0013-7227/91/1281-0532$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 1 Printed in U.S.A.
Tamoxifen Inhibition of Prolactin Action in the Mouse Mammary Gland RATNA BISWAS* AND BARBARA K. VONDERHAAR Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT. Binding of lactogenic hormones to participate and solubilized microsomal membranes isolated from mammary glands of lactating mice is inhibited by direct addition of 10~10 M or greater concentrations of triphenylethylene antiestrogens [i.e. tamoxifen (TAM), 4-hydroxy-tamoxifen, and Nafoxidine] to the binding assays. Estradiol and other antiestrogens such as BPEA (-2-(4-tert-butyl-phenoxy) ethyl diethylamine hydrochloride, LYl 17018, and LY 156758 do not have this effect. The triphenylethylene antiestrogens bind to the membrane-associated antiestrogen binding sites (AEBS). Effectiveness of binding to the AEBS parallels the effectiveness of inhibition of the lactogen binding. The effect is selective in that binding of
T
HE nonsteroidal antiestrogens such as tamoxifen (TAM) have become the first line endocrine therapy for advanced breast cancer (1-3). The mechanism of action of these antiestrogens is incompletely understood; considerable experimental data suggest that they exert their effects primarily by competing with estrogen for its cytosolic receptors in target cells (4-7). Recently another high affinity antiestrogen binding site (AEBS) to which estrogen does not bind has been localized in microsomal membrane fractions (8). This AEBS is present in both estrogen receptor-positive and negative breast cancer cells (9), but the biological function of this site is still controversial. Both the demonstration of an antiestrogen-resistant MCF-7 breast cancer cell line that has lost sensitivity to these agents but remains responsive to estrogen for growth through the estrogen receptor system (10), and another breast cancer cell line, ZR75-1, where the action of TAM on growth may be independent of estradiol under certain conditions (11) suggests the existence of another biologically relevant receptor system. This is supported by the observations by Sutherland et al. (12) using MCF-7 cells and three high affinity hydroxylated antiestrogens. Treatment of these cells with
epidermal growth factor and insulin to these same membranes is unaffected by the antiestrogens. Binding of PRL to membranes prepared from the livers of the lactating mice is also unaffected. Both the PRL receptor and AEBS are primarily localized to the microsomal membrane fraction of cells. Maximal inhibition of PRL binding by TAM is observed in the light microsomes that contain plasma membranes. In addition to inhibition of PRL binding, TAM also prevents the PRL-induced accumulation of caseins by cultured mouse mammary explants. Thus it appears that the triphenylethylene antiestrogens, acting through the AEBS, act as antilactogens in the normal mammary gland. {Endocrinology 128: 532-538, 1991)
the drugs resulted in biphasic inhibition of cell growth. At low concentrations, the relative potencies of the drugs paralleled their affinities for estrogen receptors, but at higher concentrations this relationship did not hold, suggesting another as yet undefined mechanism of antiestrogen action. Recently we have shown (13) that antiestrogens inhibit the PRL-responsive growth of rat Nb2 lymphoma cells, which are unresponsive to estrogen and devoid of the estrogen receptor. Specific lactogenic hormone binding to these cells is inhibited by antiestrogens, and the potency of the various antiestrogens in this system parallels their ability to bind to the AEBS. Hence, the objective of the present work is to correlate antiestrogen and lactogen binding sites in subcellular fractions of the normal mammary gland and to define the role, if any, of antiestrogens in the modulation of PRL-induced functional differentiation.
Materials and Methods Reagents Ovine PRL (NIADDK oPRL-17), and human GH (NIADDK hGH-I-1) were obtained through the hormone distribution program of the NIH (Bethesda, MD). TAM was purchased from ICN Pharmaceuticals, Inc (Corning, CA). Nafoxidine (NAF) was a gift from the research laboratories of the Upjohn Company (Kalamazoo, MI). LY117018 and LY156758, products of the Eli Lilly Company, were a generous gift from Dr. Gerald C. Mueller (University of Wisconsin-Madison, Madison, WI).
Received August 23, 1990. Address all correspondence and requests for reprints to: Dr. Barbara K. Vonderhaar, National Cancer Institute, National Institutes of Health, Building 10, Room 5B56, Bethesda, Maryland 20892. * Present address: Department of Tumor Immunobiology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Calcutta700026, India.
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PRL AND TAMOXIFEN IN MAMMARY GLAND BPEA, (-2-(4-tert-butyl-phenoxy) ethyl diethylamine hydrochloride, is a gift from Drs. John and Benita Katzenellenbogen (University of Illinois, Urbana, IL). Estradiol-17/3, aldosterone, and corticosterone are purchased from Sigma Chemical Company (St. Louis, MO). Culture media, antibiotics, and porcine insulin were purchased from GIBCO (Grand Island, NY). BSA (fraction V) was purchased from Miles Scientific (Naperville, IL). [125I]hGH was prepared by a lactoperoxidase method as described (14) and had a specific activity of 40-60 /iCi/^g protein. 3H-TAM (sa, 75.6 Ci/mmol) was purchased from New England Nuclear (Boston, MA). 3H-amino acids are from ICN Pharmaceuticals Inc. Purified caseins and a-lactalbumin and their specific antibodies were prepared as described previously (15,16). All other reagents used were the best analytical grade. Preparation of microsomal membranes Mammary glands were removed from C3H/HeN mice 10-12 days lactating after their first pregnancy. The tissue was homogenized in 8 vol (wt/vol) ice-cold Tris-HCl, pH 7.4, containing 0.3 M sucrose, and centrifuged at 15,000 x g for 20 min. Crude microsomal membranes were collected by centrifugation of the resulting supernatant at 105,000 x g for 60 min at 4 C. The pellet was resuspended in 25 mM Tris-HCl, pH 7.4, at a final concentration of 15-20 mg protein/ml and stored at —20 C until used (17). Membranes were solubilized with 0.5% CHAPS (Calbiochem, La Jolla, CA) as described (18). Protein determination Protein concentrations are estimated by modifications (18) of either the method of Lowry et al. (19) or Bradford (20) using BSA as standard. Subcellular fractionation Microsomes were subfractionated on a discontinuous gradient containing CsCl according to the method of Dallner (21). Briefly, 5 ml of the 15,000 X g supernatant prepared from lactating mouse mammary glands homogenized in 25 mM TrisHCl, pH 7.4, containing 0.44 mM sucrose, was underlayed first with 1 ml 0.6 M sucrose containing 15 mM CsCl and then with 4 ml 1.3 M sucrose containing 15 mM CsCl. The gradients were centrifuged at 130,000 x g for 1 h at 4 C in a 50Ti rotor (Beckman Instruments, Fullerton, CA). The material banding at the 0.6 M/1.3 M interphase is light microsomes, and the pellet contains heavy microsomes. The pellet was resuspended in 25 mM Tris-HCl, pH 7.4. NADPH cytochrome C redustase [EC 1.6.2.4], 5'Nucleotidase [EC 3.1.3.5], and Glutathione reductase [EC 1.6.4.2] were assayed by the methods of Williams and Kamin (22), Michell and Hawthorne (23), Ellman (24), and Mitchell et al. (25). Cross-contamination of the subcellular fractions based on enzymatic criteria was judged to be minimal in three separate experiments.
