(Received for publication, July 25, 1995; and in revised form, October 10, 1995)
From the
Small cell lung carcinoma (SCLC) accounts for 20-25% of
primary lung cancers and is rapidly growing, widely metastatic, and
rarely curable. Autocrine stimulation of multiple G protein-coupled
neuropeptide receptor systems contributes to the transformed growth of
SCLC. The ability of neuropeptide receptors to stimulate phospholipase
C and mobilize intracellular Ca indicates that
G
family members of heterotrimeric G proteins are a
convergence point mediating autocrine signaling by multiple
neuropeptides in SCLC. Expression of a GTPase-deficient, constitutive
active form of an
family member,
Q212L, in SCLC markedly inhibited growth of the cells
in soft agar and tumor formation in nude mice. SCLC lines expressing
Q212L exhibited 2-4-fold elevated basal
phospholipase C activity, but neuropeptide and hormone-regulated
intracellular Ca
mobilization was nearly abolished.
The data suggest that Ca
mobilization is an
obligatory signal in neuropeptide-stimulated growth of SCLC. In
addition, the proline-directed c-Jun NH
-terminal
kinases/stress-activated protein kinases, which are members of the
mitogen-activated protein kinase family, were stimulated
2-fold in
parental SCLC in response to exogenous neuropeptides and muscarinic
agonists and were constitutively activated to the same degree in
Q212L-expressing SCLC. Thus,
Q212L
expression induced desensitization of neuropeptide-stimulated
Ca
signaling and persistent activation of the c-Jun
NH
-terminal kinase/stress-activated protein kinase pathway.
We propose that the induction of discordant signaling by selective
perturbation of receptor-regulated effector systems leads to the
inhibition of SCLC cell growth.
Small cell lung carcinoma (SCLC) ()displays
neuroendocrine features exemplified by the presence of cytoplasmic
neurosecretory granules containing a wide variety of mitogenic
neuropeptides including gastrin-releasing peptide, arginine
vasopressin, neurotensin, cholecystokinin, and many
others(1, 2) . Significantly, SCLC also expresses
receptors for these neuropeptides, thereby establishing
autocrine-stimulated cell growth(3) . The number and
variability of neuropeptides released from individual small cell
carcinomas hampers effective blockade of mitogenic signaling at the
level of ligand/receptor binding using specific neuropeptide
antagonists. This redundancy at the level of receptor signaling
highlights the importance of defining the intracellular components
involved in mitogenic signal transduction in SCLC where convergence of
multiple neuropeptide receptor systems into common pathways would be
anticipated.
Molecular cloning of the receptors for
gastrin-releasing peptide, vasopressin, and gastrin/cholecystokinin
reveals that they are members of the superfamily of seven
membrane-spanning
receptors(4, 5, 6, 7, 8) .
As a class, these receptors initiate signaling in response to ligand
binding by interacting with heterotrimeric G proteins. Although the
repertoire of G proteins potentially involved in neuropeptide signaling
in SCLC is quite large, the failure of pertussis toxin to appreciably
inhibit in vitro growth of SCLC (9) suggests that the
pertussis toxin-sensitive G and G
proteins are
unlikely to be dominant components of mitogenic signaling in SCLC. In
fact, the prominence of phospholipase C activation and Ca
mobilization (10, 11, 12) by
neuropeptides in SCLC points to the pertussis toxin-insensitive
G
proteins as likely candidates for mediating autocrine
signaling in SCLC because the G
family of G proteins are
known to stimulate several of the phospholipase C
isoforms(13, 14) .
To examine the role of G proteins in SCLC growth pathways, we have used
retrovirus-mediated gene transfer to express a GTPase-deficient,
constitutively active form of the
family member,
G
, in SCLC lines. We find that expression of
GTPase-deficient G
in SCLC markedly inhibits their
growth and oppositely affected two signal transduction pathways
normally engaged by neuropeptides and muscarinic agonists in SCLC;
Ca
mobilization was inhibited, and the c-Jun
NH
-terminal kinase/stress-activated protein kinase pathway
was constitutively activated. The data indicate that derangement of
coordinated neuropeptide signaling in SCLC leads to strong inhibition
of growth.
