From the
We have reported previously mitogenic effects of gastrin on
several immortalized and neoplastic cell lines, including Swiss 3T3
fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for
a closely related peptide, cholecystokinin, were recently cloned. These
studies were undertaken to investigate if CCK-A- and CCK-B receptors
were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3
cells. Receptor antagonists that inhibit the biological effects and
binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260
(L60)) receptors were ineffective toward inhibiting the binding and
proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18
and L60 demonstrated no binding to the cells, indicating that CCK-A and
CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8,
gastrin, L18, and L60, on the other hand, demonstrated specific binding
to a pancreatic cancer cell line (AR42J cells) (used as a positive
control). In cross-linking studies the molecular mass of the major band
of gastrin receptors (GR) on Swiss 3T3 cells was determined to be
Possible mRNA expression of CCK-A and CCK-B
receptor subtypes by gastrin-responsive rodent intestinal and
fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the
methods of Northern blot analysis and reverse transcriptase-polymerase
chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum
served as positive controls. We were unable to detect CCK-A and CCK-B
receptor mRNA in the three cell lines by both the methods. The positive
control samples, on the other hand, gave the expected results. Thus the
three gastrin-responsive rodent cell lines examined in this study
lacked CCK-A and CCK-B receptors, but were positive for the gastrin
preferring GR that demonstrated physicochemical and pharmacological
attributes distinct from CCK-A and CCK-B receptors. Since colon cancers
are known to express high concentrations of non-amidated gastrin-like
peptides, a possible autocrine role of glycine extended forms of
gastrin becomes a highly relevant clinical question in the light of the
presence of novel GR on the fibroblast and intestinal cell lines.
Several gut peptides hormones, including gastrin and bombesin
(BBS),
Cholecystokinin (CCK) shares significant structural homology with
gastrin at the C-terminal end and mediates its biological effects via
CCK-A and CCK-B receptor subtypes
(8) . CCK-A and CCK-B receptor
subtypes were recently cloned from brain, stomach, and pancreatic cells
(8, 9, 10, 11) . Recent reports suggest
that CCK-A and CCK-B receptors can potentially mediate mitogenic
effects of CCK/gastrin-like peptides on normal and cancerous pancreatic
cells
(12, 13, 14) and on small cell lung
cancers
(15) . It remains to be determined if CCK-A and CCK-B
receptor subtypes are also expressed by normal and cancerous intestinal
(IEC-6, CA) and fibroblast (Swiss 3T3) cell lines, which demonstrate a
significant mitogenic response to gastrin and express high-affinity
( K
We and others have recently
reported mitogenic effects of a processing intermediate of gastrin
(glycine-extended gastrin) on human colon cancer
(17) and
pancreatic cancer
(18) cell lines, suggesting for the first
time that amidation of gastrin peptides is not a prerequisite for
measuring mitogenic effects of gastrin-like peptides. In order to
further confirm the mitogenic potency of noncarboxyamidated gastrins,
we measured the binding and mitogenic potency of Gly- and Lys-extended
forms of gastrins in comparison with that of amidated gastrins. Our
results point to a possible presence of a novel class of gastrin
preferring receptors that bind amidated and glycine extended forms of
gastrin with almost equal affinity and are not recognized by the CCK-A
and CCK-B receptor antagonists.
The
molecular mass of GR on Swiss 3T3 cells was determined by cross-linking
of
Based on the above results, it thus appears more than
likely that the Swiss 3T3 fibroblasts and the intestinal cell lines do
not express detectable levels of the CCK-A and CCK-B receptor subtypes
and perhaps express novel GR that bind and mediate the mitogenic
effects of gastrin/CCK-like peptides on these cells.
We have described for the first time the presence of
gastrin-preferring receptors (GR) on an immortalized fibroblast cell
line (Swiss 3T3 cells) that do not discriminate between amidated and
glycine-extended gastrins and apparently mediate mitogenic effects of
gastrin-like peptides on these cells. The early passages of Swiss 3T3
cells (<30), unlike the latter passage (>110), were highly
responsive to BBS, insulin-like growth factors (IGFs), and gastrin
(present studies). A subclone of Swiss 3T3 cells, enriched in cells
expressing gastrin-releasing peptide (GRP) receptors (Dr. A. Oliff)
were similarly highly responsive to BBS and gastrin (present studies).
Swiss 3T3 cells demonstrated a dual (two peak) bi-phasic response to
gastrin. Cells were maximally responsive to 0.5-1.0 n
M gastrin and slightly responsive to 0.1-1.0 µ
M gastrin. Some mouse and human colon cancer cells similarly
demonstrate a dual bi-phasic response to gastrin
(6, 16, 28, 29) . Several other human
colon cancers, however, respond only to high concentrations of gastrin
(17, 31) . High-affinity ( K
The majority of the
biological effects of gastrin-like peptides on normal target cells in
the pancreas, gallbladder, stomach, and central nervous system are
believed to be mediated by CCK-A and CCK-B receptors
(8) .
Transformed and cancerous cells arising from the pancreas and ECL cells
also express high concentrations of CCK-B and/or CCK-A R receptors
(12, 13, 37, 38) . Since the cloning of
CCK-A and CCK-B receptors, however, none of the reports to date, to the
best of our knowledge, have described the expression of CCK-A- and/or
CCK-B-R by the normal and cancerous intestinal cells. There is,
however, a large volume of literature from several laboratories
including ours, wherein trophic effects of gastrin have been described
on normal
(1, 39, 40, 41) and cancerous
(2, 5, 6, 20, 28, 29, 31) intestinal cells from both humans and rodent species. Based
on structural requirements of gastrin-like peptides for binding GR on
colon cancer cells, we reported a decade ago about the possible
presence of unique GR
(42) . Since then we have published
several studies, all suggesting the presence of unique GR on normal and
cancerous intestinal cells
(6, 16, 17, 24, 25, 41) .
The present studies confirm the presence of novel gastrin-preferring
receptors on Swiss 3T3 and intestinal cells as discussed below.
The
selective CCK-A (L-364,418) (L-18) and CCK-B (L-365,260) (L-60)
receptor antagonists had no effect on either the mitogenic potency or
the binding of gastrin to Swiss 3T3 and intestinal cells (CA and
IEC-6). Even though multiple binding states of CCK receptors have been
described
(23, 33) , all the multiple affinity states of
CCK-A-R bind L-18 with equal affinity
(23) . Thus our results
with the Swiss 3T3 and intestinal cells suggest that gastrin-preferring
receptors do not meet the pharmacological criteria of CCK-A- and
CCK-B-R. No specific binding sites for the two selective antagonists
were measured on Swiss 3T3 cells further confirming this possibility.
