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Molecular Biology and Genetics |
Sealy Center for Oncology and Hematology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25% of all lung cancers and is characterized by early
metastasis and initial responsiveness to chemotherapy and radiation.
Despite the initial marked responsiveness to therapy, the vast majority
of patients with SCLC relapse and develop drug/therapy resistance and
eventually die from the disease (1
, 2)
. Overall, the
5-year survival rate is 3–8% (3)
. Therefore, novel
approaches to the treatment of this rapidly invasive tumor are urgently
needed. It is likely that progress will require a better understanding
of the intracellular molecular events that determine the malignant
behavior of SCLC. The biological mechanisms underlying tumor progression and the development of chemoresistance in SCLC are still not understood. Clinically, approximately one-third of the recurring chemoresistant tumors exhibit an apparent transition toward NSCLC histology, with cells resembling NSCLC partially or completely replacing SCLC cells (4 , 5) . A similar in vitro phenomenon has also been observed, involving the accrual of large cell undifferentiated phenotype when SCLC cell lines are maintained for extended periods of time in continuous culture (6 , 7) . These data suggest that one component of malignant progression may be a movement of the SCLC phenotype along a differentiation continuum linking SCLC with NSCLC, and the potential transition between these phenotypes may play an important role in the development of treatment resistance in patients (8) . In support of this hypothesis, in vitro studies have demonstrated that insertion and expression of a v-Ha-Ras oncogene into SCLC cell lines with amplified Myc family oncogenes induces a phenotypic transition with the acquisition of features typical of the NSCLC phenotype (9 , 10) . These features include a profound morphological change from SCLC cells in suspension to NSCLC-like adherent monolayer cells, reductions in expression of neuroendocrine markers, and induction of expression of the NSCLC-associated growth factor/receptor genes. Furthermore, this phenotypic transition is accompanied with an acquired resistance to 2-difluoromethylornithine typical of NSCLC carcinoma, but not SCLC, in vitro (11) . However, the molecular mechanisms underlying the oncogenic Ras-mediated phenotype transition have not been elucidated.
Ras proteins function as a molecular switch that cycles between an
active GTP-bound and an inactive GDP-bound form and play a pivotal role
in regulating cell growth and differentiation (12)
.
Activated GTP-bound Ras proteins interact with downstream effectors and
promote the activation of at least two distinct serine/threonine kinase
cascades, the Raf-dependent MAPK pathway and the Raf-independent
JNK/stress-activated protein kinase pathway
(13, 14, 15)
. Ras activation of the Raf-dependent MAPK pathway
(Raf/MEK/ERK pathway) involves sequential activation of the Raf kinase,
MEK1/2, and ERK1/2. Activated ERKs phosphorylate numerous substrates
including other kinases, cytoskeletal elements, and nuclear
transcription factors such as Elk-1, resulting in immediate-early gene
expression, cell growth, and differentiation (15
, 16)
. Ras
also activates a Raf-independent serine/threonine kinase pathway
leading to JNK activation. JNK activation up-regulates c-Jun and ATF-2
transcriptional activity (17
, 18)
. Unlike ERKs that are
primarily activated by mitogens, the JNKs are potently and
preferentially activated by cellular stress (heat shock, osmotic shock,
and UV and 
-irradiation) and by inflammatory cytokines (tumor
necrosis factor 
and interleukin-1; Ref. 19
). The JNK
pathway consists of MEK kinases, JNK kinases, and JNKs, which are
sequentially activated by phosphorylation.
The Raf/MEK/ERK pathway is essential for Ras-mediated transformation (20, 21, 22) . However, increasing evidence suggests that Raf-independent signaling pathway(s) are also important for Ras-induced cellular transformation. Studies using Ras effector domain mutants suggest that the coordinated activation of Raf-dependent and Raf-independent pathways by oncogenic Ras is necessary for establishing and maintaining the transformed phenotype (23 , 24) . Furthermore, it has been demonstrated recently that activation of the JNK pathway via a Raf-independent pathway is also essential for Ras transformation (25) . Identification of multiple Ras downstream effectors raises the possibility that a very complex set of signals is generated by Ras that could cooperate to induce cellular transformation (21 , 26 , 27) . Currently, the contribution of multiple downstream effector-mediated pathways to oncogenic Ras signaling and transformation has not been fully elucidated. In particular, the signaling mechanisms by which oncogenic Ras controls proliferation of human cancer cells remain poorly characterized, despite a high frequency of activating mutations of Ras family genes associated with many types of human cancer including lung cancer (28) .
