(CANCER RESEARCH 52. 3295-3299. June 15, 1992]
Overexpression of Cholecystokinin Receptors in Azaserine-induced Neoplasms of
the Rat Pancreas1
Richard H. Bell, Jr.,2 Elna T. Kuhlmann, Robert T. Jensen, and Daniel S. Longnecker1
Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire 03756 [R. H. B., E. T. K., D. S. L.J, and Digestive Diseases Branch, NIH, Bethesda,
Maryland 20892 fR. T. JJ
7 mm, remain benign; a small subset develop further dysplastic
and anaplastic changes and are presumed to be the precursor
Cholecystokinin (CCK) is a growth factor for normal pancreas. Nu
lesions of invasive carcinoma. Well-differentiated nodules in
merous studies also suggest that CCK promotes pancreatic carcinogenesis
the size range of 3-7 mm are classified histologically as ade
in the rat. Our previous studies suggested that growth of preneoplastic
nomas, and frankly anaplastic nodules without evidence of
pancreatic foci was stimulated by CCK more than that of normal pan
creas. We hypothesized that such differential growth might be due to invasion or metastasis are classified as carcinoma in situ.
The peptide hormone CCK, which binds to specific receptors
increased numbers of CCK receptors in neoplastic tissue.
Azaserine-induced pancreatic carcinoma (DSL6) had an increased
on pancreatic acinar cells, is known to stimulate the growth of
normal pancreatic tissue (3-6). This growth effect is mediated
high-affinity CCK receptor binding capacity of 122 ±23 (SD) fmol/mg
protein compared to 12 ±2 fmol/mg protein in normal pancreas (/' <
by a high-affinity CCK receptor (7). Beyond its effect on normal
0.001). The A',,of the high-affinity site was 0.33 ±0.04 MMfor carcinoma
pancreas, several studies suggest that CCK promotes the devel
and 0.46 ±0.08 n\i for normal pancreas (/' < 0.01). The amount of
opment of pancreatic neoplasms. It was noted several years ago
Cholecystokinin octapeptide (CCK-8) bound to high-affinity receptor was that long-term consumption of diets containing trypsin inhibi
8.6 ±1.9 fmol/mg protein for DSL6 compared to 0.6 ±0.2 fmol/mg
tors in the form of raw soya flour caused the development of
protein in normal pancreas ( /' < 0.001).
adenomas and carcinoma of the pancreas in rats (8). Rats fed
Azaserine-induced premalignant nodules were compared to remaining
internodular pancreas. Nodules demonstrated a mean high-affinity CCK raw soya flour also were found to be more susceptible to
receptor binding capacity of 38 ±9 fmol/mg protein compared to 6 ±3 azaserine-induced cancers than animals on a diet of standard
fmol/mg protein in internodular pancreas (/' < 0.001). The amount of chow (9). Trypsin inhibitors are known to raise plasma CCK
CCK-8 bound to high-affinity receptor was 3.1 ±0.8 fmol/mg protein in levels (10), probably because trypsin ordinarily inactivates the
nodules compared to 0.6 ±0.3 fmol/mg protein in internodular pancreas
duodenal monitor peptide which stimulates CCK secretion (11).
(P< 0.001).
Additional support for a role for CCK in experimental pan
Overexpression of high-affinity CCK-8 receptor in premalignant and
creatic carcinogenesis comes from studies of animals undergo
malignant azaserine-induced tumors may result in a growth advantage
ing chronic surgical diversion of pancreaticobiliary secretion to
relative to normal pancreas.
the distal small intestine. Such diversion results in chronic
elevation of plasma CCK levels (12) and to the development of
INTRODUCTION
adenomatous hyperplasia and in one instance carcinoma in situ
of the pancreas (12, 13).