Additionally we did not detect mRNA for CCK-A- and CCK-B-R in the
fibroblast and intestinal cell lines by either the reverse
transcriptase-PCR method or by Northern blot analysis after low
stringency washings; the positive control samples gave the expected
results. Although we did not detect CCK-B-R mRNA in rat pancreas by the
reverse transcriptase-PCR method (Fig. 7 C), we did
detect a
Secretion of prepro, non-amidated forms of gastrin by human colon
cancers and colon cancer cell lines was recently reported (discussed in
Ref. 17). High serum concentrations of non-amidated, prepro forms of
gastrin are measured in serum under diseased states (reviewed in Ref.
46). We recently reported mitogenic effects of glycine-extended
gastrins (G1-17-Gly) on colon cancers
(17, 47) .
In the present study we measured the relative mitogenic potency of
several amidated and non-amidated gastrin-like peptides and the
relative binding affinity of the peptides for GR on fibroblast and
intestinal cells. The relative binding affinity of the amidated and
non-amidated gastrin-like peptides closely resembled the relative
mitogenic potency of the peptides. It thus became evident that
glycine-extended forms of gastrin/CCK-like peptides were equally
effective as their amidated counterparts, suggesting that the novel
gastrin receptors did not discriminate between the glycine-extended and
the amidated gastrin-like peptides. It was recently reported that
G1-17-Gly binds a unique class of receptors that do not bind
amidated gastrins and is apparently specific for binding the C-terminal
Gly moiety
(18) . Our results, however, suggest that yet another
unique class of GR is expressed by some of the target cells that bind
amidated and non-amidated gastrin-like peptides with almost equal
affinity. G1-17-Lys had a significantly lower potency compared
with G1-17-Gly, for displacing bound gastrin and for exerting
mitogenic effects on Swiss 3T3 cells. Since gastrin is an acidic
peptide, any modifications resulting in availability of a basic amino
group for binding Glu residues could potentially result in altering the
structure of the peptide which may explain the results with
G1-17-Lys.
In earlier studies a possible autocrine role of
gastrin-like peptides was speculated
(48) . We and others
(discussed in Refs. 17, 20, and 32) have since reported that amidated
forms of gastrin are not likely to play an autocrine role in colon
cancers. However, the observations of a mitogenic potency of G1-17-Gly
(present studies and Refs. 17 and 18) raises the question of a possible
autocrine role of processing intermediates of gastrin, which needs to
be examined.
Utilizing
In order to further understand the role of
gastrin-preferring receptors in proliferation of normal and cancerous
intestinal mucosal cells, we will clearly need to further characterize
the 45-kDa GR measured on these cells (Refs. 24 and 25 and present
studies). Swiss 3T3 cells should provide a useful source for further
purification and cloning of the cDNA for the unique and novel
gastrin-preferring receptors.
In two experiments (arbitrarily termed 1 and 2),
Gastrin 1-17-glycine, gastrin 1-17-lysine,
and CCK-8-glycine were kindly provided to us by Dr. Jens Rehfled
(Professor, Rigshospitalet, Department of Clinical Chemistry,
Blegdamsvej 9, Copenhagen, Denmark). Gastrin 5-17-glycine was
obtained from Dr. John DelValle (Assistant Professor, Department of
Medicine, University of Michigan, Ann Arbor, MI). L-364,718
(3-(acylamino)-benzodiazepine analogue) and L-365,260
(3-(benzoylamino)-benzodiazepine analogue) were obtained from Dr. Roger
M. Freidinger (Senior Scientist, Department of Medicinal Chemistry,
Merck Sharp and Dohme, West Point, PA). We gratefully acknowledge the
secretarial help of Kay Smith.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
45 kDa. The mitogenic potency of 0.1-1.0 n
M gastrin-like peptides on Swiss 3T3 cells was in the order of
G1-17
G1-17-Gly > G5-17
G5-17-Gly
> G2-17 > CCK-8-Gly
G1-17-Lys
CCK-8. The
relative binding affinity of the peptides (based on the dose-dependent
inhibition of binding of
I-G1-17 to Swiss 3T3
cells) was similar to the relative mitogenic potency of the peptides as
given above. Furthermore, G1-17-Gly was equally effective as
G1-17 in displacing the binding of
I-G1-17 to
the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it
became evident that the novel gastrin preferring GR, expressed by Swiss
3T3 cells, binds and mediates the mitogenic effects of not only the
mature (amidated) forms of gastrin-like peptides but also binds and
meditates the mitogenic effects of glycine-extended forms of
gastrin-like peptides.
(
)
are mitogenic for normal and cancerous
intestinal mucosal cells
(1, 2, 3, 4, 5) . In recent
studies significant mitogenic effects of gastrin
(6) and BBS
(7) were also reported on a mouse fibroblast cell line, Swiss
3T3 cells. Although BBS receptors (BBS-R) have been identified and
characterized on Swiss 3T3 cells
(7) , gastrin receptors that
may be mediating the mitogenic effects of gastrin on Swiss 3T3 cells
remain to be identified. In the present study, binding characteristics
of gastrin to Swiss 3T3 cells are presented for the first time.
= 0.1-1.0 n
M)
and low-affinity ( K
=
0.1-1.0 µ
M) forms of specific gastrin binding sites
(6, 16) . In order to investigate this possibility we
analyzed fibroblast (Swiss 3T3) and intestinal (CA and IEC-6) cell
lines for the possible expression of CCK-A and CCK-B receptors by the
methods of Northern blot and reverse transcriptase-polymerase chain
reaction (PCR). At the pharmacological level, specific binding of
radiolabeled gastrin, CCK-8, CCK-A receptor antagonist (L-364,718), and
CCK-B receptor antagonist (L-365,260) were also measured. The growth
stimulatory effects of gastrin were measured in the presence of CCK-A
and CCK-B receptor antagonists in order to identify the receptor
subtype that may be mediating the mitogenic effects of gastrin on the
intestinal and fibroblast cell lines.
Cell Culture
The three cell lines used in this
study included a mouse fibroblast cell line (Swiss 3T3), a normal rat
intestinal epithelial cell line (IEC-6 cells) (American Type Culture
Collection, Rockville, MD), and a mouse colon cancer cell lines (CA)
(established in our laboratory
(19) ). A subclone of Swiss 3T3
cells (that were enriched in GRP receptors and developed by Dr. A.
