Int. J.
Cancer: 72, 931–936 (1997)
r 1997 Wiley-Liss, Inc.
DIFFERENTIAL EXPRESSION OF THE CCK-A AND CCK-B/GASTRIN RECEPTOR
GENES IN HUMAN CANCERS OF THE ESOPHAGUS, STOMACH AND COLON
Pascal CLERC1, Marlène DUFRESNE1, Corinne SAILLAN1, Eric CHASTRE2, Thierry ANDRÉ2, Chantal ESCRIEUT1, Karen KENNEDY1,
Nicole VAYSSE1, Christian GESPACH2 and Daniel FOURMY1,*
1INSERM U 151, Institut Fédératif de Recherche Louis Bugnard, Centre Hospitalier Universitaire Rangueil,
31052 Toulouse Cedex, France
2INSERM U 55, Hôpital Saint Antoine, 75571 Paris Cedex, France
The expression of cholecystokinin (CCK) and gastrin (G)
receptors in human gastrointestinal cancers remains poorly
documented and is still of a controversial nature. We have
measured the levels of mRNA for CCK-A and CCK-B/gastrin
receptors using quantitative reverse transcription-polymer-
ase chain reaction (RT-PCR) in primary digestive cancers and
hepatic metastases. CCK-A-receptor mRNA was detected in
5 out of 8 esophageal cancers (0.1–1 fg/mg), in 5 out of 8
gastric cancers (0.05–4.2 fg/mg) and in 5 out of 12 colon
cancers (0.1–1 fg/mg RNA). CCK-B/gastrin mRNA was not
detected in esophageal cancers but was detected in 7 out of 8
gastric cancers (0.05–5.2 fg/mg), and in only 2 out of 12 colon
adenocarcinomas (0.05–1 fg/mg RNA). The expression of the
CCK-A receptor in esophageal, gastric and colon cancers and
of the CCK-B/gastrin receptor in the majority of gastric
adenocarcinomas screened may be an important indicator of
the influence of CCK and gastrin of local or systemic origin on
the growth of these cancers. Int. J. Cancer 72:931–936, 1997.
r 1997 Wiley-Liss, Inc.
The gastrointestinal peptides gastrin and CCK, which share an
identical carboxy-terminal pentapeptide critical for receptor bind-
ing and biological activity, are 2 key regulatory peptides. They act
through 2 distinct receptor sub-types, the CCK-A receptor, which
has an approximately 500-fold higher affinity for CCK than for
gastrin, and the CCK-B/gastrin receptor, which has the same high
affinity for both CCK and gastrin (Poirot et al., 1993).
CCK and gastrin regulate digestive functions and also stimulate
proliferation of normal and of neoplastic cells (Baldwin, 1995;
Rehfeld and Van Solinge, 1994). Indeed, patients presenting a
gastrinoma often exhibit hypertrophy of the gastric mucosa and
hyperplasia of acid-secreting parietal cells and enterochromaffin-
like cells (Baldwin, 1995; Rehfeld and Van Solinge, 1994).
Moreover, it has been demonstrated that the growth of numerous
cancer cell lines derived from gastric, colorectal, pancreatic and
bronchogenic carcinomas can be stimulated by gastrin or inhibited
by antagonists of the CCK and gastrin receptors (Baldwin, 1995;
Rehfeld and Van Solinge, 1994). Interestingly, the expression of
gastrin peptides has been demonstrated in several digestive and
extra-digestive cancers, suggesting that autocrine regulation of the
growth of these neoplastic cells could occur when functional
CCK-B/gastrin receptors are present (Rehfeld and Van Solinge,
1994; Ciccotosto et al., 1995).