533
mined by addition of excess unlabeled hormone) was subtracted from total binding (determined in the absence of unlabeled hormone) to yield specific binding. 3 H-TAM binding to membranes was determined by incubation overnight (16-18 h) at room temperature in the presence of 25 mM Tris-HCl, 10 mM MgCl2 and 0.1% BSA with or without a 1000-fold excess of unlabeled antiestrogen in a total vol of 500 MI as described (13). The specific 3H-TAM binding was calculated by subtracting nonspecific binding (determined by addition of excess unlabeled hormone) from total binding (determined in the absence of unlabeled hormone). Specific binding of [125I]insulin and [125I]epidermal growth factor (EGF) to the membranes was determined by the methods of Benson et al. (26) and Ginsburg and Vonderhaar (27), respectively. All binding reactions were performed in triplicate and for a minimum of three times each. Organ culture Explants from abdominal mammary glands of either primiparous mid-pregnant or 3-4-month-old virgin mice were cultured in Medium 199 containing 20 mM HEPES, pH 7.4, as described (28). Virgin mice were used 2-7 days after ovariectomy. Hormones were added to the media before sterilization by filtration through a 0.45 nm filter. Final hormone concentrations were: insulin 1 fig/ml, aldosterone 0.1 fig/m\, corticosterone 1 Mg/ml, PRL 1 /Lig/ml, 17/3-estradiol (E2) 10~6 M, and TAM 10~6 M. The culture period was 48 h for explants from pregnant mice and 96 h for tissue from virgin mice. All cultures were maintained at 37 C in the presence of an atmosphere of 5% CO2 in air with medium changes after 24 and 72 h for explants from virgin mice. Sterilized 3H-amino acid mixtures were added to the media for the entire culture period for explants from pregnant mice (1.25 ^ci/ml) and for the final 24 h of culture of explants from virgin mouse mammary glands (12.5 Immunoprecipitation assay a-Lactalbumin or casein synthesis was measured by using saturating levels of specific antibodies for the respective milk proteins (28). After incubation overnight at 4 C, the resulting antigen-antibody complex was precipitated with goat antirabbit immunoglobulin G Immunobeads (BioRad Laboratories, Richmond, CA), washed with PBS containing 0.1% BSA, and counted in a scintillation counter. Statistical analysis The significance of the difference between groups was determined by analysis of variance.
Results Effect of antiestrogens on lactogenic hormone binding
Radioreceptor binding assay 125
Binding of [ I]hGH to microsomal membranes (200-400 ng protein) and to solubilized membranes was measured as described by Vonderhaar et al. (18). Nonspecific binding (deter-
As the first step of the biological action of any hormone involves its interaction with specific receptors, we studied the influence of a series of antiestrogens on binding to membranes from lactating mouse mammary glands.
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PRL AND TAMOXIFEN IN MAMMARY GLAND
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Direct addition of the antiestrogens to the lactogen binding reaction resulted in inhibition of binding by TAM, NAF, and 4-hydroxy-tamoxifen (OH-TAM) at concentrations as low as 10~10 M (Fig. 1). Maximum inhibition was obtained at 10~6 M, although higher concentrations were not tested due to limitations in solubility. The synthetic antiestrogen BPEA (29) gave a 30% inhibition only at 10~6 M. Neither LY117018, LY156758, nor estradiol-17/3 affected lactogen binding when added directly to these membranes. These data are similar to those reported for the Nb2 rat lymphoma cells (13). In contrast to the mammary gland membranes, lactogen binding to hepatic microsomes from livers of the same mice remained unaffected by TAM at concentrations as high as 10~6 M (Fig. 2). In this particular experiment, TAM (10~8 M) inhibited specific binding of [125I] hGH to mammary gland microsomes by 90%; 10~9 M TAM gave 50% inhibition. Yet these concentrations of TAM were without effect on the hepatic membranes.
Endo«1991 Voll28«Nol
EFFECT OF TAM ON LACTOGEN BINDING TO VARIOUS MEMBRANE PREPARATIONS FROM LACTATING MICE
12 _ No antiestrogen 1.0 _
10' 9 MTAM 10'"M TAM
10 MTAM 0.8 _
E
0.6 _
0.4 _
0.2 _
3000
E a. o
FlG. 2. Relative specific binding of lactogenic hormone to mammary gland and liver membrane proteins from lactating mice in the presence of TAM. Specific binding of [126I]hGH was determined as described in Materials and Methods. TAM, at the indicated concentrations, was presented throughout the binding reaction except for pretreated membranes. In this case, membranes (200 ng protein) were incubated overnight at room temperature with or without TAM, recollected by centrifugation, and washed to remove TAM. No difference in protein recovery was observed with or without TAM. The membranes were then tested for specific binding of [125I]hGH. All experiments were performed in triplicate at least three times.
O
z
2000
Q Z
m z LU
O
o o < o U. o
Pretreatment overnight at room temperature to maximize TAM binding before lactogen binding did not increase the effectiveness of TAM in either the mammary gland or liver microsomes (data not shown). As is also shown in Fig. 2, [125I]hGH binding to solubilized microsomal membranes from mouse mammary glands is susceptible to direct inhibition by TAM (55% inhibition at 10~6 M).
1000 -
111 Q.
10
MOLAR
10
10
CONCENTRATION
FIG. 1. Effect of antiestrogens on lactogenic hormone binding to mammary gland membranes from lactating mice. Microsomal membranes from lactating mice were incubated with [125I]hGH (100,000 cpm) in the presence and absence of excess unlabeled oPRL as described in Materials and Methods. Antiestrogens at various concentrations were added directly to the binding reactions and specific binding determined as described in Materials and Methods. Binding in the presence of TAM (—A—), NAF (—O—), OH-TAM (—A—), estradiol (- - • - -), BPEA (—•—), LY117018 (—•—), LY156758 (—D—). *P < 0.01 us. binding in the absence of antiestrogen.
