ICANCER RESEARCH
46, 1214-1218,
March 1986]
Secretin/Vasoactive Intestinal Peptide-stimulated Secretion of Bombesin/
Gastrin Releasing Peptide from Human Small Cell Carcinoma of the Lung1
Louis Y. Korman,2 Desmond N. Carney, Marc L. Citron, and Terry W. Moody
Medica/ Service (151W), Veterans Administration Medical Center, Washington, DC 20422 [L. Y.K., M.L.C.]; Department of Medicine and Biochemistry George
Washington University School of Medicine, Washington, DC 20037 [T. W. M.¡;and National Cancer Institute-Navy Medical Oncology Branch National Cancer Institute
and National Naval Medical Center, Bethesda, Maryland [D. N. C.¡
ABSTRACT
autocrine factor for SCCL (12) growth. We studied the mecha
nism of BLI secretion in several SCCL cell lines by examining
the action of agents that increase intracellular cAMP.
Because of the results of these in vitro studies and the fact
that secretin stimulates hormone release in patients with gastrin
producing tumors (Zollinger-Ellison syndrome), we examined the
action of i.v. secretin infusion on plasma BLI levels in several
patients with SCCL, non-SCCL lung tumors, and patients with
out any cancer.
Bombesin/gastrin releasing peptide-like immunoreactivity (BLI)
is found in the majority of small cell carcinoma of the lung (SCCL)
cell lines examined. Because BLI is present in high concentration
in SCCL we studied the mechanism of BLI secretion from several
SCCL cell lines and in patients with SCCL. In cell line NCI-H345
the structurally related polypeptide hormones secretin, vasoactive intestinal peptide, and peptide histidine isoleucine as well as
theophylline, a phosphodiesterase inhibitor, N6,O2'-dibutyryl
cyclic adenosine 3':5'-monophosphate,
a cyclic nucleotide ana
MATERIALS AND METHODS
logue, increased BLI release by 16-120% and cyclic adenosine
3':5'-monophosphate
by 36-350%. Similar results were ob
Cell Culture. SCCL cell line NCI-H345 was established using a bone
biopsy from a patient with SCCL who had relapsed following intensive
combination chemotherapy. In culture NCI-H345 is aneuploid, clonogenic
in soft agarose, tumorigenic in nude mice, expresses elevated levels of
3,4-dihydroxy-L-phenylalanine
decarboxylase (198 units/mg protein),
tained in SCCL cell line NCI-H209. i.v. injection of secretin (2
units/kg) significantly increased plasma BLI in 2 patients with
extrapulmonary SCCL. These data suggest that SCCL cells
possess receptors for secretin/vasoactive intestinal peptide and
that receptor occupation stimulates in vitro and In vivo BLI
secretion.
neuron specific enolase (4075 ng/mg protein), and produces high quan
tities of BLI (4.7 pmol/mg protein (2). The cell line was maintained
continuously for 24 mo in a serum free defined HITES medium (13). Cells
were incubated in 75-cm2 Falcon tissue culture flasks in a humidified
atmosphere of 5% CO2/95% air at 37°C. One to 2 wk prior to each
INTRODUCTION
experiment stock cultures were passaged and then 3 days prior to use
the cells were resuspended in fresh HITES medium. Other SCCL cell
lines were maintained as described previously (2).
BLI Radioimmunoassay.
Rabbit antisera to bombesin was obtained
as described previously (14). The antiserum recognizes bombesin and
the structurally related gastrin releasing peptide equally well, because
they have the same COOH-terminal heptapeptide, and does not crossreact with secretin, VIP, or PHI (14). For the BLI determination cells or
supernatant was extracted with boiling 2 N acetic acid, centrifuged at
2,000 x g for 10 min to remove paniculate material, and the supernatant
was frozen, lyophilized, and stored at -70°C. The assay was performed
In the United States, small cell carcinoma of the lung accounts
for 25,000-30,000 new cases of primary lung cancer each year
(1). Unlike the other major forms of lung cancer, in vivo and in
vitro studies indicate that SCCL3 expresses several unique cytochemical and functional characteristics including the presence
of high levels of 3,4-dihydroxy-L-phenylalanine decarboxylase,
dense core neurosecretory granules, and the secretion of peptide
hormones (2-4). These findings suggest that SCCL may origi
nate from endocrine (Kultschitzky) cells in the lung (5). However,
recent studies indicate that SCCL may be derived from a hematopoietic stem cell (6). Although several polypeptide hor
mones including adrenocorticotropic hormone, calcitonin, physalaemin, arginine vasopressin, and neurotensin are produced by
SCCL, BLI is frequently present (4, 7-9). Prior investigations
have demonstrated that hormone secretion from cultured endo
crine cells including SCCL (10) may be mediated by the intracellular second messenger cAMP (11) and bombesin may be an
by resuspending samples in 140 mw NaCI/16 mw NaPO4 (pH 7.4)
containing 0.25% bovine serum albumin and incubating with rabbit antibombesin serum at 1:100,000 dilution for 1 h at 4°C,then adding 5000
cpm of 125l-labeled tyrosine4 bombesin for a total vol of 400 pi and
incubated for 16 h at 4°C.At the end of the incubation 200 pi of normal
rabbit serum at 1:200 dilution was added followed by 100 pi of goat antirabbit serum at 1:20 dilution. After 30 min, 200 pi of 12% polyethylene
glycol was added to enhance precipitation and the samples were centri
fuged at 1000 x g for 20 min. The supernatant was removed and the
pellet assayed for antibody-tracer complex in a LKB gamma counter
(LKB, Rockville, MD).
