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Department of Biomedical Sciences and Oncology (P.C., A.S., G.B.), Department of Animal and Human Biology and National Institute for the Physics of Matter (L.M.), and Laboratory of Cell Biology, Department of Genetics (S.D.), University of Turin, 10126 Turin, Italy; Department of Medical Sciences, University of Novara (A.G.), 28100 Novara, Italy; Consiglio Nationale per le Ricerche Cellular and Molecular Pharmacology Center (B.C.), 20129 Milan, Italy; and Department of Reproductive and Vascular Biology, Division of Reproductive and Child Health, University of Birmingham (A.A.), Birmingham Women’s Hospital, Birmingham, B15 2TG, United Kingdom
Address all correspondence and requests for reprints to: Dr. Gianni Bussolati, Department of Biomedical Sciences and Oncology, University of Turin, Via Santena 7, 10126 Turin, Italy. E-mail: bussola{at}molinette.unito.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies we observed that OT acts as a negative regulator of cell proliferation in different neoplastic tissues, including human breast carcinomas (10), rodent mammary tumors (11), human nervous tumors (12), and human osteosarcoma cell lines (13), all of which express OTR. Human endometrial carcinomas also express OTR, and OT inhibits the proliferation of endometrial cancer cells (14). This OT-induced antiproliferative effect is coupled to nonconventional OT signaling, activated via the cAMP-protein kinase A pathway (12, 14, 15). Other researchers also suggested that OT could activate this unusual pathway in breast cancer cells (16). The evidence of a growth-regulating effect of OT has also been demonstrated in human endothelial cells (17) and human uterine smooth muscle cells (18). In these cases, however, OT exerts a positive, stimulating effect on cell proliferation that is calcium (17, 18) and protein kinase C dependent (17).
As the expression of OTR at the feto-maternal interface has never been correlated to the growth-regulating properties of their natural ligand, we decided to investigate the possible role of OT as a trophoblast growth factor under both physiological and neoplastic conditions. In this study we demonstrate the expression and functionality of OTR in human normal and neoplastic trophoblast-derived cell lines, and we present experimental evidence that the OTR-OT system may play an important role in regulating the proliferation of human trophoblast and choriocarcinoma cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
Total RNA was extracted according to the guanidine
thiocyanate-cesium method (21) from ED27, ED77, and BeWo
cells. The concentration of RNA was estimated by spectrophotometry, and
RNA degradation was monitored by agarose gel electrophoresis.
Oligonucleotide primers were designed to amplify a 391-bp fragment of
human OTR complementary DNA according to the method of Takemura
et al. (6).
Five micrograms of RNA obtained from each cell line were reverse transcribed in 20 µl reaction mixture containing 200 U Superscript reverse transcriptase (Life Technologies, Inc.) and 84 pmol antisense primer for OTR. Each reaction product was then amplified by PCR buffer containing 2.5 U Polytaq Taq DNA polymerase (Polymed, Sambuca, Firenze, Italy) and 12.5 pmol each of the specific primer pair for OTR.
After 20 cycles of amplification, 25 µl of each reaction product were electrophoresed, stained with ethidium bromide on 1% agarose gels, and transferred onto nylon membranes by vacuum blotting. MCF7 breast carcinoma cells and HT29 colon carcinoma cells were used as positive and negative controls, respectively.
Southern blot analysis was performed to further test the specificity of the RT-PCR product. Membranes were hybridized at 42 C overnight with 25 pmol digoxigenin-labeled OTR oligonucleotide probe. The membranes were then washed with 2 x SSC (standard saline citrate)-0.1% SDS for 10 min at room temperature and with 0.5 x SSC-0.1% SDS for 30 min at 42 C. Digoxigenin-labeled specific hybrids were visualized using an immunological detection system (Roche Molecular Biochemicals, Mannheim, Germany) employing antidigoxigenin antibodies conjugated with alkaline phosphatase. Detection was performed using chemiluminescent substrate disodium 3-(4-methxyspiro;1,2-dioxetane-3,2-(5-chloro)tricyclo(3.3.1.3,7)decan;-4-yl)phenylphosphate CSPD (Roche Molecular Biochemicals) according to the manufacturer’s instructions. All blots were exposed to x-ray films with intensifying screens at room temperature for 3 h.