The activity of
c-Jun NH-terminal kinases was determined essentially as
described(21) . SCLC cells were collected by centrifugation (5
min, 1000
g) and lysed (4 °C, 30 min) in 0.5 ml of
25 mM HEPES (pH 7.7), 20 mM
-glycerophosphate,
0.1 mM sodium vanadate, 0.1% Triton X-100, 0.3 M NaCl, 1.5 mM MgCl
, 0.2 mM EDTA, 0.5
mM dithiothreitol, 2 µg/ml leupeptin, and 4 µg/ml
aprotinin. Following a 5-min microcentrifugation (10,000
g), aliquots of the soluble extracts containing 400 µg of
protein were incubated for 2 h at 4 °C with GST-c-Jun(1-79)
adsorbed to glutathione-agarose as described(21) . The
GST-c-Jun(1-79) beads were washed four times by repetitive
centrifugation in 20 mM HEPES (pH 7.7), 50 mM NaCl,
2.5 mM MgCl
, 0.1 mM EDTA, and 0.05%
Triton X-100 and then incubated for 20 min at 30 °C in 40 µl of
50 mM
-glycerophosphate (pH 7.6), 0.1 mM sodium
vanadate, 10 mM MgCl
, and 20 µM
[
-
P]ATP (25,000 cpm/pmol). The reactions
were terminated with 10 µl of SDS-polyacrylamide gel
electrophoresis sample buffer, boiled, and submitted to 10%
SDS-polyacrylamide gel electrophoresis. The GST-c-Jun(1-79)
polypeptides were identified in Coomassie-stained gels, excised, and
counted in a scintillation counter.
To test the role of G family members in SCLC
growth, we analyzed the effects of expression of GTPase-deficient forms
of
family members on neuropeptide-stimulated
signaling and growth in SCLC lines. We have previously used
retrovirus-mediated gene transfer as a standard method to express
exogenous gene products in many different cell
lines(17, 20) . However, repeated attempts to
establish a stable PA317 retroviral packaging line expressing the
GTPase-deficient form of
(
Q209L)
failed, suggesting that the
Q209L polypeptide was
cytotoxic in these lines and did not permit retroviral packaging.
Subsequent attempts with the GTPase-deficient form of the
family member,
Q212L, were successful such that
a stable PA317 retroviral packaging line that secreted virus encoding
the
Q212L polypeptide was established.
Retrovirus
encoding the Q212L polypeptide as well as the vector,
LNCX, lacking a cDNA insert were used to infect four SCLC lines (H69,
H345, N417, and H1048) as well as two NSCLC cell lines (H157 and
H2122). Stable populations of tumor cells were selected for resistance
to G418. Immunoblotting with an
-specific antibody
verified that an
polypeptide was indeed expressed in
the
Q212L-transfected lines (Fig. 1A)
relative to the LNCX-transfected controls. The lung cancer cell lines
lack detectable endogenous
because it is normally
restricted in expression to cells of hematopoetic origin(22) .
As predicted for an
family member, expression of the
Q212L polypeptide in the SCLC and NSCLC lines
persistently stimulated phospholipase C activity as shown by the
2-4-fold increase in total inositol phosphate content observed in
the
Q212L-expressing cells relative to the
LNCX-transfected lines (Fig. 1B). Expression of the
Q212L polypeptide in the H1048 line, a small cell
carcinoma of extrapulmonary origin(23) , stimulated basal
phospholipase C activity at least 10-fold. Repeated attempts to stably
express
Q212L in another extrapulmonary small cell
line (H510) failed, suggesting that small cell carcinomas of
extrapulmonary origin may be particularly sensitive to
Q212L. Thus, the data show that
Q212L was functionally expressed in SCLC and NSCLC
lines and constitutively activated phospholipase C activity.
Figure 1:
Functional expression of
GQ212L in lung cancer cell lines. A,
immunoblot analysis of
Q212L expression in SCLC and
NSCLC lines. Extracts from the indicated lung cancer cell lines were
resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred
electrophoretically to nitrocellulose, and immunoblotted with a rabbit
polyclonal anti-
antiserum. B, basal
phospholipase C (PLC) activation in lung cancer cell lines
expressing
Q212L. The indicated cell lines were
labeled for 16-24 h with [
H]inositol, and
the accumulation of total inositol phosphates was determined as
described under ``Materials and Methods.'' The data are
presented as the means ± S.E. of three independent experiments,
except for H69, which depicts the average of two
experiments.
