Research Article

Estrogens and Human Papilloma Virus Oncogenes Regulate Human

Ether-à-go-go-1 Potassium Channel Expression
1 3 4 1 1
Lorenza Dı́az, Irais Ceja-Ochoa, Iván Restrepo-Angulo, Fernando Larrea, Euclides Avila-Chávez,
1 1 1 7 5
Rocı́o Garcı́a-Becerra, Elizabeth Borja-Cacho, David Barrera, Elı́as Ahumada, Patricio Gariglio,
5 5 5 4
Elizabeth Alvarez-Rios, Rodolfo Ocadiz-Delgado, Enrique Garcia-Villa, Elizabeth Hernández-Gallegos,
8 1 1 3
Ignacio Camacho-Arroyo, Angélica Morales, David Ordaz-Rosado, Ethel Garcı́a-Latorre,
4 4 2,8 2
Juan Escamilla, Luz Carmen Sánchez-Peña, Milena Saqui-Salces, Armando Gamboa-Dominguez,
4 6 9 4
Eunice Vera, Marisela Uribe-Ramı́rez, Janet Murbartián, Cindy Sharon Ortiz,
4 6 4
Claudia Rivera-Guevara, Andrea De Vizcaya-Ruiz, and Javier Camacho
1
Departamento de Biologı́a de la Reproducción and 2Departamento de Patologı́a, Instituto Nacional de Ciencias Médicas y Nutrición
Salvador Zubirán; 3Departamento de Inmunologı́a, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional; 4Pharmacology
Section, 5Department of Genetics and Molecular Biology, and 6Toxicology Section, Centro de Investigación y de Estudios Avanzados;

Downloaded from http://aacrjournals.org/cancerres/article-pdf/69/8/3300/2623357/3300.pdf by guest on 23 May 2023

7
Dirección Médica de la U.M.A.E. Hospital de Gineco-Obstetricia No. 3; 8Departamento de Biologı́a. Facultad de Quı́mica, Universidad
Nacional Autónoma de México, Ciudad Universitaria; 9Departamento de Farmacobiologı́a, Centro de Investigación y de Estudios
Avanzados, Mexico D.F., Mexico

Abstract
Ether-à-go-go-1 (Eag1) potassium channels are potential tools
for detection and therapy of numerous cancers. Here, we
show human Eag1 (hEag1) regulation by cancer-associated
factors. We studied hEag1 gene expression and its regulation
by estradiol, antiestrogens, and human papillomavirus (HPV)
oncogenes (E6/E7). Primary cultures from normal placentas
and cervical cancer tissues; tumor cell lines from cervix, cho-
riocarcinoma, keratinocytes, and lung; and normal cell lines
from vascular endothelium, keratinocytes, and lung were used.
Reverse transcription-PCR (RT-PCR) experiments and South-
ern blot analysis showed Eag1 expression in all of the cancer
cell types, normal trophoblasts, and vascular endothelium, in
contrast to normal keratinocytes and lung cells. Estradiol and
antiestrogens regulated Eag1 in a cell type–dependent manner.
Real-time RT-PCR experiments in HeLa cells showed that
Eag1 estrogenic regulation was strongly associated with the
expression of estrogen receptor-A. Eag1 protein was detected
by monoclonal antibodies in normal placenta and placental
blood vessels. Patch-clamp recordings in normal trophoblasts
treated with estradiol exhibited potassium currents resembling
Eag1 channel activity. Eag1 gene expression in keratinocytes
depended either on cellular immortalization or the presence
of HPV oncogenes. Eag1 protein was found in keratinocytes
transfected with E6/E7 HPV oncogenes. Cell proliferation of
E6/E7 keratinocytes was decreased by Eag1 antibodies inhibit-
ing channel activity and by the nonspecific Eag1 inhibitors
imipramine and astemizole; the latter also increased apoptosis.
Our results propose novel oncogenic mechanisms of estrogen/
antiestrogen use and HPV infection. We also suggest Eag1 as
an early indicator of cell proliferation leading to malignancies
and a therapeutic target at early stages of cellular hyper-
proliferation. [Cancer Res 2009;69(8):3300–7]
Note: L. Diaz and I. Ceja-Ochoa contributed equally to this work.
Requests for reprints: Javier Camacho, Centro de Investigación y de Estudios
Avanzados del I.P.N., Pharmacology Section, Avenida Instituto Politécnico Nacional
2508, Mexico D.F., Mexico 07360. Phone: 52-55-57473800, ext. 5416; Fax: 52-55-
53430106; E-mail: fcamacho@cinvestav.mx.
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-2036
Introduction
Ether à go-go (EAG) potassium channels possess oncogenic
properties (1); human Eag1 (hEag1) mRNA shows restricted
distribution in healthy tissues expressed mainly in brain; slightly
in placenta, testis, and adrenal gland; and transiently in myoblasts
(1–3). Interestingly, Eag1 is abundantly expressed in tumor cells,
including cervical, lung, breast, colon, and prostate cancer (2, 4);
therefore, Eag1 is suggested as a cancer marker. In addition, an
association of Eag1 amplification with reduced overall survival has
been observed in patients with colon carcinoma (5). Inhibition of
Eag1 gene expression decreases tumor cell proliferation (1, 6, 7),
and inhibition of Eag1 channel activity by imipramine or
astemizole has been suggested to decrease cancer cell proliferation
(6–8). Specific block of Eag1 with monoclonal antibodies inhibits
tumor cell growth both in vitro and in vivo (9). Thus, Eag1 is also
suggested as a cancer therapeutic target. Because no obvious side
effects were observed in the animals treated with monoclonal
antibodies (9), the potential therapeutic value of Eag1 channels
seems to be very high.
High-risk human papillomavirus (HR-HPV) is the major risk
factor for cervical cancer (10), and estrogen use is suggested as a
likely factor contributing to this cancer (11). Actually, estrogen
receptors (ER) or aromatase enzymes provide advantages for
proliferation of tumor cells derived from mammary gland, lung,
and cervix (12, 13). Nevertheless, tamoxifen stimulates proliferation
of some cancer cells (14–16).
We reported Eag1 expression in normal cervical samples from
women with negative pap smears, including a patient with HPV
infection (4). In addition, Eag1 mRNA was detected in the tumor-
free mammary gland surrounding the breast carcinoma tissue (2),
in human diverticulitis (which has the potential to change into
colonic cancer), and in crypt cells of a colon cancer mouse model
(5). These observations suggest Eag1 expression as an early sign
of cellular hyperproliferation.
Despite the potential clinical relevance of Eag1, its regulation
by cancer etiologic factors is poorly understood. Here, we studied
hEag1 expression and regulation by some cancer-associated
factors, namely, estrogens, antiestrogens, and HPV oncogenes in
normal and tumor cells. We studied cervical cancer cells obtained
from primary cultures and cell lines from different histogenesis.

