Conditional Apoptosis Induced by Oncogenic Ras in Thyroid Cells
Jill M. Shirokawa1,
Rosella Elisei1,
Jeffrey A. Knauf,
Takeshi Hara,
Jianwei Wang,
Harold I. Saavedra and
James A. Fagin
Department of Medicine (J.M.S., R.E., J.A.K., T.H., J.W.,
J.A.F.) Division of Endocrinology and Metabolism, and
Department of Cell Biology, Neurobiology and Anatomy (H.I.S.)
University of Cincinnati College of Medicine Cincinnati, Ohio
45267-0547
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ABSTRACT
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Mutations of ras are tumor-initiating
events for many cell types, including thyrocytes. To explore early
consequences after oncogenic Ras activation, we developed a
doxycycline-inducible expression system in rat thyroid PCCL3 cells.
Beginning 34 days after H-Rasv12 expression,
cells underwent apoptosis. The H-Rasv12 effects
on apoptosis were decreased by a mitogen-activated protein kinase
kinase (MEK1) inhibitor and recapitulated by
doxycycline-inducible expression of an activated MEK1 mutant
(MEK1S217E/S221E). As reported elsewhere, acute
expression of H-Rasv12 also induces mitotic
defects in PCCL3 cells through ERK (extracellular ligand-regulated
kinase) activation, suggesting that apoptosis may be secondary to DNA
damage. However, acute activation of SAPK/JNK (stress-activated protein
kinase/Jun N-terminal kinase) through acute expression of
Rac1v12 also triggered apoptosis, without
inducing large-scale genomic abnormalities.
H-Rasv12-induced apoptosis was dependent on
concomitant activation of cAMP by either TSH or forskolin, in a protein
kinase A-independent manner. Thus, coactivation of cAMP-dependent
pathways and ERK or JNK (either through
H-Rasv12, Rac1v12, or
MEK1S217E/S221E) is inconsistent with cell
survival. The fate of thyrocytes within the first cell cycles after
expression of oncogenic Ras is dependent on ambient TSH levels. If both
cAMP and Ras signaling are simultaneously activated, most cells will
die. Those that survive will eventually lose TSH responsiveness and/or
inactivate the apoptotic cascade through secondary events, thus
enabling clonal expansion.
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INTRODUCTION
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The ras protooncogenes encode 21-kDa G proteins, which
transduce signals from a wide variety of growth factor receptors,
particularly those of the tyrosine kinase family. Oncogenic activation
of ras is a consequence of gene mutations that either impair
GTPase activity or enhance GTP binding affinity and result in a highly
active signaling form of Ras. About 30% of all human tumors contain a
mutation in a ras allele, making this the most widely
mutated human protooncogene (1, 2). Mutations in ras are
thought to be one of the initiating molecular events in thyroid
tumorigenesis (3, 4).
Although much has been learned about the interaction of Ras with its
downstream signaling effectors, the precise mechanism by which
activated Ras initiates tumorigenesis is not known. There is
considerable data demonstrating that oncogenic Ras enhances cell
proliferation and inhibits apoptosis. Equally there is much evidence
that oncogenic Ras triggers an antitumorigenic apoptotic response.
Whether Ras will inhibit or trigger apoptosis is dependent on the cell
type, signaling pathways used, and the nature of concomitant regulatory
signals.
A key cellular response to Ras is transduced through the extracellular
ligand-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)
pathway, resulting from the sequential activation of Raf, MEK, and the
p42 and p44 MAP kinases. Depending on the cell type and other
conditions, the Raf/MAPK pathway influences apoptosis either positively
or negatively (5). Strong activation of the Raf/MAPK pathway has
pro-apoptotic effects (6, 7), whereas a lesser degree of MAPK
activation protects from neurotrophic factor withdrawal-induced death
in neuronal cells (8). Ras also activates other members of the MAPK
superfamily such as Jun N-terminal kinase (JNK) (9) and p38 MAPK (p38
MAPK) (10), both of which have been implicated in apoptosis signaling
(10, 11, 12, 13). In addition, Ras activates the phosphatidylinositol 3-kinase
(PI3K) pathway and initiates an antiapoptotic response via
activation of protein kinase B (PKB)/Akt (14, 15, 16). It appears
that PKB/Akt may protect from apoptosis in part through the
phosphorylation of the proapoptotic factor Bad, which results in its
binding to 143-3 as an inactive complex (17, 18, 19).
Most studies exploring the effects of oncogenic Ras on apoptosis
have been performed in cells in which growth is negatively regulated by
cAMP. Thyroid cells are dependent on the presence of TSH for growth,
primarily through activation of adenylyl cyclase, cAMP generation, and
stimulation of protein kinase A activity. Activation of the cAMP
signaling pathway, in turn, prevents recruitment of Raf to the plasma
membrane and results in markedly decreased ERK1/ERK2 activation. In
this paper, we investigate the early biological events that take place
after illegitimate activation of H-Rasv12 using
an inducible expression system in rat thyroid PCCL3 cells. These cells
are dependent on TSH for growth as well as expression of differentiated
gene products. In the absence of TSH, acute expression of
H-Rasv12 accelerates cell proliferation. However,
in the presence of TSH, H-Rasv12 induces a
transient increase in cell proliferation but later inhibits
TSH-mediated cell growth via initiation of programmed cell death. These
effects are mediated via the ERK and JNK signal transduction pathways
and are only observed with concomitant stimulation of the cAMP
signaling cascade. Furthermore, constitutive activation of either ERK
or JNK is sufficient to trigger apoptosis in thyroid cells. The
signaling conflict between ERK or JNK and cAMP likely operates in
vivo and may serve as a protective mechanism for tumor initiation
in the thyroid.
