Interaction of Vasopressin and Oxytocin with Human Breast ... oxytocin: KD= 51.3 £â€¢±0.4 nmol/liter,

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  • (CANCER RESEARCH 50, 7882-7886, December 15, 1990]

    Interaction of Vasopressin and Oxytocin with Human Breast Carcinoma Cells A. H. Taylor, V. T. Y. Ang, J. S. Jenkins, J. J. Silverlight, R. C. Coombes, and Y. A. Luqmani1

    Divisions of Biochemical Medicine [A. H. T., K T. Y. A., J. S. J., J. J. S.] and Medical Oncology [R. C. C., Y. A. L.], and Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, England

    ABSTRACT

    The arginine vasopressin and oxytocin content of normal and cancerous human breast tissue were measured using radioimmunoassay. Both pep- tides were present in amounts greater than that found in the circulation, but no difference between normal and malignant tissues was found. Binding of (3H|oxytocin and (3H|vasopressin were characterized in human

    breast carcinoma cells (MCF7 cells). Binding of both hormones to MCF7 cells was specific and saturable, the vasopressin receptor found to be of the V, subtype. Scatchard analyses of the data were linear, indicating a single high affinity, low capacity binding site for each hormone (vaso- pressin: KD = 47.4 ±1.6 nmol/liter, A™,= 27,300 ±6,500 sites/cell; oxytocin: KD= 51.3 ±0.4 nmol/liter, B^ = 87,000 ±4,000 sites/cell). The effects of vasopressin and oxytocin on the growth of MCF7 cells were assessed using protein accumulation and cell numbers. Vasopressin at 10-1000 pmol/liter was mitogenic for MCF7 cells, but higher doses (10 nmol/liter) were growth inhibitory. Oxytocin was also mitogenic for MCF7 cells but to a lesser extent than vasopressin. In conclusion, we suggest that vasopressin and possibly oxytocin may be important modu lators of the growth of some human breast carcinomas.

    INTRODUCTION

    Vasopressin and oxytocin are closely related nonapeptides synthesized as part of larger neurophysin molecules in the hypothalamus and secreted from the pituitary (1). In addition to their classical roles in kidney function and smooth muscle contraction, recent observations have shown a wide variety of activities for both peptides in the central nervous system, as possible modulators of adrenocorticotrophic hormone and pro- lactin secretion, maternal behavior, and memory acquisition (2-6). They are also found in peripheral tissues where they may have modulating activities (7, 8).

    The normal breast is generally recognized as being a target organ for oxytocin in the process of milk ejection, but the influence of the hormone on breast carcinoma is not known (9). In the case of arginine vasopressin, involvement in breast function and growth is less certain (10). Receptors for oxytocin have been reported in normal rat and bovine mammary tissue (11, 12). Receptors for vasopressin have been described and characterized in the WRK1 rat mammary tumor cell line (13, 14), and vasopressin has been shown to increase lipid synthesis and protein accumulation by WRK1 cells (15).

    In the present study we have investigated the presence of oxytocin and vasopressin in normal and malignant human breast tissue and have sought evidence for the existence of specific receptors for both hormones in the human MCF7 breast carcinoma cell line. In an attempt to establish whether these observations have any functional relevance we have also studied the effects of vasopressin and oxytocin on the growth of MCF7 cells in vitro.

    MATERIALS AND METHODS

    Extraction of Oxytocin and Vasopressin from Breast Tissue. Tissue from carcinoma of the breast was obtained at the time of operation and

    Received 4/9/90; accepted 7/13/90. The costs of publication of this article were defrayed in part by the payment

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    ' To whom requests for reprints should be addressed.

    from normal breast during the surgical treatment of breast reduction. All samples were histologically assessed. No macroscopically discerni ble differences were apparent between the normal breast tissues used, fibrous areas being avoided. All tumors were diagnosed as intraductal carcinomas and were of approximately equivalent cellularity and did not differ significantly in the degree of stromal invasion. Samples were frozen immediately in liquid nitrogen. Prior to extraction, tissue was thawed out in small pieces and homogenized in 1 mol/liter acetic acid. Fat was removed by centrifugation at 700 x g. The aggregates of tough stromal material were removed by freezing rapidly on dry ice and allowing to thaw before being centrifugea at 2000 x g for 20 min. Oxytocin and vasopressin were extracted from the supernatant with Sep-Pak CIS cartridges (Waters Associates, Northwich, Cheshire, Eng land) and assayed as described previously (16) using the first interna tional standard for vasopressin (75/501) and the fourth international standard for oxytocin (76/575). The intraassay coefficients of variation at the level of 1-10 fmol/tube were 6.5% for the oxytocin assay and 7.5% for vasopressin. Interassay coefficients of variation were 12.8% for oxytocin and 13.0% for vasopressin. The limits of detection were 0.6 fmol/tube for oxytocin and 0.3 fmol/tube for vasopressin. An aliquot of the extract was taken for protein measurement (17).

