the origin and evolution ofnon-neoplastic lesions and carcinomas of the breast. (Am J Pathol. 1996, 14&1895-1903). Interest in the location of oxytocin receptors ...
American Journal of Pathology, Vol. 148, No. 6, June 1996 Copynight ©) American Society for Investigative Pathology
Immunolocalization and Gene Expression of Oxytocin Receptors in Carcinomas and Non-Neoplastic Tissues of the Breast
Gianni Bussolati, Paola Cassoni, Gianpiero Ghisolfi, Franco Negro, and Anna Sapino From the Depatiment of Biomedical Sciences and Oncology, University of Torino, Torino, Italy
Recent evidence indicates that oxytocin (OT), in addition to the induction of myometrial and myoepithelial ceU contraction, can influence proliferation and differentiation in developing mammary glands and in breast cancer ceUs, hence the interest in detecting and locating OT receptors (OTRs). We produced rabbit antisera and a monoclonal antibody against a synthetic peptide corresponding to the amino terminus and antisera against a peptide corresponding to the carboxy terminus of the predicted OTR sequence. We tested their specificity in immunoblots and immunocytochemical tests. AU of the antibodies specificaly stained myometrium (at term of pregnancy). In the human breast, OTRs were detected in myoepithelial ceUs along ducts of normal lobules and in sclerosing adenosis. Intraductal ceUs in benign hyperplastic lesions were also positive. OTRs were demonstrated in cases of primary and metastatic carcinomas of the breast. In the same tissues, OTR gene expression was shown by reverse transcriptase polymerase chain reaction procedures detecting the specific mRNA. These results suggest that the interaction between OT and its receptors might play a role in the origin and evolution ofnon-neoplastic lesions and carcinomas of the breast. (Am J Pathol
in breast carcinoma cell lines. The observation that oxytocin (OT) induces a net decrease in the relative number of undifferentiated cells in the developing mouse mammary gland1 led us to investigate the OT effect on cultured breast cancer cells. By reverse transcriptase polymerase chain reaction (RT-PCR) procedures we detected OTR mRNA in breast cancer cell lines, and in the same lines we described an OT-induced decrease of the proliferation rate.2 The mechanism of such an effect is not clear, but preliminary data indicate that, unlike what was observed in myometrial cells during OT-induced contraction,3 intracellular Ca2" transients are not evoked. However, breast carcinoma MDA-MB231 cells, when cultured in serum-free medium, showed a significant cAMP increase at 30 minutes of OT treatment (preliminary unpublished data). The OT-induced decrease of the proliferation is not due to cell toxicity and is not observed in colon carcinoma cells nor in immortalized MCF10A mammary epithelial cells.2 All of these data suggest that OT might interact with other growth factors and play a role in the origin and evolution of non-neoplastic lesions and carcinomas of the breast. The OTR structure can be deduced from analysis of the OTR gene, recently identified and sequenced by Kimura et al.4 The amino acid sequence suggests that the 388-amino-acid receptor protein has seven putative transmembrane domains, showing partial identities to the corresponding domains of other Gcoupled rhodopsin-type receptors. The location of OTRs is not clear. Kimura et al4 demonstrated by Northern blot procedures the presence of the specific mRNA in the uterus, ovary, and mammary gland, whereas other tissues such as cerebral cortex, lung, liver, and kidney were negative.
1996, 14&1895-1903) Interest in the location of oxytocin receptors (OTRs) in the breast stems from the recent finding that this hypothalamic nonapeptide not only induces contraction of myoepithelial cells during lactation but also influences cell proliferation in mammary glands and
Supported by grants from the Italian CNR (Milan) from the Ministry of Universities and from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan). Accepted for publication February 12, 1996. Address reprint requests to Dr. Gianni Bussolati, Department of Biomedical Sciences and Oncology, University of Torino, Via Santena 7, 10126 Torino, Italy.
