Journal of
J Cancer Res Clin Oncol (1981) 100:135-148
,Cancer Research Clinical Oncology (~) Springer-Verlag 1981
Plasma Membrane-integrated Estrogen Receptors in Breast Tissue: Possible Modulator Molecules for Intracellular Hormone Level* K. S. Z~inker 1, G.W. Prokscha z, and G. Bltimel 1 1 Institute for Experimental Surgery and 2 Dept. of Surgery and Policlinic, Technical University Munich, Ismaninger Str. 22, D-8000 Munich 80, Federal Republic of Germany
Summary.Plasma membrane fractions from tumor specimens of 14 peri- and postmenopausal primary breast cancer patients and from non-neoplastic tissues were prepared by sucrose density sedimentation. The membranes were desintegrated by Triton X-100 and the 3H-estrogen binding capacity of membrane-derived proteins was determined. The receptor system found in the plasma membranes was mainly of low affinity and high capacity, working apparently in tandem with the high affinity and low capacity system described for the cytosol. Receptor concentrations in plasma membranes of non-neoplastic and neoplastic tissue were distributed over a wide range of values, but a significantly lowered receptor capacity was found in neoplastic tissue. An association constant, K,=6.35. 101~ -1 was determined for neoplasma-derived membrane receptor proteins, whereas non-neoplastic tissue membrane proteins were not saturable, when incubated with up to 150 pmol 3H-estradiol. From a Scatchard plot analysis of some experiments a molar concentration of binding sites for membrane proteins, derived from breast cancer tissue, n(M)= 1.7 9 10-12/mg protein was extrapolated. Furthermore, ample evidence was provided by an estrogen fluorescence probe that an estrogen binding system is located within the plasma membrane. It is suggested that the estrogen binding capacity of the epithelium cell membrane, due to the phospholipid moiety, can modulate the estrogen uptake and, thus, preventing hazardous high levels of estrogens within the cytoplasm. The presented experimental data throw a new light on the use of antiestrogens in the treatment of breast cancer. Key words: Estrogen receptor - Breast cancer tissue - Plasma membrane - fluorescence probe - Antiestrogen * Shortly after accepting this manuscript for publication a review was published in: Anticancer Res 1:39-44 (1981 ) by Barnett Zummoff on the role of estrogen excess in human breast cancer. This article is within the scope of the present paper and should be noticed by the reader Abbreviations: PBS=phosphate-buffered saline; PM II=plasma membrane fraction II; Tris= tris(hydroxymethyl)aminomethane; EDTA = ethylendiaminetetraacetic acid Offprint requests to: Dr. Dr. K.S. Z~inker (address see above)
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Introduction
The es trogen-cancer hypothesis has aroused new interest in the etiologic role in the pathogenesis of cancer in estrogen-responsive tissues (Herzt 1977; Korenman 1980). A number of tumors requires estrogen for continued growth and it goes back to the earlier observations of Beatson (1898) that ovariectomy ameliorated the clinical course of breast cancer. Tumor progression, however, can eventually lead to insensibility for estrogen and, therefore, tumor growth in the absence of hormone. In man, the main indications for an endocrine contribution to the etiology of breast cancer came from epidemiological studies (MacMahon et al. 1973) and despite numerous investigations there is a lack on appropriate models for breast cancer in human beings to elucidate the development of the malignancy due to hormonal influences. It is well-founded model that estradiol enters the cell, binds to a specific, high affinity cytoplasmic receptor and is then translocated to the nucleus to stimulate R N A synthesis. In literature, there are conflicting data about the possible mechanisms of estrogen uptake in estrogen-sensitive cells. Erdos et al. (1969) have suggested that estradiol passes from the weak to the strong binding receptor proteins by an equilibrium process mainly governed by the concentration of receptor; hereby, nothing is said about the cellular localization or compartmentation of those receptor proteins. Studies on the uptake and retention of estradiol in uterus and diaphragma by Peck et al. (1973) suggested that the entry of estradiol in target and non-target tissues occurs by passive diffusion. Kinetic studies of estradiol entry into uterine cells, as carried out by Mfiller and Wotiz (1979) seemed to confirm these findings. Milgrom et al. (1973) and Pietras and Szego (1977), on the other hand, obtained data suggesting membrane-binding sites with specifities for steroid hormones and a passage of estrogen through the uterine cell membrane by protein-facilitated diffusion (Milgrom et al. 1973). Very recently, Skinner (1979) reported, with an outlook on the currently used and most reliable and reproducible methods for cytosol estrogen receptor assay that a significant amount of specific receptor proteins in breast cancer tissue is insoluble and remains firmly bound to the tissue fragments as sediments, which were usually discarded. The present paper describes studies on plasma membrane-integrated estrogen receptors of non-neoplastic and neoplastic breast tissue to make an attempt to reveal the mechanism of entry of estrogen into mammary epithelial cells. The results obtained are put into a framework of endocrine features, thus, contributing to the understanding of estrogen-dependent malignancy as well as to the development of a rational approach for the diagnostic and therapeutic procedure in human breast cancer.