Pharmacological and functional studies have shown that CCK-A
and CCK-B/gastrin receptors are widely distributed throughout the
gastrointestinal tract (Poirot et al., 1993). The cloning of the cDNA
encoding these receptors has assigned them to the superfamily of
G-protein-coupled receptors characterized by 7 putative transmem-
brane domains (Poirot et al., 1993). The cloning of these cDNA has
also provided new cell lines expressing CCK-A and CCK-B/gastrin
receptors, as well as molecular tools to investigate the involvement
of these receptors in the proliferation of neoplastic cells, and their
signalling pathways. By means of these tools, the growth-factor-
like activity of gastrin was confirmed in CHO, NIH 3T3 and Rat1
cells transfected with the CCK-B/gastrin cDNA (Ito et al., 1993;
Taniguchi et al., 1994; Seufferlein et al., 1995), while CCK was
found to inhibit the proliferation of human pancreatic cell lines
transfected with CCK-A- and CCK-B/gastrin-receptor cDNA
(Detjen et al., 1997). The proliferative action of gastrin has been
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
further supported by data demonstrating that CCK-B/gastrin-
receptor activation induces intracellular events similar to those
involved in the signal transduction of growth-factor receptors, such
as the phosphorylation of proteins p125FAK, p42MAPK, p74raf-1, the
adaptor protein Shc, and expression of the early responsive genes
c-fos, c-myc and c-jun, which are characteristic of mitogenic
signals (Taniguchi et al., 1994; Seufferlein et al., 1995; Seva et al.,
1995).
In spite of the important potential therapeutic interest of these
findings, the expression of the genes encoding CCK-A and
CCK-B/gastrin receptors in human digestive cancers remains
poorly documented. We have therefore developed a simple and
efficient method of reverse transcription-polymerase chain reaction
(RT-PCR) for the quantification of messenger RNA coding for the
CCK-A and CCK-B/gastrin receptors, and have used this method to
screen a series of primary digestive carcinomas from the esopha-
gus, stomach and colon, and hepatic metastases.
MATERIAL AND METHODS
Patients and tissue sampling
Mucosa from tumoral and adjacent tissue samples was obtained
from patients during surgery at Hôpital Saint-Antoine (Paris) and
was histopathologically examined. None of the patients had
received chemotherapy or radiotherapy before or at time of surgery.
Biopsies of normal mucosa were also obtained during a gastrointes-
tinal endoscopic procedure in patients free of malignant diseases.
The study protocols were approved by hospital ethical committees.
The 8 esophageal-tumor samples (7 epidermoid carcinomas and
1 adenocarcinoma) were all from male patients with a mean age of
61 years (range, 53–74 years). The tumors were staged according to
the TNM classification, and were distributed as follows: T3N1M0,
5 specimens, numbers 1, 2, 3, 7, 8; T3N0M0, 1 specimen, number
6; T3N0M1, 1 specimen, number 5; T2N1M0, 1 specimen, num-
ber 4.
The 8 gastric-adenocarcinoma samples were from 6 male and 2
female patients with a mean age of 63.5 years (range, 34–84 years);
3 tumors were localized in the cardia, 2 of them (numbers C2 and
C3) presenting poor or moderate degrees of differentiation, while
the third (number C1) was polymorphic; 5 tumors were from the
antrum, among them, 4 poorly differentiated (numbers A1, A3, A4,
A5) and 1 well differentiated (number A2).
The 12 specimens of colon adenocarcinomas were from 3 male
and 9 female patients with a mean age of 74.5 years (range, 61–89
years). The tumors, staged according to the Dukes’ classification as
modified by Astler and Coller (1954), were distributed as follows:
stage B, 9 specimens (numbers 1, 2, 3, 4, 8, 9, 10, 11, 12); stage C, 2
Contract grant sponsor: Association pour la Recherche sur le Cancer;
contract grant number: 6234; Contract grant sponsor: Région Midi-
Pyrénées.
*To whom correspondence and reprint requests should be addressed.
Fax: 33-5-61-32 24 03. E-mail: fourmyd@rangueil.inserm.fr.
Received 19 November 1996; revised 7 May 1997

932 CLERC ET AL.
specimens (numbers 5, 13); stage D, 1 specimen (number 14). The
villous tumor (number 7) was from a 67-year old female; the
sporadic colon adenoma (number 6) was from a 34-year old
female, and the 2 colonic adenomas from familial adenomatous
polyposis were from a 36-year old male (number 15) and a 43-year
old female (number 16).
Finally, 8 hepatic metastases from patients presenting a colon
adenocarcinoma (6 males, 2 females, mean age, 67.5 years) were
analyzed.
Cell lines
Three gastric-carcinoma cell lines (HGT-1, MNK 28, MNK 74),
3 human colonic cell lines (HT-29, CaCo2, LoVo), the neuroblas-
toma cell line CHP 212 and the lymphoma cell line Jurkat T were
grown in the presence of 10% FCS. After 5 days of culture, total
RNA was extracted.