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RESULTS
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Inducible H-Rasv12 Expression in Rat
PCCL3 Thyroid Cells
To investigate the mechanisms of tumor initiation in thyroid
cells, we studied the role of H-Rasv12 using a
doxycycline-inducible expression system in rat thyroid PCCL3 cells to
permit analysis of early biological events after illegitimate
activation of this signaling protein. As shown in Fig. 1
, clones Ras 25 and Ras 39 demonstrate
dose-dependent H-Rasv12 expression after addition
of doxycycline. H-Rasv12 expression was
detectable by Western blotting several hours after induction and peaked
at 24 h (data not shown).

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Figure 1. Titration of Doxycycline-Inducible
H-Rasv12 Expression
Doxycycline (dox) was added to Ras 25 and Ras 39 cell lines in H6
complete medium at the indicated concentration, and the cells were
harvested after 48 h. Western analysis was performed utilizing
whole-cell extracts and anti-H-Ras IgG.
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H-Rasv12 Is Initially Mitogenic but Later
Induces Programmed Cell Death
In the absence of TSH, H-Rasv12 increases
cell growth (Fig. 2A
), which is in
agreement with previous studies in rat FRTL5 thyroid cells
stably overexpressing H-Rasv12 or K-Ras (20).
Acute activation of H-Rasv12 is transiently
mitogenic in the presence of TSH, namely during the first couple of
cell cycles (24 and 48 h) after induction of oncogene expression;
however, by 96 h the number of cells abruptly decreases (Fig. 2B
).
Addition of doxycycline to the rtTACL3 control cell line does not alter
cell growth (Fig. 2
, A and B).

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Figure 2. H-Rasv12 Is Initially Mitogenic but
Later Induces Cell Death in Rat Thyroid PCCL3 Cells
Ras 25 and rtTACL3 cells were incubated with or without doxycycline (1
µg/ml) either in the absence of TSH (H5 complete medium) (panel A),
or in the presence of TSH (H6 complete medium) (panel B) for the
indicated time, and cells remaining on the dish were counted
electronically. Bars represent the mean ±
SEM of a single experiment performed in duplicate. Similar
results were obtained in a second exper-iment.
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By 36 days after the induction of
H-Rasv12 expression in the presence of TSH, the
Ras 25 and Ras 39 cells began detaching (Fig. 3A
). These cells were found to be
apoptotic as determined by DNA laddering (Fig. 3B
).
H-Rasv12-induced apoptosis was further verified
by measuring cells with DNA strand breaks via the APO-BrdU assay (Table 1
). The rate of apoptosis in response to
acute expression of H-Rasv12 was concentration
dependent (Fig. 3C
).

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Figure 3. H-Rasv12 Induces Apoptosis in Rat
Thyroid PCCL3
A, Ras 25 and rtTACL3 cells were incubated with or without doxycycline
(1 µg/ml) in H6 complete medium for the indicated time. The number of
detached cells were collected and counted. The number of attached cells
were collected after trypsin treatment, stained with trypan blue, and
counted with a hemacytometer to determine the number of viable and dead
cells. The percentage of dead cells was determined by combining the
number of cells in the detached fraction and the number of trypan
blue-stained cells in the attached fraction. Bars
represent the mean ± SEM of a single experiment
performed in triplicate. Similar results were obtained in two
additional experiments. B, Agarose gel analysis of DNA fragmentation of
Ras 39 and rtTACL3 cells incubated with or without doxycycline (1
µg/ml) for 4 days in H6 complete medium. C, Doxycycline was added to
Ras 25, Ras 39, and rtTACL3 cell lines in H6 complete medium at the
indicated concentration, and the percentage of dead cells was
determined after 6 days. Bars represent the mean ±
SEM of a single experiment performed in triplicate. Similar
results were obtained in a second experiment.
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H-Rasv12 Activates Multiple MAP Kinase
Superfamily Pathways in PCCL3 Cells
As shown in Fig. 4A
, the
classical MAPK (ERK1 and ERK2) pathway is strongly activated after
induction of H-Rasv12 expression. Activation of
the ERK pathway was further characterized with a reporter assay in
which Ras 25 cells were transiently cotransfected with a luciferase
reporter plasmid and an expression vector containing the yeast Gal4 DNA
binding domain (DBD) (residues 1147) fused to the activation domain
of human Elk-1 (residues 307428), the transcription factor target of
ERK. H-Rasv12 expression caused an approximately
10-fold increase in Elk-1 activation, as compared with a 20-fold
increase induced by expression of activated mitogen-activated protein
kinase kinase (MEK1), whereas the negative control Gal4 (DBD) was
without effect (Fig. 4D
).