Materials and Methods Patients, Benign and Malignant Breast 7~ssue
A total of 14 primary, peri- and postmenopausal breast cancer patients were studied. The carcinomas were of varioushistologicaltypes and the clinical staging, accordingto the TNM-system,was modified after the histologicalexamination;only patients with operablebreast cancer,who had not receivedsystemic cytotoxicor hormonal therapy wereincluded. All patients were treated with some form of mastectomy,which included axillary dissection. Benign mammary tissue was dissectedfrom the ipsilateral breast and verifiedby cryostat sections.
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Tissue Preparation for Estrogen Receptor Determination Fresh tissue was obtained within 15 rain after excision, Samples weighing 0.5-1.0 g were macroscopically examined for normal and malignant breast tissue and in most cases verified by cryostat sections. The carefully defatted tissues were minced, homogenized in 4 volumes of 40 m M Tris-HC1, 1.5 m M EDTA and 1.4 mM/~-mercaptoethanol buffer, pH 7.4 at 20 ~ (buffer A) using a Polytron, with 30 s intervals in ice. The homogenate was centrifuged at 105,000 g for 60 rain. The resultant supernatant was brought to 30%-satd. (NH4)~SO 4 and centrifuged at 10,000 g for 30 min. The pellet was resuspended in 2 ml of 40 m M Tris-HC1, 1.5 m M EDTA, 1.4 mM/%mercaptoethanol, 50 m M KC1 buffer, pH 7.4 (buffer B), and an aliquot was assayed for cytosol estrogen receptors. The pellet from the 105,000 g centrifugation step was resuspended in 3 mt of buffer B and gently rehomogenized in a Potter-Elvehjem with a glass pestle.
Plasma Membrane Preparation To the 3 ml of resuspended 105,000 g pellet 10 ml of sucrose having a density (d) = 1.3 was added dropwise wilh constant mixing. Successive equal portions of sucrose solution with a density of d = 1.2, l.l 8, 1.173, 1.166, and 1.16 were filled into the tubes. The gradient was centrifuged at 25,000 rev./min for 90 rain in a Beckman Rotor SW 27. Purified plasma membranes were collected as a thick rug from the l. 166/1.173 interface using a pasteur pipette. The plasma membranes were desintegrated by Triton X100 (2% final concentration). The probes were stirred overnight and dialyzed exhaustively (72 h) against buffer B with two changes of the buffer. All procedures were carried out at 0 ~ ~ if not stated otherwise. Specimens of non-neoplastic mammary tissue were treated likewise.
Enzyme Assay as Membrane Markers The ATPase activity was determined according to Jackson et al. (1977) with 50-100/xg membrane protein preparation. K+-stimulated p-nitrophenylphosphatase (K+-pNPPase) activity was measured by the method of Torriani (1968) with 100-150 gg membrane protein preparation in the incubation medium.
