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Molecular Endocrinology 17(3):450–459
Copyright © 2003 by The Endocrine Society
doi: 10.1210/me.2002-0344
Ras Stimulates Aberrant Cell Cycle Progression and
Apoptosis in Rat Thyroid Cells
GUANJUN CHENG, AURÉLIA E. LEWIS,
AND
JUDY L. MEINKOTH
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6084
Abundant evidence supports the ability of Ras to
stimulate thyroid cell proliferation. Stable expression of activated Ras enhances the sensitivity of
thyroid cells to apoptosis. We report that apoptosis
is a primary and general response of rat thyroid
cells to acute expression of activated Ras in the
absence or presence of thyrotropin, insulin, and
serum, survival factors for thyroid cells. Ras induced apoptosis in quiescent and cycling cells.
Concomitantly, Ras stimulated S phase entry in
quiescent cells and enhanced G1/S transition in
cycling cells. Ras effects on the cell cycle were
characterized by delayed progression through S
phase and an apparent failure to proceed through
G2/M phase. Unlike thyroid cell mitogens, Ras
markedly decreased cyclin D1 expression. Although acute expression of Ras decreased cyclin
D1 protein levels, cells selected to survive chronic
Ras expression exhibited a selective increase in
cyclin D1 expression. In summary, thyroid cells
harbor an apoptotic program activated by Ras that
outstrips the protective effects of thyrotropin, insulin, and serum. Apoptosis is accompanied by
dysregulated cell cycle progression, suggesting
that cell death may arise, at least in part, as a
consequence of inappropriate proliferative cues.
(Molecular Endocrinology 17: 450–459, 2003)
N
pairs thyrotropin-stimulated DNA synthesis (7, 9), indicating that Ras is an essential component of hormone-induced proliferation.
Stable expression of activated Ras leads to the morphological transformation of rat thyroid cells, where it
confers hormone-independent proliferation (10, 11).
Counterintuitively, Ras-transformed cells exhibit an enhanced sensitivity to apoptosis upon serum withdrawal
(12), deprivation of adhesion, or treatment with MAPK
kinase (MEK) or phosphatidylinositol 3-kinase (PI3K) inhibitors (13). The death-sensitizing effects of Ras in thyroid cells are not secondary to cell transformation, nor
are they restricted to rodent cells. Chronic phorbol ester
treatment induced apoptosis in Ras-expressing human
cells (14) and treatment with PI3K inhibitors stimulated
apoptosis in human thyroid cells injected with Ras protein (15). In rat PC-Cl3 cells, inducible expression of
activated Ras stimulated apoptosis, although this occurred selectively in the presence of thyrotropin (16). We
noted that Wistar rat thyroid (WRT) cells selected to
stably express Ras formed many small colonies that
perished whether grown in the presence or absence of
thyrotropin. Therefore, we examined whether the acute
effects of Ras in thyroid cells included apoptosis. Our
studies revealed that Ras is an important determinant of
thyroid cell survival. Infection of three different rat thyroid
cell lines with an adenovirus encoding activated Ras
stimulated rapid, growth factor-independent apoptosis.
Cell death was preceded by an aberrant entry into the
cell cycle, characterized by delayed progression through
S phase and a failure to enter or complete G2/M. We
conclude that activating Ras mutations predispose thyroid cells to apoptosis, and that only a subset of Ras-
ORMAL ADULT TISSUES maintain a carefully
regulated balance between cell proliferation, cell
differentiation, and cell death, cellular processes that
are tightly linked. Cell proliferation serves to produce
highly specialized cells that can undergo several fates.
In some instances, cells exit the cell cycle and survive
indefinitely as terminally differentiated cells. Alternatively, proliferating cells can undergo growth arrest
followed by apoptosis. In other instances, for example
thyroid cells, differentiated cells survive and retain the
capacity for self-renewal. Imbalances in the regulation
of these processes, such as those induced by oncogenes like Ras, can lead to tumor formation.
Thyroid follicular cells are specialized epithelial cells
that synthesize thyroid hormone. Thyroid cells retain
the capacity to divide, albeit slowly, in response to
thyrotropin. Ras mutations are frequent in thyroid tumors (reviewed in Refs. 1 and 2), and data from human
(3) and rodent (4, 5) model systems have confirmed an
important role for Ras in the regulation of thyroid cell
proliferation. When expressed in primary human thyroid cells where the proliferation index is low, Ras
stimulates proliferation for a period of weeks, ultimately followed by growth arrest and apoptosis (6).