Cancer Res 2009; 69: (8). April 15, 2009 3300 www.aacrjournals.org

Figure 1. Eag1 channels in human placenta. A, Eag1 protein was
immunolocalized (dark blue-violet staining ) in the cytoplasm, nucleus of the
syncytiotrophoblast layer (SC ), and nucleus of vascular endothelial cells (VEC ).
Magnification, 200. Insert, negative control in the absence of Eag1
antibody. Image is representative of seven placentas. In some other sections,
Eag1 was also expressed in the plasma membrane (data not shown). B,
Southern blot analysis (top ) from RT-PCR experiments of seven different
normal-term placentas. All of them displayed Eag1 expression. Columns, Eag1
mRNA relative expression after normalization with the constitutive gene
cyclophilin (cyc ).
In the case of normal cells/tissues, we selected some normal cell
lines and normal human placenta as a very interesting tissue to
study, because placental development resembles tumor growth (17)
and estrogens play a pivotal role in placental physiology, and
noteworthy, because it expresses Eag1 mRNA and the potential role
of Eag1 in healthy tissues is almost unknown. Our regulation
studies unravel potential oncogenic mechanisms of HPV infection
and estrogen/antiestrogen use, emphasizing the potential use of
Eag1 as an early indicator of cell proliferation. On the other hand,
our pharmacologic experiments suggest Eag1 as a therapeutic
target at the early stages of cell hyperproliferation.
Materials and Methods
Biological samples. Biopsies from normal human placentas (38–42 wk
of gestation, n = 7) were obtained from patients registered at Centro Médico
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Potassium Channels and Cancer Risk Factors
‘‘La Raza,’’ following the local ethical considerations. Tissue was placed in
liquid nitrogen to obtain mRNA or fixed in paraformaldehyde for
immunochemistry. For cytotrophoblast culturing, tissue was washed with
saline solution and immediately processed. Neonatal foreskins from
patients registered at Hospital ‘‘Gabriel Mancera’’ were collected following
the local ethical considerations and used to obtain normal keratinocytes
primary cultures.
Cell culture and reagents. Cytotrophoblasts were isolated and cultured
as described (18, 19). Villous tissue was enzymatically dispersed;
cytotrophoblasts were separated on a Percoll gradient, plated in DMEM-
HG with 20% fetal bovine serum (FBS) and incubated at 37jC (95%
humidity and 5% CO2 atmosphere). Human chorionic gonadotrophin
hormone (hCG) concentration was measured with an immunoassay kit
(Immunometrics Ltd.). Human cervical cancer cells were obtained from
previously established primary cultures (4). Tumor cell lines (HeLa, SiHa,
CaSki, INBL, and C33A from cervix; JEG-3 and A-549 from choriocarcinoma
and lung, respectively), as well as bronchial epithelial cells (BEAS) and
human umbilical vascular endothelial cells (HUVEC) from normal lung and
umbilical cord, respectively, were obtained from American Type Culture
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Collection and cultured according to manufacturer’s instructions. Cell
treatments were performed, as follows, to avoid participation of exogenous
estrogens present in serum or medium: cells were washed with HBSS
24 h after plating and incubated in supplemented medium (DMEM-HG
without phenol red plus 5% charcoal-stripped FBS and antibiotics). Cells
were incubated with 17h-estradiol (Sigma Chemical Co.) and/or the
antiestrogens ICI 182,780 (Zeneca Pharmaceuticals) or 4-hydroxy-tamoxifen
(Sigma Chemical Co.) during 24 h; ethanol was used as vehicle, and
incubations were stopped by aspirating the culture media and adding Trizol
reagent (Invitrogen) for RNA extraction. Normal keratinocytes were
prepared from neonatal foreskins and grown in the keratinocyte serum-
free medium (Invitrogen) supplemented with 20 mg/mL bovine pituitary
extract and 0.1 ng/mL of epidermal growth factor (Invitrogen); transformed
human keratinocytes (HaCat cells) provided by Dr. Lutz Gissmann [German
Cancer Research Center (DKFZ)] were cultured in the same medium.
Primary human keratinocytes immortalized by amphotropic retroviruses
encoding E6, E7, or E6/E7 oncoproteins of HPV-16 (provided by Dr. Frank
Rösl, DKFZ) were grown in a keratinocyte growth medium (Invitrogen) and
cultured as described (20).
Transient human ER-a transfection and ER Western blot. Expression
vector for human ER-a (pCMV5-hERa) to transfect HeLa cells was provided
by Dr. A.J. Cooney (Baylor College of Medicine). Cells were transfected using
PolyFect (Qiagen, Inc.) as described (21). Transcriptional activity of
transfected ERa was confirmed by a chloramphenicol acetyltransferase
(CAT) reporter assay (21). Western blots were performed to detect ERa in
HeLa, A-549, BEAS, and cervical cancer cells with the primary antibody anti-
ERa (Santa Cruz). HeLa cells expressing ERa (HeLa-ERa) were subjected to
the same treatments as described above.
PCR amplifications and Southern blot analysis. Total RNA was
isolated from tissues and cell cultures with Trizol reagent. RNA was
reversed transcribed, and PCR amplifications were performed with the
following primers: 5¶-GCTTTTGAGAACGTGGATGAG-3¶ and 5¶-CGAA-
GATGGTGGCATAGAGAA-3¶, which yielded 475-bp hEag1 reverse tran-
scription-PCR (RT-PCR) products. In some cases, Eag amplification was
performed using the following sense and antisense primers: 5¶-
TGGTCCTGCTGGTGTGTG-3¶ and 5¶-ACAACGAGGAGATGTAGACAG-3¶.