Estrogen Receptor Assay Estrogen receptors in the tissues were assayed by measuring the binding affinity of their cytosol and membrane-derived protein fractions for 3H-estradiol-17/~ (New England Nuclear, Boston, spec. activity 90.0 Ci/mmol). A volume of 100 gl of cytosol and/or membrane-derived protein preparation was incubated in duplicate for 18 h at 20 ~ ~ and another set of tubes at 0 ~ with increasing amounts of 3H-estradiol, ranging from 1 to 150 pmol 3H-estradiol. The free and loosely bound 3H-estradiol was removed by a preparative linear sucrose density gradient (5-15% w/v) as recommended by the NIH Consensus Development Conference (1979).
Analys& and Presentation of the Data The area under the fast sedimenting peak of the sucrose gradient was considered to be proportional to the amount of 3H-estradiol receptor complex; therefore, the area was integrated by means of the Gaussian distributed specific activities. The specific activity in each sucrose gradient fraction of the fast running peak could be computed from scintillation countings of 3H-estradiol bound, converted to desintegration per minutes, using a 3H-toluene standard and the protein measurement, according to Lowry etal. (1951), using bovine serum albumin as reference. Finally, the results were expressed as a number of picomoles 3H-estradiol bound to milligram cytosol and/or membrane-derived proteins. The differences in the binding observed between the two sets of incubation tubes were considered as specific binding. In some experiments, the results of incubating increasing amounts of 3H-estradiol with a fixed amount of protein concentration was plotted according to Scatchard in order to demonstrate binding properties of membrane-derived proteins.
Detection of Estrogen Receptors with a Fluorescent Probe Cryostat-frozen sections (10 gin) of breast cancer tissue and normal mammary epithelium were airdried for 60 min at 4 ~ rehydrated with a few drops of 2% BSA dissolved in BPS, pH 7.4; excess BSA
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was wiped off after a few seconds without further washing. The sections were covered with 0.1 ml fluorescent steroid conjugate (Fluoro-CEPtm, Zeus ScientificInc. Raritan, NJ, USA, distr, by Hyland Laboratories Munich, FRG) and incubated in a moist chamber for 120 min at room temperature. Excess conjugate was drained offby placing the slidesin a slide holder and then immersed in a large staining dish, containing PBS; the buffer was changed twice within 120 rain. The slides were examined under a UV microscope for fluorescence.A Leitz microscopewas used, equipped with a mercury high pressure HPQ 100 W lamp as a light source. Polyacrylamzde-Gel-Electrophoresis (PAGE) of Cytoplasm and Membrane-derived Proteins from Neoplastic Tissue A 7.5% polyacrylamide gel was prepared and electrophoresis was performed with protein samples of the cytoplasmic and membrane-derived proteins, containing 200-250 gg protein, mixed with 5 gt of tracking dye and i drop of glyceroI. Electrophoresis was usually carried out for 2 h in the cold and one gel was stained for proteins to locate the bands. From the other gels, protein regions were removed by slicingwith a razor blade, homogenized in buffer B and proteins were eluted by diffusion. After extensive dialysis in the cold, the homogenate was cleared by filtration through a glass frite and an aliquot was assayed for 3H-estradiol binding. Transplantatwn of Human Breast Carcinomas into Nude Mice Breast cancer tissue obtained at the time of surgery was asepticallyminced and connective and adipose tissues were trimmed off. Pieces measuring 1-2 mm in diameter were surgically transplanted s.c. between the scapulae of 8- to 10-week-oldhomozygous nude (nu/nu) female mice of Balb/c background (gift Max-Planck Institute, Martinsried, FRG). The animals were housed at 22 ~ ~ in particle-filtered air, and sterilized bedding, cages, food, and water were used. Tissue Culture of the Transplanted Breast Carcinomas The tumor was surgicallyexcised, washed a few times in lukewarm, steril PBS and cubes of 1 mm3 were seeded on the bottom of Falcon plastic flasks. Once dried the tissue blocks were carefully covered with medium 199 Earle (Seromed, Munich, FRO) and antibiotics (100 I.U./ml penicillin and 100 I~g/mldihydrostreptomycin). The cultures were incubated at 37 ~ in an atmosphere of 5% COs and 95% air of high humidity.