Venous blood samples were centrifuged in a Sorvall RC-5B refriger
ated centrifuge at 500 x g for 10 min and the plasma was removed and
stored at -70°C. Prior to assay the plasma was thawed and processed
Received 2/18/85; revised 7/30/85, 11/26/8S; accepted 12/2/85.
1This work was supported by funds from the Veterans Administration Research
Service and by National Cancer Institute grant CA-33767. Presented in part at the
Annual Meeting of the American Federation for Clinical Research, Washington DC
April 1983.
2 To whom requests for reprints should be addressed, at Gastroenterology
using a modification of the method of Bohlen ef a/. (15). In brief, 2 ml of
plasma were added to 6 ml of 5% formic acid, 15% trifluoracetic acid,
9% HCI, and 1% NaCI. The precipitated plasma proteins were centrifuged
at 2000 x g for 10 min and the supernatant was applied to a Sep-Pak
(Millipore). The peptides were eluted from the Sep-Pak using ethanol/0.2
N HCI (1/1), and the eluate was frozen, lyophilized, and assayed for BLI
(14).
Section (151 W), Veterans Administration Medical Center, 50 Irving St., NW, Wash
ington, DC 20422.
3 The abbreviations used are: SCCL, small cell carcinoma of the lung; BLI,
bombesin/gastrin releasing peptide-like immunoreactivity; cAMP, cyclic AMP; VIP,
vasoactive intestinal peptide; PHI, peptide histidine isoleucine; HITES (medium),
RPMI 1640 medium supplemented with hydrocortisone (10 nM), bovine insulin (5
fig/ml), transferrin (10 jig/ml), 17^-estradiol (10 nw), and sodium selenite (30 nM)cGMP, cyclic GMP.
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BOMBESIN/GASTRIN
RELEASING
PEPTIDE SECRETION
Cyclic AMP Radioimmunoassay. Cyclic AMP was determined using
a minor modification (16) of the procedure described by Brooker ef al.
(17). At the times specified iced (4°C)absolute ethanol was added to an
equal vol of either the cell suspension or the cell pellet, kept at 4°Cfor
15 min and then frozen and stored at -70°C. Samples were thawed and
centrifuged at 10,000 x g for 5 min, and an aliquot of supernatant was
removed and assayed for cAMP. Protein was measured by the method
described by Lowry ef at. (18).
Secretin Infusion. Secretin (2 units/kg) was injected over a 30-s
period into a large antecubital vein via a freely flowing i.v. At 15 min, just
prior to and at 2, 5,10, 30, and 60 min after secretin injection 5-ml blood
samples were obtained, added to 0.5 ml of 20 HIM ethyleneglycol bis(/3aminoethyl ether)-A/,A/,/v",A/'-tetraacetic acid and bacitracin (pH 7.4), 1
mg/ml and placed on ice. Symptoms, blood pressure, pulse, and respi
ratory rate were monitored during and for 2 h after secretin injection and
no untoward effects occurred. Informed consent, approved by the Med
ical Center Human Studies Subcommittee, was obtained from the pa
tients.