These amplifications yielded a 187-bp hEag1 product. Human Eag1-
transfected Chinese hamster ovary (CHO) cells (provided by Walter
Stühmer, Max-Planck-Institut für Experimentelle Medizin) were used as
positive control. Cyclophilin and b2-microglobulin genes were used as
constitutive genes and amplified with the following primers: 5¶-CCC CAC
CGT GTT CTT CGA CAT-3¶ and 5¶-AGG TCC TTA CCG TTC TGG TCG-3¶ for
cyclophilin yielding a 453-bp product and 5¶-ACCCCCACTGAAAA-3¶ and 5¶-
ATGATGCTGCTTACATGTCTCGAT-3¶ for b2-microglobulin yielding a 100-
bp product. Identity of the 475-bp Eag RT-PCR product has been already
determined by nucleotide sequence (4). For Southern blot analysis, PCR
products were separated in agarose gels, blotted onto nylon membranes,
and hybridized with [32P]dCTP-labeled nested probes. Probes were obtained
3301 Cancer Res 2009; 69: (8). April 15, 2009
Cancer Research
as previously described and also confirmed by sequence (4). In all cases,
filters were washed after 18 h of hybridization and exposed to X-ray films.
Real-time PCR amplifications. SYBR Green real-time PCR was
performed with 1 AL of cDNA obtained as described above. SYBR Green
reaction was conducted using a QuantiTect SYBR Green PCR reagents kit
(Qiagen) following the manufacturer’s instructions. The following primers
were used: 5¶-TGG TCC TGC TGG TGT GTG-3¶ ( forward), 5¶-ACA ACG AGG
AGA TGT AGA CAG-3¶ (reverse) for Eag1 (4) and 5¶-ACC CCC ACT GAA
AAA GAT GAG TAT-3¶ ( forward), 5¶-ATG ATG CTG CTT ACA TGT CTC
GAT-3¶ (reverse) for b2-microglobulin (used as constitutive gene and to
analyze relative Eag1 expression). Data analysis was performed by the
2 DDCt method.
Eag1 immunochemistry. Specific anti-Eag1 antibodies were kindly
provided by Walter Stühmer and Luis Pardo (Max Planck Institute). This
antibody has been validated and shown to be very specific; it does not
bind to hErg channels and discriminates between Eag1 and Eag2 (9). With
this antibody, we already reported Eag1 channel expression in human
cervical biopsies, cervical cancer cells from primary cultures, and CHO
cells transfected with Eag1 in opposite to the absence of Eag1 in
nontransfected CHO cells (4). Placental biopsies were fixed in a buffered
4% formaldehyde solution, processed as standardized for histology, and
embedded in paraffin. Serial sections (4 Am) were mounted on charged
glass slides and deparaffinized using xylene and a decreasing series of
ethanol. After washing with TBS/Tween (pH 7.4), slides were immersed in
Cancer Res 2009; 69: (8). April 15, 2009
EDTA buffer [0.01 mol/L (pH 8.0)] and boiled for antigen retrieval. Slides
were blocked for 2 h with levamisole and incubated in the presence of
1:50 anti-Eag1 antibody (Ifv001 single-chain antibody coupled with alkaline
phosphatase) for 2 h at room temperature. The slides were washed with
TBS/Tween, and the specific staining reaction was completed by
incubating the slides in the presence of 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium (Roche) in a buffer solution for 1 h at
room temperature, protected from light, and observed as a dark blue-
violet staining. Sections were counterstained with eosin. Slides were
observed with a Nikon Eclipse E600 microscope using a Nikon Plan Apo
10/0.45 objective lens. Images were acquired with a DMX1200 camera
and the Nikon ACT-1 acquisition software at room temperature.
Keratinocytes expressing HPV oncoproteins (K-E6, K-E6/E7) were pro-
cessed in a very similar manner. After antigen retrieval with EDTA, slides
were blocked with endogenous peroxidase and phospatase blocker (Dako)
for 10 min and then incubated in the presence of 1:100 anti-Eag1 antibody
for 1 h at room temperature. The slides were then incubated with MACH
4 mouse probe (Biocare) in TBS/Tween for 10 min and then incubated
with MACH 4 horseradish peroxidase polymer (Biocare) in TBS/Tween for
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10 min. The specific staining reaction was completed by incubating the
slides in the presence of diaminobencidine in buffer reaction solution
(Dako) and observed as a brown staining. Sections were counterstained
with hematoxylin (Dako), observed with a 40 objective lens, and
analyzed as above described for the placental sections.
Figure 2. Eag1 regulation by E2 in
cultured human syncytiotrophoblast and
vascular endothelial cells. Southern
blot analysis from RT-PCR experiments
shows that E2 induced Eag1 mRNA
expression in syncytiotrophoblasts with no
detectable basal Eag1 expression (A )
and antiestrogenic treatment with ICI
182,780 (ICI ) prevented such
up-regulation and decreased Eag1 levels
in a syncytiotrophoblast primary culture
with basal Eag1 expression (B). Eag1 is
endogenously expressed in HUVEC and
up-regulated by E2, and antiestrogenic
treatment prevented such up-regulation
(C) as in trophoblasts. Columns,
Eag1 mRNA relative expression after
normalization with cyclophilin (A–C); Eag1
expression in vehicle was given the
value of 1 in B and C. D, whole-cell
patch-clamp experiments showed
outward potassium currents in
syncytiotrophoblasts treated with E2 (top )
and untreated choriocarcinoma JEG-3
cells (bottom ). Currents elicited at +60 mV
were preceded by negative prepulses
( 140 or 60 mV) and displayed different
activation kinetics, suggesting Eag1
channel activity. Eh = 80 mV,
unsubtracted traces. Figures are
representative of at least three separate
experiments.
3302 www.aacrjournals.org