In this report, we investigate the signaling mechanisms by which an activated Ras oncogene induces a phenotype transformation of NCI-H82 SCLC cells. We demonstrate that, in NCI-H82 cells, oncogenic Ras preferentially activates JNK, and that activation of the JNK-dependent pathway, but not the ERK pathway, is required for the maintenance of the oncogenic Ras-induced transformed phenotype. Our results further suggest that one mechanism underlying JNK-mediated Ras transformation is stimulation of c-Jun/AP-1 activity. Our results indicate that the JNK pathway plays an important role in promoting lung carcinoma cell growth and suggest that the components of the JNK pathway may be possible targets for development of novel antineoplastic drugs in the treatment of carcinoma.
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MATERIALS AND METHODS |
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Cell Culture, DNA Transfection, and Luciferase Assay.
The NCI-H82 SCLC cell line was maintained in RPMI 1640 supplemented
with 9% (v/v) calf serum, 100 units/ml penicillin, and 100 units/ml
streptomycin. Stably transfected cells were maintained in the same
medium plus 400 µg/ml G418 or 250 µg/ml hygromycin B. Stable
transfection of NCI-H82 cells was carried out using an electroporation
method (31)
. Cells were selected in G418-containing growth
medium. For double transfectants, Ras-expressing cell lines were
secondarily transfected with pCDNA3-FLAG-dnJNK1 along with pSV-HygB2 at
the molar ratio of 5:1 using Lipofectamine and selected in
hygromycin-containing growth medium. Transient transfections were
carried out using either Lipofectamine or Lipofectin, according to the
procedures recommended by the manufacturer (Life Technologies). For
transient transfection luciferase assays, plasmid pSV-ß-gal (Promega)
was cotransfected with the luciferase reporter constructs to monitor
transfection efficiency. Cells were harvested 48 h after
transfection and lysed in 1x Reporter Lysis Buffer (Promega). For
serum starvation, cells were cultured in a serum-free medium
supplemented with 1% BSA for 24 h before lysis. Assays for
luciferase and ß-galactosidase activities were performed as described
using kits purchased from Promega and Clontech (Palo Alto, CA),
respectively. Data shown are normalized luciferase activities as a
ratio of luciferase activity (RLU/µl) to ß-gal activity (unit
ß-gal/µl).
Soft Agar Assay and Growth Studies.
Soft agar assays were carried out in six-well plates as described by
Clark et al. (32)
. Briefly, cells were seeded
in 0.3% agarose in growth medium overlaid on a base of 0.6% agarose.
Cultures were fed weekly. Colonies were scored 14 days after plating.
For growth curves, 1x105 cells/well were plated
in six-well plates in complete medium or low serum medium containing
0.5% calf serum. Media were changed every 2 days. Cell number was
determined by trypan blue exclusion. Triplicate cultures of each cell
clone were prepared and processed in all experiments.
Immunocomplex Kinase Assay for ERK and JNK.
Cells were lysed in a modified RIPA buffer [50 mM HEPES
(pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 2
mM EDTA, 2 mM EGTA, 50 mM sodium
ß-glycerophosphate, 5 mM sodium pyrophosphate,
50 mM sodium fluoride, 1 mM sodium
orthovanadate, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin]. Endogenous ERK1/2 and JNK1 were immunoprecipitated from 50
µg of cleared cell lysate using the appropriate rabbit polyclonal
antibodies in conjunction with protein A-agarose (Life Technologies,
Inc.). The immunoprecipitates were washed twice with lysis buffer and
twice with kinase assay buffer. Kinase reactions were carried out by
incubating the immunoprecipitates with substrate in 30 µl of kinase
assay buffer [25 mM HEPES (pH 7.5), 20 mM
magnesium chloride, 0.1 mM EGTA, 50 mM sodium
ß-glycerophosphate, 0.1 mM sodium orthovanadate, 1
mM DTT, and 1 µM okadaic acid] supplemented
with 20 µM ATP and 5 µCi
[-32P]ATP at room temperature (22°C) for
25 min. Ten µg of MBP and 1 µg of GST-c-Jun 1–79(1–79) were used as
substrates for ERK1/2 and JNK activities, respectively. Kinase
reactions were terminated by the addition of 30 µl of 2x Laemmli
sample buffer. The phosphorylated proteins were resolved on a 10%
polyacrylamide-SDS gel and analyzed by autoradiography and
InstantImager (Packard Instrument, Meriden, CT).