Microinjection of activated Ras protein into rat thyroid
cells stimulates DNA synthesis in the absence of thyrotropin (7) and enhances the mitogenic effects of
thyrotropin (8). Moreover, interference with Ras imAbbreviations: BrdU, Bromodeoxyuridine; FACS, fluorescence-activated cell sorting; 2H, thyrotropin-deficient medium; 3H, growth medium; MEK, MAPK kinase; m.o.i., multiplicity of infection; PI3K, phosphatidylinositol 3-kinase;
WRT, Wistar rat thyroid cells.
450
Cheng et al. • Ras Stimulates Apoptosis
expressing cells, those in which apoptosis is actively
circumvented, survive in the form of thyroid tumors.
RESULTS
Acute Expression of Activated Ras
Stimulates Apoptosis
To explore effects on cell survival, WRT cells in hormone- and growth factor-free basal medium (see Materials and Methods) were infected with an adenovirus
containing activated Ras (H-RasL61). Infection resulted
in dose-dependent Ras expression after infection at
multiplicity of infection (m.o.i.) of 10 or more (Fig. 1A).
Ectopic Ras was active in that it stimulated MAPK
phosphorylation. After infection at m.o.i. of 10, Ras
expression was detected within 24 h of infection (d 1)
and maintained for at least 4 d (Fig. 1B). Infection with
a virus containing the Escherichia coli lacZ gene resulted in time-dependent ␤-galactosidase expression.
Expression of activated Ras stimulated a timedependent increase in hypodiploid DNA content (Fig.
1C). This effect was first observed 2 d after infection
and increased over the subsequent 2 d. This time
course agrees well with other reports where expression of activated Ras stimulated apoptosis between
24 and 72 h post expression (17, 18). In contrast, cells
infected with LacZ virus exhibited a normal cell morphology and contained only intact DNA, indicating that
the effects of Ras were not secondary to viral infection.
Ras-infected cells exhibited DNA laddering (Fig. 1D),
membrane blebbing, and chromatin condensation
(data not shown), indicating that cell death was
apoptotic.
To determine whether apoptosis was a consequence of Ras expression in the absence of survival
signals, cells in growth medium (3H) were analyzed.
Cells in thyrotropin-deficient medium (2H) were also
analyzed as thyrotropin has been reported to stimulate
apoptosis in Ras-expressing PC-Cl3 thyroid cells (16).
Ras was expressed under both conditions (Fig. 2A)
and stimulated cell death to a similar extent and with
similar kinetics (data not shown) in 3H and 2H as in
basal medium (Fig. 2B).
To confirm that apoptosis was a conserved response of rat thyroid cells to activated Ras, experiments were carried out in two additional rat thyroid cell
lines, FRTL-5 and PC-Cl3 (Fig. 3). As for WRT cells,
Ras stimulated apoptotic cell death in both cell lines in
the presence and absence of thyrotropin, insulin, and
serum. Therefore, cell death is a general, early response induced by expression of activated Ras in rat
thyroid cells.
Apoptosis Is Correlated with Aberrant Cell
Cycle Progression
The magnitude of the apoptotic response was initially
surprising in that WRT cells survive microinjection of
Mol Endocrinol, March 2003, 17(3):450–459
451
Ras protein, where it stimulates DNA synthesis in assays typically measured at 48 h post injection (7).
Therefore, we examined if infection with Ras virusstimulated cell cycle progression before apoptosis.
Cells rendered quiescent by incubation in basal medium for 72 h were infected, floating and adherent cells
collected, and cell cycle distribution analyzed. A representative FACS analysis is depicted in Fig. 4A, and
the results of several experiments summarized in Fig.
4B. Mock-infected cells were arrested predominantly
in G1 with a minor proportion of cells arrested in G2/M.
At d 1 post infection, Ras-expressing cells exhibited a
cell cycle distribution similar to mock-infected cells,
except for a small decrease in G2/M and increase in
G1 phase cells. Over the next 3 d, the proportion of G1
phase cells steadily declined. A robust increase in S
phase cells was noted at d 2, and cells continued to
accumulate in S phase over the next 2 d. The FACS
data were corroborated by bromodeoxyuridine (BrdU)
incorporation, which showed a marked increase in
DNA synthesis between d 1 and 2, and stabilization
thereafter (Fig. 4C). Strikingly, the proportion of G2/M
phase cells remained constant over all 4 d, suggesting
that most cells die rather than continue through the
cell cycle.