    Characterization of Oxytocin and Vasopressin in Breast Tissue. Tissue extracts were lyophilized and subjected to HPLC2 as described before

    (18). The HPLC profiles were compared with those of authentic oxy tocin and vasopressin.

    Culture of MCF7 Cells. MCF7 cells were maintained in DMEM (Gibco, Ltd., Paisley, Scotland) supplemented with 10% fetal calf serum (batch 2044 or 1981; Tissue Culture Services, Slough, Berkshire, United Kingdom), penicillin (5000 units/ml; Flow Laboratories, Ltd., Rick- mansworth, Hertfordshire, United Kingdom), streptomycin (15 mg/ ml; Flow Laboratories) in monolayer cultures at 37°Cin humidified

    air/5% CO2 atmosphere, in T75 flasks (Flow Laboratories). Binding Studies on MCF7 Cells. Cells from stock were removed from

    the flasks by scraping with a "policeman" washed once with warm (37°C)Locke's solution consisting of 154 mmol/liter NaCl, 5.6 mmol/

    liter KC1, 5.6 mmol/liter glucose, 5 mmol/liter 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid buffer, 40 mg/liter gentamycin, 200,000 units/liter penicillin, 2 g/liter BSA, and where appropriate 1 mmol/ liter MgCl2 and 2.3 mmol/liter CaCl2. Cells were resuspended in this solution to give approximately IO6 cells/assay tube. All assays were carried out in triplicate at 4"(" and at pH 7 for 3 h in a final assay

    volume of 400 Mi- Cells were incubated with [3H]vasopressin (specific activity, 60.3 Ci/

    mmol) or [3H]oxytocin (specific activity, 36.6 Ci/mmol), obtained from

    New England Nuclear, Stevenage, Hertfordshire., United Kingdom in the presence of increasing concentrations of unlabeled ligands. At the end of the incubation period the tubes were placed on ice for 5 min and diluted with 1 ml of ice-cold Locke's/BSA solution. The cells were

    separated by rapid filtration on Whatman GF/B glass microfilters (BDH, Ltd., Dagenham, Essex, England), quickly washed twice with ice-cold Locke's/BSA solution (2 ml), and transferred to scintillation

    vials for counting. Nonspecific binding was determined by incubating cells with labeled peptide in the presence of an excess (100 /imol/liter) of unlabeled hormone. The V|-vasopressin antagonist rf(CH2)5 Tyr(Me)2 AVP was kindly supplied by Dr. M. Manning, Ohio, and the V2agonist l-deamino-8-D-AVP was supplied by Ferring AB, Malino, Sweden.

    Studies on Growth of MCF7 Cells. Cells in stock culture were grown

    2The abbreviations used are: HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; /?„„, maximum number of binding sites; AVP, arginine vasopressin; d(CH2)5Tyr(Me)2 AVP, d-(CH2), tyrosine(methyl)2 AVP; DDAVP, l-deamino-8-D-AVP.

    7882

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  • VASOPRESSIN AND OXYTOCIN IN HUMAN BREAST

    until confluent and removed with trypsin-EDTA (0.5 g/liter trypsin- 0.2 g/liter EDTA) and replated on 35-mm Petri dishes in DMEM with 2.5% fetal calf serum at an initial density of 3 x 10* or 9 x 10" cells/

    dish. After 72 h the medium was changed to one with or without the addition of vasopressin or oxytocin. Each day the cells were removed from the plates using 500 nl of trypsin/EDTA with vigorous pipeting and counted using a Coulter Counter.

    Studies on Protein Accumulation by MCF7 Cells. Stock MCF7 cell cultures were removed from the flasks with trypsin/EDTA and replated onto multiwell plates (Falcon, Marathon Laboratories, London, United Kingdom). Cells were grown for 7 days in DMEM supplemented with 2.5% fetal calf serum. The medium was changed to one without serum but containing BSA (1 g/liter) and either vasopressin or oxytocin. After 24 h the cells were placed on ice, washed once with Dulbecco's phos

    phate-buffered saline (Gibco), and solubili/.cd with 500 p\ of 0.5 mol/ liter NaOH. Protein concentration was measured by the method of Lowry et al. (17) using BSA as standard.

    Studies on the Phosphoinositide Metabolism by MCF7 Cells. To study the effect of vasopressin and oxytocin on the hydrolysis of phosphoinositides, cells in monolayer culture were incubated with myo- [3H]inositol, stimulated with peptides and the water-soluble inositol phosphates separated from nonincorporated myo-[3H]inositol using Dowex ion-exchange chromatography, as described previously (19).

    RESULTS

    Oxytoci