Immunofluorescence and flow cytometry
In a standard immunofluorescence procedure, ED27, ED77, and BeWo
cells were grown on glass coverslips for 5 days. After washing in PBS,
cells, either unfixed or fixed in methanol for 5 min at -20 C, were
incubated at room temperature for 30 min with a primary monoclonal
antibody (MAb) to human OTR (clone IF3) (22) diluted 1:20
in PBS. Cells were then rinsed in PBS and finally incubated for 30 min
at room temperature with the appropriate fluorescein-labeled secondary
antiserum (Sera-Lab Ltd., Sussex, UK) diluted 1:20 in PBS. The reaction
was evaluated with a Leitz Orthoplan fluorescence
microscope equipped with Xenon lamp and epifluorescence apparatus
(Leitz, Rockleigh, NJ). An unrelated primary MAb (Common
Leukocytic Antigen, DAKO Corp., Glostrup, Denmark) as well
as omission of primary antibody were used as negative controls.
The percentage of positive cells was evaluated by flow cytometry on both unfixed and fixed cells. Cells (5 x 106/ml) were suspended in PBS and incubated for 30 min with anti-OTR IF3 MAb and with the secondary fluorescein isothiocyanate-labeled antibody, as described for the immunofluorescence procedure. The percentage of fluorescent cells was then analyzed using a FACSort (Becton Dickinson, San Jose, CA).
OT binding
Binding assays were performed on crude membrane preparations;
briefly, cells were homogenized in a glass potter, washed twice, and
resuspended in the binding buffer (50 mM Tris-HCl and 5
mM MgCl, pH 7.4). Membrane proteins (10 mg) were incubated
with increasing concentrations of [125I]OT
antagonist (10–800 pM) for 60 min at 30 C. Nonspecific
binding was determined in the presence of 1 mM OT for each
[125I]OT antagonist concentration point. Bound
and free radioactivities were separated by filtration over
Whatman GF/C filters (Clifton, NJ) presoaked in 10 mg/ml
bovine serum. Data were fitted with a binding isotherm curve
(PRISM version 3.0, GraphPad Software, Inc., San Diego,
CA).
Calcium measurements
ED 27, ED77, and BeWo cells grown on glass coverslips were
treated alternatively with OT at different concentrations (1
nM to 10 µM), AVP (1 µM),
selective OT agonist [Thr4]OT (1
µM), and OT antagonist (1 µM). In selected
experiments EGTA was used to chelate extracellular calcium. The
intracellular calcium levels were evaluated as follows.
The cells were loaded with the acetoxymethyl ester form of indo-1
(Molecular Probes, Inc., Eugene, OR; incubation for 45 min
with 2.5 µM at 37 C). The medium was then replaced with
standard Tyrode solution, and the coverslips were placed on an inverted
IM-35 Carl Zeiss microscope (New York, NY) with a x100
fluorescence objective. Diaphragms were used to observe single cells.
Fluorescence signals were taken at an excitation wavelength of 380 nm
and emissions of 400 and 480 nm using a spectrophotometer from Cairn
Ltd. (Newnham, UK). Intracellular calcium
([Ca2+]i) values were
calculated according to the formula:
[Ca2+]i =
Kd 
(R -
Rmin/Rmax - R)
(23), where R is the ratio of the cellular fluorescence
signals (R =
F400/F480),
Kd is assumed to be 820 nM, is
defined as F480, zero Ca/F480,
saturating Ca, and Rmax,
Rmin, and were obtained by calibrations using
the calcium ionophore ionomycin (Sigma, St. Louis, MO).
Background and autofluorescence corrections were performed before each
experiment. All experiments were carried out at 22–24 C.
Tyrosine phosphorylation
ED27, ED77, and BeWo cells were serum-starved overnight and then
treated with 100 nM OT for 20 min. Control and OT-treated
cells were lysed in Laemmli solubilization buffer as previously
described (24). For each sample, 50 µg solubilized
proteins were separated on an 8% SDS-PAGE, transferred to
polyvinylidene difluoride paper (Amersham Pharmacia Biotech, Aylesbury, UK), and then decorated with
antiphosphotyrosine antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) as previously described
(24). The changes in tyrosine phosphorylation were
quantified by the Chemi Doc System using Quantity One software
(Bio-Rad Laboratories, Inc., Hercules, CA).