Isolation of total RNA
Tissue samples were cut into small pieces and frozen in liquid
nitrogen until RNA preparation. Tissue or cells were homogenized
with a polytron tissue homogenizer in RNAZOLy B (Bioprobe,
Montreuil-sous-Bois, France). Homogenized material was then
incubated in RNAZOL for 10 min at 4°C, and RNA was extracted
with chloroform for 5 min at 4°C. After centrifugation at 12,000 g
for 30 min at 4°C, the extracted RNA was precipitated with
isopropanol and washed with cold 75% ethanol. RNA concentra-
tion was measured spectophotometrically at 260 nm. The integrity
of the mRNA was controlled by analyzing ribosomal RNA content
by electrophoresis on an agarose gel and by RT-PCR amplification
of b-actin mRNA.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA (3 to 10 µg) was heated for 10 min at 70°C in the
presence of an oligo dT primer (1 mM), then incubated at 37°C in a
20-µl reaction volume consisting of reverse-transcriptase buffer (50
mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM Mg Cl2, 10 mM DTT),
20 U RNAsine (GIBCO-BRL, Cergy Pontoise, France) and 200 U
Superscript reverse transcriptase (GIBCO-BRL). The single-
stranded cDNA obtained from 1 µg reverse-transcribed total RNA
was amplified using specific primers for b-actin or for CCK-A or
CCK-B/gastrin receptors. For amplification of b-actin cDNA, the
sense primer P5 was 58-ACCACACCTTCTACAATGAGCTGC-
GTG-38 and the anti-sense primer P6 was 58-CACAGCTTCTCCT-
TAATGTCACGCACG-38. The expected amplicon had a size of
365 bp (Matsumori et al., 1995). For the CCK-A receptor, the sense
primer P1 58-CTGCTCAGCGTGCTGGGAAAC-38 and the anti-
sense primer P2 58-CGGGACTGTAAGGGTTTGCAAAT-38 were
used. P1 and P2 were analogous to nucleotides (nt) 156–177 and
the complement to nt 426–449 in the translated cDNA, and yielded
an expected amplicon of 293 bp. For the CCK-B/gastrin receptor,
the sense primer P3 was 58-CGGGACACGAGAATTGGAGC-
TGG-38 and the anti-sense primer P4 was 58-CCGTCAAAGC-
GAAGCCCTAAGTAG-38. P3 and P4 were analogous to nt
140-163 and the complement to nt 734–758, and yielded an
expected amplicon of 617 bp. Primer pair P1–P2 amplified a
fragment that crossed intron 2 of the CCK-A-receptor gene, while
primer pair P3–P4 amplified a fragment that crossed introns 2 and 3
of the CCK-B/gastrin-receptor gene, thereby allowing the detection
of contaminating genomic DNA.
The PCR reaction was carried out in a total volume of 100 µl,
composed of PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0,
0.01% Triton X-100), 0.25 mM dNTPs, 2 µM primers and 2.5 U
Taq polymerase (Promega, Charbonnieres, France). The amplifica-
tion reaction involved denaturation at 94°C for 5 min followed by
cycling as follows: 94°C for 1 min, primer annealing at 65°C for 1
min, extension at 72°C for 1.5 min. After cycling, terminal
elongation of 10 min at 72°C was performed. Routinely, amplifica-
tion required 35 cycles for b-actin, and 30 and 32 cycles for the
CCK-A and CCK-B/gastrin receptors respectively.
10970215, 1997, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0215(19970917)72:6<931::AID-IJC2>3.0.CO;2-Q by The King George Medical College, Wiley Online Library on [14/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
In preliminary experiments, we tested the specificity of the
primers by performing RT-PCR amplifications from total RNA
extracted from several reference human organs such as gallbladder,
stomach, pancreas, brain cortex and cerebellum. Amplicons of the
expected size were obtained without any non-specific PCR prod-
ucts.