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Interestingly, the same group5 using in situ hybridization procedures identified the presence of OTR mRNA in the glandular epithelial cells of the human endometrium. This suggests that OT, besides its classical role in uterine contractility during parturition, might have an important but still unknown role in non-pregnant human endometrial glands. Previous investigations aimed at identifying the location of OTRs were limited to animal tissues and to the selective binding of radioactive OT in homogenates and tissue sections. In the breast, the only reported experience deals with lactating mammary gland of rats, where specific OT binding was detected by autoradiography in myoepithelial cells.6 Novel procedures are needed to reach a precise cellular location of OTRs and to understand the functional role of OT in human non-neoplastic and neoplastic breast tissues. We have therefore produced specific antibodies against the amino- and carboxylterminal fragments of human OTR and used them in immunocytochemistry on breast tissues. A confirmatory and enlightening role was played by the detection in the same tissues of the OTR mRNA by RTPCR.
Materials and Methods Tissues Human tissue specimens immediately after surgery were subdivided into blocks, which were partly frozen in liquid N2, partly routinely fixed in 10% formalin, and embedded in paraffin for histopathological diagnosis. Some of the frozen blocks were used for cryostat sections and some were powdered in liquid nitrogen by a Mikro-dismembrator (Braun-Melsungen, Melsungen, Germany). Total RNA and proteins were extracted to be used, respectively, in RT-PCR and immunoblot procedures (see below). Samples of histologically normal mammary tissues were collected from adult (menstruating) women who were having surgery for fibroadenomatous nodules of the breast. We also collected 4 cases of benign hyperplastic lesions (2 associated with cystic disease), 3 papillomas of the ducts, 3 fibroadenomas (1 showing phylloides features), and 12 cases of carcinoma of the breast. In 7 of these last cases, in addition to the primary lesion, fresh lymph node metastases (histologically confirmed) were also collected. As positive control we used tissue blocks taken with informed consent from the uterus of two patients who underwent caesarian section at term of pregnancy.
Cell Lines The MCF7 and the MDA-MB231 human breast cancer cell lines and the HT29 colon carcinoma cells were obtained from American Type Culture Collection (Rockville, MD). MDA-MB231 and HT29 cells were routinely cultured in RPMI (GIBCO, Burlington, Ontario, Canada) supplemented with 10% fetal calf serum (GIBCO). MCF7 cells were grown in Dulbecco's modified Eagle's medium-F12 (Sigma Chemical Co., St. Louis, MO) added with low (5%) or high (10%) fetal calf serum concentrations. Cells were grown in petri dishes or on coverslips in 24-multiwell plates at 37°C in humidified air/5% CO2 atmosphere.
Production of Rabbit Anti-OTR Antisera Antibodies were produced in rabbits immunized with synthetic peptides selected following the criteria of hydrophilicity and relative location in the receptor protein. A search of GenBank murine and human data bases of different fragments revealed that the base pair sequences 1 to 42 and 368 to 388, corresponding to the predicted amino terminus and carboxy terminus of OTR, were specific. We selected therefore the amino acid amino-terminal sequence 1 to 14 (Met-Glu-Gly-Ala-Leu-Ala-Ala-Asn-Trp-Ser-AlaGlu-Ala-Ala) and the carboxyl-terminal sequence 368 to 388 (Ser-Phe-Val-Leu-Ser-His-Arg-Ser-SerSer-Gln-Arg-Ser-Cys-Ser-Gln-Pro-Ser-Trh-Ala). The peptide 1 to 14 was produced by Neosystem Laboratories (Strasbourg, France), whereas that corresponding to the carboxy terminus was kindly synthetized by Dr. Guerrini (Department of Pharmacology, University of Ferrara, Ferrara, Italy). Terminal lysine was added to peptide 1 to 14. Part of the synthesized peptides was covalently coupled (at the amino terminus) to keyhole limpet hemocyanin via bifunctional cross-linking using N-succinimidyl-6-maleimido caproic acid and 1-ethyl-3 (3'-dimethylaminopropyl) carbodiimide-HCI. The carboxy terminus was coupled to keyhole limpet hemocyanin via 0.2% glutaraldehyde in phosphate-buffered saline (PBS). To produce antibodies, albino rabbits were immunized by several subcutaneous and intramuscular injections of the keyhole-limpet-hemocyanin-coupled peptides in distilled water and homogenized with equal amounts of Titermax (Charles River, Calco, Italy); intravenous injections of the peptides followed every 15 to 20 days. To check the presence and level of antibodies in the rabbit sera, we set enzyme-linked immunosorbent assay in vitro tests. The synthetic peptides were dissolved
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phosphatase (Schleicher and Schuell, Keene, NH). The reaction was developed with p-nitro-phenyl-phosphate (Test Combination, Boehringer, Mannheim, Germany). Reading of absorbance at 405 nm was made with a Multiskan Bicromatic apparatus (Labsystem, Helsinki, Finland).