Cell proliferation
ED27, ED77, and BeWo cells were seeded in triplicate in
24-multiwell plates at a density ranging from 5000–8000 cells/ml. To
evaluate the effect of OT on cell proliferation, 24 h after
plating OT was added to culture medium at concentrations ranging from
10 nM to 1 µM. The medium was changed every
48 h. At 48 and 96 h of culture, cells were counted in double
blind by two independent investigators using a hemocytometer. Each
experiment was repeated three times. EGF (20 ng/ml) was added to
culture medium of ED27 cells and used as a positive control for its
known promoting effect on trophoblast cell growth. HT29 colon carcinoma
cells were used as a negative control.
Statistical analysis was carried out using ANOVA followed by Bonferroni correction. The cut-off for significance was 0.05.
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Results |
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, lanes 1, 3, and 4) as well as MCF7
cells (positive control, lane 2), showed a 391-bp signal corresponding
to OTR mRNA. Colon carcinoma cells (HT29) were used as a negative
control (Fig. 1A, lane 5).
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, choriocarcinoma BeWo cells; normal trophoblast ED27
and ED77 cells not shown).
By flow cytometry, OTR positivity was evident in 93% of ED77 cells,
92% of ED27 cells, and 78% of BeWo cells. No signal was detected when
the primary MAb was omitted or the unrelated MAb was used (Fig. 1C).
OT binding
Specific binding was detected in ED27, ED77, and BeWo cells tested
with the specific radiolabeled [125I]OT
antagonist (not shown). Saturation studies performed on ED77 cells
showed a Kd of 247 ± 77 pM
(n = 3) for [125I]OT antagonist, a value
comparable to those shown by other OTR-expressing cells (25, 26), and a calculated binding capacity of approximately 25
± 12 fmol/mg protein (three independent experiments). This level of
expression is similar to that recently measured in breast cancer cells
in culture (26).
Intracellular Ca2+
Application of OT in ED27, ED77, and BeWo cell lines induced a
slow, persistent, and dose-dependent increase in cytosolic free
[Ca2+]i (total of 60
cells; Fig. 2, A–C). The addition of 10
mM EGTA in the bath during the response induced a rapid and
complete decline of
[Ca2+]i to the resting
levels in all of the cell lines, showing that a calcium entry was
involved in the response (Fig. 2D). EGTA application failed to induce
any effect on [Ca2+]i in
resting conditions. Only in 2 of 60 cells tested could a release from
intracellular stores be detected (not shown). In ED27 cells the
application of 1 µM [Thr4]OT, an
OTR-selective agonist (20), caused a similar OT response
(Fig. 2E). Cell perfusion with 1 µM AVP did not trigger
any [Ca2+]i increase
(Fig. 2F). In contrast, the selective antagonist of OTR (1
µM) inhibited the response triggered by 1
µM OT in ED27 and BeWo cells (Fig. 2, G and H).
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). The increased band
intensity was quantified by the Chemi Doc system and Quantity
One software (+19%, +13%, and +20% after OT treatment for the 125-,
60-, and 45-kDa bands, respectively). In both ED27 and ED77 normal
trophoblast cells, two proteins of 125 and 60 kDa became tyrosine
phosphorylated after OT treatment (+31% and +19% for the 125- and
60-kDa bands, respectively; -2% for the 45-kDa band; Fig. 3).
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)
and fully abolished by the incubation with the selective OTR
antagonist, which inhibited cell proliferation when used alone (OT
antagonist vs. control at 48 h: 1, P <
0.05; Fig. 3C).
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, A and B) increase in cell number. Treatment with 1
µM OT produced 200% and 270% increases in
ED77 and ED27 cell number, respectively. Addition of the OTR antagonist
completely abolished the OT effect. The use of OT antagonist alone
reduced cell growth at all time points (1, P <
0.05). The addition of EGF to culture medium brought about a significant increase in ED27 cell number compared with that under basal conditions. The increase, however, was only 15% more than that caused by 1 µM OT (not significant; data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our experiments OT stimulated cell proliferation through a selective activation of OTR that led to an increase in intracellular [Ca2+]i and tyrosine phosphorylation. The OT effects on calcium uptake were dose dependent and were completely abolished by simultaneous incubation with the selective OT antagonist, which inhibited cell proliferation when used alone. Moreover, the treatment with [Thr4]OT, a selective OTR agonist, evoked a response similar to OT, whereas no effect on the [Ca2+]i increase was observed after AVP perfusion. The lack of effect on the [Ca2+]i increase after AVP perfusion ruled out the possibility of a contribution of other receptors of the AVP/OT receptor family, namely the V1a, V1b, and V2 subtypes (27), through which OT may exert a pharmacological effect, although with a lower efficacy with respect to AVP.
