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Laboratories of Virology and
* Molecular Pathology and Ultrastructure, Regina Elena Cancer Institute, 00158 Rome, Italy
1Correspondence: Laboratory of Virology, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, 00158 Rome, Italy. E-mail venuti{at}ifo.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSREFERENCES |
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Key Words: ETAR antagonists • genital tumors • tumor growth • papillomavirus • autocrine loop
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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. Human cervical epithelial
cells are a primary target for the infection by the oncogenic
papillomaviruses (HPV16 and 18), which are strongly implicated as
causative agents in the development of cervix carcinoma (2
, 3)
.
HPV16 transfection of epithelial cells may cause a specific increase in
the expression levels of growth factors and their receptors, such as
the epidermal growth factor (EGF), leading an enhanced sensitivity to
growth stimulation (4
, 5)
.
Various growth factors can be produced by epithelial cells influencing
the neighboring cells and, in some instances, acting as autocrine
growth factors, including EGF, nerve growth factor, basic fibroblastic
growth factor, tumor growth factor alpha, and endothelin
(6
7
8
9)
.
The family of endothelins (ETs), named ET-1, ET-2, and ET-3, are
21-amino acid peptides originally identified as potent vasoconstrictors
(10)
. They act through at least two major receptor
subtypes, belonging to the G-protein-coupled family receptors:
ETA receptor, which binds ET-1 and ET-2 with high
affinity and ET-3 with low affinity; and ETB
receptor, which nonselectively binds all ET isopeptides with equal
affinity (11)
. ET-1 is a potent vasoactive peptide
synthesized in vascular endothelial cells and nonvascular tissues. ET-1
acts on cell proliferation directly or synergistically with other
growth factors implicated in cancer progression. Several human cancer
cells and human tumors release ET-1, and it has been proposed that ET-1
participates in tumor development or progression by exerting autocrine
or paracrine action on neoplastic and surrounding stromal cells
(12)
.
Human keratinocytes express ETA receptors and
produce ET-1, which stimulates growth response (9)
.
Recently, a new mechanism in the growth control of immortalized
keratinocytes has been reported. ET-1 is released from HPV transfected
keratinocytes and induces increased growth response in these cell lines
in comparison with normal cells. All HPV transfected cell lines secrete
ET-1 and exhibit a twofold increase in ETA
receptors compared to parental cells in the absence of recycling
alteration (13)
. HPV transfected keratinocytes can
proliferate in the absence of any growth factor and their growth is
completely prevented by a specific ETA receptor
antagonist (14)
. These data demonstrate that selective
growth of HPV-infected cells is up-regulated by the ET-1 autocrine
loop. In the present study, we tested the hypothesis that in
HPV-associated cervical cancer cells the expression of ET-1 and its
receptors could play a pathophysiological role. Therefore, we evaluated
ET-1 production at the mRNA and protein levels and the subtype receptor
expression in human normal and cervical cancer cells. New therapeutic
strategies against HPV-infected and transformed cells need studies of
the inhibition of the autonomous growth of tumor cells. Therefore, we
evaluated the receptor subtype mediating the mitogenic effects in the
HPV-associated cancer cells and investigated whether specific ET-1
antagonists may inhibit the autocrine growth of cervix cancer cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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.
ET-1 extraction
The conditioned media were acidified with 0.1% trifluoroacetic
acid (TFA) and the supernatant was applied to Sep-Pak C18 cartridges.
After elution with 2 ml 60% acetonitrile/0.1% TFA, the material was
lyophilized and analyzed for ET-1 content by radioimmunoassay (RIA).
The recovery of synthetic ET-1 through the extraction procedure was
78%.
Radioimmunoassay
The lyophilized ET-1 samples were reconstituted in the assay
solution consisting of 0.1 M phosphate buffer (pH 7.4) containing 0.05
M NaCl, 0.1% bovine serum albumin (BSA), 0.1% Triton-X100, and 0.01%
sodium azide. Samples were equilibrated for 24 h at 4°C with
specific ET-1 antibody (Peninsula Lab., Belmont, Calif.), followed by
the addition of 20,000 cpm of [125I]ET-1 (2200
Ci/mmol; DuPont NEN, Wilmington, Del.). After incubation for additional
48 h, free and antibody-bound tracers were separated by the
addition of sheep antiserum to rabbit immunoglobulin and incubated for
another 2 h at room temperature. The cross-reactivity of the
antiserum for ET-related peptides (vs. ET-1=100%) was ET2 = 7%,
ET3 = 7%, and big-ET-1 = 17%. The sensitivity of the ET-1 RIA
was 1.9 pg/tube and the 50% intercept was 36 pg/tube. The infra- and
interassay variations were 8% and 13%, respectively.