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Figure 4. Inducible H-Rasv12 Activates the ERK,
SAPK/JNK, and p38 MAPK Signaling Cascades
Ras 25 and control cell lines rtTACL3 and rtTA7 were incubated in 0.5%
FBS/Coons media for 2 days and then treated with or without doxycycline
(1 µg/ml) in H6 complete medium for 48 h. Western analysis was
performed utilizing whole-cell lysates with the following antibodies:
panel A, anti-phospho-ERK IgG; anti-ERK IgG; panel B, anti-phospho-JNK
IgG; anti-JNK IgG; panel C, anti-phospho-p38MAPK IgG; anti-p38MAPK IgG.
Panel D, A luciferase reporter plasmid was cotransfected into Ras 25
cells with the expression vectors for Gal4-Elk1 alone,
Gal4-Elk1 and MEK1, or the negative control plasmid [Gal4(1147)].
Where indicated, cells were treated with doxycycline for 48 h.
Cell lysates were prepared 48 h after transfection, and luciferase
assays were performed. Bars represent the mean ±
SEM of a single experiment performed in triplicate. Similar
results were obtained in two additional experiments. Panel E, The
luciferase reporter plasmid was cotransfected into Ras 25 cells with
expression vectors for the Gal4-c-Jun alone, Gal4-c-Jun and MEKK, or
Gal4 (1147). Cell lysates were prepared after 48 h and
luciferase assays were performed. Bars represent the
mean ± SEM of a single experiment performed in
triplicate. Similar results were obtained in two additional
experiments.
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H-Rasv12 expression also stimulates the stress-
activated protein kinase (SAPK/JNK) and the p38 MAP kinase
cascades, but to a lesser extent than ERK (Fig. 4
, B and C). Activation
of SAPK/JNK was further quantitated with a reporter assay in Ras 25
cells cotransfected with a luciferase reporter plasmid and an
expression vector containing the Gal4 DBD fused to the activation
domain of human c-Jun (residues 1223). Induction of
H-Rasv12 expression caused an approximately
3-fold increase in c-Jun activation, as compared with a 10-fold
increase induced by expression of activated MEK kinase (MEKK)
(Fig. 4E
).
H-Rasv12 Induces Apoptosis via the ERK and
SAPK/JNK Pathways
Treatment of cells with the MEK1-specific inhibitor PD98059
partially blocks H-Rasv12-induced apoptosis. In
these experiments, PD98059, used at the minimally effective
concentration verified to block kinase activation (Fig. 5B
), inhibited doxycycline-induced
apoptosis by about 50% in Ras 39 cells (Fig. 5A
). As a further
verification of the involvement of the MAP kinase (ERK) pathway in
H-Rasv12-induced apoptosis, we generated
doxycycline-inducible activated MEK1
(MEK1S217E/S221E) cell lines (21). Two
independent stable cell lines, MEK155 and MEK165, express the
MEK1S217E/S221E mutant protein in a doxycycline-
dependent manner (Fig. 6A
). After
treatment with doxycycline, both clones activate the ERK pathway as
analyzed through Western blotting utilizing phospho-antibodies for
ERK1/ERK2 (Fig. 6A
) and Elk-1 reporter assays (
5-fold, Fig. 6B
). Six
days after addition of doxycycline, both MEK155 and MEK165 cells
become apoptotic as measured through cell detachment, DNA fragmentation
(Fig. 6
, C and D), and APO-BrdU assays (Table 1
).

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Figure 5. The MEK1 Inhibitor PD98059 Blocks
H-Rasv12-Induced Apoptosis
A, Ras 39 and control rtTACL3 cells were treated with or without
doxycycline in the presence or absence of 35 µM PD98059
for 3 days in H6 complete medium (PD98059-treated cells were pretreated
for 1 h before the addition of doxycycline). Percent dead cells
were determined as previously described. Bars represent
the mean ± SEM of a single experiment performed in
triplicate. Similar results were obtained in a second experiment. B,
Ras 39 cells were treated with PD98059 as described in panel A, and
whole-cell lysates were subject to Western analysis utilizing
anti-phospho-ERK IgG and anti-ERK IgG.
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Figure 6. An Activated Mutant of MEK1 Induces Apoptosis
A, Inducible expression of His-tagged MEK1S217E/S221E was
visualized by Western blotting of whole cell lysates from rtTACL3,
MEK155, and MEK165 cell lines with anti-His IgG. The same lysates
were analyzed for phospho-ERK expression using anti-phospho-ERK IgG. B,
The luciferase reporter plasmid was co-transfected into MEK155 cells
with expression vectors for either Gal4-Elk1 alone, Gal4-Elk1 and MEK1,
or Gal4 (1147). Cell lysates were prepared after 48 h and
luciferase assays were performed. Bars represent the
mean ± SEM of a single experiment performed in
triplicate. Similar results were obtained in a second experiment. C,
Percent dead rtTACL3, MEK155, and MEK165 cells were determined as
previously described after 6 days of incubation with or without
doxycycline in H6 complete medium. Bars represent the
mean ± SEM of a single experiment performed in
triplicate. Similar results were obtained in a second experiment. D,
Agarose gel analysis of DNA fragmentation of rtTACL3, MEK155, and
MEK165 cell lines incubated with or without doxycycline for 6 days in
H6 complete medium.
