3. Growth of MCF-7TAM tumors (mean ±SE) (n = 18) in athymic miceor beige mice. Animals treated with placebo capsules in beige mice (A) or inathymic mice (O): animals treated with TAM 2.0-cm capsules in beige mice (A)or in athymic mice (•).

12

10

E

i

Days8

Fig. 4. DNA determination of MCF-7TAM cells in 24-well plates afterdifferent times of treatment with E; IO"10M (•)or concentrations of HT at I0~'°M (A); 10"* M to 10~*M (A); (the separate curves are superimposable so only the10~" HT curve is shown). O. cells treated with control media are designated.

Zo

-11 -10 -9 -8 -7Log M Concentration of HT

-6

Fig. 5. Inhibition of estradiol stimulated MCF-7TAM cells by HT. Cells weregrown in 24-well plates and DNA content per well was measured after 8 days oftreatment. Estradiol (•,10~'°M) added to cells alone or with increasing concentrations of HT (•):Ei. HT I0~6 M alone: D, control media alone.

these tumors in an estrogenized environment is mediated bysuppression of NK cell activity. We have previously noted thisphenomenon with a slow-growing ER-negative endometrialtumor that grew more rapidly if the athymic host was treatedwith estrogen (29).

Estradiol is known to reduce NK cell activity in mice (18)and we now show that the prolonged treatment of mice with asustained release preparation of TAM can cause a depression

(at 12 weeks) in the poly(I:C)-induced increases in mouse NKcell activity. This effect is not as dramatic as seen with estradiol.Interestingly enough though, TAM does not cause an earlydecrease in NK cell activity (Table 2) at the time of the highestuterotrophic (estrogenic) activity. This again illustrates thespectrum of biological effects of TAM that appear to be targetsite specific (1).

Overall, these observations raise the question of the role of asuppression of NK cells in the growth of tumors and theirdesignation as "hormone-dependent" in an estrogen-treated

animal. A recent report (30) of the facilitated growth of laryn-geal tumors in estrogen-treated athymic mice has illustratedthe "hormone dependency" of laryngeal tumors with sugges

tions for the treatment of patients with TAM. It is possible thatan estrogen-induced depression of NK cells in their experimentshas permitted increased tumor growth.

Nevertheless, a suppression of NK cell activity is not solelyresponsible for "hormone-dependent" growth. TAM facilitates

further growth of MCF-7TAM tumors in NK cell-deficientmice and this can be interpreted as an example of the estrogenicaction of the drug. Furthermore, tumors appear to maintain adifferential or target site specificity for TAM in vivo. Theendometrial tumor, EnCalOl, will grow more rapidly inathymic mice during either estradiol or TAM treatment (31).In contrast, wild-type MCF-7 cells only grow in response to

estradiol (8, 15). Athymic mice that are bitransplanted withEnCalOl and MCF-7 cells maintain a differential sensitivity toTAM; TAM causes the endometrial tumor to grow but thebreast cancer cells do not (13). Therefore, host factors aloneare not responsible for TAM-stimulated growth.

These laboratory findings in the athymic mouse raise thequestion of whether TAM can modulate the immune functionof patients receiving adjuvant therapy for breast cancer. Previous studies (32-34) have demonstrated that TAM affects NK

cell activity in peripheral blood lymphocytes of patients. Unfortunately, there are conflicting reports as to whether TAMaugments or reduces the NK cell activity. This discrepancy maybe because patients were treated with TAM for different periods. Clearly, this is an area for further study because a clearunderstanding of the effects of TAM on immune functions mayhave a profound bearing on the overall ability of the drug tocontrol the disease in patients during long-term adjuvant therapy.

The observation that MCF-7TAM tumors grow in responseto TAM in vivo, but antiestrogens do not stimulate and in factblock estrogen-stimulated growth of the cells in culture, pointsto a complex interaction between the tumor and the host. It ispossible that TAM is metabolized to estrogenic ligands in vivowhich cause tumor growth locally but no evidence to supportthis hypothesis has been published (35, 36). Several antiestro-gen-resistant breast cancer cell lines have been reported (37-

39), but none are stimulated to grow by TAM. There is, however, a recent report (40) to document the ability of highconcentrations of HT to stimulate an endometrial cell line to

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grow in vitro. Similar concentrations of HT did not stimulateMCF-7TAM in culture.