Oliff) were obtained from Dr. E. Spindel (Division of Neuroscience
Oregon Regional Primate Center). Swiss 3T3 cells were also obtained
from ATCC at early passages of <35 and late passages of >100. All
the cell lines were grown as monolayer cultures in either
Dulbecco's modified Eagle's medium (IEC-6, Swiss 3T3) or
RMPI 1640 (CA) (Life Technologies, Inc.), supplemented with glutamine
(2 m
M) and 10% heat-inactivated fetal calf serum (FCS,
Hyclone, Logan, UT) in an atmosphere of 95% O, 5% CO
at 37 °C. The cell lines were regularly monitored for the
absence of mycoplasma, using the fluorescence stain Hoechst 33258.
Stock cultures of cells were subcultured at appropriate intervals to
maintain the cells at subconfluent densities. For cell counting and
subculturing the cells were dispersed with 0.05% trypsin and 0.02%
EDTA.
In Vitro Growth Assays
Cell numbers were either
measured directly by counting the total number of cells using a Coulter
electronic particle counter (model ZF) or measured indirectly by using
a semi-automated tetrazolium-based colorimetric assay (MTT assay). Both
the MTT and cell counting assays were conducted essentially as
described previously
(20) . Briefly, for the MTT assay, cells
(1-2 10
cells/well) were plated into 96-well
plates with 200 µl of normal growth medium containing 10% FCS.
After 24 h, the medium was changed to serum-free medium, and the cells
were cultured for 24 h in order to achieve quiescence. Cell were then
stimulated with or without various doses of the peptides in the
presence or absence of the nonpeptide receptor antagonist for
48-72 h in serum-free medium. At the end of the treatment, 0.1 mg
(50 µl of 2 mg/ml) of MTT was added to each well and incubated at
37 °C for an additional 4 h. The medium was then aspirated from the
well, leaving about 30 µl of medium in each well. One-hundred fifty
µl of dimethyl sulfoxide (Me
SO, Sigma) was added to
each well, and the plates were shaken for 10 min to solubilize the
formazan crystals. The plates were then read immediately at 540 n
M on a scanning multiwell spectrophotometer (Umax, Molecular
Devices, Melno, CA). Readings from wells containing culture medium
alone with no cells, or cells without various drugs, were used as
control. Optical absorbance readings from test wells were corrected
against basal control absorbance values. In growth assays where the
cell numbers were directly measured with the help of a Coulter counter,
an optimal number of cells (0.5-1.0
10
cells)
were plated in 35-mm dishes with 2 ml of normal growth medium
containing 10% FCS. After 24 h, the medium was changed to serum-free
medium and cells cultured for an additional 24 h. Cells were then
treated with various peptides ± receptor antagonists for 48 h in
serum-free medium. At the end of the treatment, cells were disbursed
with trypsin-EDTA solution as given above and cells counted using
either a Coulter electronic counter or with the help of a hemacytometer
under light microscope.
Binding of Radiolabeled Agonists and Receptor Antagonists
with the Swiss 3T3 Cells
For measuring specific binding of
gastrin to the Swiss 3T3 cells, we used either synthetic human gastrin
(G1-17) or human gastrin 2-17 (G2-17) (Research Plus,
Bayonne, NJ). G1-17 and G2-17 were iodinated with IODO-GEN
(Pierce) as described previously
(21) and specific activity
calculated to be 1000-2000 cpm/fmol. For measuring
specific binding sites for CCK,
I-BH-CCK-8-sulfate
(DuPont NEN) was used. For measuring specific binding sites for CCK-A
and CCK-B receptor antagonists,
H-
L-365,260
(specificity activity of 160 dpm/fmol) and
H-
L-364,718 (specificity activity of 184 dpm/fmol)
(DuPont NEN) were used, respectively. For cell binding assays, Swiss
3T3 cells were subcultured in 160-mm flasks and grown to subconfluence.
All binding assays were performed 36-48 h after cell seeding in
culture medium containing 10% FCS and 2% glutamine. Before the start of
the binding assays, the cells in culture were washed with Hanks'
balanced salt solution (HBSS, Life Technologies, Inc.) containing 0.1%
bovine serum albumin (BSA) and 25 m
M HEPES (Sigma) and
scrapped with a rubber policeman into conical tissue culture
polystyrene tubes. Cells were centrifuged at 500
g for
5 min and resuspended in HBSS at a concentration of 1-3
10
cells/ml. Aliquots (0.5 ml) of suspended cells in
polystyrene tubes were used in all binding assays. All binding assays
were conducted essentially as described previously (gastrin
(21) , CCK
(22) , L-364,718, and L-365,260
(13, 23) ). Briefly, cells in suspension were incubated
with either the
I-labeled peptide (0.5-1.0 n
M G1-17; 0.025 n
M CCK-8) or the
H-antagonist (2 n
M) in the presence or absence of
increasing concentrations (0.1 n
M to 10.0 µ
M) of
either the homologous or the heterologous peptide/antagonist. The
antagonists L-365,260 and L-364,718 were added at a final dilution of
0.01% Me
SO in the binding assay tubes. All control and
total binding tubes received the same concentration of
Me
SO. Nonspecific binding was determined in all assays in
the presence of 1000-fold excess of the nonlabeled peptide/antagonist.
Binding assays were performed generally at 37 °C for 30 min at pH
7.4 (determined to be optimal for most binding assays as described
previously
(21, 22, 23) ). At the end of the
incubation, the cells were pelleted and washed twice with 1 ml of fresh
ice-cold HBSS + 0.1% BSA. In the case of the peptide hormone
assays, cell pellets were counted for
I in a
-counter (model 5500, Beckman) with
70% efficiency for
I counting. In the case of assays for receptor
antagonists, the cell pellets were counted for
H using 10
ml of scintillation fluid, consisting of 5 g of permablend 2 (Packard
Instruments) dissolved in 1 liter of toluene and counted in a
scintillation counter (Packard TriCarb 460C, Downers Grove, IL) at
<2% counting error with conversion to disintegrations/min by
external standard ratio method. The binding affinity and total binding
capacity of gastrin for binding the Swiss 3T3 cells were determined
from a Scatchard plot of the specific binding data
(21) . The
relative binding affinity (RBA) of the amidated and non-amidated
gastrin-like peptides for binding the high-affinity GR on Swiss 3T3 and
CA cells was determined from a log-dose inhibition of specific binding
of
I-G1-17 by various peptides and receptor
antagonists as described previously
(21) .