Results P l a s m a m e m b r a n e fractions o f n o n - n e o p l a s t i c a n d p r i m a r y breast c a r c i n o m a tissue were enriched by a d i s c o n t i n u o u s sucrose step gradient. E n z y m a t i c d a t a confirmed o n 8- to 10-fold increase in the relative specific activities of A T P a s e a n d K § p N P P a s e from P M II in respect to the homogenate_ Electron-microscopic pictures revealed a visually enriched m e m b r a n e fraction with occasionally c o n t a m i n a t i n g m i t o c h o n d r i a a n d vesicles. The m o r p h o l o g i c a l a n d enzymatic features of P M II were m o r e or less u n i f o r m in m a l i g n a n t a n d b e n i g n tissues, The enriched P M II served as a basis for s t u d y i n g the estrogen b i n d i n g capacity of P M H-integrated proteins. Preparative sucrose density c e n t r i f u g a t i o n of 3H-estradiol incubated proteins f r o m T r i t o n X-100 desintegrated P M It revealed two peaks o f radioactivity (Fig. 1). T h a t peak, either from b e n i g n or m a l i g n a n t tissue, which sedimented far fi'om the top o f the tube, showed r e m a r k a b l y reduced 3H-estradiol b i n d i n g when the proteins were digested by T r y p s i n prior to 3H-estradiol i n c u b a t i o n . The p a t t e r n of radioactivity, which was f o u n d o n the very top of the tube r e m a i n e d unaffected by protease digestion. F r o m this, it is concluded that 3H-estradiol b o u n d to m e m b r a n e - d e r i v e d proteins a n d u n b o u n d 3H-estradiol were clearly separated. It is ap-
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Estrogen Receptors in Breast Cancer Tissue
Fig. 1. Preparative sucrose gradient centrifugation for separating free and bound 3H-estradiol. The open circles show the 3Hestradiol pattern, the filled circles the protein distribution. This standard procedure revealed mostly two peaks of radioactivity, the fast running peak representing the receptor protein bound to 3H-estradiol. The area under this peak is considered to be proportional to the amount of 3H-estradiol receptor complex
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Fig. 3. Examplefor the binding of 3H-estradiol to proteins derived from membranes of non-neoplastic (open circles) and neoplastic (filled circles) tissue. The inset shows the Scatchard plot of the saturation experiment with 3H-estradiol (150 pmol) to mammary neoplastic tissue. The horizontal line denotes the low affinity binding and the steeply sloping straight line represents the high affinitybinding, as calculated from the experimental curve, n(M)= molar concentration of binding sites, Ka= 6.35. 101~ i; S= specific,NS=non-specific
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horizontal line contributes to the non-specific binding of apparent unlimited capacity. In most of the experiments, proteins from PM II of non-neoplastic tissue were not saturable and gave a straight line; within two experiments, however, the results were inverse, where proteins from PM II of neoplastic tissue ended up with a straight line, when aH-estradiol binding data were tried to express according to Scatchard. The fluorescent estradiol conjugate staining of human ductual epithelial cells is shown in Fig. 4. The cellular distribution o f the bright apple green fluorescence followed the epithelial lining, indicating that these mammary ducts were composed of entirely receptor-positive cells (Fig. 4 a, 4 c). In Fig. 4 b a consecutive cryostate frozen section was stained for hematoxylin-eosin (HE). Examples for different fluorescent staining on a cellular level are illustrated by Fig. 5. One cell exhibited positive nucleus and nucleolus staining (Fig. 5 a), another cell revealed nucleus and membrane fluorescence (Fig. 5 b). An almost similar pattern for nucleolus and membrane labeling by the fluorescence conjugate is manifested in Fig. 5 c. Interestingly, two cells exposed membrane-positive fluorescence and a few cells showed a marked nucleolus staining; the cytoplasm or the plasma membrane were receptor-negative. The electrophoretic mobility of 3H-estradiol binding proteins from both cytoplasm and membrane of neoplastic tissue was different, when analyzed by PAGE. Cytoplasma proteins, accepting 3H-estradiol banded almost on top of the gels. Membrane-derived proteins exhibited at least four 3H-estradiol binding qualities, which penetrated far into the gels (Fig. 6). A close correlation between protein staining and the radioactive binding pattern of the sliced gels was not found, because the latter is assumed to be more sensitive than protein visualization by the dye. The tumorigenicity of one part of the surgically removed cancer tissue was proved for autonomous progressive growth in athymic mice as detailed by Sebesteny et al. (1979). F r o m the inocculated tumor a cell culture was establish-