Quantification of mRNA for the CCK-A and CCK-B/gastrin
receptors
For quantitative analysis of the mRNA, PCR products were
separated by electrophoresis on a 7.5% polyacrylamide gel which
was stained with ethidium bromide and then photographed under
UV light. Signals from PCR products were submitted to image
analysis (Biocom Image station, Biocom, France). Experiments
were performed to determine the range of exponential amplifica-
tion and to ensure that a linear relationship existed between the
amounts of cDNA matrix and PCR product. Quantification of the
target CCK-A-receptor mRNA and the CCK-B/gastrin receptor
was achieved by extrapolating the signal intensity yielded by the
amplicon to a standard curve obtained in a separate yet identical
PCR reaction. For the standard curves, serial dilutions of a defined
amount of double-stranded cDNA coding for the CCK-A and the
CCK-B/gastrin receptors were performed to give quantities of
matrix ranging from 0.05 to 5 fg. Double-stranded cDNA used for
the standard curves were obtained by RT-PCR amplification of
cloned cDNA using primer pairs P1–P2 and P3–P4, and were
quantified by comparing intensity of bands corresponding to serial
dilutions of these synthetic matrices with those from serial
dilutions of PGEM markers of identical size. Finally, controls were
included in PCR assays: a negative control corresponding to a PCR
reaction in which reverse-transcribed RNA was replaced by water,
and a positive control of samples used for the standard curve.
RESULTS
Setting up of the RT-PCR method for CCK-A- and
CCK-B/gastrin-receptor-mRNA quantification
We first defined the experimental conditions for quantification of
receptor mRNA. In RT-PCR, the PCR reaction represents the step
of amplification which must be standardized for quantitative
measurements. Experiments performed to determine the number of
cycles producing quantifiable signals within a linear range of
amplification showed that the appropriate number of cycles was
between 30 and 33 for the CCK-A receptor and between 32 and 36
for the CCK-B/gastrin receptor (Fig. 1). Then, using 30 cycles for
the CCK-A receptor and 32 cycles for the CCK-B/gastrin receptor,
we ensured that a linear relationship existed between the amounts
of cDNA matrix and the quantities of PCR product obtained. As
illustrated in Figure 2, these criteria were fulfilled for quantities of
cDNA matrix ranging from 0.05 to 5 fg.
Validation of RT-PCR by quantification of CCK-A- and
CCK-B/gastrin-receptor-mRNA levels in normal human tissues
and cancer cell lines
The method was first tested with normal human tissues in which
expression of the CCK-A and CCK-B/gastrin receptors has been
demonstrated or can be expected from functional studies. RT-PCR
amplification of total RNA isolated from human tissues demon-
strated the following CCK-A-receptor-mRNA levels (fg/µg RNA,
Fig. 3): gallbladder, 0.30 6 0.15 (n 5 3); pancreas, 0.05 6 0.05
(n 5 3); gastric fundus, 0.45 6 0.26 (n 5 5). For the CCK-B/
gastrin receptor, mRNA levels were as follows (fg/µg RNA, Fig. 3):
brain cortex, 5.00 6 0.80 (n 5 3); cerebellum, 7.50 6 1.20
(n 5 3); gastric fundus, 0.13 6 0.09 (n 5 5). In addition, CCK-B/
gastrin-receptor mRNA was detected in the human pancreas
(1.00 6 0.20 fg/µg RNA).
As shown in Figure 3, screening of several cell lines indicated
that CCK-A-receptor mRNA was detectable in the CHP 212
neuroblastoma cell line (0.10 6 0.05 fg/µg RNA) and mRNA for
the CCK-B/gastrin receptor in the lymphocyte cell line Jurkat T

CCK-A AND CCK-B/GASTRIN RECEPTORS IN DIGESTIVE CANCERS
FIGURE 1 – Determination of the exponential range of RT-PCR amplifi-
cation for 0.5 fg of double-stranded cDNA coding for the CCK-A and
CCK-B/gastrin receptors. The intensity of the stained bands at 293 bp
(CCK-A receptor: —d—) and 617 bp (CCK-B/gastrin receptor:
—s—) measured by image analysis was plotted against the number of
PCR cycles. A linear range of amplification was obtained with 30 to 33
cycles for the CCK-A receptor and 32 to 36 cycles for the CCK-B/
gastrin receptor, therefore, 30 cycles for the CCK-A receptor and 32
cycles for the CCK-B/gastrin receptors were chosen for the quantitative
analysis of receptor mRNA expression.
(4.00 6 0.50 fg/µg RNA). In the human colonic cell lines HT 29
(not shown), CaCo2, LoVo, and in gastric cell lines HGT-1 (not
shown), MKN28 and MKN74, no receptor mRNA could be
detected, the lower limit of detection being 0.05 fg/µg RNA for
both receptors.