Determination of the Molecular Mass of GR on Swiss
3T3
The molecular mass of GR on Swiss 3T3 cells was measured in
cross-linking experiments as described previously
(24, 25) . Aliquots of Swiss 3T3 cells (5
10
/ml) were labeled with 1 n
M
I-gastrin 2-17 as given above in the presence
of 0.1% soybean trypsin inhibitor, 0.1% bacitracin, and 100 units/ml of
Trasylol and 1 m
M phenylmethylsulfonyl fluoride and incubated
with 1 m
M disuccinimidyl suberate (DSS, Sigma) for 15 min at
22 °C, in the absence of BSA. DSS was dissolved in
Me
SO, immediately prior to use, and added to the substrate
to give a final concentration of 1% Me
SO in the binding
buffer (HBSS, pH 7.4, containing 1 m
M phenylmethylsulfonyl
fluoride). The reaction was terminated by rapid centrifugation and the
cells washed with excess binding buffer, devoid of BSA. The washed cell
pellets were solubilized by boiling at 100 °C for 3-5 min in
0.2
M Tris HCl buffer, pH 6.8, containing 6% sodium dodecyl
sulfate (SDS, w/v), 2 m
M EDTA, 10% glycerol (v/v) in the
presence or absence of 4%
-mercaptoethanol (v/v). The supernatant
was then subjected to SDS-polyacrylamide gel electrophoresis using 9.5%
acrylamide in the separating gel and 4% acrylamide in the stacking gel.
The gels were stained, destained, dried, and exposed to Kodak XAR-5
film for 5-7 days at -70 °C. The autoradiograms were
densitometrically analyzed with the help of a laser densitometer (LKB
UltroScan XL enhanced densitometer) and GelScan XL Software.
PCR and Northern Blot Analysis
Total cellular RNA
was isolated as described previously
(26) from various cell
lines. RNA was recovered by ethanol precipitation and quantitated by
its absorbance at 260 nm. Purity of RNA was determined by its
absorbance at 260/270/280 nm, and the integrity was assessed by
fractionating on 1% formaldehyde-agarose gels and observing the
intensities of 28 and 18 S RNA bands after staining with ethidium
bromide. Northern blot analysis of the samples was carried out as
described previously
(26) . Briefly the samples (30
µg of RNA) were electrophoresed through formaldehyde-agarose gels,
transferred to a nylon membrane, and cross-linked with UV light. For
analyzing the presence of CCK-A and CCK-B receptor mRNA, plasmids
containing the full-length cDNA fragments for the rat CCK-A and CCK-B
receptors were used which were kindly provided by Dr. Steven Wank. The
rat cDNA probes used included: 1) a 1.5-kilobase pair XbaI
insert of CCK-A receptor cDNA fragment inserted in pcDNA-1 plasmid and
a 2.2-kilobase pair EcoRI insert of CCK-B receptor cDNA
fragment subcloned in the vector PGEM (3ZF+), as per the
information provided by Dr. Wank. The cDNA probes were labeled by nick
translation and used for Northern hybridization of the RNA blots. The
blots were washed under less stringent conditions (2
15 min at
room temperature with 6
SSPE (1
SSPE, 0.18
M NaCl, 10 m
M sodium phosphate (pH 7.7), 1 m
M EDTA) 0.1% SDS, and 2
15 min at 48 °C with 1
SSPE, 0.1% SDS) in order to enable us to detect CCK-A and CCK-B
receptor-like transcripts that may show only partial homology with the
cDNA probes used.
cDNA Synthesis and PCR Amplification
cDNA
synthesis and PCR was conducted as described previously
(17) .
For PCR amplification of the cDNA, 10 pmol each of upstream and
downstream primers and 1 unit of AmpliTaq DNA polymerase (Perkin-Elmer)
was added to the reaction mixture. The reaction mixture was subjected
to 30 cycles of DNA polymerization (72 °C, 1.5 min), denaturation
(94 °C, 30 s), and primer annealing (58 °C, 30 s) in a
Perkin-Elmer DNA thermal cycler. PCR products were analyzed by
electrophoresis on 1.5% agarose gels. The upstream and downstream
primers were designed based on published cDNA sequences of rat CCK-A
and CCK-B receptor genes in order to flank the coding sequences for the
mature peptide. The sequences for the specific primers used are given
in the legend of Fig. 7.
Figure 7:
Detection of CCK-A and CCK-B receptor mRNA
in various cell lines/tissues by Northern blot analysis ( A and
B) and reverse transcriptase-PCR amplification ( C).
Autoradiographs of representative Northern blots for CCK-A receptors
( A) and CCK-B receptors ( B) are shown from a total of
four separate blots. Northern hybridization for CCK-A and CCK-B
receptors was conducted under low stringency conditions using the
appropriate P-labeled cDNA probes. A total of
30
µg of total RNA was loaded per lane. RNA samples in A in
lanes 1-5 are: rat pancreas ( lane 1), AR42J
cells ( lane 2), IEC-6 cells ( lane 3), CA tumors
( lane 4), and Swiss 3T3 cells ( lane 5). RNA samples
in B in lanes 1-5 are: IEC-6 cells ( lane
1), CA tumors ( lane 2), Swiss 3T3 cells ( lane
3), rat pancreas ( lane 4), and AR42J cells ( lane
5). Arrows to the right of the figures ( A and B) point toward the hybridization of the probes to
2.7-kilobase transcript for CCK-A receptors ( A) and
2.7-kilobase transcript for CCK-B receptors ( B). Reverse
transcriptase-PCR amplification of CCK-A and CCK-B receptor transcripts
from various tissues/cell lines, stained with ethidium bromide on 1.5%
agarose gels, is shown in C. cDNA was synthesized from 1
µg of total RNA and amplified by PCR for 25 (CCK-B receptor) to 30
(CCK-A receptor) cycles using the appropriate primer sets as described
under ``Experimental Procedures.'' For amplification of CCK-A
receptor cDNA, a 5`-3` 24-nucleotide sense oligomer from nucleotides
386 to 409 and an antisense 24 oligomer 5` to 3` from nucleotides 751
to 728 (based on the published sequence of rat CCK-A receptor cDNA
(56)) were used to amplify a 366-bp cDNA fragment. For amplification of
CCK-B receptor cDNA, a 5` to 3` sense 24 oligomer from nucleotides 794
to 817 and an antisense 24 oligomer 5` to 3` from nucleotides 1287 to
1264 (based on the published cDNA sequence of rat CCK-B receptors (11))
were used to amplify a 493-bp cDNA fragment. Samples in lanes
1-5 in C represent amplification with CCK-A
receptor primers and samples in lanes 6-10 represent
amplification with CCK-B receptor primers as shown. The samples in
lanes 1-10 in C are: CA tumors ( lanes 1 and 6), Swiss 3T3 cells ( lanes 2 and
7), IEC-6 cells ( lanes 3 and 8), AR42J cells
( lanes 4 and 9), and rat pancreas ( lanes 5 and 10). The position of the molecular mass markers in
base pair units is shown on the left-hand side of C.