53 Kd-
Production of Anti-OTR Monoclonal Antibody A mouse monoclonal antibody was prepared using the amino-terminal peptide sequence as immunogen. Details on the production and characterization of the antibody are to be presented elsewhere (submitted). A clone, labeled CHINA/IF3, was selected because of superior performances in terms of clone stability, antigen binding in enzyme-linked immunosorbent assay tests (see above), and immunocytochemical reactivity on formalin-fixed, paraffin-embedded sections of myometrium at term (taken as positive control). The clone was expanded in vitro and grown as tumor ascites. The purified monoclonal antibody obtained (here indicated as 1F3) proved to be an IgM.
Figure 1. Immunoblot test using anti-amino-terminal sequence antibody. In homogenate of pregnant human uterus a specific band is detected at 53 kd.
in PBS at a concentration of 10 ,ug/ml and used in the microwell strip plate high binding system (BIOHIT, Helsinki, Finland). Each well was filled with 50 ,ll of solution. After overnight incubation at 40C, the wells were washed (three times) with PBS added with 0.05% Tween 20 and then saturated with 100 ,ld of 1% bovine serum albumin (2 hours at 370C). After additional washings with PBS/Tween 20, microwells were incubated for 2 hours at 370C with 50 ,tl of a gradual dilution of immune and preimmune sera, washed (three times) in PBS/Tween 20, and finally incubated with goat antirabbit immunoglobulin serum, conjugated with alkaline
Immunoblotting Fragments of pregnant uterus were homogenized by mechanical fragmentation and solubilized in boiling
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Figure 2. Uterus at term of pregnancy. Methanol-fixed cryostat sections. Antisera against the amino-terminal sequence (a) and against the carboxyl-terminal sequence (b) both specifically react with myometrial cells. The staining is mainly decorating the cell membrane. APAAPprocedure; magnification, X 200 (a) and X 100 (b).
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Figure 3. Breast lobule showing minor degrees of adenosis in formalin-fixed, paraffin-embedded tissue. The antibody against the amino-tenninal sequence of OTR strongly stains myoepitbelial cells along tbepenphery of the ductules, whereas columnar epithelial cells are negative. ABCprocedure; slight nuclcear counterstaini; magnification, X 400.
buffer (25% 0.5 mol/L Tris-HCI at pH 6.8, 25% sodium dodecyl sulfate (SDS), 50% double-distilled water). The gel lane was loaded with 10 ,ug of total proteins in the absence of the reducing agent ,B-mercaptoethanol. After SDS-polyacrylamide gel electrophoresis (12%), proteins were transferred to nitrocellulose filters (Hybond, Amersham Corp., Buckingamshire, UK) in a Trans-blot semi-dry (BioRad Laboratories, Richmond, CA). Nitrocellulose was saturated in 5% bovine serum albumin in PBS and incubated overnight at 40C with the antisera and the antibody at proper dilution. The filter was then incubated for 1 hour at room temperature with the appropriate peroxidase-labeled secondary antibody. Specific binding was detected by the enhanced chemiluminescence system (Amersham).