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To determine whether other members of the MAP kinase family, such as
SAPK/JNK and p38MAP kinase, also participate in apoptosis signaling in
thyroid cells, we generated stable cell lines with inducible expression
of an activated Rac1 (Rac1v12) mutant protein.
The small GTP-binding protein Rac1 is known to regulate activity of
SAPK/JNK/p38MAPK pathways through MEKK and stress-activated protein
kinase/ERK kinase (SEK) (22). The Rac188 cell line inducibly
expresses the Rac1v12 protein and was used for
functional assays. Doxycycline-induced expression of
Rac1v12 was associated with activation of SEK1
and SAPK/JNK in the Rac188 cell line as demonstrated by Western
immunoblotting with phospho-antibodies for SEK1 and JNK1, with only
marginal changes in p38MAPK phosphorylation as demonstrated by Western
blotting (Fig. 7A
).

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Figure 7. Acute Expression of Rac1v12 Induces
Apoptosis
A, Rac188 and rtTA7 cells were treated with or without
doxycycline in H6 complete media for 48 h. Western blotting of
whole-cell lysates from rtTA7 and Rac188 cells was performed with the
following antibodies: anti-c-myc IgG (recognizes a
c-myc epitope tag of Rac1v12),
anti-phospho-SEK1 IgG, anti-SEK1 IgG, anti-phospho-JNK IgG, anti-JNK
IgG, anti-phospho-p38MAPK IgG, and anti-p38MAPK IgG. B, rtTA7 and
Rac188 cell lines were treated with or without doxycycline for 6 days
in H6 complete medium. MTT assays were used to quantitate cell
survival. Bars represent the mean ±
SEM of a single experiment performed in triplicate. Similar
results were obtained in two additional experiments and additionally
replicated in a second Rac1-inducible cell line.
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Acute activation of Rac1v12 induces apoptosis in
thyroid cells (Fig. 7B
) as displayed by the decrease in cell viability
upon addition of doxycycline as measured by the
3-(4,5-dimethylthiazol)-2-yl)-2,5-diphenyl tetrazolium bromide assay
(MTT). In addition, the percentage of APO-BrdU-positive Rac188
cells increased from approximately 11% in the absence of doxycycline
to approximately 80% in its presence (Table 1
).
In contrast to the MEK1 inhibitor, the p38MAPK specific inhibitor
SB203580 did not block H-Rasv12-induced apoptosis
(Fig. 8A
) when used at a concentration
(30 µM) that decreased
H-Rasv12-induced p38MAP kinase activation to
basal levels (Fig. 8B
). These results suggest that the SAPK/JNK
pathway, but not the p38MAPK pathway, mediates
H-Rasv12-induced apoptosis in PCCL3 thyroid
cells.

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Figure 8. The p38MAPK Inhibitor SB203580 Does Not Block
H-Rasv12-Induced Apoptosis
A, Ras 25 and control rtTA7 cell lines were treated with 30
µM SB203580 and incubated either with or without
doxycycline for 6 days. MTT assays were used to quantitate cell
survival. Bars represent the mean ±
SEM of a single experiment performed in triplicate. Similar
results were obtained in two additional experiments. B, Western blot of
Ras 25 cells treated as indicated above with anti-phospho-p38MAPK IgG
and anti-p38MAPK IgG.
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H-Rasv12,
MEK1S217E/S221E, and
Rac1v12-Induced Cell Death Does Not Occur via
Anoikis
Approximately 42% of H-Rasv12-expressing
cells, 50% of MEK1S217E/S221E-expressing cells,
and 80% of Rac1v12-expressing cells that
remained attached to the tissue culture plates were APO-BrdU positive
(Table 1
). This indicates that the apoptotic program in the
H-Rasv12-,
MEK1S217E/S221E-, and
Rac1v12-expressing cells is initiated before cell
detachment and, hence, that cell death does not occur by anoikis.
H-Rasv12-,
MEK1S217E/S221E-, and
Rac1v12-Induced Apoptosis Is Dependent on
TSH
TSH is required for thyroid cell growth and differentiated
gene expression. Ligand-dependent activation of the TSH receptor
results in increased levels of cellular cAMP through stimulation of
adenylyl cyclase, leading to activation of cAMP-dependent protein
kinase A (PKA), as well as other cAMP-dependent cellular intermediates.
H-Rasv12-induced apoptosis is dependent on TSH.
As shown in Fig. 9A
, Ras 25 cells in the
absence of TSH did not display inducible cell death whereas treatment
of Ras 25 cells with doxycycline in the presence of TSH evoked a 3-fold
to 4-fold increase in cell death. Similarly, activation of
H-Rasv12 expression in the presence of forskolin
induced apoptosis (Fig. 9B
). Treatment of Ras 39 and the control rtTA7
cell lines with the PKA inhibitor H89 (10 µM) did not
block H-Rasv12-induced apoptosis (Fig. 9C
). H89
was used at the minimal concentration verified to decrease cAMP
response element-binding protein (CREB) phosphorylation to near basal
levels (Fig. 9D
). Further evidence of the effectiveness of H89 at this
concentration is displayed by the growth inhibition of both the rtTA7
and Ras 39 cells lines, which are dependent on TSH and PKA activation
for growth (Fig. 9C
). H89 had no effect on survival of rTTA7 cells in
the absence of TSH, indicating that the effects of H89 were not due to
toxicity. In addition, treatment of the Ras 39 cell line with the PKA
inhibitor KT5720 (1.5 µM) did not block
H-Rasv12-induced apoptosis (data not shown).