OTR are coupled to both
G
q and
Gi subunits, which
mediate the triggering of a number of intracellular signaling pathways,
including protein kinase C activation (28). Protein
tyrosine phosphorylation is an early event of the proliferative
signaling triggered by growth factors. Recent reports suggested an
involvement of protein tyrosine phosphorylation in OT signal
transduction, as a tyrosine kinase inhibitor partially inhibits the
OT-induced calcium increase (28). The mechanism by which
OT stimulates tyrosine phosphorylation has not been elucidated, but may
be mediated by G
dissociating from Gi
(28). Tyrosine phosphorylation in OT signaling contributes
to activate both p38 mitogen-activated protein kinase and extracellular
signal-regulated kinase 2 or to participate in the intracellular
calcium increase (5, 28). Here we investigated protein
tyrosine phosphorylation induced by OT in both normal trophoblast and
choriocarcinoma cell lines. Interestingly, in both cell types we
observed a significant increase in tyrosine phosphorylation of two
proteins of 125 and 60 kDa. In BeWo choriocarcinoma cells OT treatment
also determined the phosphorylation of a 45-kDa protein. Although we
have not identified these proteins, we can suggest from their mol wt
that the first one is FAK or its related protein Pyk2. Tyrosine
phosphorylation of FAK has indeed been reported to occur upon OT
stimulation of CD8+ T lymphocytes
(29). It is possible to speculate that stimulation of FAK
and Src tyrosine kinase, which is common to several growth factors, may
contribute to the proliferative activity of OT reported here.
Very few observations have been previously reported on the possible role of OT as a factor regulating cell proliferation, and they were controversial. In previous studies on breast (10, 11, 15), nervous (12), and endometrial (14) tumor cell lines, we observed that OT inhibited cell proliferation and that this inhibitory effect was mediated via the cAMP-protein kinase A pathway, whereas [Ca2+]i and inositol phosphate were not involved. Identical demonstration of lack of an OT-dependent increase in intracellular calcium in breast cancer cells was reported by others (16).
Beside this evidence in neoplastic cells, in a recent paper Thibonnier et al. (17) documented that OTR are present in human vascular endothelial cells and that OT stimulates endothelial cell proliferation in vitro. This trophic effect is coupled to the cytosolic calcium increase and protein kinase C response, the conventional OT signaling (17). Similarly, it has been reported that OT induces the hyperplasia of human uterine smooth muscle cells in vitro through the increase in [Ca2+]i (18).
The discrepancies observed on the biological effects of OT on cell proliferation (inhibition or stimulation) and the specific coupling between signaling and effect on cell growth (Ca2+ together with increased proliferation; cAMP together with decreased proliferation) indicate that OT may activate two intracellular distinct pathways. The choice of one or the other pathway may be explained by either a cell type specificity or the activation of different OTR subtypes or even the AVP receptors, including V2 receptor subtype and its intracellular signaling, involving cAMP (27).
The data presented here are consistent with the observations reported in endothelial (17) and smooth muscle uterine cells (18), as the cellular proliferation of normal and neoplastic trophoblast cells lines is stimulated by OT through calcium and tyrosine phosphorylation. The relatively high doses of OT required to stimulate cell proliferation may depend on the presence of OT-degrading enzyme produced by trophoblast cells (30).
In conclusion, our data demonstrate that functional OTR are present in both human normal trophoblast and choriocarcinoma cell lines and that through these binding sites OT stimulates cell proliferation. The future validation of these observations on fresh human placental tissue could definitely indicate whether OT should be considered a true growth factor for trophoblast-related tissues. Even though previous studies elucidated the role of OT at the feto- maternal unit in relation to the timing of labor and parturition (3, 6, 7, 8), no data were provided before on other biological effects of OT in either normal or neoplastic trophoblast-derived cells. Interestingly, as it has been reported that OT is produced at that site through a paracrine/autocrine loop (8), it may act locally to promote cell proliferation. These data, therefore, suggest new implications for the pathogenesis of chorion-derived tumors and open new perspectives on a possible therapeutic role for OT agonists and antagonists.
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Footnotes |
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Received August 31, 2000.
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