Colony
formation in semi-solid medium was used to monitor the transformed
phenotype of SCLC and NSCLC expressing Q212L. The
ability of the
Q212L-expressing SCLC to form colonies
in soft agar was inhibited approximately 70% respective to lines
infected with the vector containing no cDNA insert (Fig. 2). In
contrast,
Q212L expression did not influence the
growth of two NSCLC lines (H157 and H2122). Note that the absolute
cloning efficiency of the different lung cancer cell lines varied
considerably, although the percentage of inhibition of soft agar
cloning efficiency by
Q212L was similar. The soft
agar cloning efficiencies of H345 cells expressing GTPase-deficient
forms of
and
were 96 and 139%,
respectively, of the cloning efficiency of H345 cells infected with
LNCX (data not shown), indicating that inhibition of SCLC growth is
specific for the
family members. This finding is
consistent with the inability of pertussis toxin to significantly
stimulate or inhibit the in vitro growth of SCLC
lines(9) . As a more stringent indicator of the transformed
growth properties of SCLC lines expressing
Q212L, the
H1048 lines infected with LNCX or
Q212L were
implanted in the flanks of nude mice. Of the three mice injected with
H1048-LNCX cells (10
cells per mouse), all formed large
tumors after 6-8 weeks. In contrast, none of the three mice
injected with H1048-
Q212L cells developed tumors
within 8 weeks. Thus, expression of
Q212L in SCLC but
not NSCLC significantly inhibited their in vitro and in
vivo growth properties.
Figure 2:
Soft agar cloning efficiencies of control
and Q212L-expressing lung cancer cell lines. Single
cell suspensions of the indicated cell lines were prepared and cultured
in growth medium containing 0.3% agar nobel as described under
``Materials and Methods.'' The data are presented as cloning
efficiencies (percent of seeded cells that formed a colony) and are the
means of two independent experiments where each experiment was assessed
in triplicate.
To define the mechanism by which
Q212L inhibits SCLC growth, the influence of
Q212L expression on neuropeptide signaling was
examined. A prominent indicator of neuropeptide signaling in SCLC is
intracellular Ca
mobilization(10, 11, 12) , which can be
readily detected by changes in fluorescence of the
Ca
-sensitive dye, Indo-1. The H1048 and H345 SCLC
lines infected with the LNCX vector alone show marked Ca
responses following exposure to exogenous cholecystokinin (Fig. 3A) and the muscarinic agonist, carbachol (Fig. 3C), respectively. The H1048-LNCX cells also
exhibit a measurable Ca
mobilization response to
bradykinin (Fig. 3B). The H1048 and H345 SCLC lines
expressing
Q212L exhibited markedly blunted
Ca
mobilization responses following application of
cholecystokinin, bradykinin, and carbachol. The data are consistent
with a mechanism whereby
Q212L expression
desensitizes hormone and neuropeptide-regulated Ca
mobilization in a heterologous fashion. Previously, workers have
shown that chronic activation of phospholipase C
systems induces
desensitization or down-regulation of the the inositol
trisphosphate-gated Ca
channel in the endoplasmic
reticulum (24) as well as induction of polyphosphatidyinositol
phosphatases. (
)
Figure 3:
Influence of GQ212L on
the regulation of intracellular Ca
levels by
neuropeptides and hormones in SCLC lines. LNCX and
Q212L-expressing H345 and H1048 cells in HITES medium
were loaded with the calcium indicator Indo-1 AM, and the changes in
intracellular Ca
levels were determined as described
under ``Materials and Methods.'' The data are representative
of three independent experiments with the indicated cell
lines.
Besides a blunted growth rate, SCLC H345
cells expressing Q212L are significantly increased in
cell size relative to LNCX-infected controls. In addition, both H345
and H1048 exhibited increased adherence to tissue culture plastic upon
expression of
Q212L. Thus, expression of
Q212L alters the morphologic properties of some of
the SCLC lines. Interestingly, a rat neuroendocrine tumor cell line,
PC12 pheochromocytoma cells, undergoes marked growth arrest and
neuronal differentiation upon introduction of
Q212L
by retrovirus-mediated gene transfer. (
)The inhibition of
SCLC growth observed with
Q212L expression is likely
to be related, at least in part, to loss of Ca
signaling, indicating that the Ca
mobilization
is a major mitogenic signal in SCLC. However, it seemed possible that
additional signal pathways involved in regulation of growth and
differentiation were being regulated in response to expression of
Q212L, especially in relation to altered cellular
morphology. The activity of the p42/44 MAP kinases was assessed because
they have been previously shown to be persistently stimulated in
morphologically differentiated PC12 cells(20) . Extracts from
LNCX and
Q212L-expressing H345 cells were
fractionated on MonoQ fast protein liquid chromatography and analyzed
for MAP kinase activity with an EGFR
peptide
phosphorylation assay. The data in Fig. 4reveal that increased
p42/44 MAP kinase activity relative to control cell extracts was not
observed in fractionated extracts from
Q212L-expressing H345 cells. In addition, incubation
of the various SCLC lines with neuropeptides including bombesin and
vasopressin failed to significantly stimulate p42/44 MAP kinase
activity (data not shown), although treatment of H345 cells with the
phorbol ester 12-O-tetradecanoylphorbol-13-acetate markedly
increased MAP kinase activity (Fig. 4).