Effect of TAM on ^IJinsulin binding or binding to mammary gland microsomes
f^IJEGF
A 30% inhibition of lactogen binding was consistently obtained at TAM concentrations as low as 10~9 M. The final extent of inhibition by higher concentrations of TAM varied with the microsomal preparation. However, as shown in Fig. 3, TAM at all concentrations tested (up to 10"6 M) had no effect on specific EGF binding to its receptors on lactating mouse mammary gland microsomes. This was true for all membrane preparations
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PRL AND TAMOXIFEN IN MAMMARY GLAND
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M
6.0
N M Mt C HM LM
5.0
b
CO
4.0
b
N
M
SPECIFIC i
l> u E E microsomes
light
heavy
plasma membranes
800
600
400
200
Q. U
1000
1200
ng oPrl 3
FIG. 6. Specific binding of H-TAM to AEBS in lactating mouse mammary gland membranes in the presence of PRL. Specific binding of TAM was determined as described in Materiab and Methods in the absence or presence of various concentrations of oPRL. The experiment was performed four times with similar results. 3000 r |
| ovx 1 week
^ H mid-pregnant 3 GO co 2000
-X.
o
u 2 111
.X-
X -
1000 -
heavy
plasma membranes
FRACTION FIG. 5. The reciprocal effects of TAM and PRL on specific binding to membrane preparations from lactating mouse mammary glands. A, Specific binding of [125I]hGH to crude, light, and heavy microsomes as well as plasma membranes was determined in the presence of 10~8 M TAM as described in Materials and Methods. Binding in the absence of TAM (D), binding in the presence of TAM (M). B, Specific binding of 3H-TAM to crude, light, and heavy microsomes as well as plasma membranes was determined in the presence of 1 ng PRL as described in Materials and Methods. Binding in the absence of PRL (•), binding in the presence of PRL (B).
mary gland explants cultured with insulin, aldosterone, corticosterone, and PRL (IACPRL). A dose-dependent inhibition was observed in tissue from pregnant mice (not shown). However, the presence of endogenous estrogens in the tissue and the possible supportive role of these steroids in the biological response of glands from pregnant animals (30) makes the interpretation of these data difficult. This is emphasized by the results shown
IAC
IAC I Prl
IAC < Prl •< E2
IAC IAC t Prl i Prl i TAM ) E2 i TAM
IAC
IAC i Prl
IAC i Prl i E2
IAC IAC i Prl i Prl i TAM i E2 t TAM
FIG. 7. Effect of TAM on PRL-induced casein synthesis in mammary gland explants from pregnant and ovariectomized virgin mice. Pooled explants from 8 ovariectomized (7 days post surgery) virgin (•) and 5 pregnant (•) mice were cultured in serum-free medium 199 containing the indicated hormones as described in Materials and Methods. Cultures were exposed to 3H-amino acid mixture (virgin = 12.5 fid/ml for the final 24 h of a 96-hr culture period; pregnant = 1.25 fid/ml for entire 48-hr culture period), after which tissue was collected and analyzed for casein synthesis by specific radioimmune precipitation as described in Materials and Methods. Data shown are the mean ± SEM for three separate determinations. I, insulin (1 /ig/ml); A, aldosterone (0.1 ng/ ml); C, corticosterone (1 /xg/ml); PRL, oPRL (1 /ig/ml); E2, estradiol (10~6 M); TAM (10~6 M). Similar results were obtained when E2 as low as 10~9 M was tested in explants from pregnant mice.
in Fig. 7. TAM (10~6 M) inhibited the PRL-induced casein synthesis by over 80%, and this effect was not
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PRL AND TAMOXIFEN IN MAMMARY GLAND reversed by estrogen. When both estrogen and TAM are present there was nearly complete inhibition of PRLinduced casein synthesis. In an attempt to eliminate the problem of the presence of endogenous estrogens and any resulting estrogen responsiveness of the mammary tissue in culture, virgin mice were ovariectomized at least 48 h before the onset of culture. Figure 7 also shows that mammary gland explants from ovariectomized virgin mice showed significant inhibition (>80%) of casein synthesis by TAM when grown in media containing IACPRL. When both PRL and estrogens are present, TAM still inhibited casein synthesis by over 60%. Discussion Previous studies have shown that antiestrogens have a binding site in the microsomal fraction of mammary carcinoma cells (8, 31), which is distinct from the estrogen binding site. However, the biological role(s) of this AEBS is still unknown. Our recent results (13) however, have suggested that in some cases TAM, acting through the AEBS, may modulate PRL action. PRL is a key hormone in mammary gland development, and PRL receptors are also localized in the microsomal fraction of the cell. The mammary gland is also one of the principal target organs of estrogens and antiestrogens. We have now shown that antiestrogens, such as TAM, inhibit lactogen binding to the microsomal membranes as well as to the solubilized form of the receptor from normal mouse mammary glands. Prebinding of TAM to membranes causes the inhibition of lactogen binding to the same extent as when added simultaneously with the ligand. Inhibition occurs at 0 C as well as at room temperature, suggesting that the action of the antiestrogen through the AEBS is not enzymatic (Das, R., and B. K. Vonderhaar, in preparation). The AEBS is present in both estrogen target and nontarget tissues such as liver (32). However, even though liver microsomes from lactating mice are rich in lactogen binding activity, antiestrogens had no effect on the lactogen binding. The reason for this lack of an effect is not clear at this point. Purification of the AEBS and cloning of the gene from mammary glands and from liver may help clarify this observation. Inhibition of binding to mammary gland microsomes by antiestrogens is specific for lactogenic hormones, as TAM did not affect the EGF or insulin binding to mammary gland microsomes. Both EGF and insulin receptors are also located in microsomes. This specificity for lactogen receptors may be indicative of an interrelationship between the AEBS and the lactogenic hormone receptor. Both TAM and NAF inhibited specific lactogen binding, but another antiestrogen, LY117018, had little effect
537
on the mouse mammary gland microsomes. This antiestrogen has little or no affinity for the AEBS (29), suggesting that the effects of the antiestrogens on PRL action and binding are through the AEBS, not through the estrogen receptor. This close relationship of the two membrane-associated binding sites is emphasized by the reciprocal effect of PRL on the binding of TAM to the AEBS. A complex interrelationship of the two binding sites (i.e. lactogenic hormone and antiestrogen) becomes evident from these studies. How this reciprocal effect is brought about and what its biological function may be remains to be investigated. The phenomenon may reflect the close proximity and biochemical similarity of the two binding sites located on the same membrane protein (Das, R., and B. K. Vonderhaar, unpublished). Subcellular fractionation of mammary gland microsomes shows that both lactogen and antiestrogen binding are localized in the same fractions. Total microsomes and heavy microsomes are rich in both lactogen and antiestrogen binding sites, whereas light microsomes also contain some of both activities. At this point we are not sure whether this is due to contaminating heavy microsomes or is inherent to the light microsomes. Watts et al. (8) also localized AEBS at heavy microsome of mammary carcinoma cells. Lactogen binding in both light and heavy microsomes was inhibited by TAM (10~6 M), whereas the antiestrogen binding in heavy microsomes was enhanced by PRL. In light microsomes lactogen inhibited the antiestrogen binding. Because crude microsomes are subfractionated to obtain light and heavy microsomes, the overall effect of PRL on TAM binding in any given membrane preparation depends on the ratio of light and heavy microsomes present. Likewise, the extent of inhibition of PRL binding by antiestrogen is the combined effects on the different microsomal fractions contained therein. The biochemical relationship of lactogen binding and antiestrogen binding to the AEBS has biological significance, because TAM inhibited PRL-induced mammary gland differentiation just as it inhibits PRL-induced growth of the estrogen receptor-negative Nb2 cells (13). Production of the casein family of milk proteins, which is dependent on the presence of PRL in the culture medium, was inhibited by TAM. This action of TAM is not through the estrogen receptor, because it is not completely reversed by the addition of estradiol to the culture media, and a similar effect was obtained in tissue from ovariectomized mice where there is little residual estrogen and the level of the estrogen receptor is very low. Similar effects of the antiestrogen TAM have recently been obtained on PRL-induced protein kinase C activity in the NOG-8 mouse mammary cell line (Das, R., and B. K. Vonderhaar, manuscript in preparation). Like the Nb2 cells, these normal cells are devoid of
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estrogen receptors but contain membrane-associated AEBS. Hence, we believe that the ability of TAM to inhibit binding of lactogenic hormones to receptors is mediated through the membrane associated AEBS, and this biochemical action is translated to biological function in the blocking of PRL action during normal functional differentiation of the mammary gland. What the relationship of the AEBS is to the PRL receptor and how they interact with each other is the subject of our further investigations.