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Figure 9. H-Rasv12-Induced Apoptosis Is Dependent
on Concomitant Activation of cAMP by TSH or Forskolin
A, The percentage of dead Ras 25 and rtTACL3 cells after incubation
with or without doxycycline for 4 days in the presence or absence of
TSH in complete medium was determined as previously described.
Bars represent the mean ± SEM of a
single experiment performed in triplicate. Similar results were
obtained in two additional experiments. B, Percentage of dead Ras 39
and rtTACL3 cells after induction with or without doxycycline in the
presence or absence of TSH or forskolin (20 µM) in
complete medium for 3 days. Bars represent the mean
± SEM of a single experiment performed in triplicate.
Similar results were obtained in a second experiment. C, Ras 39 and
rtTA7 cell lines were treated with or without doxycycline (1 µg/ml)
in the presence or absence of 10 µM H89. Both media and
the respective inhibitors were replenished daily. MTT assays were
performed to determine cell viability. Bars represent
the mean ± SEM of a single experiment performed in
triplicate. Similar results were obtained in two additional
experiments. D, Ras 39 cells were treated with the indicated
concentrations of H89 in the presence or absence of TSH in complete
medium. Western analysis was performed utilizing whole-cell extracts
and phospho-CREB IgG.
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As demonstrated after acute activation of
H-Rasv12, MEK1S217E/S221E
mutant-expressing cell lines become apoptotic in the presence, but not
in the absence, of TSH or forskolin (Fig. 10
, A, B, and D). In the absence of
TSH, MEK1S217E/S221E increases mitogenesis, but
in the presence of either TSH or forskolin apoptosis is induced (Fig. 10
, A, B, and D). Similarly, Rac1v12-induced
apoptosis is TSH dependent, as displayed by the decrease in viability
measured by MTT assays (Fig. 10C).

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Figure 10. MEK1S217E/S221E and
Rac1v12-Induced Apoptosis Is Dependent on TSH
rtTA7 and MEK155 (panel A) or MEK165 (panel B) cells were grown in
the presence or absence of TSH and were treated with or without
doxycycline for 6 days in complete medium. MTT assays were used to
quantitate cell survival. C, Rac188 and rtTA7 cells were grown in the
presence or absence of TSH and treated with or without doxycycline for
6 days in complete medium. D, rtTA7, MEK155, and MEK165 cell lines
were treated with or without doxycycline for 6 days in the presence or
absence of forskolin (20 µM). Bars
represent the mean ± SEM of a single experiment
performed in triplicate. Similar results were obtained in a second
experiment.
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Although H-Rasv12-induced apoptosis is dependent
on TSH, H-Rasv12 activates the ERK, SAPK/JNK, and
PI3 kinase signaling cascades with a similar magnitude in the presence
or absence of TSH (Fig. 11
, AC). TSH
did not modulate the ability of doxycycline to induce
H-Rasv12 in these cell lines (not shown).

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Figure 11. Inducible H-Rasv12 Activates the ERK,
JNK, and PI3 Kinase Signaling Pathways in the Presence and Absence of
TSH
Ras 25 and the control rtTA7 cell lines were treated with or without
doxycycline for 24 h in the presence or absence of TSH in complete
medium. Western analysis was performed utilizing whole-cell lysates
with the following antibodies: panel A, anti-phospho ERK IgG; anti ERK
IgG; panel B, anti-phospho JNK IgG; anti-JNK IgG; panel C, anti-Akt
IgG.
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DISCUSSION
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Activating mutations of Ras are involved in the pathogenesis of
about 30% of human cancers. Unregulated Ras activity leads to cell
proliferation in most cell types, but its effects on apoptosis vary
considerably between cells of different lineage (5). A simplistic
prediction would be that Ras would inhibit apoptosis as part of its
role in promoting expansion of the neoplastic clone. This is often the
case in epithelial (14, 23) and myeloid cells (24), whereas in T
lymphocytes and fibroblasts, constitutive Ras activity can induce cell
death (25). Ras has the potential to interact with multiple signaling
systems, many of which have effects on apoptosis. Cell-specific
differences in the action of Ras have been attributed to preferential
signaling through specific downstream intermediates. Prominent
among them is the Raf-MAP kinase pathway, which has been found to
stimulate apoptosis in several cell types, particularly when the signal
intensity is high and its duration prolonged (26). Activated Ras can
also interact with JNK, and with Jun itself (27). This may occur
through activation of Rac (22, 28), MEKK1 (29), or through a direct
interaction between Ras and JNK (27). The role of JNK in cell death is
less clear, although this intermediate has been implicated in apoptotic
pathways triggered by microtubule disruption and radiation-induced
apoptosis (30). Disruption of microtubules also results in activation
of JNK through Ras and the apoptosis signal-regulating kinase (ASK1)
(31). Conversely, Ras-mediated activation of PI3 kinase appears to
provide protection from cell death in most cell types.