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Fig.4a-c. Fluorescence staining for estrogen and hematoxylin-eosin staining of an intraductual carcinoma. The tumor cells grow diffusely along the ducts and show a bright apple-green fluorescent image a, e, indicating a high amount of estrogen receptors. The tumor cells remain for a long time within the ducts so that evidence of invasion is absent in b. X 176
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Fig. 5 a-e. Fluorescence photographs of mammary epithelial cells. In a, a positive fluorescence is only seen in the nucleus and the nucleolus, whereas in b the nucleus and the plasma membrane is positively stained for estrogen. Most of the labeled estrogen in this cell is located peripherally in the area of membrane ruffling (• 1,025). A nest of mammary epithelial duct cells with plasma membrane fluorescence (1) and fluorescent nucleoli is demonstrated in e. •
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~2 Fig. 6. PAGE of estrogen accepting molecules from both cytoplasmic proteins (open columns) and plasma membrane-derived proteins (closed columns) of neoplastic mammary tissue. The 3H-estradiol binding qualities are clearly separated. When an aliquot (200 gg) of the plasma membrane-derived protein preparation is electrophoresed alone, no binding of 3Hestradiol is observed in that region of the gel (slices 1 3), where cytoplasmic proteins, binding to 3H-estrogen, are normally banding
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ed (Fig. 7). After 2 or 3 days in culture the cells started to grow out of the cube (Fig. 7 a), and appeared as a single cell suspension (Fig. 7 b). After the cells were attached to the surface of the Falcon plastic flasks, they began to differentiate themselves morphologically, forming triangled or polygonal cell shapes, and searched for contact with each other with a tendency for assembling gland-like, remembering the architecture of mammary ducts, as normally seen in histological sections (Fig. 7 c). A similar picture of the morphogenesis of mammary epithelial cells in culture was recently reported by Bennett (1980). Discussion
The results reported in the present study provide evidence that membrane-extracted proteins from benign and malignant mammary tissue bind to estradiol, a result in congruence with that of Pietras and Szego (1977) and Suyemitsu and Terayamy (1975), obtained for endometrial cells and rat liver cells. In agreement with other workers (Sebesteny et al. 1979; Sprang-Thomsen 1976) we found that the fraction of primary breast carcinomas growing well as heterotransplants is quite low (5/14). Nevertheless, it is a must to prove tumorigenicity of the biologic material which is assumed to be neoplastic. The concentration of receptor found is highly variable, a finding mainly influenced by the actual content of the normal and malignant epithelium component in the tissue, the percentage of receptor extractable from an inhomogenous cellular composition, above all from fibrous stroma, frequently adipose tissue, hyperplastic ductual epithelial cells and inflammatory cells. Two independent methods are applied to substantiate the view on membrane-integrated estrogen receptor proteins and estrogen uptake by a high affinity (cytosol receptor) and a low affinity system [plasma membrane compound(s)] associated with a specific plasma membrane-integrated binding activity (high affinity), which may work in tandem with the cytosol receptor. The application of a recently available fluorescence conjugate to estrogen receptor of mammary tissue (Lee 1978, 1979, 1980) exhibits a selective staining of the ductual epithelial cells of the mammary glands and, on a subcellular level, the cytoplasm, the plasma membrane, and the nucleus. In some cases, it can be observed that only the nucleus and, more often the nucleolus is heavily labeled with fluorochrome. Control experiments on cryostat-frozen sections of non-neoplastic mammary tissue showed, of course, strong fluorescence. However, it is out of the scope of this paper