Levels of mRNA coding for CCK-A and CCK-B/gastrin receptors
in primary digestive carcinomas and hepatic metastases
We analyzed a total of 43 specimens, distributed as follows: 18
colorectal adenocarcinomas, 1 villous tumor, 8 gastric adenocarci-
nomas, 8 esophageal cancers and 8 hepatic metastases from
colorectal adenocarcinomas. In addition, 1 sporadic colon adenoma
and 2 adenomas from familial adenomatous polyposis were
analyzed. Control tissues were composed of tissues surrounding the
tumor or of biopsies from patients without a cancer.
The RNA preparations from all tumoral-tissue samples were
submitted to RT-PCR determination of b-actin mRNA levels, to
ensure that intact mRNA was present in the samples studied and
that contaminating genomic DNA was absent. We found an almost
constant level of b-actin mRNA, and no fragments from the loci of
the b-actin gene (not shown).
In esophageal cancers, CCK-A-receptor mRNA was detected in
5 tumor samples out of 8, at levels ranging from 0.1 to 1 fg/µg; 6
samples of normal mucosa from patients presenting an esophagus
cancer also contained CCK-A-receptor mRNA, and only one
sample presented a low level of CCK-B/gastrin mRNA (Fig. 4).
The 3 esophageal tumors presenting the highest levels of CCK-A-
receptor mRNA (0.3, 1.0 and 4.0) were classified as T2N1 for the
first (number 4) and T3N0 for the other 2 (numbers 6 and 5). The
mean level of CCK-A-receptor mRNA in the esophageal tumors
(0.70 fg/µg) was 2-fold higher than that in the surrounding mucosa
(0.35 fg/µg) and 7-fold higher than that in mucosa from patients
who did not present a cancer (0.10 fg/µg). In contrast, CCK-B/
gastrin-receptor mRNA was undetectable in all samples except one
from patients with cancer of the esophagus, but was well expressed
10970215, 1997, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0215(19970917)72:6<931::AID-IJC2>3.0.CO;2-Q by The King George Medical College, Wiley Online Library on [14/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
933
(0.3, 0.6, 4.0 fg/µg RNA) in 3 out of 5 biopsies of normal mucosa
from patients who did not present a cancer.
We analyzed 8 gastric adenocarcinomas for CCK-A- and
CCK-B/gastrin-receptor-mRNA expression: 3 tumors were local-
ized in the cardia and 5 in the antrum. Transcripts for CCK-A and
CCK-B/gastrin receptors were present in 5 out of 8 and 7 out of 8
specimens respectively (Fig. 5). Levels of mRNA were higher in
the tumor than in the adjacent mucosa for the CCK-A receptor in 2
patients (numbers A1, A2) and for the CCK-B receptor in 4 patients
(numbers A1, A3, A5, C1). The opposite situation occurred in 4
patients for the CCK-A receptor (numbers A4, A5, C2, C3) and 3
patients for the CCK-B/gastrin receptor (numbers A4, C2, C3). In
fact, the mean levels of receptor mRNA in the tumors were very
similar to those in the surrounding mucosa from the same group of
patients. Interestingly, for patients with a cancer localized in the
antrum, the mean level of CCK-B/gastrin-receptor mRNA in
tumors (1.8 fg/µg RNA) and in adjacent tissue (0.9 fg/µg RNA)
were respectively 18- and 9-fold higher than that in normal antral
mucosa from patients without a cancer (0.10 fg/µg RNA). For
patients with a cancer localized in the cardia, the mean level of
CCK-A-receptor mRNA in tumors (0.33 fg/µg RNA) and in
adjacent tissue (2.1 fg/µg RNA) were respectively 4- and 25-fold
higher than that in normal cardiac mucosa from patients without a
cancer (0.08 fg/µg RNA). The differences were less pronounced for
CCK-B/gastrin-receptor mRNA, since the levels in the tumors (1.4
fg/µg RNA) and in the adjacent mucosa (2.3 fg/µg RNA) were
respectively 2- and 4-fold higher than that in mucosa from patients
without a cancer (0.7 fg/µg RNA).