Western Blot Analysis.
Whole-cell lysates (50–100 µg) each sample were resolved on a
polyacrylamide-SDS gel and electroblotted onto a nitrocellulose
membrane. Protein expression was determined by immunoblotting with
appropriate antibodies. Briefly, the membrane was blocked in TBST [20
mM Tris-HCl (pH 7.5), 500 mM sodium chloride,
and 0.05% Tween 20] containing 5% nonfat dry milk at 37°C for
2 h and then incubated with primary antibody in TBST containing
1% BSA (fraction V) at room temperature for 2 h, followed by
incubation with secondary antibody conjugated with horseradish
peroxidase in TBST containing 5% nonfat dry milk. Visualization of the
protein was by the enhanced chemiluminescence detection system
(Amersham, Arlington Heights, IL).
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RESULTS |
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Val)
(34)
, was introduced into NCI-H82 cells by stable
transfection. Clonal populations were generated from individual
G418-resistant colonies. Northern blot analysis was used to determine
steady-state levels of Ras mRNA. Elevated p21-Ras protein
expression was observed in those clonal populations that have increased
steady-state levels of Ras mRNA (Fig. 1B
and data not shown). The Ras-expressing clonal cell lines
underwent profound morphological changes from floating aggregates of
loosely packed cells to adherent monolayer cells, whereas the
morphology of vector-transfected cells [Vector(Neo)], a pool of
G418-resistant colonies from the vector transfected NCI-H82 cells, was
indistinguishable from the parental cells (Fig. 1A
). The
morphological changes in Ras-expressing cells were accompanied by
marked alterations in growth parameters. Ras-expressing cells showed
greatly reduced serum dependence, proliferating well in medium
containing 0.5% calf serum (Fig. 1D
), and had an increased
colony-forming activity on soft-agar (Fig. 1C
). Consistent
with a previous report (9)
, these data indicate that
expression of an activated Ras oncogene in NCI-H82 cells
promotes transforming activities and induces changes in cell
morphology.
|
. Oncogenic Ras causes morphological transformation of
NCI-H82 cells. A, morphology of clones stably expressing
an activated c-Ha-Ras(V12) oncogene (x200).
B, Western blot analysis of p21-Ras protein expression.
Whole-cell lysate (50 µg) from representative cell clones was
separated on a 12% polyacrylamide-SDS gel, transferred to
nitrocellulose membranes, and immunoblotted with a monoclonal antibody
against rat Ha-Ras. C, anchorage-independent growth.
Cells were seeded in six-well plates in growth medium containing 0.3%
agarose over a base layer of 0.6% agarose. Colonies were scored 14
days after seeding. The efficiency of colony formation (%) represents
the ratio of the number of colonies formed to the total number of cells
seeded. D, growth curves in low serum (0.5% calf serum)
medium. Cells were plated at 1x105 cells/well in six-well
plates. Cell number was determined by trypan blue exclusion at day 3,
7, and 10. Data shown are the means of triplicate cultures from one
representative experiment; bars, SD. At least three
independent experiments were done with similar results. RasV12-9,
RasV12-11, and RasV12-27 are clonal populations of
c-Ha-Ras(V12)-transfectants of NCI-H82 cells. Vector
(Neo) is a pool of vector-transfected NCI-H82 cells.\.
three Ras-expressing clonal cell lines (RasV12-27, RasV12-11, and
RasV12-9) that express roughly equivalent levels of activated Ras (Fig. 1B
) were used in all studies. Raf-1 kinase activity was
assayed using the immunoprecipitated Raf-1 from whole-cell lysates to
measure its ability to phosphorylate the Raf-1 substrate, a kinase-dead
MEK-1, in the presence of [-32P]ATP
(35)
. Surprisingly, the Ras-expressing cells showed no
significant increase in the Raf-1 kinase activity (data not shown).