To further investigate the effects of Ras on cell cycle
regulation, molecular markers of G1/S and G2/M
phase cell cycle transit were examined (Fig. 5). Quiescent WRT cells respond to 3H with increased expression of cyclins D1, E, A, and p21 expression, and only
a modest decrease in p27 expression as they progress
through G1 (Lewis, A. E., manuscript in preparation).
Ras expression in quiescent cells elicited very different
effects. At 48 h post infection, Ras expression resulted
in a marked decline in cyclin D1 protein levels. Ras
stimulated time-dependent increases in p21 and cyclin E and A expression, together with a decrease in
p27 levels. Interestingly, Ras failed to stimulate cyclin
B expression, a marker of G2/M progression in WRT
cells. These data corroborate those obtained by FACS
analysis and indicate that Ras-stimulated cell cycle
progression is aberrant.
To determine whether the unusual effects of Ras on
cell cycle progression were secondary to the absence
of other mitogenic signals, quiescent cells were infected and subsequently stimulated with 3H (Fig. 6).
Cells were analyzed at times corresponding to early
G1 (6 h), S (20–24 h), and G2/M (30 h) phase in 3Htreated parental cells. As expected, mock- and LacZinfected cells exhibited a time-dependent decrease in
G1 cells accompanied by an increase in S phase cells,
and a later increase in cells in G2/M. Ras stimulated
G1 to S phase transition in the growth-arrested cells (0
h 3H in Fig. 6); however, little further evidence of cell
cycle progression was evident in 3H-treated cells.
These data indicate that the aberrant effects of Ras on
cell cycle progression are not a consequence of Ras
expression in the absence of thyroid cell mitogens.
Because the previous studies were performed after
infection of growth-arrested cells, we also analyzed
452 Mol Endocrinol, March 2003, 17(3):450–459
Cheng et al. • Ras Stimulates Apoptosis
Fig. 2. Ras Stimulates Apoptosis in the Presence of Thyrotropin and Other Growth Factors
A, Cell lysates (25 ␮g) prepared from WRT cells 48 h
following infection with Ras in 3H and 2H medium were
analyzed by Western blotting for Ras expression. Fas ligand
expression was assessed to document equal protein loading.
B, Cells were infected in 3H, 2H, or basal medium and floating and adherent cells collected and analyzed by FACS analysis 48 h post infection. Results shown are summarized from
three independent experiments. Error bars less than 0.5 are
not shown.
Fig. 1. Acute Expression of Ras Stimulates Apoptosis
A, Quiescent WRT cells were infected with Ras adenovirus
at the m.o.i. indicated. At 48 h post infection, cell lysates were
prepared and 25 ␮g cell protein analyzed by Western blotting
with antibodies to Ras, activated MAPK (MAPK-P), and actin
as a loading control. B, Western blot showing Ras and ␤galactosidase expression over time after infection (m.o.i. 10)
of quiescent WRT cells. C, Quiescent WRT cells infected with
Ras or LacZ virus (both at m.o.i. 10) were harvested at d 1–4
post infection. Floating and adherent cells were collected and
analyzed by FACS analysis. Results shown (% hypodiploid
Ras effects in cycling cells (Fig. 7). As seen in quiescent cells, Ras dramatically reduced cyclin D1
expression, stimulated cyclin E and A expression,
and failed to increase cyclin B expression. Moreover, cyclin E and A expression were sustained over
4 d, suggesting that the majority of the cells analyzed at these times were arrested in S phase. These
results indicate that the aberrant effects of Ras on
DNA content) are from three independent experiments performed for Ras and a single experiment for the LacZ virus.
Error bars less than 0.2 are not shown. D, DNA isolated from
Ras-infected WRT cells at 2 and 4 d post infection and from
mock-infected cells at d 4 was analyzed on agarose gels.
Cheng et al. • Ras Stimulates Apoptosis
Mol Endocrinol, March 2003, 17(3):450–459
453
with parental cells and to cells acutely infected with
Ras. Similarly, either acute or stable expression of Ras
increased p21 expression. Very different results were
obtained for cyclin D1 (Fig. 8B). Although D1 protein
levels were markedly decreased upon acute expression of activated Ras, they were increased in Rastransformed cells (chronic Ras) vs. parental WRT cells.