Adenocarcinoma of the pancreas presents a formidable clin
Two studies of azaserine-induced carcinogenesis in the rat
ical challenge. It has become the fifth most common cause of pancreas strongly support a role for CCK as a relevant tumor
cancer death and remains nearly incurable, with an average
growth factor in vivo. In each study, all rats received a single
survival from diagnosis until death of approximately 3 months
injection of the carcinogen azaserine at 2 weeks of age. Rats
(1). For this reason, there has been considerable interest in subsequently received s.c. injections of either CCK or control
trying to identify factors involved in the growth of pancreatic
diluent for 16 weeks and were then sacrificed. In both studies,
cancer, particularly those which may be manipulable. Studies
the number of AACN in the pancreas of CCK-treated rats was
of pancreatic carcinogenesis take advantage of animal models
quantitatively measured and compared to controls. The quan
of the disease, of which one well described example is azaserinetitative measurement of AACN (14) is an accepted and stand
induced pancreatic carcinoma in the rat (2). In response to a ardized short-term bioassay for the development of carcinoma
single injection of the carcinogen azaserine (30 mg/kg body in the azaserine model. In one study (15), the administration of
weight) at age 2 weeks, approximately 50% of Lewis rats CCK to azaserine-treated rats led to an 8-20-fold increase in
develop acinar cell adenocarcinomas of the pancreas after a AACN as a percentage of pancreatic area. In the second study
latency period of 18 months. The course of azaserine-induced
(16), the administration of CCK caused an 8-fold increase in
carcinogenesis is characterized initially by the development of AACN as a percentage of pancreatic area. In addition, the
microscopic foci of atypical acinar cells (AACN),4 some of
administration of the CCK receptor antagonist CR-1409 sig
which become grossly visible about 6 months after exposure to nificantly reversed this effect, lowering AACN development to
azaserine. Most of these nodules, which range in size from 1 to levels not significantly different from those of controls not
treated with CCK.
Received 9/18/91; accepted 4/3/92.
The costs of publication of this article were defrayed in part by the payment
In a previous report from this laboratory (17), feeding of the
of page charges. This article must therefore be hereby marked advertisement in
synthetic trypsin inhibitor camostate (FOY-305) increased the
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This project was supported by a grant from the Hitchcock Foundation (No.
number and size of AACN present 4 months after treatment
250-94) and by NIH Grants ES-03687, CA 47327, and CA 23108, USPHS.
with
azaserine. Of particular interest in that study was the
2 Present address: Department of Surgery (ML 558), University of Cincinnati
demonstration by quantitative stereological techniques that ca
College of Medicine. 231 Bethesda Avenue, Cincinnati, OH 45267.
3 To whom requests for reprints should be addressed, at Department of
mostate had a greater effect on the growth of AACN than on
Pathology. Box 7600. Dartmouth Medical School. Hanover, NH 03756.
the intervening histologically normal pancreas. This suggested
'The abbreviations used are: AACN, acidophilic neoplastic foci; MES, 4that the preneoplastic foci were more sensitive to CCK than
morpholineethanesulfonicacid;
CCK, Cholecystokinin; '"I-BH-CCK, '"I-BoltonHunter-Iabeled CCK.
normal pancreas with regard to stimulation of growth. This
3295
ABSTRACT
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CCK RECEPTORS IN PANCREATIC CARCINOMA
might reflect more avid binding of CCK, either because of
increased receptor number or because of increased receptor
affinity for CCK. The current study was designed to test the
hypothesis that premalignant and/or malignant pancreatic tu
mors exhibit increased binding of CCK compared to normal
pancreas because of increased receptor number or affinity or
both. If, in fact, pancreatic tumors possess an enhanced ability
to bind CCK, this may provide a growth advantage leading to
tumor promotion or progression.
MATERIALS AND METHODS
Preparation of Tissues for CCK Binding Studies. Four types of tissue
were examined in this study. In the first experiment, binding of CCK
to a transplantable azaserine-induced carcinoma (DSL6) was compared
to normal rat pancreas. DSL6 was harvested from donor animals,
minced, and injected s.c. in the interscapular area of 1-2-month-old
male Lewis rats (Charles River Breeding Laboratories, Wilmington,
MA). After the implanted tumor had grown to 1-2 cm in diameter,
animals were decapitated under ether anesthesia and the tumor was
harvested. At the same time, the normal pancreas of the tumor-bearing
animals was harvested via a midline laparotomy. Six animals bearing
DSL6 tumor were studied.