References 1. Cole MP, Jones CTA, Tod IDH 1971 A new antiestrogenic agent in late breast cancer: an early clinical appraisal of ICI 46474. Br J Cancer 25:270-275 2. E.O.R.T.C Breast Cancer Group 1972 Clinical trial of nafoxidine an oestrogen antagonist in advanced breast cancer. Eur J Cancer 8:387-389 3. Lerner LJ, Jordan VC 1990 Development of antiestrogens and their use in breast cancer: eighth Cain memorial award lecture. Cancer Res 50:4177-4189 4. Horwitz KB, McGuire W 1978 Antiestrogen: mechanism of action and effects in breast cancer. In: McGuire W (ed) Breast Cancer Advances in Research and Treatment. Plenum Publishing Co., New York, vol 2:155-204 5. Coezy E, Borgna JL, Rochefort H 1982 Tamoxifen and metabolites in MCF-7 cells. Correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res 42:317-323 6. Eckert RL, Katzenellenbogen BS 1982 Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cell: differences with estrogen and antiestrogen. J Biol Chem 257:88408846 7. Lippman M, Bolan G, Huff K 1976 The effect of estrogens and antiestrogens on hormone responsive human breast cancer in long term tissue culture. Cancer Res 36:4595-4607 8. Watts CKN, Murphy LC, Sutherland RL 1984 Microsomal binding sites for nonsteroidal antiestrogens in MCF-7 human mammary carcinoma cells. J Biol Chem 259:4223-4229 9. Katzenellenbogen BS, Miller MA, Mullick A, Sheen YY 1985 Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis and interaction with estrogen receptors and additional antiestrogen binding sites. Breast Cancer Res Treatment 5:231-243 10. Miller MA, Lippman ME, Katzenellenbogen BS 1984 Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells. Cancer Res 44:5038-5045 11. Allerga JC, Lippman ME 1978 Growth of human breast cancer cell line in serum free hormone supplemented medium. Cancer Res 38:3823-3829 12. Sutherland RL, Watts CKW, Ruenitz PC 1986 Definition of two distinct mechanisms of action of antiestrogens on human breast cancer cell proliferation using hydroxytriphenylethylenes with high affinity for the estrogen receptor. Biochem Biophys Res Commun
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140:523-529 13. Biswas R, Vonderhaar BK 1989 Antiestrogen inhibition of prolactin-induced growth of the Nb2 rat lymphoma cell line. Cancer Res 49:6295-6299 14. Liscia D, Vonderhaar BK 1982 Purification of a prolactin receptor. Proc Natl Acad Sci USA 79:5930-5934 15. Bhattacharjee M, Vonderhaar BK 1983 Purification and characterization of mouse a-lactalbumin from lactating mammary glands. Biochim Biophys Acta 755:279-286 16. Smith GH, Vonderhaar BK 1981 Functional differentiation in mouse mammary epithelium is attained through DNA synthesis inconsequent of mitosis. Dev Biol 88:167-179 17. Bhattacharya A, Vonderhaar BK 1979 Phospholipid methylation stimulates lactogenic binding in mouse mammary gland membranes. Proc Natl Acad Sci USA 76:4489-4492 18. Vonderhaar BK, Bhattacharya A, Alhadi T, Liscia DS, Andrew EM, Young JK, Ginsburg E, Bhattacharjee M, Horn TM 1985 Isolation, characterization and regulation of the prolactin receptor. J Dairy Sci 68:466-488 19. Lowry OH, Rosebrough NJ, Farr AC, Randall RJ 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265275
20. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 21. Dallner G 1978 Isolation of microsomal subfractions by use of density gradients. Methods Enzymol 52:71-83 22. Williams Jr CH, Kamin H 1962 Microsomal triphosphopyridine nucleotide-cytochrome c reductase in liver. J Biol Chem 237:587595 23. Michell RH, Hawthorne JN 1965 The site of diphosphoinositide synthesis in rat liver. Biochem Biophys Res Commun 21:333-338 24. Ellman GL 1959 Tissue sulfhydryl groups. Arch Biochem Biophys 82:70-77 25. Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB 1973 Acetaminophen-induced hepatic necrosis IV. Protective role of glutathione. J Pharmacol Exp Ther 187:211-217 26. Benson EA, Holdaway M 1982 Regulation of insulin binding to human mammary carcinoma. Cancer Res 42:1137-1141 27. Ginsburg E, Vonderhaar BK 1985 Epidermal growth factor stimulates the growth of A431 in athymic mice. Cancer Lett 28:143150 28. Vonderhaar BK, Nakhasi HL 1986 Bifunctional activity of epidermal growth factor on the expression of a- and /c-caseins in rodent mammary glands in vitro. Endocrinology 119:1178-1184 29. Katzenellenbogen BS, Miller MA, Mullick A, Sheen YY 1985 Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis and interaction with estrogen receptors and additional antiestrogen binding sites. Breast Cancer Res Treatment 5:231-243 30. Bolander Jr FF, Topper YJ 1980 Stimulation of lactose synthetase activity and casein synthesis in mouse mammary explants by estradiol. Endocrinology 106:490-495 31. Sutherland RL, Murphy LC, Foo MS, Green MD, Whybonne AM 1980 High affinity antiestrogen binding sites distinct from estrogen receptors. Nature 288:273-275 32. Sudo K, Monsma FJ, Katzenellenbogen BS 1983 Antiestrogen binding sites distinct from estrogen receptors: subcellular localization, ligand specificity, and distribution in tissues of rat. Endocrinology 112:425-434
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Vol. 128, No. 1 Printed in U.S.A.