Binding assay
Cells were cultured in 6-well plates until confluent
(8x105 cells/well) and then growth factors were
starved for 24–48 h. After a wash with the assay buffer composed of
Hank’s balanced salt solution, 0.2% BSA, and 100 µg/ml bacitracin,
cells were incubated in 500 µl assay buffer containing 40 pM
[125I]ET-1 for time periods ranging from 0 to
180 min at temperatures of 4°C, 22°C, and 37°C. For equilibrium
binding studies with [125I]ET-1, cells were
incubated at 22°C for 60 min with increasing concentrations of the
radioactive tracer in the presence or absence of an excess (1 µM) of
unlabeled ET-1 (Peninsula Lab, Belmont, Calif.). At the end of
incubation, cells were washed three times with cold phosphate-buffered
saline (PBS) solution containing 0,2% BSA, then solubilized with 1 M
sodium hydroxide and analyzed for bound radioactivity in a 
spectrometer. The nonspecific binding, determined in the presence of
10-6 M unlabeled peptide, was 5% of the added
radioligand.
RNA extraction and reverse transcriptase-polymerase chain reaction
(RT-PCR)
Total RNA was extracted and purified from cell cultures by a
commercial kit RNAzol (Biotecx Lab, Houston, Tex.) that uses
the guanidinium-thiocyanate method. One microgram of RNA was reverse
transcribed to cDNA and amplified by the RNA PCR Kit (Perkin Elmer Co.
Oak Brook, Ill.) according to the manufacturer’s instructions with the
specific primers for GAPDH, ET-1, and ET receptor mRNAs described by
Pekonen et al. (16)
. Briefly, reverse transcription was
carried out at 42°C for 15 min with the downstream primers.
Incubation at 99°C for 5 min and then chilling in ice stopped the
reaction. The cDNA was amplified with 2.5 U Amplitaq (Roche, Nutley,
N.J.) for 30 cycles in the presence of up- and downstream primers. In
all experiments two control reactions, one containing no RNA and
another containing no reverse transcriptase, were included. The
specificity of the amplified products was verified by the presence of
products of the expected size and by molecular hybridization with the
digoxigenin-labeled internal oligonucleotide probe.
Thymidine incorporation assay
Cells were seeded in 96-well plates (2x104
cell/well) and incubated in growth factor-free medium for 24 h.
Selected concentrations of ET agonists and/or antagonists (Peninsula
Lab.) and EGF (Collaborative Biomedical Products, Bedford, Mass.) were
then added; after 18 h, 1 µCi
[3H]thymidine (6.7 Ci/mmol; DuPont, NEN) was
added to each well. Six hours later the culture media were removed and
cells were washed three times with PBS, fixed with 10% trichloroacetic
acid, washed twice with 100% ethanol, and solubilized with 0.4 N NaOH.
The cell-associated radioactivity was then determined by liquid
scintillation counting.
Growth curve
Cells were seeded in a 96-well plates at a density allowing an
exponential growth rate for the following 5 day incubation
(2x104 cells/well). After overnight incubation,
the medium was replaced with a serum-free medium containing the ET-1
antagonists. At 24 h intervals the cellular monolayers were
stained with methanol/1% crystal violet. The cell density was
evaluated by reading O.D. of the eluted dye at 540 nm. Eight replicas
for each point were assayed.
Statistical analysis
All statistical analyses were performed by the INSTAT software
system. The number of binding sites and the Kd
values were determined by using the LIGAND program.