Among the 12 colon-adenocarcinoma samples presenting compa-
rable levels of b-actin mRNA, CCK-A-receptor mRNA was found
both in the tumor and in adjacent mucosa in 5 patients (Fig. 6). In 4
patients, (3 cancers at Dukes’ stage B, numbers 2, 3, 4 and one at
stage C, number 5), CCK-A-receptor mRNA levels were low, (0.10
to 0.15 fg/µg RNA), while one patient (number 1) with an
adenocarcinoma at stage B demonstrated a higher CCK-A-receptor
mRNA level both in the tumoral tissue (1.0 fg/µg RNA) and in the
adjacent mucosa (0.5 fg/µg RNA). A low level (0.05 fg/µg) of
CCK-B/gastrin-receptor mRNA was found in only 2 tumor speci-
mens (numbers 1 and 7), a colon adenocarcinoma and a villous
tumor (Fig. 6). Finally, a low level of CCK-A-receptor mRNA (0.1
fg/µg RNA) was detected in a sporadic colon adenoma (number 6),
but not in the colon familial adenomatous polyposis adenomas (not
illustrated). Together, these results indicate that the mean level of
CCK-A-receptor mRNA in the tumors (0.30 fg/µg) was in the same
range as in the adjacent mucosa from the same group of patients
(0.20 fg/µg) and was 3-fold higher than that in the normal colonic
mucosa from patients free of malignant disease (0.10 fg/µg).
Among 8 hepatic metastases from patients with colon cancers, 7
specimens contained no detectable mRNA for the CCK-A and the
CCK-B/gastrin receptors. One metastasis demonstrated a low level
of CCK-B/gastrin-receptor mRNA (0.1 fg/µg RNA, not illustrated).
DISCUSSION
We here report RT-PCR analysis of a wide variety of human
digestive cancers for expression of mRNA coding for the CCK-A
and CCK-B/gastrin receptors. The RT-PCR method used by us to
measure receptor transcript levels is distinct from Northern-blot
analysis and analytical RT-PCR in that it is highly sensitive and
quantitative, and can be used on amounts of human tissues as low
as those present in biopsies. In spite of its inability to specifically
detect mRNA splice variants which may be present in normal and
malignant tissues, this method allowed determination of the levels
of receptor mRNA potentially of biological significance. Indeed, 2
splice variants of the CCK-B/gastrin receptor have been identified,
one which differs in the third intracellular loop, and one which has
a truncated N-terminus (Song et al., 1993; Miyake, 1995). In fact,
the first splice variant was demonstrated to present coupling
features identical to those of the CCK-B/gastrin receptor, and we
 10970215, 1997, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0215(19970917)72:6<931::AID-IJC2>3.0.CO;2-Q by The King George Medical College, Wiley Online Library on [14/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
934 CLERC ET AL.

FIGURE 2 – Calibration curves for RT-PCR quantification of CCK-A and CCK-B/gastrin receptor mRNA. Using 30 cycles for the CCK-A receptor
and 32 cycles for the CCK-B/gastrin receptor, we amplified increasing quantities of double-stranded cDNA. The signal was proportional to the
quantity of matrix for quantities from 0.5 to 5 fg cDNA. RT-PCR quantification of the target mRNA in tissue samples was achieved by
extrapolating the signal intensity obtained for the amplicon to this typical standard curve obtained in a separate but identical PCR reaction.

FIGURE 3 – Validation of the RT-PCR method using RNA extracts from human normal tissues and cancer cell lines. Total RNA from various
human tissues and tumoral cell lines were RT-PCR-amplified using specific primers in the conditions described in ‘‘Material and Methods’’. The
upper part of the picture corresponds to the b-actin signal, the middle and lower parts correspond to signals for CCK-A and CCK-B/gastrin
receptors, respectively. The following tissues were analyzed: lane 2, gallbladder; lane 3, brain cortex; lane 4, cerebellum; lane 5, pancreas; lane 6,
gastric fundus; cell lines analyzed: lane 7, CHP 212 neuroblastoma; lane 8, CaCo2; lane 9, Jurkat T; lane 10, LoVo; lane 11, MKN28; lane 12,
MKN74.

have observed, in separate experiments, that this splice variant was
present in minority in all tissues tested, including tumor samples
(unpublished data). Finally, the truncated CCK-B/gastrin receptor
was demonstrated to bind gastrin and CCK with decreased
affinities (Miyake, 1995).
The RT-PCR method was first validated by measuring mRNA
levels in normal human tissues known or expected to contain
CCK-A- and/or CCK-B/gastrin receptors. The distribution of
CCK-A- and CCK-B/gastrin-receptor mRNA found by our RT-
PCR method agrees with results from studies that used other
methods, including Northern blotting, radioligand binding, and
functional studies (Adler et al., 1995; Dietl and Palacios, 1989; Ito
et al., 1993; Lloyd and Debas, 1994; Monstein et al., 1996;
Schjoldager et al., 1989). Importantly, we obtained a quantitative
distribution of the 2 receptor transcripts in several human tissues.