Immunofluorescence MCF7 and MDA-MB231 cells, grown for 5 days, were tested by an indirect immunofluorescence procedure. After washing in PBS, cells were treated with normal pig serum (diluted 1:50 in PBS for 20 minutes) and then incubated at 370C for 60 minutes with antiserum (diluted 1:100 with PBS) or with the monoclonal antibody (diluted 1:10 in PBS). Preimmune
and preabsorbed sera and unrelated monoclonal antibodies were used as controls. The appropriate fluorescein-labeled or rhodamine-labeled secondary antiserum (Sera-Lab, Sussex, UK) diluted 1:10 in PBS was used for 30 minutes at room temperature. Cells were examined with a Leitz Orthoplan fluorescence microscope equipped with a Xenon lamp and epifluorescence apparatus.
Immunohistochemistry Sections were collected on polylysine-coated slides and processed for immunoperoxidase using the avidin-biotin peroxidase complex (ABC).7 Endogenous peroxidase activity was blocked with hydrogen peroxide in methanol. Normal goat serum (diluted 1:10 in PBS) was followed by overnight incubation with the anti-OTR antisera (diluted 1:1000 in PBS) or with IF3 monoclonal antibody (diluted 1:200 in PBS). Preimmune sera, sera incubated with the synthetic peptide (1 mg/ml), and unrelated monoclonal antibodies were used as controls. Biotinylated goat anti-rabbit IgG or anti-mouse IgM antisera (Vector Laboratories, Burlingame, CA; diluted 1:200) were subsequently incubated for 30 minutes, followed by 15 minutes of ABC. The reaction product was developed with hy-
Oxytocin Receptors in Human Breast
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Figure 4. Sclerosinig adenosis of the breast informalin-fixed, paraffinembedded tissue. Elongated cells qf the niyoepitbelial tipe, Jbrming the vast majority of cell populationt, are OTR positive. ABC procedure, haevmalulm. counterstain, magnfihcationl, x200
drogen peroxide and diaminobenzidine for 5 minutes in the dark. Haemalum was used to counterstain nuclei. On methanol-fixed frozen sections of selected cases, we used immunoalkaline phosphatase with the alkaline phosphatase-anti-alkaline phosphatase (APAAP; diluted 1:50) method (Dako, Glostrup, Denmark). The reaction was developed using naphtholphosphate and diazotized new fuchsin.
Detection of OTR mRNA For detecting OTR mRNA, total cell RNA was extracted by the guanidinium-isothiocyanate procedure from cultured cells brought to confluence and from tissue blocks.8 Total RNA (10 to 5 ,ug) was subjected to RT-PCR amplification according to a previously published procedure.5 Amplified DNA was electrophoresed on a 2% agarose gel and directly stained with ethidium bromide or transferred to nylon membrane9 and hybridized to a 32P-end-labeled synthetic 48-bp oligonucleotide probe.5 As positive control we used total RNA extracted from uterus.
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Figure 5. Benign intraductal hyper?plasia in formalin-fixed, paraffinembedded tissue. The anti-OTR monoclonal antibody decorates the cell borders of spindle-shaped cells filling the lumen. In the surrouniding ductules, only the myoepithelial cells are OTR positive whereas coluimtzar cells are negative. ABC procedutre; magnification, X 400.
Results Rabbit sera and the monoclonal IF3 antibody were first tested by immunoblots on extracts of pregnant uterus at term. The antibody recognized a band migrating at 53 kd, corresponding to the predicted OTR sequence plus bound N-glycosylation and phosphorylation residues3 (Figure 1). By immunofluorescence on cultured cells, the antisera and the IF3 antibody raised against the aminoterminal sequence stained OTR as multiple positive spots on the cell membrane of breast cancer MDAMB231 and MCF7 cells, whereas colon carcinoma HT29 cells were negative. Specific immunocytochemical staining was also detected on paraffin sections of histologically normal mammary gland and uterus. The antisera against the carboxyl-terminal sequence were unreactive on unfixed breast cancer cells and on paraffin sections. On methanol-fixed cryostat sections of uterus and breast tissues, they showed the same reactivity of the antibodies against the amino-terminal sequence (Figures 2a and b). Preimmune and preabsorbed sera and unrelated antibodies were tested as negative controls.