Figure 4:
MonoQ
fast protein liquid chromatography analysis of p42/44 MAP kinase
activity in H345 cells expressing GQ212L. Extracts of
H345 cells expressing
Q212L or the empty LNCX vector
were fractionated on MonoQ fast protein liquid chromatography.
Fractions were collected and assayed for MAP kinase activity with
EGFR
peptide. The results are from one
experiment that is representative of two other independent
experiments.
The recently defined
c-Jun NH-terminal kinases or stress-activated protein
kinases are members of the MAP kinase family that exhibit homology to
the p42/44 MAP kinases (26, 27) and are strongly
activated by exposure to heat shock and ultraviolet light (21) . Analysis of c-Jun NH
-terminal kinase
activity using an immobilized GST-c-Jun(1-79) assay revealed a
statistically significant 1.8-fold increase in c-Jun
NH
-terminal kinase/stress-activated protein kinase activity
in extracts from H345 cells expressing
Q212L relative
to control cells (Fig. 5A). The magnitude of c-Jun
NH
-terminal kinase activation by
Q212L is
similar to the level of activation observed in response to acute
treatment of H345 cells with a neuropeptide, bombesin, or a muscarinic
agonist, carbachol (Fig. 5B). Furthermore, c-Jun
NH
-terminal kinase activity is strongly stimulated
(
10-fold) by exposure of H345 cells to ultraviolet irradiation
(UV-C, 96 J/M
), but not by strong activation of protein
kinase C with 12-O-tetradecanoylphorbol-13-acetate (Fig. 5C) that leads to marked p42/44 MAP kinase
activation (Fig. 4). Thus, the findings demonstrate a
significant activation of protein kinases characteristic of the c-Jun
NH
-terminal kinases/stress-activated protein kinases but
not the p42/44 MAP kinases following expression of
Q212L in a SCLC line.
Figure 5:
Regulation of c-Jun amino-terminal kinase
activity in H345 cells by GQ212L, hormones, and
ultraviolet light. A, H345 cells expressing the LNCX vector or
Q212L were incubated for 10 min at 37 °C and
soluble extracts were prepared and assayed for c-Jun
NH
-terminal kinase activity as described under
``Material and Methods.'' B, H345 cells were
incubated at 37 °C for 15 min in the presence or the absence of 100
nM bombesin or 10 µM carbachol. The cells were
collected by centrifugation, lysed, and assayed for c-Jun
NH
-terminal kinase activity as in A. C,
H345-LNCX cells were UV irradiated (192 J/M
), incubated for
30 min at 37 °C or incubated for 10 min with 100 nM
12-O-tetradecanoylphorbol-13-acetate, and assayed for c-Jun
NH
-terminal kinase activity as described above. The data
are the mean of five, three, and three independent experiments for A, B, and C, respectively. * indicates p < 0.05.
Elevated cAMP and activation
of cAMP-dependent protein kinase are growth inhibitory in many cell
systems. It has been previously established that expression of
Q212L in Swiss 3T3 fibroblasts significantly
stimulates cAMP-dependent protein kinase through a protein kinase
C-dependent mechanism(17) , a finding that may be partially
responsible for the growth inhibition observed in Swiss 3T3 cells.
However, the basal activity of cAMP-dependent protein kinase in
Q212L-expressing H345 and H69 cells was 85 and 117%
of LNCX-expressing H345 and H69 cells, respectively, indicating that
the cAMP-dependent protein kinase pathway is not involved in the
Q212L-induced growth inhibition observed in SCLC
lines.
The results show that cellular expression of
Q212L inhibits the growth of SCLC but not NSCLC. The
selective inhibition of SCLC growth relative to NSCLC growth is likely
to be explained by the different cellular pathways that exert dominance
in growth regulation in the two lung cancer cell types. Oncogenic
activation of Ras is frequently observed in NSCLC. In fact, H157, a
squamous cell carcinoma, and H2122, an adenocarcinoma, have been
previously shown to express mutated forms of Ki-Ras(28) . In
addition, NSCLC frequently overexpress specific receptor tyrosine
kinase systems such as the epidermal growth factor
receptor(29) . In contrast, SCLC is characterized by the
notable absence of GTPase-deficient forms of Ras, and overexpression of
epidermal growth factor receptors is rare(28, 29) .