The rank order for CCK-A-receptor mRNA expression was: gastric
fundus . gallbladder . pancreas; for the CCK-B/gastrin receptor,
it was cerebellum . brain cortex . pancreas . gastric fundus.
Interestingly, the levels of CCK-B/gastrin-receptor mRNA in the 2
areas of the central nervous system were at least 10-fold higher
than in the peripheral organs. Finally, results showing the presence
of mRNA for the CCK-A receptor in CHP 212 cells and for the
CCK-B/gastrin receptor in Jurkat T cells are in perfect agreement
with those showing the presence of functional receptor-binding
sites in the 2 cell lines (Barrett et al., 1989; Lignon et al., 1991). In
contrast to the findings of Miyake (1995), we did not detect any
mRNA for the CCK-B/gastrin receptor in LoVo cells. The different
PCR conditions used (number of cycles) as well as the nature of the
starting material (total RNA vs. Poly A1 RNA in the cited study)
could explain the different results. Alternatively, LoVo cells
analyzed in our laboratory could have lost expression of the
CCK-B/gastrin-receptor gene. For the other cell lines screened, the

CCK-A AND CCK-B/GASTRIN RECEPTORS IN DIGESTIVE CANCERS
FIGURE 4 – Levels of CCK-A- and CCK-B/gastrin-receptor mRNA in
esophageal cancers. We analyzed esophageal cancers (7 epidermoid
carcinomas and 1 adenocarcinoma), distributed as follows: T3N1M0, 5
specimens; numbers 1, 2, 3, 7, 8; T3N0M0, 1 specimen, number 6;
T3N0M1, 1 specimen; number 5; T2N1M0, 1 specimen; number 4.
NM (normal mucosa) corresponds to biopsies from patients not
presenting a cancer (n 5 5). T under the histograms represents tumoral
tissues; M corresponds to adjacent mucosa from the same patient. Dark
bars and closed circles corresponds to CCK-A receptor, open bars and
circles to CCK-B/gastrin receptor.
FIGURE 5 – Levels of CCK-A- and CCK-B/gastrin-receptor mRNA in
gastric adenocarcinomas. Of the 8 tumors, 3 were from the cardia,
(numbers C2 and C3) 2 with poor or moderate degrees of differentia-
tion while the third (number C1) was polymorphic; 5 tumors were from
the antrum, 4 (numbers A1, A3, A4, A5) poorly differentiated and 1
(number A2) well differentiated. CNM (cardiac normal mucosa) and
ANM (antral normal mucosa) correspond to biopsies from patients not
presenting a cancer (n 5 5); T under the histograms represents tumoral
tissues, M corresponds to adjacent mucosa from the same patient. Dark
bars and closed circles correspond to CCK-A receptor, open bars and
circles to CCK-B/gastrin receptor.
fact that no receptor transcripts were detected by RT-PCR corre-
lates with the lack of evidence for the presence of receptor-binding
sites in these cell lines (Baldwin, 1995).
We have shown that the majority of esophageal cancers screened
(62%) express mRNA coding for the CCK-A receptor but not for
the CCK-B/gastrin receptor. We also found that the majority of
gastric adenocarcinomas screened express mRNA coding for the
CCK-A and CCK-B/gastrin receptors (62 and 87% respectively).
Finally, we demonstrated expression of mRNA for the CCK-A and
CCK-B/gastrin receptors in 42 and 17%, respectively, of the colon
adenocarcinomas that were screened. In addition, the current study,
albeit performed on a relatively modest number of cancers,
revealed that receptor-mRNA levels often differed between tumor
cells and adjacent mucosa in the same patient. Patient-to-patient
variations were found in tumors and adjacent cells of the same
10970215, 1997, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0215(19970917)72:6<931::AID-IJC2>3.0.CO;2-Q by The King George Medical College, Wiley Online Library on [14/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
935
FIGURE 6 – Levels of CCK-A- and CCK-B/gastrin-receptor mRNA in
colon adenocarcinomas. We analyzed 12 colon adenocarcinomas. Data
are shown only for the 7 specimens that presented detectable levels of
CCK-A- or CCK-B/gastrin-receptor mRNA. The tumor samples were
staged as follows: stage B, 4 specimens (numbers 1, 2, 3, 4); stage C, 1
specimen (number 5). Results from 1 villous tumor (number 7) and 1
sporadic colon adenoma (number 6) are also shown. NM (normal
mucosa) corresponds to biopsies from patients not presenting cancer
(n 5 5); T under the histograms represents tumoral tissues, M corre-
sponds to adjacent mucosa from the same patient. Dark bars and closed
circles correspond to CCK-A receptor, open bars and circles to
CCK-B/gastrin receptor.