Tamoxifen Inhibition of Prolactin Action in the Mouse Mammary Gland RATNA BISWAS* AND BARBARA K. VONDERHAAR Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT. Binding of lactogenic hormones to participate and solubilized microsomal membranes isolated from mammary glands of lactating mice is inhibited by direct addition of 10~10 M or greater concentrations of triphenylethylene antiestrogens [i.e. tamoxifen (TAM), 4-hydroxy-tamoxifen, and Nafoxidine] to the binding assays. Estradiol and other antiestrogens such as BPEA (-2-(4-tert-butyl-phenoxy) ethyl diethylamine hydrochloride, LYl 17018, and LY 156758 do not have this effect. The triphenylethylene antiestrogens bind to the membrane-associated antiestrogen binding sites (AEBS). Effectiveness of binding to the AEBS parallels the effectiveness of inhibition of the lactogen binding. The effect is selective in that binding of
T
HE nonsteroidal antiestrogens such as tamoxifen (TAM) have become the first line endocrine therapy for advanced breast cancer (1-3). The mechanism of action of these antiestrogens is incompletely understood; considerable experimental data suggest that they exert their effects primarily by competing with estrogen for its cytosolic receptors in target cells (4-7). Recently another high affinity antiestrogen binding site (AEBS) to which estrogen does not bind has been localized in microsomal membrane fractions (8). This AEBS is present in both estrogen receptor-positive and negative breast cancer cells (9), but the biological function of this site is still controversial. Both the demonstration of an antiestrogen-resistant MCF-7 breast cancer cell line that has lost sensitivity to these agents but remains responsive to estrogen for growth through the estrogen receptor system (10), and another breast cancer cell line, ZR75-1, where the action of TAM on growth may be independent of estradiol under certain conditions (11) suggests the existence of another biologically relevant receptor system. This is supported by the observations by Sutherland et al. (12) using MCF-7 cells and three high affinity hydroxylated antiestrogens. Treatment of these cells with
epidermal growth factor and insulin to these same membranes is unaffected by the antiestrogens. Binding of PRL to membranes prepared from the livers of the lactating mice is also unaffected. Both the PRL receptor and AEBS are primarily localized to the microsomal membrane fraction of cells. Maximal inhibition of PRL binding by TAM is observed in the light microsomes that contain plasma membranes. In addition to inhibition of PRL binding, TAM also prevents the PRL-induced accumulation of caseins by cultured mouse mammary explants. Thus it appears that the triphenylethylene antiestrogens, acting through the AEBS, act as antilactogens in the normal mammary gland. {Endocrinology 128: 532-538, 1991)
the drugs resulted in biphasic inhibition of cell growth. At low concentrations, the relative potencies of the drugs paralleled their affinities for estrogen receptors, but at higher concentrations this relationship did not hold, suggesting another as yet undefined mechanism of antiestrogen action. Recently we have shown (13) that antiestrogens inhibit the PRL-responsive growth of rat Nb2 lymphoma cells, which are unresponsive to estrogen and devoid of the estrogen receptor. Specific lactogenic hormone binding to these cells is inhibited by antiestrogens, and the potency of the various antiestrogens in this system parallels their ability to bind to the AEBS. Hence, the objective of the present work is to correlate antiestrogen and lactogen binding sites in subcellular fractions of the normal mammary gland and to define the role, if any, of antiestrogens in the modulation of PRL-induced functional differentiation.
Materials and Methods Reagents Ovine PRL (NIADDK oPRL-17), and human GH (NIADDK hGH-I-1) were obtained through the hormone distribution program of the NIH (Bethesda, MD). TAM was purchased from ICN Pharmaceuticals, Inc (Corning, CA). Nafoxidine (NAF) was a gift from the research laboratories of the Upjohn Company (Kalamazoo, MI). LY117018 and LY156758, products of the Eli Lilly Company, were a generous gift from Dr. Gerald C. Mueller (University of Wisconsin-Madison, Madison, WI).
Received August 23, 1990. Address all correspondence and requests for reprints to: Dr. Barbara K. Vonderhaar, National Cancer Institute, National Institutes of Health, Building 10, Room 5B56, Bethesda, Maryland 20892. * Present address: Department of Tumor Immunobiology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Calcutta700026, India.
532
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PRL AND TAMOXIFEN IN MAMMARY GLAND BPEA, (-2-(4-tert-butyl-phenoxy) ethyl diethylamine hydrochloride, is a gift from Drs. John and Benita Katzenellenbogen (University of Illinois, Urbana, IL). Estradiol-17/3, aldosterone, and corticosterone are purchased from Sigma Chemical Company (St. Louis, MO). Culture media, antibiotics, and porcine insulin were purchased from GIBCO (Grand Island, NY). BSA (fraction V) was purchased from Miles Scientific (Naperville, IL). [125I]hGH was prepared by a lactoperoxidase method as described (14) and had a specific activity of 40-60 /iCi/^g protein. 3H-TAM (sa, 75.6 Ci/mmol) was purchased from New England Nuclear (Boston, MA). 3H-amino acids are from ICN Pharmaceuticals Inc. Purified caseins and a-lactalbumin and their specific antibodies were prepared as described previously (15,16). All other reagents used were the best analytical grade. Preparation of microsomal membranes Mammary glands were removed from C3H/HeN mice 10-12 days lactating after their first pregnancy. The tissue was homogenized in 8 vol (wt/vol) ice-cold Tris-HCl, pH 7.4, containing 0.3 M sucrose, and centrifuged at 15,000 x g for 20 min. Crude microsomal membranes were collected by centrifugation of the resulting supernatant at 105,000 x g for 60 min at 4 C. The pellet was resuspended in 25 mM Tris-HCl, pH 7.4, at a final concentration of 15-20 mg protein/ml and stored at —20 C until used (17). Membranes were solubilized with 0.5% CHAPS (Calbiochem, La Jolla, CA) as described (18). Protein determination Protein concentrations are estimated by modifications (18) of either the method of Lowry et al. (19) or Bradford (20) using BSA as standard. Subcellular fractionation Microsomes were subfractionated on a discontinuous gradient containing CsCl according to the method of Dallner (21). Briefly, 5 ml of the 15,000 X g supernatant prepared from lactating mouse mammary glands homogenized in 25 mM TrisHCl, pH 7.4, containing 0.44 mM sucrose, was underlayed first with 1 ml 0.6 M sucrose containing 15 mM CsCl and then with 4 ml 1.3 M sucrose containing 15 mM CsCl. The gradients were centrifuged at 130,000 x g for 1 h at 4 C in a 50Ti rotor (Beckman Instruments, Fullerton, CA). The material banding at the 0.6 M/1.3 M interphase is light microsomes, and the pellet contains heavy microsomes. The pellet was resuspended in 25 mM Tris-HCl, pH 7.4. NADPH cytochrome C redustase [EC 1.6.2.4], 5'Nucleotidase [EC 3.1.3.5], and Glutathione reductase [EC 1.6.4.2] were assayed by the methods of Williams and Kamin (22), Michell and Hawthorne (23), Ellman (24), and Mitchell et al. (25). Cross-contamination of the subcellular fractions based on enzymatic criteria was judged to be minimal in three separate experiments.