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Fig. 7 a--e. Evidence for tumorigenicity of mammary specimens in nude mice and establishing of cell cultures. After 2 days, polygonal cells start to grow out of the cube in tissue culture, when raised in Medium 199 Earle, supplemented with 10% fetal calf serum a. The predominant picture for a few days is a singlecell suspension and the beginning of cell attachment to the bottom of the Falcon plastic tube b. After about 1 week the cells have contact with each other c. Phase microscopy x 125 to validate fluorescent p a t t e r n o f neoplastic a n d n o n - n e o p l a s t i c m a m m a r y tissue. O n e o f the established procedures for visualizing estrogen receptors with estrogen c o n j u g a t e d fluorescent dyes (Lee 1978, 1979, 1980) or a n i m m u n o c y t o c h e m i c a l m e t h o d (Walker et al. 1980) was only chosen to give c o r r o b o r a t i v e evidence for m e m b r a n e - i n t e g r a t e d estrogen receptors. C a u t i o n m u s t be exercised in the inter-
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pretation of the staining of lipid-rich cell membranes because such compounds may return the estrogen-coupled fluorophore preferentially. The simultaneous biochemical determination of ~H-estradiol binding to plasma membrane-derived proteins is bridging this fallacy. It is likely that there are steroid-accepting proteins with a high affinity and low capacity and with a low affinity but high capacity system, the substrate of which might be phospholipids (Z/inker et al. 1980) within the plasma membrane; the biologic significance of these two systems is far from clear and subjected to teleologic speculations. Intensive and prolonged dialysis of the Triton X-100 desintegrated plasma membranes results in a loss of small molecular weight substances, but unspecific lipoprotein aggregates are not to rule out, above all formed by phospholipids. An anlysis of DNA content (Burton 1956) carried out from PM II was negative, excluding the possibility of errors from significant DNA3H-estradiol complex formation. A decreased binding capacity for 3H-estradiol to PM II proteins is found in neoplastic mammary tissue, neglecting the histomorphological typing, compared to non-neoplastic tissue. This is one of the first attempts to quantify estrogen binding capacity in subcellular structures of normal and malignant breast tissue besides the paper of Jungblut et al. (1976), and it may, therefore, contribute some explanations for the role of steroid hormones in human breast cancer. One of the major criticisms against plasma membrane-integrated estrogen receptors is the possible contamination with cytosol receptor or plasma steroid-binding globulins (Kreitmann et al. 1978) during the preparation procedure of the tissue. We cannot exclude this objection completely, but it seems unlikely, because no remarkable protein-dependent estrogen binding was found in the fat layer, the crude mitochondria and vesicle fractions. Moreover, PAGE of cytoplasmic proteins and membrane-derived proteins from neoplastic tissue show a different electrophoretic mobility. Membrane-derived proteins are separated in at least four proteins, accepting 3H-estradiol, whereas cytoplasmic proteins reveal one peak of 3H-estradiol binding protein(s), located near the top of the gel. If the membrane preparation procedure turns out to contaminate the "membrane proteins", then one should expect a 3H-estradiol binding peak at the same region of the gel, where the cytoplasmic protein(s) band(s). This is not the case and, therefore, exogenous contamination with estradiol binding proteins is neglectable in our membrane preparations. It is obvious that the extracellular estrogen level varies in a wide range in the life-span of man. It depends on the sex, the age, the metabolizing capacity of the liver, and the excretorial function of the kidneys. Cumulative empiric evidence is provided that the estrogen level is exogenously increased by the nutrition behavior. Alfalfa meal, e.g., can contain 340-560 ppm of coumestrol biologic equivalent to 113-186 ppb of the active synthetic non-steroid estrogen diethylstilbestrol (Bickoff et al. 1969). Most of the steroids, stilbene and phenolic and triphenolic types of estrogenic compounds in plants and/or meat, have been found to be carcinogenic. One of the postulated reasons for the lower incidence of breast cancer in Japan as compared to most Western countries is believed to be the different estrogen levels between the two populations which is due to different nourishment (Segi et al. 1969). Unphysiologic high concentrations of endogenous and/or exogenous estrogen may be trapped by receptors in the plasma membrane of non-neoplastic, estrogen-sensitive cells and come to an equilibrium with the intracellularly distributed steroid receptors as lately proposed by Sheridan et al.