Figure 3. Estrogenic regulation of Eag1 expression in HeLa cells and
participation of ERa. A, real-time RT-PCR experiments show that wild-type HeLa
cells display endogenous Eag1 expression (as previously reported in ref. 1), but
E2 up-regulates Eag1 expression at some concentrations without the effect of
antiestrogens. However, when HeLa cells were forced to express ERa, Eag1
was strongly up-regulated by both E2 and antiestrogens (B). Columns, mean;
bars, SD. *, P < 0.05 compared with control (no E2 treatment).
Electrophysiology. Whole-cell recordings were acquired from isolated
cells with the patch-clamp technique (22) using an EPC-9 amplifier
(HEKA Electronics) and analyzed with Igor Pro (WaveMetrics). Patch
pipettes (2–3 MV) were obtained by double-pulling Kimax capillaries.
Internal solution contained (in mmol/L) 140 KCl, 10 EGTA, and 10
HEPES/KOH (pH 7.2). External solution contained (in mmol/L) 2.8 KCl,
115 NaCl, 2 CaCl2, 2 MgCl2, and 10 HEPES/NaOH (pH 7.2); in some
experiments, we used external solutions containing 10 mmol/L MgCl2.
Holding potential was 80 mV, unless indicated. Experiments were
performed at room temperature (20–22jC).
Metabolic activity and apoptosis. K-E6/E7 cells were seeded in 96-well
plates, and cell proliferation was assayed by the colorimetric method based
on the conversion of the tetrazolium salts to formazan crystals by
dehydrogenase activity in active mitochondria [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) cell proliferation kit I, Boehringer
Mannheim GmbH]. Cells were incubated during 96 h in culture medium
alone or in the presence of astemizole (10 Amol/L), imipramine (10 Amol/L),
DMSO as vehicle (all of these reagents were purchased from Sigma
Chemical Co.), or the anti-Eag1 monoclonal antibody 56 (133 nmol/L, also
provided by Walter Stühmer and Luis Pardo) known to inhibit Eag1 channel
activity (9). MTT (0.5 mg/mL) was added 4 h before completing the whole
incubation time. Absorbance data were obtained from the resulting colored
solution with a microplate photometer (Sunrise Touchscreen). Apoptosis
was determined with the Annexin V-FITC kit (Invitrogen Co.) binding to
phosphatidylserine and DNA staining by propidium iodide (PI). Campto-
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Potassium Channels and Cancer Risk Factors
thecin (apoptosis inductor) and methanol (necrosis inductor) were used as
controls. Experiments were carried out with the flow cytometer FACS-SYAN
ADP with nine colors (DAKO). Percentages of viable (FITC and PI negative),
apoptotic (FITC positive and PI negative), and late apoptotic (FITC and PI
positive) cells were obtained by quadrant analysis using WinDMI software
version 2.8.
Statistical analysis. Student’s t test or ANOVA, followed by Dunnett’s or
Tukey-Kramer test, was used to compare data between different groups;
P values of <0.05 or <0.01 for the respective tests were considered to be
statistically significant. Analysis was made using GraphPad Prism software
version 3.0.
Results
Regulation of Eag1 by Estradiol in Normal and Tumor
Cells
Eag1 channels in normal human placenta and vascular
endothelium and their regulation by estradiol. We investigated
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Eag1 protein expression in normal human placenta and studied the
likely regulation of Eag1 gene expression by 17h-estradiol (E2) in
primary cultures from normal human trophoblasts. Eag1 channels
were strongly expressed in the syncytiotrophoblast layer in both
the cytoplasm and the nucleus in normal placenta (Fig. 1A, dark
blue-violet staining ). Interestingly, we also observed Eag1 channels
in the vascular endothelium, especially in the nucleus (Fig. 1A).
Whereas Eag1 protein expression pattern was very similar among
placentas, relative Eag1 mRNA levels were very different. Figure 1B
shows some placentas with low Eag1 mRNA expression and some
with higher levels. Despite such differences in mRNA levels, we
observed both Eag1 protein and mRNA expression in all of the
placentas studied.