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Table 1. Immunocytochemical and RT-PCR Evaluation of OTR on Breast Carcinomas and Lymph Node Metastases
ICC Case 2 3 4 5
Diagnosis
Grade
(MAb)
IDC Tubulo-lobular ILC ISDC* IDC IDC
3 1 3
-
1 3
ISDCt
6 7
8 9
10 11
12
Figure 6. Benign intraductal hyperplasia in a methanol-fixed cryostat section. 7The antiserum against the carboxyl-terminal sequence strongly stains the myoepithelial cells along the basement membrane but is also positive alonig the cell membrane of intraductal cells. APAAP procedure; magnification, X200.
All of the antibodies tested in histologically normal tissues and in mild adenosis revealed OTRs mainly on the cell membrane and in the cytoplasm of the basally located myoepithelial cells, whereas epithelial columnar cells were negative (Figure 3). In areas of sclerosing adenosis, elongated myoepithelial OTR-positive cells constituted the major cell component (Figure 4). In foci of benign intraductal and intralobular hyperplasia (subcutaneous epitheliosis), composed of branches of mixed epithelial and spindle cells delimiting peripheral slit-like spaces, elongated cells were OTR positive (Figures 5 and 6). In other benign lesions showing epithelial hyperplasia, such as papillomas and a case of phylloides tumor, besides the myoepithelial cells, also epithelial cells arranged in multiple layers showed OTR positivity along the cell membrane. The intensity of staining was lower than that of surrounding myoepithelial cells, and the reaction was more strictly located at the cell membrane. Ten out of twelve cases of carcinomas of the breast showed positive immunocytochemical staining (Table 1). Cellular heterogeneity was a common mammary
IDC LN-MTS IDC LN-MTS IDC LN-MTS IDC LN-MTS IDC LN-MTS IDC LN-MTS ILC LN-MTS
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ICC (MAb), immunocytochemical tests using the anti-OTR monoclonal antibody IF3 (tests were performed on formalin-fixed paraffin sections (cases 1 to 7) or on methanol-fixed cryostat sections (cases 8 to 12)); IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ISDC, in situ ductal carcinoma; LNMTS, lymph node metastases; ND, not done. *Cribriform type. tComedo type.
feature, although one completely negative case and cases in which the majority of the neoplastic population was OTR positive were encountered. The location was mainly on the cell membrane and at the intercellular boundaries of neoplastic cells (Figure
7). In situ carcinomatous foci were mostly positive (Figure 8). In OTR-negative areas, residual, strongly positive myoepithelial cells were detected at the periphery of the lesion along the basement membrane (Figure 9). In all of the non-neoplastic tissues, specific OTR mRNA was detected by RT-PCR. The immunocytochemical results and the RT-PCR results correlated in the twelve cases of breast carcinomas studied (Table 1). In seven of these cases, both the primary tumor and the lymph node metastases were collected for RT-PCR procedure. Four cases were positive in both primary and secondary lesions, and one case was negative in both lesions; in two cases that were positive in the primary tumor, the lymph node metastases were negative (Figure 10; Table 1). Unaffected lymph nodes, used as controls, were negative. In two cases, the fresh metastatic lymph nodes could be tested only with the RT-PCR procedure, due to the small size of the lesions, whereas the other
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Figure 8. In situ and infiltrating carcinoma of the brcast informalinfixed, paraffin-embedded tissue. Both the intraductal and the infiltratintg cell component are positively stained for 0TR. ABC procedure;
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lymph nodes were also examined by immunocytochemistry on cryostat sections using the IF3 monoclonal antibody (Figure 11). The RT-PCR data partly correlated with the immunocytochemical results (Table 1).