Materials. RPM11640 with glutamine was obtained from Grand Island
Biological, Grand Island, NY; VIP, PHI, secretin, bombesin, physalaemin,
neurotensin, and cholecystokinin-octapeptide
from Peninsula Laborato
ries, Belmont, CA; bovine plasma albumin (Fraction V) from Armour
Pharmaceutical, Phoenix, AZ; all other chemicals were from Sigma
Chemical, St. Louis, MO. cGMP antiserum (preconjugated to a second
antibody) and 125l-labeled 2'-O-succinyl(tyrosine methyl ester)cyclic GMP
was from New England Nuclear, Boston, MA; 125l-labeled 2'-O-succi-
FROM HUMAN SCCL
mg protein by 5 min which declined to 39 pmol/mg protein by
60 min. Similarly, 1 I¿M
secretin, a gastrointestinal peptide with
significant sequence homology to VIP (20), increased cAMP from
17 to 42 pmol/mg protein by 60 min. Without additions cAMP
varied between 10 and 25 pmol/mg protein during the 60-min
incubation. Equivalent results were obtained for cell line NCIH209 (data not shown).
Fig. 2 shows that 1 »Msecretin increased BLI in the superna
tant from 300 to 480 fmol/mg protein after 5 min. Secretion then
remained constant in the presence of 1 /IM secretin. In contrast,
without additions supernatant BLI remained constant at 250
fmol/mg protein throughout the time course of the experiment.
These data indicate that 1 /.M secretin stimulated a rapid release
of BLI into the supernatant and this release coincided with the
secretin-stimulated increase in cAMP. Similar results were ob
tained using VIP. Because both the cAMP levels and secretion
of BLI was maximal after 5 min this time point was used in all
subsequent in vitro experiments. Table 1 shows that at 5 min,
when secretagogue-stimulated cAMP was maximal, 1 ^M VIP,
PHI, or secretin significantly (21) increased BLI release by 25,
SECRETIN
500
lluM)
nylftyrosine methyl ester) cyclic AMP was from Melloy Laboratories,
Springfield, VA; goat anti-cyclic AMP antibody was a gift from Dr. Gary
Brooker, Georgetown University School of Medicine, Washington, DC;
and the infused secretin was a gift from Dr. L. Gabel, Pharmacia
Laboratories, Piscataway, NJ.
300
CONTROL
RESULTS
; 200
Previous studies suggest that SCCL cell lines of the classic
morphology enriched in dense core neurosecretory granules
have high levels of BLI (7,19). To explore the response of SCCL
cell lines to agents that increase cAMP, we examined the action
of 1 UM VIP and 1 UM secretin on cellular cAMP in SCCL cell
lines NCI-H345 and H209. NCI-H345 and NCI-H209 are SCCL
cell lines which contain BLI. Cell line NCI-H345 was grown in
HITES medium, whereas cell line NCI-H209 was grown in HITES
supplemented with 10% fetal bovine serum. Fig. 1 shows that in
cell line NCI-H345 1 MMVIP increased cAMP from 14 to 90 pmol/
100
10
TIME
ii-.m
20
30
Fig. 2. Time course of secretin-stimulated BLI secretion in SCCL cell line NCIH345. Results are mean ±SE (bars) of BLI in the supernatant. Each value was
determined in triplicate and the results are representative of 3 separate experi
ments.
Table 1
EHect of agents on cellular cyclic AMP and BLI release in SCCL
cell line NCI-H345
90
For cAMP the results are the mean ±SE of the number of experiments in
parentheses and in each experiment each value was determined in duplicate. For
BLI release the results are the mean ±SE of 5 separate experiments and in each
experiment each value was determined in triplicate. Routinely, the cell pellet
contained approximately 4.7 pmol BLI/mg protein in the absence of additions.
Without additions cellular cAMP was 37.9 pmol/mg protein and BLI in the super
natant was 260 fmol/mg protein.
80
_70
C
1
5 60
Û.
|
50
% of control
I«
AdditionNoneVIP
Q.
¡30
u
20
(1 „Ml
,/M)Secretin
PHI (1
(1 /*M)
N,,02'-dibutyryl cAMP (1 mut)
10
0
10
20
30
TIME iminl
40
50
Prostaglandin E, (10 JIM)
Theophylline
+VIP
(10 mw)
60
(1 pM)Cellular
release100
±11 (10)
462 ±58 (5f
243 ±19 (5f
(10)"111
136 ±20
±10
125 ±16"
131 ±12"
115 ±4C
141 ±9"
±39(5)
454 ±79 (5f
1180±152 (5)aBLI
111 ±10
219 ±30*
244 ±42*
* P < 0.005 by Student's paired ( test (21).
b P < 0.01 by Student's paired f test (21).
0 P < 0.05 by Student's paired f test (21).
Fig. 1. Time course of VIP- and secretin-stimulated cAMP in SCCL cell line NCIH345. Results are expressed as mean ±SE (bars) and each value was determined
in duplicate. Results are representative of 3 separate experiments.