In the second experiment, the objective was to compare CCK binding
in premalignant pancreatic nodules to that in the remaining interno
dular pancreas. To that end, male Lewis rats were treated with a single
i.p. injection of azaserine (30 mg/kg body weight) at age 2 weeks and
followed for 12-18 months, at which time they were sacrificed as above
and the pancreas was removed. Grossly visible pancreatic nodules in
the size range 2 to 5 mm were then hand-dissected from the pancreas
and pooled. The remaining internodular pancreas was prepared for
comparison to the nodule pool. Nodules were harvested from 8 animals.
From each animal 10-20 nodules were pooled. In one animal, the
pancreas was nearly completely replaced by nodules and there was
insufficient internodular tissue to harvest.
All tissues were immediately frozen on dry ice and were then trans
ferred to a -70°C freezer. For binding assays, tissue sections (20 /¿m)
were cut at -20°Con a cryostat microtome, mounted on gelatin-coated
microscope slides, and dried for 18 h at -22°C.
Studies of Binding of '"'I-CCK-8 to Tissue Sections. Binding assays
were performed using the method of von Schrenck et al. (18). Tissue
sections were preincubated in 50 mM MES buffer containing 0.5%
albumin for 20 min at pH 6.0 and 22°C.Sections were then incubated
for 4 h at 22°C,pH 6.0, unless otherwise stated, in 50 ITIMMES buffer
better than one-site fits (P < 0.05). All data in the experiments below
are presented as mean ±SD unless otherwise indicated. Differences
between tissues were analyzed with Student's t test for unpaired vari
ables. All tests of significance were two-tailed.
Receptor binding capacity was normalized to both protein and DNA
by cutting additional tissue sections which were analyzed for protein
content by the MicroBio-Rad method (Bio-Rad Laboratories, Rich
mond, CA) using an albumin standard and for DNA by the diphenylamine method (20) using calf thymus DNA as the standard. Finally,
additional tissue sections were cut at 5 ^m for histológica!analysis.
Protocols were approved by the appropriate institutional animal care
and radiation safety committees.
RESULTS
Preliminary Studies. Initial studies were conducted with sec
tions of normal pancreas and of DSL6 carcinoma to determine
optimum time, temperature, and pH conditions for receptor
binding. Binding tests were conducted at half-pH increments
from 5.0 to 7.0 and at 4°C,22°C,and 37°Con the basis of
previous studies (18). For both normal and malignant tissues,
optimum binding occurred at pH 6.0 and 22°C,the same
conditions found to maximize binding in normal guinea pig
pancreas by von Schrenck et al. (18). The addition of excess (1
MM)unlabeled CCK-8 at pH 6.0 and 22'C decreased binding to
normal pancreas by 81% and to carcinoma by 96%. For normal
pancreas, half-maximal binding of 25 pM U5I-BH-CCK oc
curred at 30 min and binding was maximal by 120 minutes.
For carcinoma, half-maximal binding occurred at 60 minutes
and maximal binding was first reached at 180 min (Fig. 1). All
subsequent experiments were conducted at pH 6.0, at 22°C,for
240 min.
Comparison of Normal Pancreas to DSL6 Carcinoma in Com
petitive Binding Assays. In competitive binding assays, tissue
sections were exposed to 25 pM '"I-BH-CCK and to 18 differ
ent concentrations of unlabeled CCK-8 ranging from a low of
zero to a high of 1 x 10~5 M, the latter concentration repre
senting a 400,000-fold excess of unlabeled ligand. The compet
itive binding curve and Scatchard plot for DSL6 carcinoma is
shown in Fig. 2. Analysis of binding curves for normal pancreas
(n = 6) and for DSL6 (n = 6) revealed that both contain highaffinity and low-affinity receptor sites for CCK-8 (Table 1). The
containing 0.5% albumin, 0.025% bacitracin, 4 ¿ig/mlleupeptin, 2 ¿ig/
ml chymostatin, 130 mM NaCl, 7.7 mM KC1, 5 mivi MgCl2, 1 mM
ethyleneglycoltetraacetic acid, and 25 p\i '"I-BH-CCK (specific activ
ity, 2000 Ci/mmol), obtained from Amersham Corporation, Arlington
Heights, IL. Incubation volume was 4 ml for four slides. For competitive
binding assays, sections were exposed in addition to graded concentra
tions of unlabeled CCK-8 (BACHEM, Inc., Terranee, CA) ranging
from 1.0 x 10~" M to 1.0 x IO"5M. At the conclusion of the incubation,
tissue sections were rinsed three times in cold (4°C)MES buffer with
0.5% albumin and then wiped from the slides with a filter paper. The
filter papers were then placed in tubes for counting in a gamma counter.