RESULTS
Production of ir-ET-1 by normal and tumor cervical cell lines
The time course release of immunoreactive ET-1 (ir-ET-1) into the
medium was measured in all cell lines examined. The production of ET-1
was detectable as early as 2 h after replacement of the culture
medium in all cell lines but the C33A. In this cell line, over a period
of 48 h no detectable ET-1 was present in the medium. The ET-1
release increased over a 48 h period for HaCaT and Caski cells,
with a maximum of production for tumor cells that was twofold over the
normal epithelial HaCaT cells (90 fmol/106 cells
vs. 40 fmol/106 cells at 48 h). The release
of ET-1 by SiHa cells was at a lower level and decayed early during the
first 10 h (Fig. 1
).
|
Expression of ET receptors in cervical cancer cells
The ET receptor saturation studies showed that
[125I]-labeled ET-1 was saturable in all three
cervical cancer cell lines and in normal epithelial cells (HaCaT). The
approximated Kd ranged from 0.02 to 0.11 nM in the
HPV-positive cell lines, clearly indicating the presence of
high-affinity receptors (Table 1
). HPV-associated carcinoma cell lines (CaSki and SiHa) showed an
increased number of binding sites (5478±148 and 4020±92,
respectively) compared to the normal cell line HaCaT (1647±82). On the
contrary, the high Kd value in C33A cells indicated
that binding of the [125I]ET-1 was mostly
accounted for by the presence at high levels of low-affinity binding
sites such as the ETB receptor. Competition for
[125I]ET-1 binding to the CaSki cell line by
unlabeled ET-1, ET-3, the selective EtB receptor
agonist sarafotoxin 6c (S6c), and the ETA
selective antagonist BQ 123 showed differences in the displacement
capacity of ET-1 compared to ET-3 and S6c, suggesting that the
predominant functional receptor in CaSki cells was the
ETA subtype (data not shown).
|
Expression of mRNA for ET-1 and its receptor subtypes
Data from the binding assay indicate that HaCaT, the normal cell
line, C33A, the tumoral cell line without the presence of HPV
sequences, and CaSki cells, the cancer cells possessing several copies
of transcriptional active HPV16 genome, have a different expression of
ET receptors. To clarify this, RNA was extracted from active
proliferating cells and RT-PCR was performed to assess the presence of
specific mRNA for ET-1 and the receptors ETA and
ETB. RT-PCR detected RNA transcripts for ET-1,
ETAR, and ETBR in normal
and cervical cancer cells, as shown in Fig. 2
. The amplified products were of the predicted length and the identity
of the amplified DNA was confirmed by Southern blot with internal
probes (data not shown). The expression of ET-1 mRNA in cervical cancer
cells is mirrored in their ET-1 secretion, and the mRNA for the ET-1
was detected only in HaCaT and Caski cells. C33A cells were unable to
produce ET-1 peptide, and the absence of ET-1 mRNA demonstrated that
the inhibition of the ET-1 production is at the transcriptional level.
The RT-PCR analysis of the ET-1 receptor confirmed binding data,
showing that the HPV-positive tumor cells (CaSki) expressed
ETAR mRNA and that the only receptor expressed by
the C33A cells (HPV-negative) was ETBR.
|
Proliferative effects of ET-1 in cervical cancer cells
Assays for ET-1-induced mitogenic responses were performed in
order to elucidate the role of this peptide in the growth of these
tumor cell lines. In all normal and tumoral cervical cell lines, we
found that increasing concentrations (1 nM to 100 µM) of ET-1 induced
a marked dose-dependent increase in
[3H]thymidine incorporation in serum-free
medium (data not shown). ET-1 was able to induce a marked increase in
[3H]thymidine incorporation in HPV-associated
tumor cell lines compared to the normal cells HaCaT
(P<0.01) (Fig. 3
). CaSki cells exhibited the highest proliferation rate, apparently
reflecting the major number of ETA receptors on
the cell surface. To examine whether the mitogenic action of ET-1 was
mediated by ETAR, we co incubated the cells with
ET-1 and ETAR antagonist BQ123. The stimulatory
action of 100 nM ET-1 on [3H]thymidine
incorporation by tumor cells was completely blocked in the presence of
BQ123. The basal [3H]thymidine uptake of CaSki,
SiHa, and HaCaT cells was reduced by the presence of BQ123, suggesting
that endogenous ET-1 may act as an autocrine regulator of cell
proliferation. In the same experiment, 100 nM of selective
ETB agonists ET-3 and S6c had no mitogenic
effect. Proliferation of EGF-stimulated cells was not inhibited by
BQ123, indicating that the inhibitory effect induced by the
ETA receptor antagonist was specific and was not
due to cytotoxicity. The inhibitory effect of the
ETAR antagonist on ET-1-mediated cell
proliferation in the cell lines expressing high-affinity ET receptors,
namely, CaSki and SiHa, confirmed that in these cell lines the
functional binding site is the ETA receptor.