Discussion The sole presently acknowledged function of OT in the breast is to induce contraction of myoepithelial cells during lactation, to help milk secretion. Accordingly, OT-binding sites have been detected in myoepithelial cells by autoradiography in lactating mammary gland of the rat.6 We produced specific antibodies and developed immunocytochemical procedures to confirm our previous data on the presence of OTR in breast carcinoma cell lines,2 to localize the receptors in nonneoplastic lesions and in carcinomas of the breast, and to identify the potential cellular target of the hormone. Antisera produced against both the amino-terminal and the carboxyl-terminal sequences stained se-
lectively myoepithelial cells of the breast and other OTR-rich sites such as myometrium at term of pregnancy. However, only the sera produced against the amino-terminal sequence were positive on paraffin sections. Such a sequence was therefore selected as the reagent of choice to prepare a monoclonal antibody recognizing OTRs on routinely fixed and embedded tissues. The specificity of this antibody, proved by in vitro tests, and its extent of reactivity in other nonmammary tissues will be reported elsewhere (submitted). Antibodies, both mono- and polyclonal, raised against the amino-terminal sequence revealed multiple dot-like antigenic sites over the cell membrane of fresh (unfixed) MCF7 and MDA-MB231 breast cancer cells, whereas colon carcinoma cells were negative. This confirms and extends our reported data on the OTR gene expression in these cell lines. Antisera against the carboxyl-terminal sequence, which lays behind the cell membrane, were completely negative on the same cell preparations. The OTR in normal (nonlactating) and non-neoplastic mammary lesions were mainly located on the myoepithelial cells. This confirms our previous study
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tion.10 Interestingly, such cells are known to lack
Figure 9. In situ lesion of a lobular carcinoma in a methanol-fixed cryostat section stainzed with antiserum against the carboxyl-terminal sequtence of OTR. Only the myoepithelial cells are positive, uhereas intradu.ctal canicer cells are negative. APAAP proceduire; nuclear counterstain with haemttaluim; magtinfication, X 200.
on the effect of OT in the developing rat mammary gland1 and suggests that OT is likely to play a role not only during lactation but also in the organogenetic process and the histogenesis of different lesions. In sclerosing adenosis we observed that OTRpositive spindle cells formed the vast majority of the cell population. These findings are in line with the classical interpretation of sclerosing adenosis as related to an overwhelming myoepithelial prolifera-
estrogen and progesterone receptors, although they express epidermal growth factor receptors."1 This suggests that myoepithelial cell proliferation is likely regulated by nonsteroid hormones and that OT might interact with other mammotrophic hormones and play a role in the histogenesis of such lesions. In areas of benign hyperplasia, spindle-shaped intraductal cells were positive, in contrast with epithelial columnar cells, which were negative. This observation is fitting with that of Bocker et al,12'13 who demonstrated that these spindle cells exhibit an immunophenotype different from classical epithelial cells and likened such elongated cells to modified myoepithelial cells. Previously, Kimura et a14 detected by Northern blot procedures the presence of OTR mRNA in normal mammary tissues. We have here confirmed this report by RT-PCR procedures and extended the study to non-neoplastic tissues, to benign lesions, and to a series of primary and metastatic carcinomas of the breast. OTR gene expression was detected in all noncancer tissues and in some but not all carcinoma cases. A possible bias of the data obtained on primary tumors (ie, the possible interference of residual myoepithelial cells) was overcome by the positive results obtained in lymph node metastases. Immunocytochemical staining using anti-OTR antibodies in primary and metastatic carcinomas of the breast showed a marked heterogeneity, but definite positivity, mainly over the cell surface. A correlation was observed between the immunocytochemical
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Figure 10. Detection of 0TR mRNA by RT-PCR. Amplified DNA was rin on1 a 1.2%o agarose gel anid hYbridized to a 32P-labeled oligonotcleotide probe. Lanes a to i: DNA obtaiiedfromi hbuman tissues. Lanes a and b. pregnianit uteruis (10 anid 5 jig, respectively); lanes c anld d, invasive breast carcinoma (10 and 5 .g, respective4l); lane e, water; lanes f toi two node metastases of the same case(case 7 in Table 1; 10, 5, 10, and 5 tg, respectively). Both the primary tumor and its metastases showz OTR genie eApression.