The arrows on the right-hand side of C point
toward amplification of 1) a 409-bp CCK-A receptor cDNA fragment
( C), 2) amplification of a 493-bp CCK-B receptor cDNA fragment
( B), and 3) a 690-bp fragment ( A). The 690-bp
fragment was amplified in the presence of the CCK-B receptor primers in
all the samples, suggesting that the primers amplified contaminating
gDNA in the samples which probably has a small
200-bp
intron.
Statistical Analysis
Differences between means
were compared by the use of the unpaired Student's t test and were considered statistically significant at the level of
p < 0.05.
Proliferative Effects of Gastrin on Swiss 3T3 Cells in
Vivo
The growth response of Swiss 3T3 cells to increasing
concentrations of gastrin is presented in Fig. 1. The
proliferation of Swiss 3T3 cells increased sharply in response to
0.5-1.0 n
M gastrin and declined in the presence of
higher concentrations (5-10 n
M) of gastrin. A second
smaller peak of activation was obtained between 0.1 and 0.5 µ
M gastrin, which was once again reduced to control levels in the
presence of 1-10 µ
M gastrin. Gastrin at doses of
0.5-1.0 n
M was most effective and increased the total
number of Swiss 3T3 cells significantly above control values within 48
h of stimulation ( n = 8). The maximum growth
stimulatory effect of 0.5 n
M gastrin was measured to range
from 40 to
110% over and above control values
in different experiments. The proliferative effects of the maximum
effective dose of insulin-like growth factor-I (IGF-I) and BBS (that
are mitogenic for the subclone of Swiss 3T3 cells), were also measured
simultaneously. Both IGF-I and BBS demonstrated a significant mitogenic
effect at the maximum effective dose of 15 ng and 10 n
M,
respectively. IGF-I (2 n
M) was only slightly more potent than
0.5 n
M gastrin, and the difference was not statistically
significant. BBS (10 n
M), on an average, was slightly less
potent than 0.5 n
M gastrin, but once again the difference was
not statistically significant. At higher doses, the mitogenic potency
of IGF-I (6.6 n
M) and BBS (50 n
M) was similar to that
measured at the maximally effective concentrations of the two peptides
(data not shown). Similarly, in previous studies we have measured a
plateauing in the growth response of colon cancer cells to BBS
(4) and IGF-I
(27) in response to increasing
concentrations of the two peptides with no evidence of a sharp decline
in the potency of the peptides at higher concentrations as measured in
the case of gastrin.
Figure 1:
Dose-dependent growth effects of
gastrin, IGF-I, and bombesin on Swiss 3T3 cells. Swiss 3T3 cells in
culture were stimulated with or without various concentrations of
G1-17 and two optimally effective doses of IGF-I and bombesin as
shown. Growth was measured in terms of optical density/well in an MTT
assay as described under ``Experimental Procedures.'' The
optical density values measured in control (nontreated) wells was
arbitrarily assigned a zero value, and all changes in the OD
measurements from the peptide-stimulated wells were converted as a
percent value of the measurements in the control wells. Each bar
graph represents mean ± S.E. values from six observations
from a representative experiment; the asterisk = p < 0.05 versus control (0)
value.
Binding Affinity and Molecular Mass of Gastrin Receptors
on Swiss 3T3 Cells
In preliminary studies we established that
specific binding of I-gastrin increased linearly with
increasing doses of the radiolabeled ligand as we have reported
previously for gastrin binding to colonic mucosal membranes
(24) . Based on these preliminary results, it became evident
that there were at least two classes of specific gastrin binding sites
present on Swiss 3T3 cells, arbitrarily named Type I and Type II sites,
just as we have reported previously for gastrin binding sites on normal
and cancerous colonic cells
(6, 16, 24, 28) . In order to determine
if the binding affinity of the Type I and Type II sites on Swiss 3T3
cells was similar to that we have previously measured on other
intestinal cell lines, the specific gastrin binding data was re-plotted
as a Scatchard plot (Fig. 2 A), and equilibrium dissociation
constants ( K
) for the Type I and Type II
sites calculated. Based on these calculations, the
K
of Type I sites was determined to be
0.83 ± 0.56 n
M ( n = 3) with a binding
capacity of 48.3 ± 23.0 fmol/10
cells. The total
number of high affinity gastrin binding sites measured on Swiss 3T3
cells varied from 0.5 to 6.0
10
sites/cell,
depending upon the passage number of the cells, confluence of the cells
in culture dish, and age of the cells in culture. The highest number of
gastrin binding sites were measured in 70-80% confluent cells
that were <30 days in culture and were taken from early passage
numbers. A very large number of Type II sites, with a
K
of 64.7 ± 22.0 n
M, were
measured (
20-40). The total number of Type II sites
measured was 1.19 ± 0.135 pmol/10
cells.
I-gastrin 2-17 to the Swiss 3T3 cells as
described under ``Experimental Procedures,'' in the presence
or absence of 1000-fold excess gastrin (Fig. 2 B). GR
cross-linked to
I-gastrin 2-17 moved as a single
major band of
45 kDa. As can be seen from
Fig. 2B, >90% of labeled gastrin bound to the 45-kDa
receptor proteins could be displaced by excess nonlabeled gastrin. The
autoradiographic profile was similar under reducing and nonreducing
conditions, indicating that the
45-kDa binding proteins did
not represent subunits linked by disulfide bonds. At longer exposure
times of >14 days we could measure additional bands of
70-80 kDa and
32 kDa cross-linked to
I-G2-17, which represented <10% of the total
specific binding sites present on the Swiss 3T3 cells (data not shown).