Unexpectedly, Eag1 mRNA was not detected in most of the
trophoblasts primary cultures but was induced by estradiol.
Figure 2A shows Eag1 mRNA up-regulation by E2 at picomolar
concentrations whereas a higher E2 concentration originated
only a faint Eag1 signal. The antiestrogen ICI 182,780 prevented
such up-regulation. In cultures displaying endogenous Eag1
mRNA (Fig. 2B), E2 induced a slight increase in Eag1 expression
and antiestrogen treatment down-regulated endogenous Eag1
expression both in the presence and absence of E2 (Fig. 2B). As
expected, we also found endogenous Eag1 expression in the
placental choriocarcinoma cell line JEG-3; however, in this case, we
did not find a significant estrogenic regulation (data not shown).
Eag1 channel expression in vascular endothelium prompted us to
study Eag1 mRNA expression and regulation by estradiol in
endothelial cells. Endogenous Eag1 mRNA was found in normal
HUVECs supplemented with 10% FBS (Fig. 2C). Eag1 was up-
regulated by E2 ; this effect was inhibited by ICI 182,780, which also
down-regulated endogenous Eag1 levels (Fig. 2C). The effect of
estrogens and antiestrogens on Eag1 expression was very similar in
both normal cell types (trophoblasts and vascular endothelium).
Endogenous Eag1 channel activity has not been reported for
most of the normal tissues where it is expressed, including the
brain. Therefore, we investigated if Eag1 channel activity could be
recorded in placental-derived cells. Whole-cell patch-clamp record-
ings showed no outward voltage-activated potassium currents in
trophoblast primary cultures in the absence of E2 (data not shown).
However, when incubated with E2 (1 pmol/L), normal human
trophoblasts displayed noninactivating outward currents resem-
bling Eag1 channel activity (Fig. 2D, top). Eag1 channels display a
strong Cole-Moore shift (the current elicited with a depolarizing
pulse is slower at a more negative prepulse voltage) and a very slow
3303 Cancer Res 2009; 69: (8). April 15, 2009
Cancer Research
activation in the presence of extracellular magnesium (23). Figure
2D shows that outward currents elicited at +60 mV are slower
when preceded by a very negative prepulse ( 140 mV) compared
with a more positive prepulse ( 60 mV), recordings were obtained
in the presence of 10 mmol/L MgCl2. Because no other potassium
channels have been reported to display such a strong Cole-Moore
shift, our data suggest Eag1 channel activity in normal human
trophoblasts incubated with E2. Similar currents were elicited in
the choriocarcinoma JEG-3 cells in the absence of estradiol (Fig. 2D,
bottom), although, in this case, the current was not that accelerated
with a more positive prepulse, as in normal human trophoblasts,
despite of a lower MgCl2 concentration (2 mmol/L) used.
Estrogenic regulation of Eag1 in cancer cells and the
participation of ERa. We studied estrogenic regulation of Eag1
in HeLa cells by real-time RT-PCR. We found Eag1 regulation by E2
only at two-hormone concentrations (Fig. 3A) with no effect of the
antiestrogens in these HeLa wild-type cells (Fig. 3A). ER-a mRNA
expression in these cells was detected by RT-PCR; however,
Western blot experiments failed to show the presence of ERa or
ERh (data not shown). Therefore, we transfected these cells with
human ERa (HeLa-ERa cells) and studied again Eag1 regulation by
E2. ER-mediated transcription was confirmed by CAT assays (21) in
HeLa-ERa cells cotransfected with a CAT reporter plasmid (data
not shown). Figure 3B shows a remarkable Eag1 up-regulation by
E2 in HeLa-ERa cells. ICI 182,780 and tamoxifen, alone or in
combination with E2, produced a very high Eag1 up-regulation
(Fig. 3B). These results suggest ERa activation as one of the
mechanisms of the estrogenic regulation of Eag1.
Eag1 regulation was also studied in cervical cancer cells from
three human cervical biopsies. Lung cell lines A549 and BEAS-2B
from cancer and normal tissue, respectively, were used to study
both Eag1 expression and estrogenic regulation in cells from a very
common cancer influenced by estrogenic signaling (12) but