Binding of radiolabeled ligand in the absence of unlabeled ligand was
taken to represent maximum binding. Binding of labeled ligand in an
excess of unlabeled ligand (1 x IO"5 M) was considered to represent
nonsaturable binding. Nonsaturable binding averaged 10.0 ±7.7% of
maximum binding in the experiments reported below. In order to rule
out the possibility that CCK binding was due to nonspecific adherence
to rat pancreas, the binding of gastrin-17 to normal rat pancreas was
examined. Gastrin is closely related to CCK, exhibiting homology in
the COOH-terminal pentapeptide. Over a wide range of pH and tem
perature conditions, no specific binding of gastrin to normal rat pan
creas was detected. For Scatchard analysis of competitive binding
curves, raw counts minus background were entered into the LIGAND
software package (19); two-site fits were accepted only if significantly
•¿o
•¿
•¿a
TJ
•¿
f
a
o
n
u
U
0.5
0.0
60
120
180
240
300
360
420
480
MINUTES
Fig. 1. Time course of saturable binding of '"I-BH-CCK to normal pancreas
(D, n = 2) and pancreatic carcinoma DSL6 (*, n = 3). Values represent percentage
of total added counts bound. In each experiment each value was determined in
duplicate. The nonsaturable binding component (defined as binding in the pres
ence of 1 JIM CCK-8) was 19% for normal tissue and 4% for DSL6 carcinoma
(not shown).
3296
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CCK RECEPTORS IN PANCREATIC CARCINOMA
A
_
E
3
100-
e
*
o
z
3
o
o
ü
-11
CCX-B
-10
-9
-• -7
CONCENTRATION
U-11
2»-11
BOUND
imol««/lll»rl
-• -5
(log
-4
M)
3»-11
Fig. 2. A, competitive binding curve using unlabeled CCK-8 in DSL6 pancreatic
carcinoma. Ordinate, percentage of maximum binding, where maximum binding
is defined as counts bound in absenceof unlabeled CCK-8. Abscissa,concentration
of added unlabeled CCK. Data points are the mean ±SEM of 6 experiments,
with each point measured in duplicate in each experiment. B, Scatchard transfor
mation of the displacement data demonstrating the presence of high-affinity (K,;
= 0.33 nM) and low-affinity (Krf = 82 nivi) receptors.
Kj for the high-affinity site appears to be physiologically rele
vant since it is close to the concentration of CCK-8 required
for half-maximal amylase release previously reported (0.1 nM)
(18). Binding capacity (ßmax)
of the high-affinity receptor was
122 ±23 fmol/mg protein for carcinoma versus 12 ±2 fmol/
mg protein for normal tissue (P < 0.001 ). At the tracer concen
tration of 25 pM used in this experiment, the combination of
increased affinity and higher binding capacity for CCK-8 in
DSL6 carcinoma led to a highly significant increase in actual
amount of CCK-8 bound to high-affinity receptor in the carci
noma (8.6 ±1.9 versus 0.6 ±0.2 fmol/mg protein, P < 0.001)
(Fig. 3).
Kj for the low-affinity CCK receptor was 1.1 ±0.4 /¿Mfor
normal pancreas and 0.1 ±0.1 pM for DSL6 carcinoma (P <
0.001). In contrast to the high-affinity receptor, normal pan
creas contained more low-affinity sites than DSL6 (8163 ±
3576 versus 423 ±320 fmol/mg protein; P < 0.001). The data
suggest a possible difference in the receptor-binding behavior
of normal pancreas and carcinoma. In normal pancreas, 77%
of total binding is to the high-affinity site and 23% to the lowaffinity site. In contrast, 98.5% of binding in DSL6 carcinoma
is to the high-affinity site and only 1.5% to the low-affinity site.