Here we report that acute activation of Ras in a clonal rat thyroid
cell line triggers a massive wave of apoptosis. The ability to control
the temporal expression of Ras in PCCL3 cells has allowed us to examine
the sequence of events after oncogenic activation and to recapitulate
cellular changes that may ensue after thyroid tumor initiation in
vivo. Expression of H-Rasv12 initially
stimulates cell proliferation, but by 46 days the majority of cells
are dead. Although thyroid cells detach as they die, the effects of Ras
on apoptosis cannot be attributed to anoikis, as Ras expression evokes
a proportional increase in the number of TUNEL-positive cells that
remain attached to the dish. We report separately that during the 46
day lag period before the onset of apoptosis, cells display evidence of
DNA damage, manifesting as chromosome misalignment in mitosis,
micronuclei formation, and centrosome amplification (32). The
triggering events resulting in apoptosis may thus in part emanate from
a response to DNA damage. However, as will be discussed below, the
signaling effectors associated with genomic destabilization differ in
part from those that induce apoptosis, indicating that DNA damage is
not solely responsible for activating the death program after acute Ras
expression. Apoptosis induced by H-Rasv12 was
apparent even at concentrations of doxycycline that stimulated
oncoprotein levels that were just above the detection limit by Western
blotting. This argues that the cellular changes resulting from acute
activation of H-Rasv12 are likely to be due to
persistence of the signal rather than nonspecific responses secondary
to its overexpression.
H-Rasv12 stimulates the MAPK-ERK, JNK, and
p38MAPK pathways in PCCL3 cells, although to a different extent.
Activation of MAP kinase is particularly prominent, and induced by
about 10-fold by H-Rasv12. Several lines of
evidence indicate that constitutive MAP kinase signaling is sufficient
to induce cell death in cells grown in the presence of TSH.
Pretreatment of Ras 25 or Ras 39 cells with MAP kinase inhibitors at
the minimal concentration needed to block
H-Rasv12-induced ERK phosphorylation markedly
inhibited apoptosis. Furthermore, acute expression of a constitutively
active mutant MEK1 also induced cell death. The mutant of MEK1 used in
these experiments was derived by Ser/Glu substitutions at positions 217
and 221, thus replacing the residues phosphorylated by Raf-1 and
rendering the mutant constitutively active and not subject to
additional stimulation by exogenous growth factors (33). Constitutive
activity of MEK1S217E/S221E generates about 0.5%
of the maximal activity obtained by treating wild-type MEK1 with Raf-1
kinase (21). Thus, signaling by this mutant, even when overexpressed,
is unlikely to result in supramaximal activation of MAP kinase. More
likely, the cellular effects are due to persistent, sustained
stimulation of this enzymatic cascade, which normally is seen only
transiently after ligand-induced activation.
Apoptosis induced by acute expression of H-Rasv12
or MEK1S217E/S221E was only apparent when cells
were grown in the continued presence of TSH. Signaling cross-talk
between TSH and Ras-activated effectors has received considerable
attention, as these two pathways are implicated in thyroid cell growth
(34, 35, 36), tumorigenesis, and differentiated gene expression (20). The
action of TSH on Ras-induced apoptosis may be related to interference
by cAMP-dependent effectors (i.e. PKA, Rap1, or others) on
the distribution of the Ras signal to its various downstream
intermediates (35). cAMP inhibits Ras signaling through Raf and MAPK in
many cell types. Indeed, PKA phosphorylates Raf at several residues and
decreases its affinity for Ras, resulting in inhibition of Raf kinase
activity (37). However, in Wistar rat thyroid cells, this appears to be
a transient effect, as treatment with TSH only inhibits the
constitutive activation of MAP kinase by H-Rasv12
for at most 10 min after addition of the growth factor, or of 8-Br-cAMP
(35). Furthermore, we observed that acute expression of
H-Rasv12 resulted in similar levels of MAP kinase
activation in PCCL3 cells regardless of whether they were grown in the
continued presence or absence of TSH. This again may be due to the fact
that inhibitory effects of PKA or other cAMP-dependent effectors on the
MAP kinase pathway are short lived when the latter is constitutively
activated. Nevertheless, simultaneous activation of these two pathways
is likely inconsistent with orderly cell cycle progression. A recent
report by Miller et al. (35) supports these findings, as
colonies formed by Wistar rat thyroid cells transfected with a
RasS35 mutant (that signals preferentially
through Raf and MAPK) were larger in the absence of TSH than in its
presence. TSH signaling through cAMP is likely to be critical for
Ras-induced apoptosis, as treatment of cells with forskolin or
8-Br-cAMP evokes similar effects. This was not abrogated by
pretreatment with PKA antagonists, suggesting that other cAMP-dependent
effectors are involved. In addition, the TSH receptor also couples to
protein kinase C (PKC) isozymes through PLC
. Susceptibility of
fibroblasts (25) and thyrocytes (38) to Ras-induced apoptosis is
enhanced after down-regulation of PKC activity. Similarly, treatment of
Ras 25 cells with phorbol esters before induction of
H-Rasv12 expression is associated with cell
death even in the absence of TSH (data not shown). However,
pretreatment of Ras 25 or Ras 39 cells with PKC inhibitors in the
presence or absence of TSH failed to prevent Ras-induced cell
death.