CANCER RESEARCH
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BOMBESIN/GASTRIN
RELEASING
PEPTIDE SECRETION
31, and 15%, respectively, and that 1 ITIMN6,O2'-dibutyryl cAMP
FROM HUMAN SCCL
Because secretin increased cAMP and BLI secretion in vitro
and is used to stimulate gastrin release from gastrinomas in
patients with Zollinger-Ellison syndrome (22), we examined the
time course of secretin-stimulated BLI secretion in several pa
increased BLI release by 41 %. Neither 10 UM prostaglandin E1
nor 1 mw nitroprusside (data not shown) significantly increased
BLI release. VIP, PHI, and secretin increased cAMP levels by
360, 140, and 40%, respectively, whereas prostaglandin EÕhad
no effect. Theophylline (10 HIM) alone caused a 119% increase
in BLI release and a 350% increase in cellular cAMP. Adding 1
UMVIP slightly increased the theophylline-stimulated BLI release
but dramatically augmented the theophylline-stimulated cellular
tients with SCCL including two with extensive extrapulmonary
disease. As described previously, basal levels of plasma BLI
were significantly greater in the patients with extrapulmonary
SCCL than in the patients with non-SCCL tumors or normal
controls (23). In the two patients with extensive SCCL the
injection of secretin (2 units/kg) rapidly increased plasma BLI
from 60 to 210 PM by 2 min and 91 pw by 1 h, respectively (Fig.
3). In one patient with non-SCCL limited to the thorax, secretin
cAMP (Table 1). Since the agents tested might also increase BLI
release by increasing cGMP we measured cellular cGMP under
the same conditions as described in Table 1 and in the presence
of 1 rriM nitroprusside. Only 10 mw theophylline and 1 mw
nitroprusside increased cellular cGMP. Furthermore, 1 UM bom-
stimulated a transient increase in plasma BLI. The reason for
this small response is not clear. In contrast, in three untreated
patients with SCCL limited to the thorax (Fig. 3A) and in two
patients without any evidence of tumor (data not shown) secretin
infusion did not alter plasma BLI. Furthermore, after chemother
apy-induced remission in the patient with extrathoracic SCCL
besin, 1 MMcholecystokinin, 1 MMphysalaemin, 1 MMneurotensin,
1 MMcalcitonin, 10 MMphenylephrine, and 10 MMcarbamylcholine
did not increase either cGMP or cAMP.
To determine whether the secretagogue-stimulated increase
in cAMP and BLI secretion was unique to cell line NCI-H345, we
examined the action of VIP, secretin and theophylline on another
SCCL cell line (Table 2). In NCI-H209,1 MMVIP and 1 MMsecretin
basal plasma BLI was reduced by approximately 50% (36 versus
74 pw) and there was no increase in secretin-stimulated plasma
BLI (Fig. 38).
increased cAMP from 5.3 to 19.6 and 9.9 pmol/mg protein,
respectively, and BLI secretion from 567 to 699 and 787 fmol/
mg protein, respectively. Although the theophylline-stimulated
increase in cAMP was equal to that of VIP, the theophylline-
DISCUSSION
VIP and secretin are widely distributed peptide hormones
which mediate a variety of physiological responses including
gastrointestinal secretion, relaxation of gastrointestinal, vascular,
and respiratory smooth muscle, lipolysis in adipocytes, glycogenolysis in hepatocytes, pituitary hormone secretion, and excit
ation and hyperthermia after injection into the central nervous
system (21, 24). VIP receptors, some of which bind VIP with
high affinity and secretin with low affinity (25, 26), and secretin
receptors are coupled to adenylate cyclase in normal and malig
nant intestinal epithelium, pancreas, gastrointestinal and cardio
vascular smooth muscle, adipocytes, hepatocytes, a pituitary
tumor cell line, brain, and leukocytes (21,24,27,28). The present
studies suggest that VIP and secretin receptors are also present
on several SCCL cell lines and that occupation of these receptors
increases adenylate cyclase activity and stimulates BLI release
via a cAMP-mediated pathway. Furthermore, other agents that
stimulated increase in BLI was significantly greater, 1134 versus
699 fmol/mg protein, respectively.
Table 2
Action of agents on cellular cAMP and BLI secretion in cell line NCI-H209
For cAMP and BLI the results are the mean ±SE of 2 separate incubations and
are representative of 3 separate experiments. For cAMP each value was determined
in duplicate and for BLI each value was determined in triplicate. Routinely the cell
pellet contained 18.3 pmol BLI/mg protein.