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(1979). The decreased estrogen binding capacity of neoplastic breast tissue holds for the hypothesis that the diffusion process of estrogen molecules and analogues through the plasma membrane may be facilitated, if the saturation level for the plasma membrane integrated hormone receptor is reached. The low affinity system in the plasma membrane is considered to be a common route of uptake for steroids, regulating the intracellular hormone level. Estrogens are more readily accumulated and retained in estrogen-responsive cells than in cells that are not their targets; examples for membrane binding in uterus, liver, and intestine were given by Pietras and Szego (1977). From these studies it is also likely that there exists a two-binding system for estrogens in the plasma membrane, i.e., a specific one represented by protein(s) and a non-specific; the binding capacity of the latter can be modulated in vitro by phospholipids which are common in plasma membranes (Zfinker et al. 1980). The antiestrogens in the treatment of breast cancer were reviewed by Tagnon (1977), and the validity for estrogen receptor determination to apply an appropriate therapeutic regimen is summarized by Leclercq et al. (1975). The overall response rate in patients with breast cancer treated with antiestrogens alone or in combination with other modalities is between 28% and 35%, with a medium duration of 9 months of remission. This rate is relatively constant (Cole et al. 1972; Brewin et al. 1974), except in the case of Ward's study (Ward 1974). At a dosage of 10 mg tamoxifen twice daily he observed a remission rate of 60%, and at a dosage of 20 mg twice daily a remission rate of 77%. We are aware of the ill-defined parameters and difficulties of measurements for "partial remission", "no change", and "progression" of the disease in these trials; however, the contradictory data may be explained in the light of our experimental results. The effect of treatment of a fraction of cells may be influenced by the high and low capacity estrogen binding system which is located in the plasma membrane. It can be argued that the high capacity estrogen binding system of the membrane has to be saturated by the administered antiestrogens. A fraction of dose of the antiestrogen is then able to penetrate the plasma membrane more rapidly and easily, ensuing the depletion of cytoplasmic estradiol receptors (Nicholson et al. 1976), and, thus, leading to remarkably decreased nucelar transfer activity of the cytosol estrogen receptors (Rochefort et al. 1980). Onthe basis of these considerations it appears reasonable to focus the attention on estrogen receptors bound to subcellular structures in mammary breast tissue and, moreover, to differentiate between cytosol, nuclear and plasma membrane bound receptors to reflect a plausible theoretical background when administering antiestrogens. Since the presence of estradiol receptor in tumors has prognostic value in predicting the proper treatment for breast cancer, those women who have receptors though masked by endogenous estradiol (Thorsen 1980) or integrated in plasma membranes might not be given the treatment that would be of greatest benefit. Acknowledgement. The
competent technical assistance of Ms. E. Sincini is gratefully acknowledged.
References Beatson GT (1898) On the treatment of inoperable cases of carcinoma of the mamma: Suggestions for a new method of treatment with iItustrative cases. Lancet II: 104-107:162-165
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Received July 29, 1980/Accepted February 27, 1981