histopathological group. The variations of mRNA levels between
tumor cells and adjacent mucosa can be explained by an effect of
neoplastic transformation on receptor-gene expression. It has been
shown that neoplastic transformation of a cell can lead to increase
or decrease (or even loss) of expression of a given gene. In
addition, as reported for breast carcinomas, adjacent tissue having
normal histology can contain genetic abberations (Deng et al.,
1996). Taken together, these facts can account for the variability of
mRNA levels between different populations of cells in the same
patient as well as between cells demonstrating identical histology
but sampled from different patients. Despite these variations, our
data suggest that the development of esophageal cancers leads to
increased CCK-A-receptor-gene expression both in tumor and in
surrounding cells. In the same way, the development of gastric
adenocarcinomas from the antrum leads to increased expression of
the CCK-B/gastrin-receptor gene both in the tumor and in the
surrounding cells.
The expression of the CCK-A receptor in multiple esophageal,
gastric and colon cancers and of the CCK-B/gastrin receptor in the
majority of gastric adenocarcinomas may be an important indicator
of the influence of CCK and gastrin of either local or systemic
origin on the growth of these cancers. Indeed, CCK and gastrin are
regularly released into the blood circulation by endocrine cells in
response to different stimuli, including meals. More importantly,
CCK and gastrin can be constitutively released in the tumoral
mucosa by neoplastic cells themselves, as demonstrated for a wide
variety of digestive and extra-digestive cancers (ovarian, broncho-
genic) which express gastrin-related peptides (Rehfeld and Van
Solinge, 1994). Although gastrin peptides synthesized by tumoral
non-endocrine cells may often be poorly matured, it is likely that a
sufficient amount of biologically active gastrin is locally secreted
and may therefore act as an autocrine factor to contribute to tumor
development. The relative abundance of mature vs. non-mature
gastrin in cancers was recently documented in a study on 32
colorectal cancers which demonstrated the presence of fully
processed gastrin in 69% of the tumors and of progastrin in 100%
of the tumors (Ciccotosto et al., 1995). The question of whether
CCK, which is present in numerous neuro-endocrine tumors, is
also produced by digestive cancers, remains unresolved (Rehfeld
and Van Solinge, 1994). The answer to this question will help to

936 CLERC ET AL.
evaluate whether an autocrine loop involving CCK exists in certain
digestive cancers expressing CCK-A and/or CCK-B/gastrin receptors.
Until very recently, the predominant view was that the CCK-B/
gastrin receptor but not the CCK-A receptor could play a role in the
proliferation of digestive cancers, especially colon cancers. This
view was essentially deduced from studies on cancer cell lines;
however, the presence of CCK-B/gastrin receptors in human colon
cancer was by no means clear. In one study, very low specific
high-affinity binding of a gastrin radioligand was detected in some
primary tumors from human colon, and was claimed to be of
prognostic significance, whereas several other binding studies
failed to demonstrate the expression of CCK-B/gastrin-receptor
sites in the majority of colon cancers (Upp et al., 1989; Imdahl et
al., 1995). However, in a study that examined 102 colorectal
cancers, CCK-B/gastrin transcripts were detected in only 11% of
the colorectal carcinomas, a rate which is consistent with the results
of our study, demonstrating expression of CCK-B/gastrin mRNA in
17% of colon cancers (Imdahl et al., 1995). With respect to gastric
cancers, the expression of CCK-B/gastrin receptors in these
cancers was still speculative before the current study.
From a therapeutic point of view, the presence in digestive
cancers of CCK-A and/or CCK-B/gastrin receptors, which might
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ACKNOWLEDGEMENTS
This study was supported by grants from the Association pour la
Recherche sur le Cancer (6234) and from Région Midi-Pyrénées.
We thank Drs. Louis Buscail and Philippe Pagès for their help and
advice.
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