These data were further validated by the stronger stimulating effect of
the ETB agonists on C33A cells, which are missing
the high-affinity receptor for ET-1. Taken together, these results
demonstrate that the mitogenic signaling by ET-1 is mainly mediated by
the ETAR subtype in HPV-positive cervical cancer
cells.
|
Growth inhibition by ETA receptor antagonist
In a previous study we demonstrated that human keratinocytes
transfected by HPV16 DNA sequences were able to growth in media without
any growth factor (13)
. To determine whether endogenous
ET-1 production by the tumor cells could act as an autocrine growth
factor, tumor cells were cultured for up to 4 days in the absence of
any growth factor. HaCaT, CaSki, and SiHa cell lines were able to
proliferate in growth factor deprivation (data not shown). CaSki cells
showed the highest efficiency whereas the C33A failed to growth in
these media (Fig. 4
). As shown in Fig. 5
, the spontaneous growth of CaSki cells was significantly and
dose-dependently inhibited in the presence of the
ETAR antagonist BQ123. The addition of an
ETBR antagonist, BQ788, was unable to affect the
basal growth rate of the cells although in CaSki cells
ETBR mRNA could be detected. These results are
consistent with those obtained in the HPV transfected keratinocytes,
confirming that the predominant functional receptor present on HPV
tumorigenic cervical cells was the ETAR subtype.
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DISCUSSION
Growth factors are involved in the development and maintenance of
cancer cells. The up-regulation or alteration of their physiological
function may lead to an unbalanced growth of the cell
(17)
. ET-1 is an important growth stimulator in various
neoplastic diseases, including breast, ovarian, and prostate cancer
(18
19
20
21)
. Autocrine ET-1 activity is also involved in the
autonomous growth of HPV immortalized keratinocytes (9)
.
This last finding led us to formulate our hypothesis on the
pathophysiological role of ET-1 autocrine loop in HPV-associated
cancer.
As postulated for the EGF receptor, the increase of ET-1
receptors enhances the proliferation rate of HPV-infected cells in the
presence of a normal amount of growth factor in the microenvironment
and could therefore confer a selective advantage to the infected cells
(13
, 14)
. We speculate that in this immortalized
population, a second event could account for the generation of tumor
cells, suggesting a role for ET-1 in the regulation and promotion of
cervical tumor growth. This growth promotion may result from the
ability of ET-1 to act alone or with various growth factors, such as
EGF, potentiating cellular transformation, and/or proliferation
(22)
. As a direct consequence of this hypothesis,
compounds that antagonize the action of the ET-1 would be able to
affect the growth of the HPV-infected or tumor cells. In this study, we
first investigated the role of ET-1 in the growth of cervical cancer
derived cell lines. Our data indicate that ET-1 is an
autocrine/paracrine growth factor for cervical carcinoma cells. This
conclusion is based on several observations. All cervical carcinoma
cell lines, except C33A, released immunoreactive ET-1 in amounts within
the biological range of this peptide. There were differences in the
amount and in the timing of production that may reflect differences in
the tumor growth activity of these cells or at the least in their
ability to proliferate in growth factor-free media. The C33A did not
produce ET-1, and therefore the absence of an autocrine loop of ET-1
may explain the lack of growth in media without growth factors. ET-1
mRNA expression confirmed the ET-1 protein production data. The absence
of ET-1 mRNA in C33A cells could be mostly accounted for by a block at
transcriptional level.
