The Gamma-Aminobutyric Acid A Receptor

Sarah K Johnson, Randy S Haun
Department of Pathology, University of Arkansas for Medical Sciences.
Little Rock, Arkansas, USA
ABSTRACT
Context The identification of genes involved
in tumorigenesis is essential for the
development of new treatment strategies or
diagnostic approaches for pancreatic cancer.
Objective To identify genes overexpressed in
pancreatic cancer we employed differential
display, a PCR-based method of differential
expression cloning. Using this method, we
identified a PCR product that was consistently
overexpressed in pancreatic tumors relative to
normal pancreatic tissues.
Setting
Five
pancreatic
ductal
adenocarcinomas and 5 bulk pancreatic ducts
isolated from independent pancreatic
specimens without malignancies were
analyzed by differential display. A panel of
12 pancreatic tumors at various stages of
differentiation and a set of 6 pancreatic ducts
without malignancies were then used to verify
expression by RT-PCR.
Results Sequence analysis of a cDNA
detected by differential display revealed that
it was a portion of the recently cloned
gamma-aminobutyric acid A receptor
π subunit. RT-PCR analysis of a panel of
RNAs prepared from pancreatic ducts isolated
from organs without malignancies and
pancreatic tumors confirmed that that the
gamma-butyric acid A receptor π subunit was
significantly overexpressed in pancreatic
carcinomas. Analysis of 12 pancreatic tumors
revealed that the π subunit was overexpressed
in 10 of the tumors (83%). The expression
varied among the tumors, however,
overexpression was observed in tumors of
each histopathological grade. In contrast,
none of the normal pancreatic tissues
analyzed displayed high levels of expression.
Conclusions The expression of the GABAA
receptor π subunit may thus play an important
role in the pathogenesis of pancreatic cancer.
INTRODUCTION
Pancreatic cancer is the fifth most common
cause of cancer death. Conventional
therapeutic approaches have not had much
impact on the course of this aggressive
neoplasm. The dismal overall 5-year survival
associated with pancreatic cancer is 3% and is
largely a result of diagnosis late in the course
of the disease. Currently, the only curative
intervention is surgery, however only 15-20%
of patients present with resectable lesions. A
better understanding of the pathogenesis of
pancreatic cancer and more effective
screening techniques are required to increase
the proportion of patients presenting with
early resectable disease and to improve
current survival rates [1].
Studies have begun to uncover the molecular
alterations that are most prevalent in

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pancreatic cancer. Among these, activating
mutations in K-ras and inactivating mutations
in the tumor suppressors p53p16, and DPC4
are the most common alterations [2]. Other
important events in pancreatic tumorigenesis
include changes in expression of receptor
tyrosine kinases including the epidermal
growth factor receptor and HER2/neu
oncoprotein. Both are overexpressed in
pancreatic cancer and are capable of
transducing extracellular growth stimulatory
signals [3].
Pancreatic cancers are resistant to both
chemo- and radiation therapy. The exact
mechanisms of resistance remain poorly
understood. Early studies suggested a role for
P-glycoprotein, the product of the multidrug
resistance gene (MDR-1), but later studies
could not show upregulation of P-
glycoprotein in pancreatic cancer cell lines.
However, another transmembrane ATP-
dependent transporter, multidrug resistance
associated protein (MRP) is upregulated in
these cell lines [3].
The identification of genes involved in
tumorigenesis is essential for the development
of new treatment strategies or diagnostic
approaches. Thus, we tried to identify genes
differentially expressed between pancreatic
cancer and pancreatic tissues without
malignancies. To investigate those genes
overexpressed in pancreatic cancer we
employed differential display, a PCR-based
method of differential expression cloning.
Using this method, we found that the gamma-
aminobutyric acid (GABA) A receptor π
subunit was overexpressed in pancreatic
tumors.