Moreover, ERK activity was only slightly elevated (1.5–2.2-fold)
relative to parental and vector-transfected control cells (Fig. 2A
), consistent with the lack of up-regulated Raf-1 kinase
activity in these cells. These data indicate a modest activation of the
Raf/MEK/ERK pathway by oncogenic Ras in NCI-H82 cells.
|
-32P]ATP. As shown in Fig. 2B,
JNK activity was robustly increased (26–33-fold induction) in response
to oncogenic Ras expression. All of three Ras-expressing clonal cell
lines display a very high level of sustained JNK activity.
JNK Activation Is Required for Oncogenic Ras Transformation of
NCI-H82 Cells.
The role of JNK signaling in Ras transformation of NCI-H82 cells was
explored further using a FLAG-epitope-tagged dominant-negative JNK1
mutant called JNK1(APF) (Ref. 17
). Transient expression of
this mutant has been shown to inhibit JNK-mediated transcriptional
regulation (17
, 36)
. To evaluate the long-term effect of
inhibition of JNK signaling, an expression plasmid for FLAG-JNK1(APF)
was introduced into Ras-expressing cells by stable transfection.
Multiple clones expressing different levels of FLAG-JNK1(APF) were
generated (Fig. 3A
). JNK1(APF)-expressing cell clones were morphologically
reverted, growing as floating aggregates of packed cells similar to the
parental NCI-H82 cells (compare Figs. 1A
and 3B
;
data not shown). Furthermore, JNK1(APF)-expressing cell clones showed a
complete loss of their ability to form colonies on soft agar (Fig. 3C
) and a marked reduction in their ability to grow in low
serum medium (Fig. 3D
). Expression of JNK1(APF) had little
effect on cell growth in complete medium (data not shown). Therefore,
the inhibitory effect of JNK1(APF) on Ras-induced changes in cell
growth properties appears to be specific.
|
). However, PD98059 inhibited the low-serum growth of
Ras-transformed cells (Fig. 4B
). The reduction in cell
growth in low serum medium was 60–80%, compared with cells treated
with the vehicle (0.1% DMSO). The inhibitory effect of PD98059 on
Ras-induced low-serum growth seems to be less specific, because PD98059
also causes a significant reduction (50–60%) in cell growth when they
were cultured in complete medium (Fig. 4B
).
|
AP-1-dependent luciferase activity was elevated
3–6-fold in the Ras-expressing cells, indicating a stimulation of
AP-1 activity by oncogenic Ras.
|
). Although no significant
changes in c-Fos protein levels were observed, the c-Jun level was
substantially increased in Ras-expressing cells. More importantly,
perhaps, the level of phosphorylated c-Jun on the serine 63 was
significantly increased in these cells. JNKs can phosphorylate c-Jun on
serine 63/73 to stimulate c-Jun transcriptional activity that may also
contribute to AP-1 induction (39
, 41)
. Thus, the
transcriptional activity of c-Jun was determined in a transient
transfection assay. In this assay, GAL4-dependent luciferase reporter
gene expression is mediated by the coexpression of either GAL4-c-Jun
1–233(1–233) (wild type) or GAL4-c-Jun (1–233; A63/73) (mutant). The
mutant GAL4-c-Jun fusion protein contains serine to alanine mutations
at residues 63 and 73 in the transactivation domain of c-Jun and thus
cannot be phosphorylated by JNK (29)
. Consistent with the
increased level of phosphorylated c-Jun on serine 63, transient
transfection results (Fig. 5C
) indicate that the c-Jun
transcriptional activity is elevated in the Ras-expressing cells
transfected with wild-type GAL4-c-Jun (3-fold induction over
Vector(Neo) cells). However, c-Jun transcriptional activity was not
increased when measured in cells transfected with the mutant GAL4-c-Jun
fusion protein (Fig. 5C
), suggesting that the increased
c-Jun transcriptional activity involves phosphorylation of c-Jun on
serine 63/73. Additionally, Northern blot and nuclear run-on
transcription analysis demonstrate that the steady-state level of
c-Jun mRNA as well as rates of c-Jun gene
transcription were increased in Ras-expressing cells (data not shown).
These results indicate that Ras activation of JNK results in c-Jun
phosphorylation in its NH2-terminal
transactivation domain, which stimulates its transcriptional activity
and leads to enhanced c-Jun gene expression. The data
suggest that the elevated c-Jun in association with constitutively
expressed c-Fos may contribute to the Ras-induced stimulation of AP-1
activity.