ET-1 possesses at least two different receptors with different affinity
for the ET peptides. The binding data clearly demonstrate that these
receptors are differently expressed in HPV-positive (CaSki, SiHa) and
HPV-negative (C33A) cell lines. High-affinity receptors are mostly
present in HPV-positive cancer cells. The functional data recorded by
thymidine incorporation assay demonstrate that ET-1 stimulates DNA
synthesis in all HPV-associated cervical carcinoma cell lines examined.
Moreover, in these cell lines the ET-1 stimulation was higher than in
normal HaCaT cells, indicating that ET-1 is able to induce a
proliferative advantage in tumor cells. The effect of specific
ETA inhibitor or ETB
agonists seems to indicate that this growth stimulation is mediated by
ETA receptor in HPV-infected cervical cells and
by ETB receptor subtype in C33A. Further, the
RT-PCR analysis indicates that the functional differences are accounted
for the total absence of ETA mRNAs in
HPV-negative cervical cancer cells. The highest level of functional
ETA receptor expression was detected in CaSki
cells, as demonstrated by binding data. In many normal cells, the
secretion and action of ET-1 are regulated by the subtype nonselective
ETBR, which mediates a variety of compensatory
activities including ET-1 clearance, inhibition of ET-1 secretion, and
activation of signal transduction pathways that counter-regulate ET-1
(12
, 23)
. Previous studies have demonstrated a diminished
ETBR expression in various tumors (24
, 25)
. In the present study, competitive binding data revealed
that HPV-positive cervical carcinoma cells predominantly express
functional ETAR, supporting the hypothesis that
the expression and function of ETAR in malignant
cervical cells is likely to become the predominant form, representing a
relevant mechanisms in tumor proliferation (26)
. The
endogenous production of ET-1 and the increased number of
ETAR may enhance the ability of these cell lines
to growth in serum-free media, as demonstrated for HPV transfected
keratinocytes (13
, 14)
. Thus, in the absence of growth
factors, the cell lines containing HPV sequences and expressing
ETA receptor were able to proliferate with a
growth rate that correlates with the number of the
ETA binding sites. In particular, CaSki cells,
which were the most responsive to the ET-1 action and produced the
largest amount of the peptide, showed the highest growth rates. On the
contrary, the C33A failed to growth in serum starvation condition,
indicating that ET-1 is one of the autocrine growth factors involved in
the growth control of HPV-associated cervical cancer cells.
We now hypothesize that the presence of HPV viral sequences may enhance the activity of the ET-1 autocrine loop.
About 90% of the cervical cancers contain DNA sequences of HPV type 16
or 18, and our data indicate that the presence of viral sequence is
associated with an active ET-1 autocrine loop. Therefore, the use of a
selective ETA antagonist blocking ET-1 effects
may result in suppression of tumor growth and of progression of the
HPV-associated cancer. In our experiments, the presence of a specific
antagonist of the ETA receptor, BQ 123,
dramatically inhibited the growth of CaSki cells. The inhibition of the
growth is dose-dependent; moreover, BQ788, an ETB
antagonist, failed to affect the cell growth, confirming that the
ET-1/ETA loop is one of the drivers of tumor
growth. The activity of ET-1 and the inhibitory effect of an
ETA receptor antagonist on HPV-associated
carcinoma cell proliferation indicate that the up-regulation of ET-1
autocrine loop might provide a selective growth advantage for
HPV-associated tumor cells. Early clinical trials using specific
antagonist for ETAR suggest the potential to
treat cancer patients (27)
.
Taken together, these findings not only support a role for ET-1 in the HPV-associated cancer, but also provide a therapeutic target for endothelin receptor blockade in the treatment of cervical carcinoma.
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ACKNOWLEDGMENTS |
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Received for publication February 3, 2000.
Revision received April 24, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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),
C33A (
), HaCaT (
), and SiHa (
) cell lines was evaluated as a
function of time. Cells were incubated for 24 h in growth
factors-free medium prior to the experiment. The conditioned media were
collected at the indicated times and the ir-ET-1 concentration was
determined. Each point represents the mean value of three different
conditioned media: bars indicate ± SD.
), and in the
presence of an ETB antagonist (BQ 788) at 1 µM (
). At
different times the cell density was evaluated as described in Methods
and reported as O.D. Each assay was done in eight replicas; bars
represented mean ± SD of data from three independent
experiments. *P<0.01; **P<0.001.