GABA is the major inhibitory
neurotransmitter in the brain and is essential
for the overall balance between neuronal
excitation and inhibition. GABA influences
neurons via a large number of receptor
subtypes which are grouped on the basis of
their pharmacology under three major classes
of receptors: GABAA, GABAB, and GABAC
receptors. GABAand GABAare ligand-
gated ion channels, while GABAare G-
protein coupled receptors. GABAreceptors
are GABA-gated chloride ion channels that
cause inhibition of neuronal firing [4].
In mammals, there are 14 known subtypes of
GABAreceptor subunits that are thought to
assemble in different pentameric complexes.
In addition to their location on central neurons
and astroglia, functional GABAreceptors
have been detected on peripheral neurons and
non-neuronal cells. The non-neuronal cells
include endocrine cells of the pituitary pars
intermedia, adrenal medulla, islets of
Langerhans, placenta, and smooth muscle
cells of the urinary bladder and uterus. The
precise function of GABAreceptors in non-
neuronal cells is unclear. In endocrine cells
they have been implicated in regulation of
hormone secretion and in the uterus their
function appears to be related directly to
tissue contractility. The receptor π subunit
was identified recently by searching a
database of expressed sequence tags (ESTs)
with a peptide consensus sequence of known
GABAA
receptor family members. The
receptor π subunit cDNAs were expressed as
recombinant proteins and shown to assemble
with known GABAreceptor subunits and
confer unique ligand binding properties to the
resulting recombinant receptor [5]. Here we
describe the identification and subsequent
characterization of the expression of the π
subunit of the GABAreceptor family in
pancreatic cancer.
METHODS
Human Tissue Samples
Tissue samples from patients with
adenocarcinoma of the pancreas were
provided by the Cooperative Human Tissue
Network (CHTN) which is funded by that
National Cancer Institute. For RT-PCR
analysis of GABAA
receptor π subunit
expression in pancreatic tumors of various
histopathological grades, 12 tumors were used
and included 1 well-differentiated, 4
moderately, 4 moderately-to-poorly, and 3
poorly differentiated tumors. A set of 6
pancreatic ducts without evidence of

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malignancy from organ donors without
suitable recipients were obtained from the
Department of Surgery, University of
Arkansas for Medical Sciences (UAMS).
RNA Isolation
RNA from tissue samples less than 250 mg
was isolated by selective absorption on to
silica
gel-based
membranes
using
commercially available spin columns
(Qiagen, Valencia, CA, USA). For samples
greater than 250 mg, such as donor pancreas,
RNA was isolated using standard guanidine
methods for total RNA preparation [6]. Total
RNA (50 µg) was treated with 10 units of
DNase I, phenol extracted, and ethanol
precipitated. The treated RNAs were
suspended with diethyl pyrocarbonate-treated
water (1.0 µg/µL, final concentration) and
stored at -80°C until use.
Differential Display
Messenger RNA expression in 5 pancreatic
ductal adenocarcinomas (3 poorly and 2
moderately-to-poorly differentiated) and bulk
pancreatic ducts isolated from 5 independent
pancreatic specimens without malignancies
was analyzed by differential display.
Differential display was performed essentially
as described by Liang and Pardee [7] using
RNAimage Kits (GenHunter, Nashville, TN,
USA). Complementary DNA fragments
detected by differential display in at least 3
out of the 5 tumors analyzed were isolated,
reamplified, and cloned into the pGEM-T
Easy vector (Promega, Madison, WI, USA).
DNA sequences of the cloned PCR products
obtained in this manner were used to search
GenBank databases (e.g., non-redundant and
EST) to determine if they represent known or
novel gene products.