Expression of JNK1(APF) Leads to a Decrease in c-Jun/AP-1 Activity.
Because c-Jun is a downstream target of the JNK pathway and its
induction is correlated with JNK activation in Ras transformed NCI-H82
cells, the effect of JNK1(APF) on c-Jun expression was also examined.
c-Jun expression (Fig. 3A
) was lower in the
JNK1(APF)-expressing cell clones 1, 8, 11, and 22 than in the
vector-transfected and untransfected RasV12-11 cells. As expected,
c-Fos expression was not changed in these cells (data not shown). These
results suggest that c-Jun induction by Ras is mediated by JNK.
Genetic evidence demonstrates that AP-1 activity is crucial for Ras
transformation and that c-Jun expression is required for activation of
AP-1 by Ras (42)
. Because Ras transformation of NCI-H82
cells results in c-Jun induction and JNK1(APF) suppresses this effect,
it is predicted that JNK1(APF) may also suppress AP-1 induction by
oncogenic Ras. Consistent with this prediction, Fig. 6A
shows that AP-1-dependent luciferase activity in
JNK1(APF)-expressing cells is 70% lower in clones 1 and 8 and 36%
lower in clone 22 than that in the vector-transfected cells
(RasV12-11 + Vector). The degree of decreases in the AP-1
activity correlates well with the ability of these cells to grow in
low-serum medium (Figs. 3D
and 6A
). These results
suggest that c-Jun is the key component of the AP-1 complex, and its
induction can directly contribute to increased AP-1 transcriptional
activity.
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),
suggesting that the activation of ERKs is not required for the
induction of AP-1 activity. Consistent with this data, treatment of
Ras-transformed NCI-H82 cells with PD98059 (50
µM, up to 72 h) did not cause
down-regulation of c-Jun expression (Fig. 6C
). Thus, the JNK
pathway is the major Ras-dependent signaling pathway leading to
stimulation of c-Jun/AP-1 activity. |
DISCUSSION |
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The role for JNK in Ras transformation has been suggested by several studies. Expression of oncogenic Ha-Ras leads to phosphorylation of c-Jun on the same sites (serines 63 and 73) phosphorylated by JNK and a c-Jun transdominant inhibitor protein, c-Jun (S63A, S73A) in which two serine sites were mutated to alanines, blocks Ras transformation (43) . Expression of a dominant-negative mutant of JNK kinase, an upstream activator of JNKs, causes a significant inhibition of oncogenic Ras-induced focus-forming activity (25) . Studies have demonstrated that members of the Rho family proteins, Rac1/2 and Cdc42, selectively regulate the activity of the JNK pathway and play essential roles in mediating Ras transformation (29 , 44) . Furthermore, a role for JNK in cell transformation induced by other oncoproteins has also been suggested (19) . It has been shown that the JNK pathway is constitutively activated in cells transformed by Bcr-Abl, and expression of dominant negative c-Jun or JIP-1, a cytoplasmic inhibitor of JNK, markedly inhibits transformation of pre-B cells by Bcr-Abl (45 , 46) . Additionally, transformation of NIH 3T3 cells mediated by v-Crk is inhibited by dominant negative JNK kinase 1 (47) . These data suggest that JNK may be a universal mediator of cell transformation in response to different transforming agents. However, whether the JNK pathway contributes to oncogenic Ras transforming activity in human cancer cells is unclear. Our study provides mechanistic evidence that suggests a role for JNK in mediating oncogenic Ras action in human lung cancer cells. This is supported by recent studies from Bost et al. (48) , which demonstrate a significant growth-promoting role for JNK in human lung cancer cells.
In contrast to the JNK activation, overexpressing the activated
Ras oncogene does not efficiently activate the Raf/MEK/ERK
pathway in NCI-H82 cells. One explanation might be that the ERK
activity in the immortal cancer cells may be already elevated, and
therefore expression of an activated Ras has no additional effect.
Indeed, we observed a high basal level of ERK activity in serum-starved
NCI-H82 cells. Alternatively, the lack of ERK activation may be
attributable to down-regulation of ERK activity by modulating the
expression of dual specificity
phosphatase(s).4
The existence of negative regulatory mechanisms that repress ERK
activity has been suggested in fibroblast cell lines transformed by
different oncoproteins including oncogenic Ras (49)
.