Comparison of Premalignant Nodules to Internodular Pancreas
in Competitive Binding Assays. Scatchard analysis of binding
curves for premalignant nodules and for internodular pancreas
revealed that both tissue types contained both high-affinity and
low-affinity receptors (Table 1). Kj for the high-affinity receptor
was 0.28 ±0.04 nM for nodules and 0.27 ±0.12 nM for
internodular pancreas (not significant). Nodules demonstrated
a significant increase in binding capacity of the high-affinity
receptor (37.6 ±8.6 fmol/mg protein for nodules versus 6.4 ±
2.8 fmol/mg protein for internodular pancreas, P < 0.001).
As was the case with the comparison of the DSL6 carcinoma
to normal pancreas, binding of CCK-8 to nodules at a tracer
concentration of 25 pM was predominantly to the high-affinity
receptor (94.5% compared to 48.3% for internodular pancreas,
P < 0.05). The increase in total binding of CCK-8 to nodules
was due entirely to increased binding to the high-affinity site
(3.1 ±0.8 fmol/mg protein versus 0.6 ±0.3 fmol/mg protein
in internodular pancreas, P < 0.001) (Fig. 4). There was a
corresponding reduction in amount of low-affinity site binding
in nodules compared to internodular pancreas (0.18 ±0.14
fmol/mg protein versus 0.59 ±0.51 fmol/mg protein, P< 0.05).
Normalization of Receptor Binding Capacity to DNA Content.
Although receptor binding capacity is typically expressed in
relation to protein content, this presented some difficulties in
this study since it might be expected that neoplastic pancreas
would contain less protein in the form of secretory enzymes
than normal pancreas. For this reason, binding capacity was
also normalized to DNA content of the tissue slices. Normali
zation to DNA content would be expected to potentially under
estimate receptor capacity in the transplantable carcinoma since
its cells are aneuploid with hyperdiploid DNA content; nodules
and adenomas have normal diploid DNA content (21). Despite
this, the fundamental findings were unchanged, namely that the
high-affinity receptor binding capacity of DSL6 carcinoma was
significantly greater than that of normal pancreas (492 ±115
versus 245 ±61 fmol/mg DNA, P < 0.01). The high affinity
binding capacity of nodules likewise significantly exceeded that
of internodular pancreas (416 ±83 versus 90 ±39 fmol/mg
DNA,P<0.001).
Histológica! Examination of Tissue. Sections of DSL6 carci
noma revealed moderately well differentiated acinar adenocarcinoma. Sections of "nodules" consisted entirely of AACN and
adenomas. Sections of internodular pancreas consisted of ap
proximately 80-90% normal pancreatic tissue and 10-20%
small nodules (<1 mm).
DISCUSSION
Carcinogenesis is believed to be a multistage process. The
process is generally divided into an initiation phase, in which
cellular DNA damage occurs, and a promotion phase during
Table I CCK receptor characteristics
High-affinity
TissueNormal
pancreas
DSL6 carcinoma
Nodules
Internodular pancreas(nin)0.46
50149'
Mean ±SD.
Low-affinity
protein)12.4
±0.08°
0.33 ±0.04
0.28 ±0.04
0.27 ±0.12(fmol/mg
±2.2
122.1 ±22.9
37.6 ±8.6
6.4 ±2.8(nM)1098
protein)8163
±450
82 ±86
83 ±43
314 ±172(fmol/mg
±3576
423 ±320
5857 ±3467
73858 ±
3297
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CCK RECEPTORS
HIGH
AFFINITY
NORMAL
IN PANCREATIC
RECEPTOR
DSL6
they detected two classes of binding sites with half-maximal
binding inhibition of 0.2 nM and 0.3 ßM,
results very similar to
ours with the DSL6 carcinoma. We have also performed a
limited number of competitive binding assays with AR42J and
agree that there are two classes of binding sites (data not
shown). Scemama's group has also suggested that the high-
CA
Fig. 3. Amount of ligand bound to the high-affinity CCK receptor in normal
pancreas (n = 6) and DSL6 carcinoma (n = 6) at tracer concentration of 25 pM.