histogenically different from placental or cervical tissue. Western
blot analysis showed that cervical and lung cells express ERa (data
not shown). Cervical cancer cells from primary cultures were
previously characterized to express Eag1, HPV, and cytokeratins (4),
supporting their cancer epithelial nature. Table 1 shows screening
of Eag1 estrogenic regulation in these cells. Effect of E2 and
antiestrogens on Eag1 expression in primary cultures from cervical
cancer was similar between two patients (cervical cancer 1 and 2);
interestingly, in a sample from another patient (cervical cancer 3),
such regulation occurred in the opposite direction in all of the
treatments studied. As expected, Southern blot analysis showed the
absence of Eag1 gene expression in the normal human lung cells
(BEAS-2B). Eag1 was not induced by E2 or antiestrogens in these
Table 1. Relative Eag-1/cyclophilin expression in cancer cells treated with E2 and/or antiestrogens (from Southern blot
analysis)
Cell type E2 (10 nmol/L) E2 + ICI 182,780
Cervical (1) 1.42 1.24
Cervical (2) 1.21 2.95
Cervical (3) 0.21 0.61
Lung (A549) 1.59 0.94
NOTE: Relative Eag-1/cyclophilin expression, in comparison with vehicle-treated cells which were given an arbitrary value of 1. Abbreviation: Tx,
tamoxifen.
Cancer Res 2009; 69: (8). April 15, 2009
cells (data not shown). In contrast, Eag1 was present in the lung
cancer cell line A549 and up-regulated with most treatments,
especially by antiestrogens (Table 1). Thus, E2 also regulates Eag1
expression in cancer cells in a cell type–dependent manner. Then,
we wondered whether a well-identified etiologic factor for some
cancers could also affect Eag1 expression.
Expression and Potential Role of Eag1 in Cells
Expressing HPV Oncogenes
Eag1 in cervical cancer cell lines and keratinocytes
expressing HPV oncogenes. We studied hEag1 gene expression
in normal keratinocytes and keratinocytes immortalized with the
oncogenes E6, E7, or both (K-E6, K-E7, K-E6/E7), as well as in
immortalized keratinocytes lacking HPV and cervical cancer cell
lines presenting or lacking HR-HPV.
Normal keratinocytes did not express Eag1 (Fig. 4A); however, a
prominent Eag1 expression was found in keratinocytes immortal-
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ized with HR-HPV oncogenes, with K-E7 showing the highest
expression. Eag1 was also found in spontaneously transformed
keratinocytes lacking HR-HPV (HaCat cells). These data suggest
that Eag1 might be regulated by different oncogenic pathways. In
accordance, Eag1 expression was found in cervical cancer cell lines
expressing either HPV-16 (CaSki, SiHa) or HPV-18 (INBL) and in a
cell line lacking HPV (C33A).
Potential role of Eag1 channels in cell proliferation and
apoptosis of keratinocytes expressing HPV oncogenes. Immu-
nocytochemistry experiments showed an important Eag1 channel
protein expression in keratinocytes immortalized with HPV
oncogenes (Fig. 4B, brown staining ). We wondered whether Eag1
channels might have a role in the proliferation and apoptosis of
these cells. Cell proliferation (assayed by metabolic activity studies)
was decreased in K-E6/E7 cells by astemizole and imipramine, two
widely used nonspecific Eag1 inhibitors (Fig. 4C). Specific Eag1
inhibition can be achieved by incubating the cells with the
monoclonal Eag1 antibody 56. Because this antibody does not have
an effect on hErg channels or even on the more closely Eag1-related
protein, namely Eag2 (9), this is a very fine and specific tool to
study potential physiologic roles of Eag1 channels. This antibody
also decreased metabolic activity of K-E6/E7 cells (Fig. 4C).
Astemizole increased K-E6/E7 apoptosis (Fig. 4D).
Discussion
Eag1 is a potential tool for cancer detection and therapy (1, 2,
4, 9), and several findings suggest Eag1 as an early marker for
breast, cervical, and colon cancer (2, 4, 5).
E2 + Tx ICI 182,780 (100 nmol/L) Tx (100 nmol/L)
0.62 1.44 0.93
0.08 1.26 0.95
1.11 0.74 1.03
1.48 2.83 2.35
3304 www.aacrjournals.org
 Potassium Channels and Cancer Risk Factors