Furthermore, the lack of Raf/ERK activation in Ras-transformed NCI-H82
cells suggests that effectors other than Raf may mediate the oncogenic
Ras function. The observation that inhibition of ERK activation via a
pharmacological approach using a MEK inhibitor PD98059 is unable to
fully reverse the Ras-induced transformed phenotype suggests that
constitutively active ERK is not essential for maintenance of the
Ras-induced transformed state in NCI-H82 cells. Supporting our
findings, recent works from Luo and Sharif (50)
and
Yip-Schneider et al. (51)
show that expression
of oncogenic Ki-Ras does not lead to constitutive ERK
activation in human astrocytoma cells and pancreatic carcinomas.
However, that the administration of PD98059 leads to inhibition of cell
growth under conditions of exponential growth or serum starvation (Fig. 4B
) suggests that ERK activity appears to be required for
normal proliferation of Ras-transformed NCI-H82 cells.
Our results reveal distinct differences in the signaling pathways that Ras uses to cause transformation of fibroblasts and the epithelial cell-derived cancer cells. In rodent fibroblasts, expression of an activated Ras oncogene has been reported to constitutively activate the Raf-dependent MAPK pathway. Consequently, inhibition of ERK activation by PD98059 reverses the Ras-transformed phenotype including morphological reversion (38) , suggesting a dominant role for the Raf/ERK pathway in mediating Ras function. However, Ras effector mutants with an impaired ability to bind Raf were still capable of causing transformation in fibroblasts (23) . When coexpressed with activated Raf-1, these mutants transform cells synergistically, suggesting that both Raf-dependent and Raf-independent signals are required for Ras transformation. There is increasing evidence that effector-mediated signaling pathways that contribute to oncogenic Ras function are regulated in a cell type-specific manner. For example, although oncogenic Ras and constitutively active Raf mutants each induced transformation of NIH 3T3 fibroblasts, activation of the Raf/MEK/ERK pathway alone was unable to cause potent morphological and growth transformation of RIE-1 rat intestinal epithelial cells (52) . Recent works have demonstrated that transformation of RIE-1 cells is Raf independent and appears to be mediated by an autocrine-dependent mechanism (53) . This indicates that Ras transformation associated with a certain MAPK module can be cell type specific, and cell type-specific factors, such as developmental background and cell cycle regulation, may be important determinants of the biological outcome of Ras signaling. However, it is worth noting that some common features in Ras transformation, such as up-regulation of AP-1 activity (see below), do exist in both fibroblasts and epithelial cells, suggesting that different MAPK module signals can converge.
Induction of c-Jun expression in Ras-transformed NCI-H82 cells seems to be mediated primarily by the JNK pathway. The mechanisms by which Ras-mediated JNK activation lead to c-Jun induction may include the transcriptional regulation of c-Jun gene expression as well as the regulation of c-Jun protein stability (19) . JNK-mediated c-Jun phosphorylation at serines 63 and 73 within its NH2-terminal transactivation domain can stimulate c-Jun transcriptional activity and increase the half-life of c-Jun protein by inhibiting ubiquitination, thereby leading to c-Jun induction. Our results demonstrate that the enhanced c-Jun transcriptional activity in Ras-expressing NCI-H82 cells is dependent on the phosphorylation status of c-Jun at serines 63/73. Furthermore, Ras-expressing cells show an increase in the steady-state level of c-Jun mRNA and an elevated c-Jun transcription rate, indicating that c-Jun induction is in part attributable to JNK-mediated transcriptional regulation of c-Jun gene expression. The elevated c-Jun gene expression may result partially from positive autoregulation of a TRE/AP-1 site in its promoter that is recognized by c-Jun-ATF2 heterodimers (39 , 54) . Similar to c-Jun, the transcriptional activity of ATF2 is stimulated by phosphorylation of sites in its transactivation domain by JNK (17) . A direct role for JNK in c-Jun induction is further supported by the fact that expression of JNK1(APF) decreases c-Jun protein expression. However, it is unknown whether Ras activation of JNK also increases the half-life of c-Jun protein (55) .