The amount of ligand bound was calculated from the binding capacity (/>,,,.,,)and
the dissociation constant (Kj) (determined from Scatchard analysis) according to
the equation
—¿
—¿
AB = Bm„¿
A + Kt
where A is the concentration of labeled ligand. i.e., 25 pM. *, P< 0.001.
O £
X •¿
U •¿
a E
•¿
HIOH AFFINITY
EÕLOW AFFINITY
•¿
TOTAL BOUND
t
I!
CARCINOMA
INTERMODULAR
affinity receptors for CCK on AR42J cells can be divided into
two subsets: one binding CCK preferentially; and a second
binding CCK and gastrin interchangeably. They have demon
strated that occupation of these latter receptors results in stim
ulation of ornithine decarboxylase activity, an event associated
with cellular proliferation (24).
As noted above, there is considerable in vivo evidence of a
role for CCK in pancreatic tumor promotion in rats. However,
the relevance of acinar cell carcinoma in rats to carcinomas in
humans is questioned since the majority of human pancreatic
carcinomas have a ductal phenotype. Recently, evidence for a
role of CCK in human pancreatic cancer has also emerged.
CCK has been shown to stimulate the growth of several human
pancreatic carcinoma cell lines in culture (25) and in xenografts
into nude mice (26). In addition, in nude mice, the CCK
receptor antagonist L364,718 has been shown both to inhibit
the basal growth of human pancreatic carcinoma and to block
the tumor-promoting effects of a high-fat diet (27, 28).
In the azaserine model in the rat, neoplastic pancreatic
changes are clearly focal, with well-defined nests of acinar cells
developing hyperplastic, dysplastic, or neoplastic changes while
other pancreatic cells appear relatively unchanged. There may
be several reasons why some cells progress through the oncogenic sequence while others show little or no change from the
normal state. We believe that the current study demonstrates
that overexpression of high-affinity CCK receptors character
izes the cells in the neoplastic pathway. The possibility that
CCK receptor overexpression confers a growth advantage on
such cells will be examined in future studies.
Fig. 4. Comparison of CCK-8 binding between nodules (n = 8 experiments)
and internodular pancreas (n = 1 experiments) in azaserine-treated rats. Total
binding represents sum of amount of ligand bound to high and low affinity sites
at a tracer concentration of 25 pM and was significantly higher for nodules (P <
0.001) than for internodular pancreas. Note that nodules exhibit significant (P<
0.001 ) increase in amount of binding to high-affinity receptor with corresponding
decrease (P < 0.05) in amount of binding to low-affinity receptor in comparison
to internodular pancreas.
ACKNOWLEDGMENTS
which further genotypic and phenotypic alterations take place
which ultimately lead to the development of the frankly malig
nant state. In the case of azaserine-induced pancreatic carci
noma in the rat, changes presumably occur in some initiated
cells which give those cells at first a growth advantage and
ultimately an unrestrained growth pattern characteristic of ma
lignancy. Studies in our laboratory and others' (15, 16) show
REFERENCES
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pancreas (17) suggests the hypothesis that cells in the neoplastic
pathway overexpress CCK receptors.
Other investigators have previously demonstrated the pres
ence of CCK receptors on pancreatic carcinoma cells. Logsdon
(22) demonstrated high-affinity CCK receptors in the AR42J
cell line, which like DSL6 is derived from an azaserine-induced
acinar cell carcinoma. He also demonstrated that the binding
capacity of the high-affinity receptor could be increased ap
proximately 3-fold by dexamethasone treatment and that the
receptor was functional as assessed by amylase release.
Scemama et al. (23) have also examined CCK receptors on
the AR42J cell. Using the CCK receptor antagonist L364,718,
The authors would like to thank Truls Brinck-Johnsen, Ph.D., De
partment of Pathology, Dartmouth Medical School, for help with the
radioisotope work and Frederick S. Whaley, Ph.D., Biostatistics and
Epidemiology Group, Dartmouth Medical School, for assistance with
the statistical analysis.
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Overexpression of Cholecystokinin Receptors in
Azaserine-induced Neoplasms of the Rat Pancreas
Richard H. Bell, Jr., Elna T. Kuhlmann, Robert T. Jensen, et al.
Cancer Res 1992;52:3295-3299.
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