Figure 2:
A,
Scatchard plot of specific binding of I-G1-17 to
Swiss 3T3 cells. Swiss 3T3 cells were incubated with 0.5 n
M
I-gastrin-17 in the presence or absence of
increasing concentrations of unlabeled gastrin as described under
``Experimental Procedures.'' The specific binding data, when
analyzed by nonlinear regression analysis by the least squares method,
demonstrated the presence of two classes of binding sites, labeled Type
I and Type II sites. The specific binding data from the two sites was
re-plotted as Scatchard plot with the x axis, indicating the
femtomoles of specifically bound gastrin/10
cells for the
two sites. B, autoradiographic profile of
I-G2-17 cross-linked to gastrin receptors on Swiss
3T3 cells. Swiss 3T3 cells were incubated with
I-G2-17 ± 1000-fold excess unlabeled
G1-17 and cross-linked with 1 m
M DSS as described under
``Experimental Procedures.'' Solubilized membrane proteins
were subjected to SDS-PAGE and analyzed autoradiographically. The data
are representative of more than six experiments. Lanes 1 and
2 = total binding under nonreducing conditions;
lanes 3-6 = binding under reducing conditions
(+4%
-mercaptoethanol); lanes 2, 5, and 6 =
I-G2-17 + 1 µ
M G1-17. The position of the molecular mass markers in
kilodalton units is shown on the left-hand side of the figure.
The arrow points to the 45-kDa gastrin-binding protein band
that represented >90% of the specifically bound gastrin binding
sites.
Effect of CCK-A and CCK-B Receptor Antagonists on the
Proliferative Effects of Gastrin on Swiss 3T3 Cells
Since
0.5-1 n
M gastrin demonstrated maximum growth stimulatory
effects on Swiss 3T3 cells, we chose 0.5 n
M gastrin as an
optimal stimulatory dose for measuring possible inhibitory effects of
CCK-A (L-364,718) (L-18) and CCK-B (L-365, 260) (L-60) receptor
antagonists. Cells were stimulated with or without 0.5 n
M gastrin in the presence or absence of increasing concentrations of
L-18 and L-60 and the percent change in the cell numbers measured in a
growth assay as described under ``Experimental Procedures.''
As can be seen from Fig. 3, neither L-18 or L-60 had any significant
effect on the basal growth of the cells in the absence of gastrin. L-18
and L-60, at all doses examined, had no significant effect on the
growth stimulatory effect of 0.5 n
M gastrin. Surprisingly, 10
n
M L-60, on an average, appeared to augment the growth of
gastrin-stimulated cells, but the difference was not statistically
significant.
Proliferative Effects of Amidated and Non-amidated
Gastrin-like Peptides on CA, Swiss 3T3, and IEC-6 Cells
In order
to measure the relative mitogenic potency of amidated and non-amidated
gastrin-like peptides, we measured the proliferation of the fibroblast
and intestinal cell lines in response to the physiological
concentrations (0.1-10.0 n
M) of the various peptides.
Proliferative effects of gastrin-like peptides on Swiss 3T3 cells are
presented in , and the proliferative effects of the
peptides on CA and IEC-6 cells are presented in Table II. As can be
seen from these results, in all cases glycine extended forms of gastrin
were as effective as the amidated forms of gastrin in stimulating the
growth of Swiss 3T3 () and intestinal cell lines
(). Based on the results with Swiss 3T3 cells, the
relative mitogenic potency of the various peptides examined was in the
order of G1-17 = G1-17-Gly > G5-17 =
G5-17-Gly > G2-17 > CCK-8-Gly CCK-8 =
G1-17-Lys. Glycine-extended gastrins were similarly found to be
equally effective as amidated gastrins in stimulating the growth of CA
and IEC-6 cells (). In order to further compare the
mitogenic potency of glycine extended gastrin and amidated gastrin, the
growth response of Swiss 3T3 cells to the two peptides was compared at
several doses ranging from 0.1 to 10.0 n
M (Fig. 4).
G1-17-Gly was only slightly less potent than G1-17 at doses
<1.0 n
M, but at doses 1.0-10.0 n
M the growth
response profile of Swiss 3T3 cells to the two peptides was almost
identical. The lowest effective dose of the two peptides ranged from
0.1 to 0.25 n
M, and the highest effective dose ranged from 0.5
to 1.0 n
M, in three separate experiments. Thus the effective
growth stimulatory concentrations of the gastrin-like peptides was
within a narrow range of concentrations (
1 log dose), with
the stimulatory effects sharply declining thereafter. This was an
intriguing phenomenon which has been measured with other biological
effects of gastrin/CCK-like peptides and is certainly worthy of further
investigation.
Figure 4:
Dose-dependent proliferative response of
Swiss 3T3 cells to G1-17 and G1-17-Gly. Swiss 3T3 cells in
35-mm Petri dishes were stimulated with or without increasing
concentrations of the two peptides as shown, and the percent increase
in the growth of the cells in treated dishes versus that in
the control (arbitrarily assigned zero level) dishes was measured as
described in the legend to Fig. 1. Each value is the mean ± S.E.
of six observations from two separate experiments (1 and 2). In
experiments 1 and 2, a total of 8.0 10
cells and
6.0
10
cells were seeded, respectively. At the end
of the assay, on an average, 9.86
10
and 7.8
10
cells were measured in the control dishes of
experiments 1 and 2, respectively (with <5% variation in individual
values).
RBA of Gastrin-like Peptides and Receptor Antagonists for
GR on Swiss 3T3 and CA Cells
In initial experiments we used
tumor cell membranes prepared from CA tumors as substrate for the
binding assays. Specific binding of I-gastrin in the
presence or absence of increasing concentrations of various test
peptides and receptor antagonists to the CA membrane aliquots was
measured in a binding assay as described under ``Experimental
Procedures.'' The binding of 1 n
M
I-gastrin
was completely displaced (inhibited) in the presence of 100-1000-fold
excess of nonlabeled gastrin. The percent inhibition of binding in the
presence of various doses of the peptides and the receptor antagonists
is shown in Table III. Glycine-extended gastrins were almost as
effective as amidated gastrins in displacing the binding of 1 n
M
I-gastrin to the CA membranes. The CCK-A and CCK-B
receptor antagonists (L-18 and L-60) were ineffective toward displacing
the specific binding of
I-gastrin to the CA membranes
(I). Based on the dose-dependent displacement of the
binding in the presence of increasing concentrations of the various
peptides, the RBA was calculated as described under ``Experimental
Procedures'' and was
100, 40, 8, 1.3, and 0.6 for the
five peptides G1-17, G1-17-Gly, G5-17, CCK-8, and
CCK-8-Gly, respectively (wherein RBA of G1-17 was arbitrarily
assigned a value of 100). The receptor antagonists, L-18 and L-60,
demonstrated no significant affinity for the specific gastrin binding
sites on Swiss 3T3 cells (Fig. 5 A). In order to ascertain that
the L-18 and L-60 being used by us were biologically effective, we used
AR42J cells as a positive control. Both L-18 and L-60, as expected,
displaced specifically bound radiolabeled CCK-8 to AR42J cells in a
dose-dependent manner (Fig. 5 B). Since gastrin is known
to primarily bind CCK-B receptor subtype on the AR42J cells, L-60
displaced
70% of the specifically bound radiolabeled
gastrin to AR42J cells, whereas L-18 was not as effective (Fig.