533
mined by addition of excess unlabeled hormone) was subtracted from total binding (determined in the absence of unlabeled hormone) to yield specific binding. 3 H-TAM binding to membranes was determined by incubation overnight (16-18 h) at room temperature in the presence of 25 mM Tris-HCl, 10 mM MgCl2 and 0.1% BSA with or without a 1000-fold excess of unlabeled antiestrogen in a total vol of 500 MI as described (13). The specific 3H-TAM binding was calculated by subtracting nonspecific binding (determined by addition of excess unlabeled hormone) from total binding (determined in the absence of unlabeled hormone). Specific binding of [125I]insulin and [125I]epidermal growth factor (EGF) to the membranes was determined by the methods of Benson et al. (26) and Ginsburg and Vonderhaar (27), respectively. All binding reactions were performed in triplicate and for a minimum of three times each. Organ culture Explants from abdominal mammary glands of either primiparous mid-pregnant or 3-4-month-old virgin mice were cultured in Medium 199 containing 20 mM HEPES, pH 7.4, as described (28). Virgin mice were used 2-7 days after ovariectomy. Hormones were added to the media before sterilization by filtration through a 0.45 nm filter. Final hormone concentrations were: insulin 1 fig/ml, aldosterone 0.1 fig/m\, corticosterone 1 Mg/ml, PRL 1 /Lig/ml, 17/3-estradiol (E2) 10~6 M, and TAM 10~6 M. The culture period was 48 h for explants from pregnant mice and 96 h for tissue from virgin mice. All cultures were maintained at 37 C in the presence of an atmosphere of 5% CO2 in air with medium changes after 24 and 72 h for explants from virgin mice. Sterilized 3H-amino acid mixtures were added to the media for the entire culture period for explants from pregnant mice (1.25 ^ci/ml) and for the final 24 h of culture of explants from virgin mouse mammary glands (12.5 Immunoprecipitation assay a-Lactalbumin or casein synthesis was measured by using saturating levels of specific antibodies for the respective milk proteins (28). After incubation overnight at 4 C, the resulting antigen-antibody complex was precipitated with goat antirabbit immunoglobulin G Immunobeads (BioRad Laboratories, Richmond, CA), washed with PBS containing 0.1% BSA, and counted in a scintillation counter. Statistical analysis The significance of the difference between groups was determined by analysis of variance.
Results Effect of antiestrogens on lactogenic hormone binding
Radioreceptor binding assay 125
Binding of [ I]hGH to microsomal membranes (200-400 ng protein) and to solubilized membranes was measured as described by Vonderhaar et al. (18). Nonspecific binding (deter-
As the first step of the biological action of any hormone involves its interaction with specific receptors, we studied the influence of a series of antiestrogens on binding to membranes from lactating mouse mammary glands.
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Direct addition of the antiestrogens to the lactogen binding reaction resulted in inhibition of binding by TAM, NAF, and 4-hydroxy-tamoxifen (OH-TAM) at concentrations as low as 10~10 M (Fig. 1). Maximum inhibition was obtained at 10~6 M, although higher concentrations were not tested due to limitations in solubility. The synthetic antiestrogen BPEA (29) gave a 30% inhibition only at 10~6 M. Neither LY117018, LY156758, nor estradiol-17/3 affected lactogen binding when added directly to these membranes. These data are similar to those reported for the Nb2 rat lymphoma cells (13). In contrast to the mammary gland membranes, lactogen binding to hepatic microsomes from livers of the same mice remained unaffected by TAM at concentrations as high as 10~6 M (Fig. 2). In this particular experiment, TAM (10~8 M) inhibited specific binding of [125I] hGH to mammary gland microsomes by 90%; 10~9 M TAM gave 50% inhibition. Yet these concentrations of TAM were without effect on the hepatic membranes.
Endo«1991 Voll28«Nol
EFFECT OF TAM ON LACTOGEN BINDING TO VARIOUS MEMBRANE PREPARATIONS FROM LACTATING MICE
12 _ No antiestrogen 1.0 _
10' 9 MTAM 10'"M TAM
10 MTAM 0.8 _
E
0.6 _
0.4 _
0.2 _
3000
E a. o
FlG. 2. Relative specific binding of lactogenic hormone to mammary gland and liver membrane proteins from lactating mice in the presence of TAM. Specific binding of [126I]hGH was determined as described in Materials and Methods. TAM, at the indicated concentrations, was presented throughout the binding reaction except for pretreated membranes. In this case, membranes (200 ng protein) were incubated overnight at room temperature with or without TAM, recollected by centrifugation, and washed to remove TAM. No difference in protein recovery was observed with or without TAM. The membranes were then tested for specific binding of [125I]hGH. All experiments were performed in triplicate at least three times.
O
z
2000
Q Z
m z LU
O
o o < o U. o
Pretreatment overnight at room temperature to maximize TAM binding before lactogen binding did not increase the effectiveness of TAM in either the mammary gland or liver microsomes (data not shown). As is also shown in Fig. 2, [125I]hGH binding to solubilized microsomal membranes from mouse mammary glands is susceptible to direct inhibition by TAM (55% inhibition at 10~6 M).
1000 -
111 Q.
10
MOLAR
10
10
CONCENTRATION
FIG. 1. Effect of antiestrogens on lactogenic hormone binding to mammary gland membranes from lactating mice. Microsomal membranes from lactating mice were incubated with [125I]hGH (100,000 cpm) in the presence and absence of excess unlabeled oPRL as described in Materials and Methods. Antiestrogens at various concentrations were added directly to the binding reactions and specific binding determined as described in Materials and Methods. Binding in the presence of TAM (—A—), NAF (—O—), OH-TAM (—A—), estradiol (- - • - -), BPEA (—•—), LY117018 (—•—), LY156758 (—D—). *P < 0.01 us. binding in the absence of antiestrogen.