Figure 11. Methanol-fixed cryostat section of a lymph node metastasis from carcinoma of the breast (case 8). APAAP procedure using antiOTR monoclonal LF3 antibody. A cluster of positive cancer cells is located among negative lymphocytes. Magnification, X 400.
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and the RT-PCR data. The number of cases reported here is too small to draw conclusions on the biological and clinical significance of OTRs in carcinomas of the breast, but the present study paves the way to more extensive investigations. We already reported on the inhibitory effect of OT and of the OT analogue F314 on OTR-positive human breast cancer cells.2 Recently, similar inhibitory effects were demonstrated on cell proliferation and tumor growth of experimental mammary carcinomas.14 All of these data suggest that OT should join the list of mammotrophic hormones. The involvement of OT in the origin and the evolution of non-neoplastic and cancerous lesions of the breast, both as a biological point of interest as well as a possible clinical interest, is suggested by these observations.
References 1. Sapino A, Macri L, Tonda L, Bussolati G: Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland. Endocrinology 1993, 133:838-842 2. Cassoni P, Sapino A, Negro F, Bussolati G: Oxytocin inhibits proliferation of human breast cancer cell lines. Virchows Arch 1994, 425:467-472 3. Arnaudeau S, Lepretre N, Mironneau J: Chloride and monovalent ion-selective cation currents activated by oxytocin in pregnant rat myometrial cells. Am J Obstet Gynecol 1994, 171:491-501 4. Kimura T, Tanizawa 0, Mori K, Brownstein MJ, Okayama H: Structure and expression of a human oxytocin receptor. Nature 1992, 356:526-529 5. Takemura M, Nomura S, Kimura T, Inoue T, Onoue H, Azuma C, Saji F, Kitamura Y, Tanizawa 0: Expression and localization of oxytocin receptor gene in human uterine endometrium in relation to the menstrual cycle. Endocrinology 1993, 132:1830-1835
6. Soloff MS, Rees HD, Sar M, Stumpf WE Autoradiographic localization of radioactivity from 3H oxytocin in the rat mammary gland and oviduct. Endocrinology 1975, 96:1475-1 477 7. Hsu SM, Raine L, Fanger H: The use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase technique. J Histochem Cytochem 1981, 29:577-580 8. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162:156-159 9. Reed KC, Manor DA: Rapid transfer of DNA from agarose gel to nylon membranes. Nucleic Acid Res 1985, 13:7207-7221 10. Hamperl H: The myothelia (myoepithelial cells): normal state; regressive changes; hyperplasia; tumors. Curr Top Pathol 1970, 53:161-220 11. van Agthoven T, Timmermans M, Foekens JA, Dorssers LCJ, Henzen-Logmans SC: Differential expression of estrogen, progesterone, and epidermal growth factor receptors in normal, benign, and malignant human breast tissues using dual staining immunohistochemistry. Am J Pathol 1994, 144:1238-1246 12. Bbcker W, Bier B, Freytag G, Brbmmelkamp B, Jarach E-D, Edel G, Dockhorn-Dworniczak B, Schmid KW: An immunohistochemical study of the breast using antibodies to basal and luminal keratin, a-smooth muscle actin, vimentin, collagen IV and laminin. I. Normal breast and benign proliferative lesions. Virchows Arch A 1992, 421:315-322 13. Bocker W, Bier B, Freytag G, Brbmmelkamp B, Jarach E-D, Edel G, Dockhorn-Dworniczak B, Schmid KW: An immunohistochemical study of the breast using antibodies to basal and luminal keratin, a-smooth muscle actin, vimentin, collagen IV and laminin. II. Epitheliosis and ductal carcinoma in situ. Virchows Arch A 1992, 421:323-330 14. Cassoni P, Sapino A, Papotti M, Bussolati G: Oxytocin and oxytocin-analogue F314 inhibit cell proliferation and tumor growth of rat and mouse mammary carcinomas. Int J Cancer 1996 (in press)