5 C). The fact that L-60 did not displace 100% of the
specifically bound G1-17 to the AR42J cells (even at 10
times excess concentration) suggested that
30% of
specific gastrin binding sites on AR42J cells were perhaps distinct
from CCK-B receptors (and may be similar to the gastrin preferring
receptors on the intestinal and Swiss 3T3 cells
(6, 16, 29, 30) ). G1-17-Gly was
once again as effective as amidated gastrin in displacing the binding
of radiolabeled gastrin to Swiss 3T3 cells (Fig. 5 A). Unlike
the CA cells, however, the RBA of G1-17-Gly was almost identical
to the RBA of G1-17 for GR on Swiss 3T3 cells (Fig. 5 A).
Since the majority of the gastrin binding sites on Swiss 3T3 cells
appeared to be present on the 45-kDa protein, we measured the relative
potency of amidated and non-amidated gastrin-like peptides for
displacing the binding of 1 n
M
I-G2-17
cross-linked to the 45-kDa protein. In Fig. 6 A is shown an
autoradiogram demonstrating the presence of 45-kDa GR proteins
cross-linked to
I-G2-17 in the presence or absence
of increasing concentrations of the various peptides. Autoradiographic
data from four separate blots were densitometrically analyzed and is
presented in Fig. 6 B. As can be seen from this figure,
G1-17-Gly was as effective as G1-17 in displacing the
binding of radiolabeled G2-17 from the 45-kDa GR protein on Swiss
3T3 cells. At concentrations of 10 and 100 n
M, G5-17-Gly
was significantly less effective compared with G1-17. At all
concentrations, CCK-8 was significantly less effective than
G1-17. Interestingly CCK-8 at 10 and 100 n
M concentrations appeared to augment the binding of G2-17, but
the difference was not statistically significant.
Figure 5:
RBA
of the CCK receptor antagonists for gastrin and CCK binding sites on
Swiss 3T3 and AR42J cells. Specific binding of
I-G1-17 ( A, B) or
I-BH-CCK-8
( C) to Swiss 3T3 ( A) and AR42J ( B, C) cells
was measured in the presence or absence of increasing concentrations of
either the homologous peptide or the receptor antagonists L-18 and L-60
as described under ``Experimental Procedures.'' The total
specific binding of radiolabeled ligands was arbitrarily assigned 100%
value, and the displacement of the specifically bound counts in the
presence of the indicated concentrations of the test substances was
calculated as a percent inhibition of specific binding as described in
Table IV. From the linear part of the log-dose inhibition curve, the
RBA of the peptides and nonpeptide receptor antagonists was calculated
as described previously (21). On the x axis the log molar
concentration of the peptides/nonpeptide receptor antagonist is shown
for Fig. 4, A-C. Additionally the molar excess
concentration of the unlabeled peptide/antagonist used for displacing
the binding of either 1 n
M
I-G1-17 (Fig.
4, A and B) or 10 p
M
I-BH-CCK-8 (Fig. 4 C) is also indicated.
Each data point in Fig. 4 A represents mean ± S.E. of
six independent measurements from two separate experiments. The data
are representative of several similar experiments. Data in Fig. 4,
B and C, are from a representative experiment of a
total of two to three similar experiments, and each data point is the
mean of four measurements, wherein the variation was <10% between
the separate measurements.
Expression of CCK-A and CCK-B Receptors at the Protein
Level by Swiss 3T3 and AR42J Cells
In order to measure the
presence of specific binding sites for the CCK-A and CCK-B receptor
subtypes, we chose the direct approach of measuring specific binding of
the agonists (I-G1-17 and
I-BH-CCK-8)
and the receptor antagonists (
H-L-18 and
H-L-60) by the two cell lines. AR42J cells were chosen as a
positive control, since these cells are known to express significant
concentrations of both CCK-A and CCK-B receptors
(8) . As can be
seen from Table IV, both Swiss 3T3 cells and AR42J cells demonstrated
the presence of specific binding sites for the agonists. However,
specific binding sites that could recognize the radiolabeled
antagonists were not detected on Swiss 3T3 cells but were detected in
significant concentrations on the AR42J cells. These results thus
further confirmed the possibility that the gastrin/CCK-like peptides
bind to a unique or novel class of GR on Swiss 3T3 cells that are
apparently distinct from the CCK-A and CCK-B receptors.
Expression of CCK-A and CCK-B Receptor mRNA by Fibroblast
and Intestinal Cells
To further confirm the absence of
expression of CCK-A and CCK-B receptors by Swiss 3T3 cells and other
gastrin-responsive intestinal cell lines, RNA from several cell lines
and tissues was screened for the possible presence of CCK-A and CCK-B
receptor mRNA by the methods of Northern blot analysis (Fig. 7,
A and B) and reverse transcriptase-PCR
(Fig. 7 C), as described under ``Experimental
Procedures.'' In all cases RNA from rat antrum, rat pancreas, and
AR42J cells were analyzed as positive controls. As can be seen from
Fig. 7
, A and B, the cDNA probes for the CCK-A
and CCK-B receptors hybridized with the expected transcript sizes for
the two receptors in the positive control samples (pancreas, AR42J
cells). Hybridization with CCK-A and CCK-B receptor cDNA was not
detected in either the intestinal cell lines (IEC-6, CA) or the Swiss
3T3 fibroblasts (Fig. 7, A and B). We next used
the highly sensitive method of reverse transcriptase-PCR to detect
possible presence of a low number of copies of the CCK-A and CCK-B
receptors in the three test cell lines (CA, Swiss 3T3, IEC-6). Once
again we were unable to measure an amplification of the CCK-A and CCK-B
receptor message in either of the three cell lines (Fig. 7 C).