This was true both in quiescent cells in basal medium
as well as in growing cells in 3H. Moreover, this effect
was specific to cyclin D1. Expression of cyclins D2 and
D3 were reduced in Ras-transformed cells compared
with parental cells (Fig. 8C). These results indicate that
cyclin D1 expression is differentially regulated by
acute vs. chronic Ras expression, and that D1 may
contribute to the ability of thyroid cells to survive
chronic expression of activated Ras.
DISCUSSION
Fig. 3. Ras Stimulates Growth Factor-Independent Apoptosis in FRTL-5 and PC-Cl3 Cells
A, FRTL-5 and PC-Cl3 cells were infected with Ras (m.o.i.
10) and maintained in basal, 2H, or 3H medium. At 48 h post
infection, cell lysates were prepared and 25 ␮g of total cell
protein analyzed by Western blotting with a Ras-specific
antibody. B, Floating and adherent cells infected as described in A were collected and hypodiploid DNA content
analyzed by FACS analysis. Apoptosis was significantly
greater in FRTL-5 cells in 3H (P ⬍ 0.05; Student’s t test) as
well as in 2H (P ⬍ 0.01) than in basal medium. The results
shown are from three independent experiments.
cell cycle progression are not due to infection of
growth-arrested cells or a consequence of hormone
or growth factor deprivation.
Acute vs. Chronic Effects of Ras on Cyclin
D1 Expression
Although Ras-transformed thyroid cells exhibit an enhanced sensitivity to apoptotic insults, in the absence
of such signals the cells survive and undergo rapid,
hormone-independent proliferation (11). To gain insight into factors that might contribute to the survival
of Ras-transformed cells, acute and chronic effects of
Ras on p27 and cyclin D1 expression were compared
(Fig. 8). These proteins were selected for analysis
based on their differential regulation by Ras vs. the
thyroid cell mitogen, 3H. Acute infection of cells in 3H
with Ras decreased p27 levels compared with mockinfected cells (acute Ras in Fig. 8A). In cells selected to
survive stable Ras expression (chronic Ras), p27 protein levels were also significantly reduced compared
Although poorly understood, cellular context is an important determinant of the phenotypic consequences of
Ras activation. Ras elicits two unusual features in thyroid
cells. In primary human thyroid cells, activated Ras stimulates proliferation (3, 6) rather than growth arrest as is
seen in primary fibroblasts (reviewed in Ref. 19). Unlike
its effects in many established cell lines where Ras promotes survival, Ras-transformed thyroid cells exhibit an
enhanced sensitivity to apoptosis (12–14). The ability of
Ras to stimulate proliferation in primary cells implies that
selective pressure to eliminate thyroid cells harboring
Ras mutations is essential to limit the expansion of neoplastic cells. Our findings support the existence of such
mechanisms in rat thyroid cells, where the acute response to Ras activation was apoptosis. Intriguingly,
apoptosis occurs concomitantly with, or perhaps as a
consequence of, aberrant cell cycle progression.
Infection of three different rat thyroid cell lines with
a virus expressing H-RasL61 resulted in massive cell
loss, which was confirmed to be a result of apoptotic
cell death. Apoptosis was detected within 24 h following Ras expression and increased over the next 48 h.
This time course agrees well with earlier reports where
Ras-induced apoptosis was first detected at 14–16 h
following microinjection of expression vectors encoding activated Ras (17) and was maximal 48–72 h following transient transfection of Ras-encoding expression vectors (18). Although Ras expression decreased
as apoptosis increased, this most likely reflects the
delayed and asynchronous manner through which
cells become committed to apoptosis (20). Cell death
was not secondary to growth factor deprivation as Ras
stimulated apoptosis in the presence and absence of
thyrotropin, insulin, and serum, important growth factors for thyroid cells. These results differ somewhat
from a previous report where Ras stimulated apoptosis selectively in the presence of thyrotropin (16) but
agree well with those reported in other cells where
Ras stimulates growth factor-independent apoptosis
(17, 18).
454 Mol Endocrinol, March 2003, 17(3):450–459
Cheng et al. • Ras Stimulates Apoptosis
Fig. 4. Acute Infection with Ras Stimulates G1 to S Phase Cell Cycle Progression
A, Quiescent WRT cells were infected with Ras or LacZ adenovirus (m.o.i. 10) in basal medium. At the times indicated (days),
floating and adherent cells were collected and analyzed by FACS analysis. The results from three independent experiments for
Ras and a single experiment with LacZ are shown in panel B. C, Quiescent WRT cells plated on coverslips were infected with Ras
as described in A. At the indicated times, the cells were fixed and stained for BrdU incorporation (see Materials and Methods).