Instead, neuropeptide autocrine loops utilizing seven membrane-spanning
receptors and heterotrimeric G proteins are proposed to be the primary
mitogenic inputs in SCLC. Previous findings that
Q209L
expression in NIH 3T3 fibroblasts leads to cellular transformation (30, 31) is consistent with a role for G
proteins, phospholipase C
, and Ca
mobilization in mitogenic signaling. However, other studies
revealed that
Q212L expression in Swiss 3T3
fibroblasts profoundly inhibited bombesin (gastrin-releasing peptide)
signal transduction including calcium mobilization, MAP kinase
activation, and arachidonic acid release. The net effect was a nearly
complete inhibition of bombesin (gastrin-releasing peptide)-stimulated
DNA synthesis(17) . Similar to the latter studies, our findings
show that expression of
Q212L in SCLC lines resulted
in strong inhibition of neuropeptide-regulated Ca
mobilization (Fig. 3) and dramatically reduced the ability
of the cells to growth in soft agar (Fig. 2). These conflicting
results concerning cellular actions of GTPase-deficient G
proteins may reflect different levels of expression of the
-subunits in the various cell lines where low expression results
in transformation and high expression leads to negative modulation of
intracellular signaling pathways and growth arrest. Alternatively, the
ability of G
, phospholipase C
, and Ca
mobilization to stimulate DNA synthesis may be quite variable
among different cell types.
Expression of GTPase-deficient
Q212L oppositely affected two signal transduction
pathways normally engaged by neuropeptides and muscarinic agonists in
SCLC; Ca
mobilization was inhibited, and the c-Jun
NH
-terminal kinase/stress-activated protein kinase pathway
was constitutively activated. Thus,
Q212L expression
induced discordant signaling in SCLC. We have found that
Q212L expression also constitutively activates the
c-Jun NH
-terminal kinase/stress-activated protein kinase
pathway in PC12 cells where growth arrest and neuronal differentiation
is observed.
The failure of phorbol esters to significantly
activate the c-Jun NH
-terminal kinase/stress-activated
protein kinase pathway (Fig. 5C) in SCLC indicates that
the phorbol ester-regulated forms of protein kinase C are not likely to
mediate the activation of the c-Jun NH
-terminal
kinase/stress-activated protein kinase pathway, although the potential
involvement of nonphorbol ester-sensitive protein kinase C isoforms
cannot be eliminated. Constitutive activation of the c-Jun
NH
-terminal kinase/stress-activated protein kinase pathway
in the absence of Ca
mobilization and p42/44 MAP
kinase activation is correlated with a strong growth arrest in two cell
types, SCLC and PC12 cells. It will be of interest to observe the
spectrum of tumor cells where this response is growth inhibitory.
Ultraviolet irradiation of H345 cells strongly activates the c-Jun
NH-terminal kinase/stress-activated protein kinase pathway (Fig. 5C) and leads to a rapid apoptotic-like cell
death. (
)The 2-fold activation of the c-Jun
NH
-terminal kinase/stress-activated protein kinase pathway
with
Q212L expression relative to the greater than
10-fold activation by UV irradiation may, in addition to inhibited
Ca
mobilization, explain the strong growth arrest
observed in the SCLC lines. We hypothesize that high level constitutive
activation of the c-Jun NH
-terminal kinase/stress-activated
protein kinase pathway in the absence of neuropeptide-stimulated
Ca
mobilization may have lethal consequences in SCLC.
Consistent with this possibility was our inability to isolate
Q212L-expressing clones in some SCLC lines such as
H510, despite repeated attempts. We have found that a proximal
regulator of c-Jun NH
-terminal kinases is a MEK kinase
distinct from Raf(25, 32) . MEK kinase activation is
Ras-regulated and independent of Raf(32) . Expression of
activated MEK kinase-1 leads to cell death in several fibroblast
systems and differentiation of PC12 cells (
)similar to the
phenotype observed with activated
members such as
Q212L. Thus, the MEK kinase/c-Jun
NH
-terminal kinase regulatory pathways may contribute to
growth inhibition and possibly cell death, a sharply contrasting
phenotype from that observed with activated Raf expression.
These
findings indicate that it is possible to inhibit cell growth of
selected tumor cell types by selectively disrupting coordinated signal
transduction pathways. Increasing awareness that signals controlling
growth and apoptosis are overlapping is insightful in regards to these
results. We suggest that the balance and magnitude of specific signal
transduction pathways regulated by hormones and growth factors can
determine commitment to tumor cell growth or arrest. Identifying tumors
that have a strong growth inhibitory response resulting from activated
expression will allow a new pharmacologic and genetic
approach to cancer therapeutics. The ability of G
family
members of G proteins to regulate the c-Jun NH
-terminal
kinase/stress-activated protein kinase pathway and to cause growth
inhibition in SCLC and some fibroblasts focuses our attention on the
effectors capable of inhibiting tumor cell growth.