Effect of TAM on ^IJinsulin binding or binding to mammary gland microsomes
f^IJEGF
A 30% inhibition of lactogen binding was consistently obtained at TAM concentrations as low as 10~9 M. The final extent of inhibition by higher concentrations of TAM varied with the microsomal preparation. However, as shown in Fig. 3, TAM at all concentrations tested (up to 10"6 M) had no effect on specific EGF binding to its receptors on lactating mouse mammary gland microsomes. This was true for all membrane preparations
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PRL AND TAMOXIFEN IN MAMMARY GLAND
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M
6.0
N M Mt C HM LM
5.0
b
CO
4.0
b
N
M
SPECIFIC i
l> u E E microsomes
light
heavy
plasma membranes
800
600
400
200
Q. U
1000
1200
ng oPrl 3
FIG. 6. Specific binding of H-TAM to AEBS in lactating mouse mammary gland membranes in the presence of PRL. Specific binding of TAM was determined as described in Materiab and Methods in the absence or presence of various concentrations of oPRL. The experiment was performed four times with similar results. 3000 r |
| ovx 1 week
^ H mid-pregnant 3 GO co 2000
-X.
o
u 2 111
.X-
X -
1000 -
heavy
plasma membranes
FRACTION FIG. 5. The reciprocal effects of TAM and PRL on specific binding to membrane preparations from lactating mouse mammary glands. A, Specific binding of [125I]hGH to crude, light, and heavy microsomes as well as plasma membranes was determined in the presence of 10~8 M TAM as described in Materials and Methods. Binding in the absence of TAM (D), binding in the presence of TAM (M). B, Specific binding of 3H-TAM to crude, light, and heavy microsomes as well as plasma membranes was determined in the presence of 1 ng PRL as described in Materials and Methods. Binding in the absence of PRL (•), binding in the presence of PRL (B).
mary gland explants cultured with insulin, aldosterone, corticosterone, and PRL (IACPRL). A dose-dependent inhibition was observed in tissue from pregnant mice (not shown). However, the presence of endogenous estrogens in the tissue and the possible supportive role of these steroids in the biological response of glands from pregnant animals (30) makes the interpretation of these data difficult. This is emphasized by the results shown
IAC
IAC I Prl
IAC < Prl •< E2
IAC IAC t Prl i Prl i TAM ) E2 i TAM
IAC
IAC i Prl
IAC i Prl i E2
IAC IAC i Prl i Prl i TAM i E2 t TAM
FIG. 7. Effect of TAM on PRL-induced casein synthesis in mammary gland explants from pregnant and ovariectomized virgin mice. Pooled explants from 8 ovariectomized (7 days post surgery) virgin (•) and 5 pregnant (•) mice were cultured in serum-free medium 199 containing the indicated hormones as described in Materials and Methods. Cultures were exposed to 3H-amino acid mixture (virgin = 12.5 fid/ml for the final 24 h of a 96-hr culture period; pregnant = 1.25 fid/ml for entire 48-hr culture period), after which tissue was collected and analyzed for casein synthesis by specific radioimmune precipitation as described in Materials and Methods. Data shown are the mean ± SEM for three separate determinations. I, insulin (1 /ig/ml); A, aldosterone (0.1 ng/ ml); C, corticosterone (1 /xg/ml); PRL, oPRL (1 /ig/ml); E2, estradiol (10~6 M); TAM (10~6 M). Similar results were obtained when E2 as low as 10~9 M was tested in explants from pregnant mice.
in Fig. 7. TAM (10~6 M) inhibited the PRL-induced casein synthesis by over 80%, and this effect was not
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PRL AND TAMOXIFEN IN MAMMARY GLAND reversed by estrogen. When both estrogen and TAM are present there was nearly complete inhibition of PRLinduced casein synthesis. In an attempt to eliminate the problem of the presence of endogenous estrogens and any resulting estrogen responsiveness of the mammary tissue in culture, virgin mice were ovariectomized at least 48 h before the onset of culture. Figure 7 also shows that mammary gland explants from ovariectomized virgin mice showed significant inhibition (>80%) of casein synthesis by TAM when grown in media containing IACPRL. When both PRL and estrogens are present, TAM still inhibited casein synthesis by over 60%. Discussion Previous studies have shown that antiestrogens have a binding site in the microsomal fraction of mammary carcinoma cells (8, 31), which is distinct from the estrogen binding site. However, the biological role(s) of this AEBS is still unknown. Our recent results (13) however, have suggested that in some cases TAM, acting through the AEBS, may modulate PRL action. PRL is a key hormone in mammary gland development, and PRL receptors are also localized in the microsomal fraction of the cell. The mammary gland is also one of the principal target organs of estrogens and antiestrogens. We have now shown that antiestrogens, such as TAM, inhibit lactogen binding to the microsomal membranes as well as to the solubilized form of the receptor from normal mouse mammary glands. Prebinding of TAM to membranes causes the inhibition of lactogen binding to the same extent as when added simultaneously with the ligand. Inhibition occurs at 0 C as well as at room temperature, suggesting that the action of the antiestrogen through the AEBS is not enzymatic (Das, R., and B. K. Vonderhaar, in preparation). The AEBS is present in both estrogen target and nontarget tissues such as liver (32). However, even though liver microsomes from lactating mice are rich in lactogen binding activity, antiestrogens had no effect on the lactogen binding. The reason for this lack of an effect is not clear at this point. Purification of the AEBS and cloning of the gene from mammary glands and from liver may help clarify this observation. Inhibition of binding to mammary gland microsomes by antiestrogens is specific for lactogenic hormones, as TAM did not affect the EGF or insulin binding to mammary gland microsomes. Both EGF and insulin receptors are also located in microsomes. This specificity for lactogen receptors may be indicative of an interrelationship between the AEBS and the lactogenic hormone receptor. Both TAM and NAF inhibited specific lactogen binding, but another antiestrogen, LY117018, had little effect
537
on the mouse mammary gland microsomes. This antiestrogen has little or no affinity for the AEBS (29), suggesting that the effects of the antiestrogens on PRL action and binding are through the AEBS, not through the estrogen receptor. This close relationship of the two membrane-associated binding sites is emphasized by the reciprocal effect of PRL on the binding of TAM to the AEBS. A complex interrelationship of the two binding sites (i.e. lactogenic hormone and antiestrogen) becomes evident from these studies. How this reciprocal effect is brought about and what its biological function may be remains to be investigated. The phenomenon may reflect the close proximity and biochemical similarity of the two binding sites located on the same membrane protein (Das, R., and B. K. Vonderhaar, unpublished). Subcellular fractionation of mammary gland microsomes shows that both lactogen and antiestrogen binding are localized in the same fractions. Total microsomes and heavy microsomes are rich in both lactogen and antiestrogen binding sites, whereas light microsomes also contain some of both activities. At this point we are not sure whether this is due to contaminating heavy microsomes or is inherent to the light microsomes. Watts et al. (8) also localized AEBS at heavy microsome of mammary carcinoma cells. Lactogen binding in both light and heavy microsomes was inhibited by TAM (10~6 M), whereas the antiestrogen binding in heavy microsomes was enhanced by PRL. In light microsomes lactogen inhibited the antiestrogen binding. Because crude microsomes are subfractionated to obtain light and heavy microsomes, the overall effect of PRL on TAM binding in any given membrane preparation depends on the ratio of light and heavy microsomes present. Likewise, the extent of inhibition of PRL binding by antiestrogen is the combined effects on the different microsomal fractions contained therein. The biochemical relationship of lactogen binding and antiestrogen binding to the AEBS has biological significance, because TAM inhibited PRL-induced mammary gland differentiation just as it inhibits PRL-induced growth of the estrogen receptor-negative Nb2 cells (13). Production of the casein family of milk proteins, which is dependent on the presence of PRL in the culture medium, was inhibited by TAM. This action of TAM is not through the estrogen receptor, because it is not completely reversed by the addition of estradiol to the culture media, and a similar effect was obtained in tissue from ovariectomized mice where there is little residual estrogen and the level of the estrogen receptor is very low. Similar effects of the antiestrogen TAM have recently been obtained on PRL-induced protein kinase C activity in the NOG-8 mouse mammary cell line (Das, R., and B. K. Vonderhaar, manuscript in preparation). Like the Nb2 cells, these normal cells are devoid of
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estrogen receptors but contain membrane-associated AEBS. Hence, we believe that the ability of TAM to inhibit binding of lactogenic hormones to receptors is mediated through the membrane associated AEBS, and this biochemical action is translated to biological function in the blocking of PRL action during normal functional differentiation of the mammary gland. What the relationship of the AEBS is to the PRL receptor and how they interact with each other is the subject of our further investigations.
