(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
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 Â±1.6 nmol/liter, Aâ„¢,= 27,300 Â±6,500 sites/cell;
oxytocin: KD= 51.3 Â±0.4 nmol/liter, B^ = 87,000 Â±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.
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.
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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 37Â°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
(37Â°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,
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; /?â€žâ€ž,
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.
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