Relative Quantitative PCR
The relative expression level of GABAA
receptor π subunit was examined by
quantitative PCR essentially as described by
Tanimoto et al. [8]. Sequence-specific
primers used to amplify the GABAreceptor
π
subunit
were
5′-
CGTCGAGGTCGGCAGAAGT-3′ (sense)
and 5′-GCGGGCATCCAGAGTGAAG-3′
(antisense) which amplifies a sequence that
corresponds to nucleotides 237-487 of the π
subunit mRNA sequence (accession #
NM_014211). Primers for beta-actin, 5´-
GCATGGGTCAGAAGGAT-3´ (forward)
and
5´-CCAATGGTGATGACCTG-3′
(reverse), were included as an internal
control. First-strand cDNAs were synthesized
from 2 µg of DNase I-treated total RNA from
pancreatic ducts and adenocarcinomas by
Moloney murine leukemia virus reverse
transcriptase (Promega, Madison, WI, USA)
using oligo (dT) and random primers. Two
microliters of each cDNA product (about 50
ng) were amplified in a mixture containing 5
pmol of GABAreceptor π subunit-specific
primers, 2.5 pmol of beta-actin-specific
primers, 200 µM dNTPs, 5 µCi [alpha-32P]
dCTP, and 1 unit Taq DNA polymerase with
reaction buffer in a final volume of 25 µL.
The PCR amplification was carried out for 35
cycles of 94°C for 30 sec, 55°C for 30 sec,
and 72°C for 1 min. The linearity of the PCR
reaction for 35 cycles of PCR was confirmed
with the two sets of primers. Reaction
products were separated on 1.5% agarose gels
containing ethidium bromide and the level of
amplification was determined using a
PhosphorImager (Molecular Dynamics,
Sunnyvale, CA, USA). The relative
expression was measured as a ratio of
GABAreceptor π subunit expression to
beta-actin expression.
STATISTICS
The overexpression cutoff value was defined
as the mean value for pancreatic duct
expression +2 standard deviations (SD). An
unpaired t test was used for the comparison of
the mean values of normal pancreatic ducts
with tumors (Prism software, GraphPad
Software, San Diego, CA, USA). A
significant difference in the relative mean
expression is defined using as a two-tailed P

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value less than 0.05. Expression in each RNA
sample was measured 3 to 5 times.
ETHICS
All research using human tissues was
reviewed and approved by the UAMS Human
Research Advisory Committee.
RESULTS
Differential Display of Pancreatic Tissues
A cDNA fragment, 12A3, was identified by
differential display that was consistently
overexpressed in pancreatic tumors compared
to its expression in pancreatic ducts from
normal organs (Figure 1). The cDNA
fragment was reamplified with the primers
used in differential display and the resulting
250-bp PCR product was cloned and
sequenced. GenBank database searches
revealed that the clone corresponded to a
portion of the GABAreceptor π subunit.
RT-PCR Analysis of GABAReceptor π
Subunit Expression in Pancreatic Tissues
To verify the overexpression of the GABAA
receptor π subunit observed in the differential
display profile, the expression level of the
receptor π subunit was measured in RNA
prepared from pancreatic ducts isolated from
organs without malignancies and pancreatic
adenocarcinomas. Gene-specific primers were
designed from the GABAreceptor π subunit
cDNA sequence and used in a RT-PCR assay
to quantify its expression relative to beta-
actin. A comparison of π subunit expression
in pancreatic adenocarcinomas with
pancreatic ducts from donor organs indicated
that the GABAA
receptor π subunit is
expressed at significantly higher levels in the
pancreatic tumors (Figure 2).
To assess the correlation of GABAreceptor
π subunit expression in pancreatic tumors
with progressing stages of tumor
differentiation, RT-PCR analysis was
performed on RNA isolated from tumors with
various histopathological grades (Figures 2
and 3). Using a cutoff level for
overexpression of the mean expression in
nontumor pancreatic duct +2SD, 10 of 12
(83%) adenocarcinoma cases were above the
cutoff value. The expression of the GABAA
receptor π subunit varied among the tumors,
however, overexpression was observed in
tumors at each stage of differentiation. In
contrast, none of the normal tissues displayed
high levels of expression. The expression
Figure 1. Portion of a representative differential
display gel comparing pancreatic duct (D) and tumor
(T) cDNAs amplified with arbitrary primers (11, 12,
13) and anchored oligo (dT) primers (A, G, C) as
described in Methods. Tumor cDNA 12A3 (arrow) was
cloned and identified as GABAreceptor π subunit.