References 1. Cole MP, Jones CTA, Tod IDH 1971 A new antiestrogenic agent in late breast cancer: an early clinical appraisal of ICI 46474. Br J Cancer 25:270-275 2. E.O.R.T.C Breast Cancer Group 1972 Clinical trial of nafoxidine an oestrogen antagonist in advanced breast cancer. Eur J Cancer 8:387-389 3. Lerner LJ, Jordan VC 1990 Development of antiestrogens and their use in breast cancer: eighth Cain memorial award lecture. Cancer Res 50:4177-4189 4. Horwitz KB, McGuire W 1978 Antiestrogen: mechanism of action and effects in breast cancer. In: McGuire W (ed) Breast Cancer Advances in Research and Treatment. Plenum Publishing Co., New York, vol 2:155-204 5. Coezy E, Borgna JL, Rochefort H 1982 Tamoxifen and metabolites in MCF-7 cells. Correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res 42:317-323 6. Eckert RL, Katzenellenbogen BS 1982 Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cell: differences with estrogen and antiestrogen. J Biol Chem 257:88408846 7. Lippman M, Bolan G, Huff K 1976 The effect of estrogens and antiestrogens on hormone responsive human breast cancer in long term tissue culture. Cancer Res 36:4595-4607 8. Watts CKN, Murphy LC, Sutherland RL 1984 Microsomal binding sites for nonsteroidal antiestrogens in MCF-7 human mammary carcinoma cells. J Biol Chem 259:4223-4229 9. Katzenellenbogen BS, Miller MA, Mullick A, Sheen YY 1985 Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis and interaction with estrogen receptors and additional antiestrogen binding sites. Breast Cancer Res Treatment 5:231-243 10. Miller MA, Lippman ME, Katzenellenbogen BS 1984 Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells. Cancer Res 44:5038-5045 11. Allerga JC, Lippman ME 1978 Growth of human breast cancer cell line in serum free hormone supplemented medium. Cancer Res 38:3823-3829 12. Sutherland RL, Watts CKW, Ruenitz PC 1986 Definition of two distinct mechanisms of action of antiestrogens on human breast cancer cell proliferation using hydroxytriphenylethylenes with high affinity for the estrogen receptor. Biochem Biophys Res Commun
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140:523-529 13. Biswas R, Vonderhaar BK 1989 Antiestrogen inhibition of prolactin-induced growth of the Nb2 rat lymphoma cell line. Cancer Res 49:6295-6299 14. Liscia D, Vonderhaar BK 1982 Purification of a prolactin receptor. Proc Natl Acad Sci USA 79:5930-5934 15. Bhattacharjee M, Vonderhaar BK 1983 Purification and characterization of mouse a-lactalbumin from lactating mammary glands. Biochim Biophys Acta 755:279-286 16. Smith GH, Vonderhaar BK 1981 Functional differentiation in mouse mammary epithelium is attained through DNA synthesis inconsequent of mitosis. Dev Biol 88:167-179 17. Bhattacharya A, Vonderhaar BK 1979 Phospholipid methylation stimulates lactogenic binding in mouse mammary gland membranes. Proc Natl Acad Sci USA 76:4489-4492 18. Vonderhaar BK, Bhattacharya A, Alhadi T, Liscia DS, Andrew EM, Young JK, Ginsburg E, Bhattacharjee M, Horn TM 1985 Isolation, characterization and regulation of the prolactin receptor. J Dairy Sci 68:466-488 19. Lowry OH, Rosebrough NJ, Farr AC, Randall RJ 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265275
20. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 21. Dallner G 1978 Isolation of microsomal subfractions by use of density gradients. Methods Enzymol 52:71-83 22. Williams Jr CH, Kamin H 1962 Microsomal triphosphopyridine nucleotide-cytochrome c reductase in liver. J Biol Chem 237:587595 23. Michell RH, Hawthorne JN 1965 The site of diphosphoinositide synthesis in rat liver. Biochem Biophys Res Commun 21:333-338 24. Ellman GL 1959 Tissue sulfhydryl groups. Arch Biochem Biophys 82:70-77 25. Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB 1973 Acetaminophen-induced hepatic necrosis IV. Protective role of glutathione. J Pharmacol Exp Ther 187:211-217 26. Benson EA, Holdaway M 1982 Regulation of insulin binding to human mammary carcinoma. Cancer Res 42:1137-1141 27. Ginsburg E, Vonderhaar BK 1985 Epidermal growth factor stimulates the growth of A431 in athymic mice. Cancer Lett 28:143150 28. Vonderhaar BK, Nakhasi HL 1986 Bifunctional activity of epidermal growth factor on the expression of a- and /c-caseins in rodent mammary glands in vitro. Endocrinology 119:1178-1184 29. Katzenellenbogen BS, Miller MA, Mullick A, Sheen YY 1985 Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis and interaction with estrogen receptors and additional antiestrogen binding sites. Breast Cancer Res Treatment 5:231-243 30. Bolander Jr FF, Topper YJ 1980 Stimulation of lactose synthetase activity and casein synthesis in mouse mammary explants by estradiol. Endocrinology 106:490-495 31. Sutherland RL, Murphy LC, Foo MS, Green MD, Whybonne AM 1980 High affinity antiestrogen binding sites distinct from estrogen receptors. Nature 288:273-275 32. Sudo K, Monsma FJ, Katzenellenbogen BS 1983 Antiestrogen binding sites distinct from estrogen receptors: subcellular localization, ligand specificity, and distribution in tissues of rat. Endocrinology 112:425-434
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