Ras-induced apoptosis may be triggered, at least in part, as a result
of DNA damage associated with disruption of critical cell cycle
checkpoints. In a separate report, we show that doxycycline-initiated
H-Rasv12 or MEK1S217E/S221E
expression induces large-scale chromosomal abnormalities within the
first cell cycle after activation (32). This is associated with
accelerated passage through the G2-M phase of the cell cycle and,
later, stabilization of p53 and activation of a p53-dependent apoptotic
cascade. Although these mechanisms are likely significant, they do not
fully explain the observed changes after Ras activation for the
following reasons. 1) Acute effects of H-rasv12
or uMEK1Glu217/221 on micronuclei formation
and chromosome misalignment do not require concomitant activation by
TSH, indicating that the degree of DNA damage induced by
unregulated signaling through these effectors is not sufficient to
evoke an apoptotic response of this magnitude; 2) activation of p38MAP
kinase and JNK (i.e. through acute expression of
Rac1v12) does not induce genomic instability but
triggers an apoptotic response in a TSH-dependent manner. We cannot
exclude, however, that after initiation of oncogenic Ras expression the
JNK signaling cascade is further activated or sustained as a
consequence of a DNA damage response; 3) genomic instability induced by
acute H-Rasv12 or
MEK1S217E/S221E expression is detected in about
10% of the cell population after 2 days, whereas almost all cells
undergo apoptosis after a 4- to 5-day period. Taken together, these
data indicate that although DNA damage may contribute to generate
apoptotic signals after Ras activation, it is not sufficient to explain
the phenomenon in its entirety.
Thyrocytes represent an exception among cells of epithelial
lineage in their response to oncogenic Ras activation. Among tumors of
endocrine cells whose growth is positively regulated by cAMP
(i.e. pituitary somatotrophs, gonadotrophs, ovarian
granulosa, or adrenocortical cells), thyroid neoplasms are the only
ones in which mutations of Ras are prevalent. This may be
explained by the fact that in pituitary or adrenal cells expression of
a mutant Ras may result in rapid deletion of the cell from the
population through apoptosis. As opposed to other G protein-coupled
receptors, the unliganded TSH receptor is silent in follicular cells
(39), which may allow survival of a cell in the first cell cycles after
acquiring a Ras mutation if ambient TSH levels are relatively low.
Subsequently, the genomic instability evoked by Ras may allow some
cells with secondary genetic defects to develop a resistance to the
apoptotic stimulus and thus to initiate the tumor clone.
 |
MATERIALS AND METHODS
|
---|
Materials
The following antibodies used for Western blotting were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA): H-Ras rabbit polyclonal (sc-520); MEK-1 rabbit polyclonal
(sc-219); JNK1 rabbit polyclonal (sc-571); phospho-JNK mouse monoclonal
(sc-6254); p38MAPK rabbit polyclonal (sc-535); phospho-p38MAPK mouse
monoclonal (sc-7973); phospho-ERK mouse monoclonal (sc-7383); ERK1
rabbit polyclonal (sc-94); MEK4(SEK1) rabbit polyclonal (sc-837);
His-probe rabbit polyclonal (sc-803); antirabbit-horseradish peroxidase
(HRP) and antimouse-HRP. Phospho-SEK rabbit polyclonal, Akt rabbit
polyclonal, and phospho-CREB rabbit polyclonal antibodies were
purchased from New England Biolabs, Inc. (Beverly, MA).
The c-myc mouse monoclonal antibody was purchased from
Invitrogen (Carlsbad, CA). The inhibitors PD98059,
SB203580, H89, and KT5720 were purchased from Calbiochem
(San Diego, CA). TSH, forskolin, insulin, apo-transferrin,
hydrocortisone,
glycyl-L-histidyl-L-lysine-acetate,
and MTT were purchased from Sigma (St Louis, MO). Coons
medium/F12 high zinc was purchased from Irvine Scientific (Irvine, CA).
FBS and penicillin-streptomycin-L-glutamine were
purchased from Life Technologies, Inc. (Gaithersburg,
MD).
Cell Culture
All PCCL3 cell lines were maintained in H6 complete medium
consisting of Coons medium/F12 high zinc supplemented with 5% FBS,
0.3 mg/ml L-glutamine, 10 mIU/ml TSH, 10 µg/ml insulin, 5
µg/ml apo-transferrin, and 10 nM hydrocortisone, 10 ng/ml
somatostatin, 10 ng/ml
glycyl-L-histidyl-L-lysine-acetate, and
penicillin/streptomycin. H5 complete medium was identical to H6
complete medium but without addition of TSH.
Cell Transfections
The expression system we used was developed by Bujard and
co-workers (40, 41) to deliver doxycycline-inducible expression based
on the high specificity of the Escherichia coli tet
repressor-operator-doxycycline interaction. Stable transfections were
performed first to establish clonal lines constitutively expressing the
transactivator rtTA (composed of a fusion of the rtetR DBD and the VP16
activation domain). Individual rtTA-expressing clones were then
explored for doxycycline-inducible expression by transient transfection
with a luciferase reporter construct under control of a tet-operator.