Figure 2. RT-PCR analysis for the mRNA expression
of GABAA
receptor π subunit and beta-actin in
pancreatic ducts without malignancies (Control) and
pancreatic
adenocarcinomas
of
various
histopathological grades (Well, Moderate, Mod-Poor,
Poor). RT-PCR products were electrophoretically
separated on agarose gels and stained with ethidium
bromide as described in Methods.

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level of GABAreceptor π subunit was
highest in the well-differentiated tumor,
however the significance of this observation
is tempered by the analysis of only a single
tumor at this stage of differentiation. Seventy-
five percent of the moderately and
moderately-to-poorly differentiated adeno-
carcinomas overexpressed the π subunit as
did all three of the poorly (G3) differentiated
tumors that were analyzed.
DISCUSSION
The GABAreceptor family is considered to
be the most complicated of the GABA
receptor families due to the large number of
receptor subtypes and the variety of ligands
that interact with specific sites on the
receptors. No selective agonist of the GABAA
receptors has been identified that does not
also have significant agonist action on the
ligand-gated ion channels of the GABAC
receptors. There are at least 11 proposed
structurally specific sites on GABAA
receptors that can be bound by oleamide,
thyroid hormones, peptide hormones (e.g.,
somatostatin-14), amyloid-beta protein, and
even insulin which promotes the rapid
translocation of GABAreceptors from the
intracellular compartment to the plasma
membrane in transfected human embryonic
kidney cells. Other chemicals acting on
GABAA
receptors include nitric oxide,
flavonoids, terpenoids, and many therapeutic
agents [4].
Transepithelial solute transport and
bicarbonate secretion are major functions of
pancreatic duct cells and both functions are
thought to involve the presence of chloride
channels in the apical membrane of the cell.
In the pancreatic adenocarcinoma cell line
Capan-1, a high density of chloride-selective
channels was identified. These cells express
vasoactive intestinal peptide receptors
associated with adenylyl cyclase that may be
involved in the secretion of ions. There is also
a high basal level of cAMP in Capan-1 cells
that may account for the existence of ion
transport in the absence of hormone
stimulation [9]. In pancreatic tumor tissues,
we identified a subunit of the GABAA
receptor that may associate with other
GABAA
receptor subunits to form a
functional chloride channel. GABAreceptor
subunits were identified in the carcinoma cell
line P19. Neurons derived from the
embryonal carcinoma cell line P19 were
found to express mRNAs for alpha, beta and
gammasubunits and to possess GABA
receptor-activated chloride currents [10]. The
association of malignancy with elevated
diamine oxidase (DAO), an enzyme
producing GABA, is well documented.
Elevated urine GABA levels were observed in
ovarian cancer patients providing evidence for
the association of a GABA producing enzyme
in malignant tissues and the possibility of
functional GABA-activated chloride channels
in these tissues [11].
Chloride channels regulated by different
mechanisms have been associated with
several types of cancer. The multidrug
resistant gene (MDR-1) which produces the P-
glycoprotein, a drug efflux pump, is often
considered in relation to ion channels
overexpressed in malignant tissues. The P-
glycoprotein and the multidrug resistance
associated protein (MRP) have been
implicated in cancers that are highly resistant
to chemotherapy such as pancreatic
Figure 3. Quantitation of GABAreceptor π subunit
expression relative to beta-actin. Expression of the
receptor π subunit mRNA was measured by RT-PCR
in pancreatic ducts without malignancies (Duct, n=6)
and pancreatic adenocarcinomas (Tumor, n=12) and
defined relative to beta-actin mRNA expression in each
tissue. GABAreceptor π subunit mRNA expression
levels were significantly elevated in tumors compared
to normal pancreatic ducts (*P=0.026).