Figure 4. Eag1 in cervical cancer cells and

effect of Eag1 inhibitors in cell proliferation and
apoptosis of keratinocytes expressing HPV
oncogenes. A, duplex PCR experiments
and densitometric analysis for Eag1 and
b2-microglobulin showed no Eag1 expression
in normal keratinocytes (NK ); in contrast,
keratinocytes expressing HPV oncogenes
(K-E6, K-E7 , or both, K-E6/E7 ) expressed
Eag1 . Eag1 was also found in transformed
keratinocytes without HPV (HaCat and C33A )
and in cervical cancer cell lines with either
HPV-16 (CaSki and SiHa ) or HPV-18 (HeLa
and INBL ). Columns, mean of Eag1 mRNA
relative expression after normalization with
h2-microglobulin (b2-m); bars, SD. Eag1

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expression in HeLa cells was given the value
of 1. B, Eag1 protein (brown staining ) in
K-E6/E7 and K-E6 cells can be observed in the
plasma membrane (CM ), nuclear membrane
(Nuc ), or cytoplasm (Cyt ). Negative controls in
the absence of the Eag1 antibody are shown
in the insets. Magnification, 800. C,
nonspecific Eag1 inhibitors astemizole (Astem )
and imipramine (Imip ; 10 Amol/L, 96 h)
decreased cell proliferation of keratinocytes
expressing both HPV oncogenes (K-E6/E7 ),
assayed by metabolic activity; the anti-Eag1
monoclonal antibody 56 (mAb56 ), a very
specific inhibitor of Eag1, also decreased
metabolic activity. D, astemizole increased
apoptosis in these K-E6/E7 cells. Columns,
mean; bars, SD. *, P < 0.05 compared with
control (medium or vehicle).

Placental development resembles malignant tumor behavior
(17). We found Eag1 protein in the syncytiotrophoblast layer and
vascular endothelium of placental villi at the plasma membrane, in
the cytoplasm, and in the nucleus. This distribution was expected
because Eag1 has a bipartite nuclear targeting signal in its carboxy
terminus (24) and perinuclear localization of Eag1 channels has
been observed in tumors (2, 6). A COOH terminal fragment of a
voltage-gated calcium channel translocates to the nucleus and
regulates transcription (25), a similar role for Eag1 remains elusive.
Eag1 mRNA was rarely detected in trophoblast primary cultures
despite cell viability confirmation by basal and 8-bromo-cyclic
AMP–stimulated placenta hCG secretion (data not shown).
Nevertheless, Eag1 mRNA expression was induced by E2. Chorio-
carcinoma JEG-3 cells displayed endogenous Eag1 gene expression;
however, Eag1 expression was only slightly regulated by estrogens
and unaffected by ICI 182,780. Our results suggest that Eag1 might
participate in the proliferation and/or fusion of trophoblast (as in
myoblasts fusion; ref. 3), but this deserves further investigations.
Eag1 protein was found in placental blood vessel endothelium,
accordingly; endogenous Eag1 mRNA was found in normal
HUVECs, up-regulated by E2 and down-regulated by ICI 182,780.
This is important because estradiol is also considered as an
angiogenic factor (26–28). Interestingly, it has been recently found
that Eag1 promotes vascular endothelial growth factor secretion
and angiogenesis in tumors (29). The effect of other angiogenic
factors on Eag1 expression in both normal and cancer cells remains
to be elucidated. We show the first recordings suggesting Eag1
channel activity in normal human trophoblasts, but more
pharmacologic studies are needed to determine the potential
contribution of other potassium channels to the outward currents
recorded in placental cells.
Human ERa is necessary for a clear estrogenic regulation of Eag1
gene expression in HeLa cells. Only when forced to express ER-a did
estradiol and antiestrogens clearly up-regulated Eag1. Actually, this
is a potential explanation for the lack of estrogenic regulation of
Eag1 in JEG-3 cells, because these cells express very low amounts of
ER-a (30, 31). Thus, concerning ER-a expression, JEG-3 cells could be
compared with HeLa wild-type cells not transfected with ER-a.
When screening estrogenic regulation of Eag1 mRNA in cells from
cervical cancer primary cultures derived from three patients, we
found similar effects in two cases but opposite effects in another
culture. Accordingly, tamoxifen opposite effects have been reported
in cervical cancer. Tamoxifen treatment inhibited growth of cervical
cancer cell lines, including HeLa cells (32); but in the cervical cancer
cell line SFR lacking ERs, tamoxifen stimulated HPV-16 gene
expression and cell proliferation (16). Cervical cancer biopsies

www.aacrjournals.org 3305 Cancer Res 2009; 69: (8). April 15, 2009
Cancer Research