In summary, JAM causes the growth of a selected MCF-7tumor in vivo; however, these cells are sensitive to the inhibitoryeffects of TAM in vitro. Although these studies may only beapplicable to the laboratory models used, the results point tothe possibility that the estrogen-like properties of TAM couldpromote tumor growth in vivo. There are anecdotal reports thatcomment upon the stimulatory effects of TAM during therapy(41-43); however, it is interesting to note that one patient whodid not respond to TAM (after 3 months of therapy) providedthe cells (from a massive ascites) to establish the ER-positivecell line, ZR-75-1 (44). TAM inhibits estrogen-stimulatedgrowth of ZR-75-1 cells in vitro (45) so the pharmacologyreported in this paper may occur in the clinics.

ACKNOWLEDGMENTS

We are grateful to Drs. Ray Weigand and Don Lynch of AbbottLaboratories for generously supplying the ER-EIA kits used in thesestudies. Dr. Mary Lindstrom of the University of Wisconsin Biostatis-tics Center for LU computer analysis, Sue Langan-Fahey for doing thetamoxifen assay, and to Dr. Edward Balish and Cindy Sulkowski forlending their experience on discussions of mouse NK function. Wewould also like to thank Kathy Edge for her expert technical typing.

REFERENCES

1. Furr, B. J. A., and Jordan, V. C. The pharmacology and clinical uses oftamoxifen. Pharmacol. Ther.. 25: 127-205, 1984.

2. Jordan. V. C. (ed.) Eslrogen/Anlieslrogen Action and Breast Cancer Therapy. Madison, WI: University of Wisconsin Press, 1986.

3. Tormey, D. C., and Jordan, V. C. Long-term tamoxifen adjuvant therapy innode-positive breast cancer: a metabolic and pilot clinical study. BreastCancer Res. Treat., 4: 297-302. 1984.

4. Fisher. B., et al. Prolonging tamoxifen for primary breast cancer: findingfrom the National Surgical Adjuvant Breast and Bowel Project Clinical Trial.Ann. Int. Med., 106: 649-654, 1987.

5. Breast Cancer Trials Committee, Scottish Cancer Trials Office (MRC).Adjuvant tamoxifen in the management of operable breast cancer: the Scottish trial. Lancet, 2: 171-175, 1987.

6. Jordan, V. C., Allen, K. E., and Dix, C. J. Pharmacology of tamoxifen inlaboratory animals. Cancer Treat. Rep., 64: 745-759, 1980.

7. Sutherland, R. L.. Green, M. D., Hall, R. E., Reddel, R. R.. and Taylor, I.W. Tamoxifen induces the accumulation of MCF-7 human mammary carcinoma cells in Go/G, phase of the cell. Eur. J. Cancer Clin. Oncol., 19: 615-621, 1983.

8. Osborne, C. K., Hobbs, K., and Clark, G. M. Effect of estrogens andanticstrogens on the growth of human breast cancer cells in athymic nudemice. Cancer Res., 45: 584-590. 1985.

9. Jordan, V. C., Robinson, S. P.. and Welshons. W. V. Resistance to anties-trogen therapy. In: D. Kessel (éd.).Resistance to Antineoplastic Drugs, pp.403-427. Boca Raton. FL: CRC Press. 1988.

10. Robinson. S. P., and Jordan, V. C. Reversal of the antitumor effects oftamoxifen by progesterone in the 7.12-dimethylbenzanthracene-induced ratmammary carcinoma model. Cancer Res.. 47: 5386-5390. 1987.

11. Cormier, E. M., and Jordan, V. C. Contrasting ability of antiestrogens toinhibit MCF-7 growth stimulated by estradici or epidermal growth factor.Eur. J. Cancer Clin. Oncol., 25: 57-63. 1989.

12. Robinson, S. P., and Jordan. V. C. The paracrine stimulation of MCF-7 cellsby MDA-MB-231 cells: possible role in anticstrogen failure. Eur. J. CancerClin. Oncol., 25: 493-497, 1989.

13. Gottardis, M. M.. Robinson. S. P., Satayaswaroop, P. G.. and Jordan, V. C.Contrasting actions of tamoxifen on endometrial and breast tumor growthin the athymic mouse. Cancer Res.. 48: 812-814, 1988.

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1989;49:4765-4769. Cancer Res   Marco M. Gottardis, Randall J. Wagner, Ernest C. Borden, et al.  

in Vitro and in VivoCell (MCF-7) Growth Differential Ability of Antiestrogens to Stimulate Breast Cancer

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