AdditionsNoneVIP
UM)Secretin
(1
cAMP
(pmol/mg
protein)5.3
secretion
(fmol/mg
protein)567
±92
699 ±59°
787 ±66"
1134 ±154"
±0.3
19.6 ±2.0a
9.9 ±1.5a
17.4 ±0.9*BLI
(1 UM)
Theophylline (10 mM)Cellular
" P < 0.01 by Student's paired f test (21).
" P < 0.05 by Student's paired ( test (21).
A.
B.
X SECRETIN|2U/kg)
200
Fig. 3. A, secretin-stimulated plasma BLI in
patients with SCCL and non-SCCL tumors. Se
cretin infusion and plasma BLI determinations
were performed as described in "Materials and
Methods." Patient M. G. (•),
treated, recurrent,
extrapulmonary
SCCL; Patient J. P. (O),
treated, metastatic. well-differentiated adenocarcmoma; Patients L. R. (A), F. P. (T), and E.
R. (•)
untreated SCCL limited to the thorax. B,
secretin-stimulated plasma BLI in a patient with
extrathoracic
SCCL. Secretin infusion and
plasma BLI determinations were performed as
described in "Materials and Methods." Patient
W. T. pretreatment (•)and after 4 courses of
chemotherapy (A.) resulted in a 90% reduction
in extrathoracic tumor burden.
SECRETIN (2U/kg)
500
160
400
H120
I 300
80
200
40
100
-15
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60
BOMBESIN/GASTRIN
RELEASING
PEPTIDE SECRETION
so that tumor BLI release may not be detectable, and (c) the
absence of BLI in the tumor cells. Secretin-stimulated BLI release
increase cAMP by inhibiting phosphodiesterase activity or by
simulating an increase in intracellular cAMP also increase BLI
release (10). Thus, SCCL cells, like other cultured endocrine
cells, possess an intact cAMP-mediated pathway for stimulating
hormone release. Because binding studies were not performed
the present results do not distinguish between two separate
classes of receptors for VIP and secretin or one class of recep
tors for VIP/secretin which recognizes each with different affini
ties (25, 26); nor do these studies define the pharmacological
characteristics of the VIP/secretin receptor.
That secretion from SCCL cell lines is also calcium dependent
is suggested by prior studies (10, 29) as is the finding that
theophylline-stimulated BLI release is significantly greater than
VIP-stimulated BLI release, whereas there is no difference be
tween VIP- and theophylline-stimulated cellular cAMP (Table 1).
Previous studies indicate that carbamylcholine and methylxanthine phosphodiesterase inhibitors increase pancreatic enzyme
secretion by modifying cellular calcium fluxes (16). In addition,
carbamylcholine stimulates BLI release (10) but does not in
crease cAMP or cGMP production. Thus, BLI may be secreted
from SCCL cells by altering the intracellular cAMP levels or by
altering calcium flux.
In classic SCCL cell lines one of the characteristic findings is
the presence of BLI. Of the polypeptide hormones examined
prior work indicates that 94% (32 of 34) of the SCCL cell lines
and none (0 of 13) of a variety of non-SCCL cell lines contained
is unlikely to be of use in the differential diagnosis of disease
limited to the thorax. However, the presence of a positive re
sponse to secretin may be useful in predicting response to
therapy, diagnosing extrathoracic spread, or documenting recur
rence. Clearly, larger numbers of normal patients and patients
with limited and extensive non-SCCL tumors need to be studied
to determine the clinical utility of this provocative test. Another
potential clinical application for these findings is in the treatment
of SCCL. Recent observations suggest that changes in cellular
cAMP alter the growth rate and metabolism of SCCL and other
cells in vitro (38, 39). Since BLI acts as an autocrine growth
factor for SCCL, regulation of BLI release from SCCL may play
a role in regulating tumor growth (12).
ACKNOWLEDGMENTS
We thank Drs. L. Recant, A. Gazdar, J. Minna, M. Cohen, and S. Krasnow for
their support and suggestions and V. Bertness and G. Lebovic for technical
assistance. We thank Paula Kittrell and Jan Parker for preparing this manuscript
for publication.
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10.
11.
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patients with limited SCCL may be due to (a) rapid degradation
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12.
13.
14.
15.
16.
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CANCER
SECRETION
VOL.
46 MARCH
1986
1218
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
Secretin/Vasoactive Intestinal Peptide-stimulated Secretion of
Bombesin/Gastrin Releasing Peptide from Human Small Cell
Carcinoma of the Lung
Louis Y. Korman, Desmond N. Carney, Marc L. Citron, et al.
Cancer Res 1986;46:1214-1218.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.