The correlation between JNK-mediated c-Jun/AP-1 activation and Ras transforming activity suggests that one mechanism underlying JNK-mediated Ras transformation is up-regulation of AP-1 activity. The constitutive activation of AP-1 transcription factors is thought to be a critical event in Ras-mediated transformation. Inhibition of AP-1 activity by dominant negative c-Jun mutants reverts the transformed phenotype of Ras-overexpressing NIH 3T3 fibroblasts (56 , 57) . Furthermore, c-Jun-/- cells cannot be transformed by activated Ras protein and are markedly impaired in their AP-1 transcriptional response (42) , suggesting that AP-1 complexes containing c-Jun are essential downstream effectors of Ras. Other studies show that c-Jun activity/expression is necessary for the initiation and/or maintenance of the Ras-transformed state (42 , 55 , 58) . The data presented here are consistent with these reports; Ras transforming efficiency correlates with the steady-state level of c-Jun protein, and stable expression of JNK1(APF) inhibits the effects of oncogenic Ras and results in decreased c-Jun/AP-1 activity, but the MEK inhibitor PD98059 does not. These results suggest that JNK activation is the major Ras-dependent signaling pathway leading to increased c-Jun/AP-1 activity.
The up-regulation of AP-1 activity appears to be a common feature in the in vitro transition from SCLC to NSCLC phenotype. A recent work from Risse-Hackl et al. (59) shows that the H-Ras/c-Myc-mediated transition of SCLC to NSCLC phenotype is accompanied by a strong induction of AP-1-binding activity. Interestingly, it was found that AP-1 is abundantly present in NSCLC cells but not in SCLC cells. Moreover, the induction of AP-1-binding activity in phenotypically converted SCLC cells is intimately linked to the up-regulation of AP-1 target genes, such as CD44, which is preferentially expressed in NSCLC-type tumor and cell lines (60) . These data suggest that AP-1 may be an important mediator during lung cancer development/progression. Therefore, by modulating AP-1 activity, and probably that of other transcription factors, the JNK pathway may mediate the long-term effects of Ras in oncogenic transformation and tumorigenesis. It should be noted that in contradiction to our results, a recent report (61) suggests that elevated JNK or AP-1 activities are not required to maintain the Ras-induced transformed state in NIH 3T3 fibroblasts. This result suggests that the dependence of Ras-induced transformation on the JNK/c-Jun/AP-1 pathway is cell specific.
In summary, the data presented here provide direct evidence that JNK plays an essential role in Ras transformation of NCI-H82 human lung carcinoma cells. Our results suggest that persistent JNK activation may promote proliferation and transformation of cancer cells. With respect to the cell type-specific responses to JNK activation (19) , it is likely that the effect of JNK signaling is cell context dependent and may be modified by the activation state of other cellular signaling pathways. Future studies in a range of carcinoma cells harboring Ras-activating mutations are required to further define the roles of the JNK and ERK pathways in mediating Ras actions.
ACKNOWLEDGMENTS
We are grateful to Dr. R. J. Davis for kindly providing
expression constructs for JNK1 and JNK1(APF) and to Dr. M. Karin for
providing expression constructs for GAL4-c-Jun and 5xGLA4-LUC. We also
thank Drs. W. S. May, M. P. Carroll, and B. Davis for critical
comments on the manuscript and Greg Tyler for technical expertise and
administrative assistance.
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FOOTNOTES |
|---|
1 Supported by the American Cancer Society
Institutional Research Grant and a start-up fund from the Sealy Center
for Oncology and Hematology, University of Texas Medical Branch at
Galveston, Texas. 
2 To whom requests for reprints should be
addressed, at Sealy Center for Oncology and Hematology, University of
Texas Medical Branch at Galveston, Galveston, Texas 77555-1048. Phone:
(409) 747-1939; Fax: (409) 747-1938; E-mail: lxiao{at}utmb.edu
3 The abbreviations used are: SCLC, small cell
lung cancer; NSCLC, non-SCLC; MAPK, mitogen-activated protein kinase;
JNK, c-Jun NH2-terminal kinase; ERK, extracellular
signal-regulated kinase; MEK, MAPK/ERK kinase; AP-1, activation
protein-1; GST, glutathione S-transferase; PD98059,
2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one; MBP, myelin basic
protein; ß-gal, ß-galactosidase.
Received 7/ 7/99. Accepted 11/10/99.
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