(16%) from patients treated with tamoxifen had a significant
decrease in the number of mitotic figures (33). Thus, the variable
estrogenic regulation of Eag1 in cervical cancer primary cultures
might have several explanations, including treatment conditions,
differences in HPV copy number, and different amounts of ERs or ER
phosphorylation; actually, tamoxifen can switch from ERa antago-
nist to ERa agonist if ERa is phosphorylated (34).
Estrogens stimulate growth and progression of lung tumors (35),
and aromatase inhibitors suppress tumor growth in mice with
A-549 lung tumor xenografts (12). Eag1 was not found in BEAS-2B
cells in accordance with the absence of Eag1 in normal bronchial
epithelium (2). We found Eag1 expression in A-549 cells in
compliance with the findings in lung cancer biopsies (2); in these
cells, almost all treatments up-regulated Eag1. A combined therapy
of antiestrogens and epidermal growth factor receptor (EGFR)
inhibitor has been suggested for lung cancer (36) because of the
functional linkage between estrogens and EGFRs (37). Therefore,
at early stages of cell hyperproliferation, for example, in cells initially
transformed by HPV.
Eag1 channel activity is increased by arachidonic acid in IGR1
melanoma cells (46), and insulin-like growth factor-I increases both
channel activity and gene expression of Eag1 in MCF-7 breast
cancer cells (47). Here, we show for the first time Eag regulation by
cancer risk factors in normal cells. A highlighted feature in this
study was that Eag1 can be regulated by both estrogens and HPV
oncoproteins. Eag1 up-regulation by E2 in HeLa cells and some
cervical cancer primary cultures suggest that estrogens and HPV
might have a synergistic effect on Eag1 expression. Actually, it has
been shown that estrogens contribute to the onset, persistence,
and malignant progression of cervical cancer in a HPV-transgenic
mouse model (48). A very interesting issue to address is to know
the potential participation of Eag1 channels in the mouse cervical
cancer model mentioned. Another interesting issue is to investigate
the differential concentration-dependent effect of estrogens.

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Eag1 up-regulation by antiestrogens might be related to the cross-
talk between different signal transduction pathways.
Different cervical cancer cell types and keratinocytes expressing
HPV oncogenes displayed Eag1 expression in contrast to normal
keratinocytes. HPV oncoproteins E6 and E7 influence cell prolifer-
ation by targeting p53 and Rb proteins, respectively (38). The cell
lines used present different molecular alterations. HeLa cells
overexpress c-Myc and Bcl-2 proteins, SiHa cells have high levels
of pRb and BclXL proteins (39), and keratinocytes expressing E7
display high c-Myc levels (20). Eag1 was also found in transformed
keratinocytes lacking HPV (HaCat and C33A cells). Interestingly, p53
and Rb are mutated in C33A cells (40) and HaCat cells have two
mutations in p53 (41). Probably Eag1 is down-regulated, at least,
through p53 and Rb pathways. Because Eag1 was found in cells
presenting or lacking HPV, these results suggest that Eag1 gene
expression is regulated by different factors, which is in accordance
with the Eag1 overexpression observed in very different types of
tumors (2, 4–6); our results also offer possible alternative
explanations for the oncogenic mechanisms of HPV infection.
Eag1 protein was found in K-E6/E7 and K-E6 cells, and Eag1 channel
inhibitors affected cell proliferation and apoptosis. Astemizole was
more effective than impramine in inhibiting cell proliferation and
increasing apoptosis in keratinocytes expressing HPV oncogenes.
Both drugs have other targets different from Eag1; thus, a plausible
explanation is a higher affinity of other proteins participating in cell
proliferation/apoptosis to astemizole. One of such proteins might be
hErg channels, which are strongly inhibited by astemizole and have
also been suggested to play a role in tumorigenesis (42–45). Further
mechanistic studies are required to understand the effect of
astemizole on cell proliferation and apoptosis. Nevertheless, despite
of some nonspecific effects of imipramine and astemizole that might
be involved, the effect of these drugs and the monoclonal Eag1
antibody on proliferation and/or apoptosis suggests Eag1 as a target
Concentration-dependent dual effects of estrogens have been
already observed, for example, on growth hormone synthesis and
interleukin-induced proteoglycan synthesis (49, 50).
Our experiments unravel novel potential oncogenic mechanisms
of estrogen/antiestrogen use and HPV infection. This proposes that
Eag1 might be expressed in early stages potentially leading to
cervical transformation, for example, in patients infected with HPV
or those undergoing estrogen treatment or subjects with low-grade
intraepithelial lesions.
The data presented herein suggest Eag1 as an early marker of
cellular proliferation leading to malignancies, enhancing potential
benefits of using Eag1 in early detection programs. Because cancer is
a multifactorial disease, detecting the expression of oncogenic genes
or proteins regulated by several cancer risk factors, like Eag1, might
lead to improved diagnosis methods and early detection of
malignancies. Finally, our pharmacologic experiments propose
Eag1 as a therapeutic target at early stages of cell hyperproliferation.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 6/2/08; revised 1/14/09; accepted 2/16/09; published OnlineFirst 4/7/09.
Grant support: CONACyT grants 45753 (J. Camacho) and 45953 (P. Gariglio).
E. Garcı́a-Latorre is a fellow of EDI and COFFA-IPN.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Walter Stühmer and Luis Pardo (Max Planck Institute) for providing
Eag1-transfected CHO cells and the Eag1 antibodies and for their comments on the
manuscript; Dr. Lutz Gissmann and Dr. Frank Rösl (DKFZ) for providing HaCat cells
and immortalized human keratinocytes expressing E6, E7, or E6/E7 HPV oncoproteins;
Dr. Austin J. Cooney (Baylor College of Medicine) for providing the expression vector
for ERA (pCMV5-hERA); Verónica Cadena for her secretarial work; and Guadalupe
Montiel for her technical assistance.

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