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carcinoma. Other anion channels have also
been associated with cancer; in particular, the
volume-sensitive chloride currents that are
stimulated by cell swelling. In multidrug-
resistant lung cancer cells chloride channels
that do not require ATP and are not associated
with MDR-1 gene expression were observed
[12]. Volume-sensitive chloride channels are
also expressed in colon adenocarcinoma,
transformed tracheal epithelium, and cervical
cancer among others [13, 14].
In many epithelial cells, chloride channels are
essential for the transport of salt and water
across the membrane bilayer. Three distinct
chloride currents, regulated by cAMP, Ca2+,
and cell volume have been demonstrated in
airway epithelial cells and in the T84 colonic
carcinoma cell line [14]. In cervical cancer,
cell swelling was shown to activate ATP-
dependent Clcurrents but not in normal
cervix suggesting that activation of volume-
activated chloride currents is associated with
malignant transformation of human cervical
squamous epithelium. Volume-sensitive
chloride channels have been reported to be
important for regulation of cellular volume
during mitosis and osmotic challenge,
activation of the transport of amino acids and
other organic substrates, and also are related
to the cytoskeleton or motility of cells. From
these studies it was suggested that activation
of Clchannels might confer on the cells a
selective advantage for continuous growth
and survival [14].
Volume-regulated anion channels (VRAC)
have been linked with cell cycle progression
in cervical cancer. During cell cycle
progression, cells undergo a significant
increase in size (especially at the G1/S
transition) that perturbs cell volume
homeostasis and is counterbalanced by
regulatory volume decrease. Several studies
suggest that differential expression of K+
channels and concomitant changes in
membrane potential are critical for cell cycle
checkpoints. Indeed, when the VRAC was
investigated in cervical cancer, arrest of cell
growth in G0/Gphase was accompanied by a
marked decrease of VRAC current density
and that activity recovered upon re-entry into
the cell cycle [15].
These studies implicate a role for anion
channels in carcinogenesis. In the present
study, we have observed overexpression of
the GABAreceptor π subunit in pancreatic
tumors. These findings are consistent with the
original identification of the π subunit from a
pancreatic carcinoma cDNA library [5]. The π
subunit was overexpressed in tumors from all
histopathological grades analyzed, including
well to poorly differentiated tumors. This
suggests that increased transcription of the π
subunit gene is an early and sustained event in
the tumorigenic process. Expression of this
anion channel in tumor tissues indicates it
may have a role in pancreatic carcinogenesis
by as yet undiscovered mechanisms. Further
studies will have to be performed to
determine if other GABAsubunits are
expressed which can interact with the π
subunit, associate, and form a functional
chloride channel. In addition, confirmation of
increased levels of protein expression in these
tumors must await the development of
specific antisera directed against this GABA
receptor subunit for immunohistochemical
analysis of tumor tissues. This study,
however, has provided a new avenue for
study of pancreatic carcinogenesis by
determining another mechanism by which
pancreatic tumor cells may enhance
proliferation and growth in this aggressive
disease.
Received November 18th, 2004 - Accepted
January 31st, 2005
Keywords
Gene Expression; Gene
Expression Profiling; Reverse Transcriptase
Polymerase Chain Reaction
Abbreviations CHTN: Cooperative Human
Tissue Network; DAO: diamine oxidase;
EST: expressed sequence tag; GABA:
gamma-aminobutyric acid; MDR-1: multidrug
resistant gene; MRP: multidrug resistance
associated protein; UAMS: University of

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Arkansas for Medical Sciences; VRAC:
volume-regulated anion channels
Acknowledgments We thank Dr. Gary
Barone for acquisition of normal and chronic
pancreatitis tissues. This work was supported
in part by a grant from the UAMS College of
Medicine (R.S.H.) and Graduate Student
Research Fund (S.K.J.).
Correspondence
Randy S Haun
Department of Pathology
University of Arkansas for Medical Sciences
4301 W. Markham St., Slot 753
Little Rock, AR 72205-7199
USA
 
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