The positive control samples, on the other hand, gave the expected
results for the CCK-A and CCK-B receptors. In the absence of reverse
transcriptase no amplification of the receptor message was measured in
AR42J cells (data not shown), confirming amplification of cDNA in these
samples. In the presence of the primers for CCK-B receptor cDNA, we
additionally measured a weak band of 690 bp in all the samples which
probably represented amplification of gDNA (which is usually present as
<1% contaminant in RNA samples, untreated with DNase
(17) ).
This suggests that the gDNA for rat CCK-B receptor has at least one
small intron of 200 bp. The sense and antisense primers for CCK-B
receptors used by us for amplification of cDNA were also used for PCR
amplification of gDNA from the same cell lines. Once again we measured
amplification of a 690-bp gDNA fragment from all the cell lines (data
not shown), indicating that a similar CCK-B receptor gene was present
in all the cell lines examined. cDNA expression, however, could only be
detected in the positive control samples, confirming the absence of
expression of CCK-B receptors mRNA by the intestinal and fibroblast
cell lines.
=
1 n
M) and/or low-affinity
( K
=
0.1
µ
M) gastrin binding sites are present on normal and
cancerous intestinal cell lines
(6, 16, 28, 31, 32) and Swiss
3T3 cells (present studies), arbitrarily termed Type I and Type II
sites. Since gastrin was mitogenic within the dose ranges compatible
with binding Type I and Type II sites, occupation of Type I and Type II
sites may result in the two peak mitogenic effects of gastrin.
Endogenous and recombinant CCK receptors exist in multiple binding
states
(23, 33) . Glycosylation
(34) and
association with G proteins and guanyl nucleotides
(35, 36) are believed to effect the affinity status and/or ligand
specificity of CCK receptors. It remains to be seen if Type I and Type
II GR on Swiss 3T3 cells are different binding states of the same
receptor or are two separate receptor subtypes.
2.7-kilobase band hybridized to the CCK-B-R cDNA
in the Northern blots of rat pancreatic samples. We believe this band
represents CCK-A-R mRNA and not CCK-B-R mRNA, since the two have
54% homology
(8) and can be expected to hybridize
under the low stringency conditions of washing. Since gastrin is
mitogenic for human colon cancers
(2, 5, 6, 16, 29, 31, 32) ,
Northern blots containing 5 µg of poly(A)
RNA from
representative human colon cancer cell lines (Colo-320, CaCo2) (that
are responsive to gastrin in vitro) were analyzed by Dr. Kopin
at our request. The gastrin-responsive human colon cancer cells were
analyzed to be devoid of CCK-B-R mRNA. We have additionally utilized
sense and antisense primer sets from the most conserved portions of the
CCK-A- and CCK-B-R transmembrane domains and have once again failed to
detect significant amplification of PCR products from either the normal
or the cancerous intestinal cells from rat and human (data not shown).
These results suggest that the novel class of gastrin-preferring
receptors, expressed by the fibroblasts and normal and cancerous
intestinal cell lines, may not belong to the family of
G-protein-coupled receptor superfamily. In recent years several other
investigators have similarly concluded that trophic effects of gastrin
on colon cancer cell lines are probably mediated by a separate class of
gastrin-preferring receptors that do not meet the criteria of either
CCK-A- or CCK-B-R
(29, 31, 32, 35, 43, 44, 45) .
Nonspecific inhibitory effects of receptor antagonists at high
concentrations, however, have been reported that are independent of
binding CCK receptors
(32, 43, 44) .
I-G2-17 as the ligand GR
of several molecular sizes, ranging from 30 to 200 kDa have been
described
(24, 49) . A predominant 75-80-kDa
protein was measured on the parietal/fundic cells
(50, 51) . On rodent and human colon cancer cells a
32-kDa GR protein was predominately measured
(25) . In the
present studies, GR on Swiss 3T3 cells were predominately
45 kDa in size, similar to that on colonic mucosal
membranes from rodent species
(24) . The 45-kDa protein on
colonic mucosal membranes was reduced to an molecular mass of
32 kDa on deglycosylation
(24) . The 45-kDa GR on
Swiss 3T3 cells was also glycosylated and on digestion with
endo-
- N-acetylglucosaminidase F reduced to
30
kDa.
(
)
The molecular masses of the deglycosylated
protein core of CCK-A and CCK-B receptors from different species and
tissues have been reported to range from 37 to 42 kDa (discussed in
Ref. 52), which is close to the theoretical mass (
50 kDa)
of the cloned proteins
(8) . The cloned CCK-A- and CCK-B-R
universally contain three to four potential sites for
N-glycosylation
(8) . CCK-A-R are heavily glycosylated
(53) , whereas CCK-B-R are variably glycosylated (discussed in
Ref. 52). CCK-B-R from calf pancreas
(52) have an molecular
mass similar to that of GR on Swiss 3T3 cells (present studies). The
major difference, however, is in the pharmacology of the two receptors.
CCK-B-R from the calf pancreas demonstrate an equal binding affinity
for the agonists CCK-9 and G1-17 and a relatively high binding
affinity for the CCK-B receptor antagonist L-60, whereas GR on Swiss
3T3 cells and CA cells demonstrate
10 times higher affinity
for G1-17 versus CCK-8 and do not bind L-60 (present
studies).
Table:
Proliferative effects of gastrin-like peptides
on Swiss 3T3 cells
5
10
and
7
10
cells
were seeded in each 35-mm dish. At the end of the growth assay, on an
average 5.51
10
and 7.99
10
cells were measured in control dishes of experiments 1 and 2,
respectively, with <5% variation in individual values within an
experiment. Values are a mean ± S.E. of eight observations from
the two experiments. Cells measured in control wells were arbitrarily
assigned a 100% value. Cell numbers in peptide treated wells are
expressed as a percent of cells in the control (nontreated) wells.
Letters a-h indicate that the values are significantly different
( p < 0.05) compared with the indicated values for the
corresponding dose. In order to ensure that increase in cell number
reflected proliferation of cells, nuclear labeling of the cells with
[
H]thymidine was measured in a few control and
treated dishes (treated with G1-17). Only 5-10% of cells
demonstrated nuclear labeling in control dishes, whereas
30% of
the cells treated with 0.5 and 1.0 n
M gastrin were labeled,
when examined autoradiographically (data not shown).
Table:
Percent inhibition of I-gastrin
(1 n
M) binding to CA cells by gastrin-like peptides and
nonpeptides antagonists
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.