Clones of rtTA demonstrating very low or undetectable basal luciferase
activity and marked induction (i.e. >100 fold) by
doxycycline were selected as hosts for secondary stable transfection
with constructs consisting of a minimal cytomegalovirus (CMV) promoter
containing tet-operator sequences cloned upstream of the signaling
protein cDNA to be expressed.
The H-Rasv12 and the
Rac1v12-myc-tagged cDNAs were a generous gift
from Dr. Kenji Fukasawa. The MEK1S217E/S221E cDNA
was kindly provided by Dr. Christopher Marshall. The
constructs were subcloned into the pUHG 103 vector
downstream of the heptamerized tetO and a minimal CMV promoter. The
pUHG 103 vector was developed by Bujard and co-workers as previously
described (40, 41). The constructs were then cotransfected with
Tkhygro, which contains the hygromycin gene under control of a minimal
thymidine kinase (TK) promoter, into the rtTA7 cell line. This cell
line was created by stably transfecting well differentiated rat thyroid
cell line, PCCL3, with pUHD1721neo, which directs high level
expression of rtTA. The rTTACl3 cell line was created by cotransfecting
rtTA7 cells with the pUHG 103 vector and Tkhyrdro and was used as a
control.
Transient Transfections and Luciferase Assay
Transient transfections were performed as specified by the
manufacturer of the Pathdetect signaling assay
(Stratagene, La Jolla, CA). Briefly, 2 µg of the
luciferase reporter plasmid (pFRLuc) were cotransfected using the
Superfect Reagent (QIAGEN, Valencia, CA) into 2.5 x
105 Ras 25 cells with 100 ng of the appropriate
expression plasmids (pFA-Elk1, pFA-cJun, pFC-MEK1, pFC-MEKK, pFCdbd).
After the transfection, doxycycline was added to the appropriate
samples at 1 µg/ml. Cell lysates were prepared after 48 h, and
luciferase assays were performed according to the manufacturers
procedure (Stratagene).
Western Blotting
Western blots of total cell lysates treated with and without
doxycycline were performed as previously described (42) and probed with
the indicated antibody. Preparation of total cell lysates from PCCL3
cells has been described previously (42).
Cell Detachment Assays
Cells were plated in H6 complete media at 200,000 cells per well
(six-well plate) and allowed to recover for 23 days at 37 C. After 2
days, doxycycline was added at 1 µg/ml, and the cells were incubated
at 37 C for the indicated time (36 days). Detached cells were
harvested by removal of the media supernatant from the plates followed
by centrifugation. Supernatants were aspirated away, and the detached
cell pellet was resuspended in PBS and counted utilizing a
hemacytometer. Attached cells were washed with PBS, removed from the
plates by trypsin treatment, recovered by centrifugation, and
resuspended in PBS. The attached cell fraction was stained with Trypan
Blue, and the number of alive and dead cells were counted utilizing a
hemacytometer. Percent dead cells were calculated by combining the
number of cells in the detached fraction and the number of trypan
blue-stained cells in the attached fraction.
MTT Assays
The viability of cells treated with and without doxycycline was
analyzed by the MTT assay as previously described (42).
DNA Fragmentation Analysis
Cells were harvested by spinning at 1,000 x g
for 10 min. The pellet was resuspended in a solution containing 10
mM Tris, pH 8, 10 mM EDTA,
and 150 mM NaCl. Cells were then incubated with
0.1 mg/ml proteinase K and 1% SDS at 50 C overnight. The samples were
extracted in phenol-chloroform-isoamyl alcohol and ethanol
precipitated, and the pellets were washed with 70% ethanol and dried.
The pellets were resuspended in Tris-EDTA (TE), pH 7.5. The DNA
was electrophoresed in 1% agarose gels and ethidium bromide
stained.
APO-BrdU Assays
APO-BrdU assays were performed following the manufacturers
procedure (PharMingen, San Diego, CA). Briefly, cells were
treated in 1% paraformaldehyde followed by fixation in 70% ethanol
and stored at -20 C. For the DNA labeling procedure, the cells were
washed and resuspended in the DNA labeling solution containing TdT
enzyme and Br-dUTP in a reaction buffer and incubated at 37 C for 60
min. After the labeling reaction, the cells were washed and stained
with fluorescein-labeled anti-BrdU antibody for 30 min and treated with
propidium iodide and RNAse A. APO-BrdU positive cells were analyzed by
flow cytometry.
 |
FOOTNOTES
|
---|
Address requests for reprints to: James A. Fagin, M.D., Division of Endocrinology and Metabolism, Vontz Center for Molecular Studies, 3125 Eden Avenue, First Floor North, Cincinnati, Ohio 45267-0547. E-mail:
James.Fagin{at}uc.edu
This work was supported in part by NIH Grants CA-72597 and CA-50706
(J.A.F.), NRSA Grant F32CA80389 (J.M.S.), and K01DK02781 (J.A.K.).
1 These two investigators contributed equally to this work and should
be considered joint first authors. 
Received for publication April 19, 2000.
Accepted for publication August 9, 2000.
 |
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