Mean % BrdU incorporation from three independent experiments was as follows: mock-infected cells, 3% at d 1, 2, 3;
Ras-infected cells, 4% at d 1, 39% at d 2, 45% at d 3, and 53% at d 4.
Ras stimulated apoptosis in both cycling and
growth-arrested cells. Nonetheless, a correlation between apoptosis and cell cycle progression was observed. Ras stimulated cell cycle progression after
infection of quiescent cells, and accelerated G1/S pro-
gression in cycling cells. Although Ras stimulated exit
from G1 and entry into S phase, the proportion of
G2/M phase cells remained constant, suggesting that
most cells failed to complete the cell cycle. As anticipated, Ras increased the expression of cyclins E and
Cheng et al. • Ras Stimulates Apoptosis
Fig. 5. Ras Effects on G1/S Phase Cell Cycle Progression
Quiescent WRT cells infected with Ras (m.o.i. 10) were
harvested at the times indicated (days). Cell lysates (prepared
from floating and adherent cells) were prepared and analyzed
by Western blotting with the indicated antibodies. Actin expression was assessed to determine equal protein loading.
A, markers of G1 to S phase transition. However, Ras
failed to increase the expression of cyclin B, a marker
of G2/M phase, even in mitogen-treated cells. Moreover, cyclin A expression, which normally declines at
mitosis, remained high for as long as 4 d post infection. These data suggest that cell cycle progression
induced by Ras is sensed as aberrant, and as a consequence, triggers an apoptotic response. The tight
linkage between cell cycle progression and apoptosis
is further supported by the similar dose response and
kinetics with which Ras stimulated cell cycle progression and apoptosis.
We attempted to block cell cycle progression using
pharmacological inhibitors that impair proliferation in
Ras-transformed thyroid cells (21). However, treatment
with the MEK1 inhibitors, PD98059 and UO126, greatly
attenuated Ras expression, whereas treatment with the
PI3K inhibitor, LY294002, enhanced it. We also examined whether cell cycle arrest using the DNA polymerase
␣ inhibitor, aphidicolin, blocked Ras-stimulated apoptosis. Although aphidicolin blocked Ras-stimulated cell cycle progression in the absence of apparent effects on
apoptosis, it also stimulated hypodiploid DNA content in
cycling WRT cells. These results precluded interpretation
of aphidicolin effects on Ras-induced apoptosis but further support the notion that inappropriate cell cycle progression results in apoptosis in these cells.
There are several potential explanations as to why
Ras-stimulated cell cycle progression might be sensed
as aberrant in thyroid cells. Several aspects of cell cycle
regulation are unusual in thyroid cells. Unlike fibroblasts
and other cells where cAMP impairs proliferation, cAMP
stimulates thyroid cell proliferation (reviewed in Refs. 9
and 22). As differentiated cells, thyroid cells cycle slowly.
Mol Endocrinol, March 2003, 17(3):450–459
455
Ras is well known for its potent mitogenic effects in many
cell lines that, unlike thyroid cells, are poised to respond
to mitogenic stimuli with rapid proliferation. Ras elicits
effects including a robust decrease in cyclin D1 and p27
levels that may be interpreted as inappropriate in the
context of the thyroid cell and are certainly distinct from
the action of mitogens in these cells. The idea that aberrant cell cycle progression results in apoptosis is supported by other data in thyroid cells and fibroblasts.
Overexpression of the high mobility group protein,
HMGA1, in PC-Cl3 cells resulted in apoptosis accompanied by accelerated progression into S phase and
delayed G2/M transition, similar to the effects reported
here (23). In murine embryonic fibroblasts lacking p21cip1
and p27kip1, Ras stimulated incomplete cell cycle progression characterized by DNA re-replication and failure
of cytokinesis (24). Similar to this report, we observed
aberrant nuclei and multinucleate cells after acute Ras
expression in thyroid cells. Similar results have been
reported in PC-Cl3 cells where acute expression of Ras
induced chromosomal aberrations within a single cell
cycle (25).
The high frequency of Ras mutations in thyroid tumors suggests that either human cells respond differently to Ras than do rodent cells, or that secondary
changes that allow the survival and expansion of thyroid cells with Ras mutations are frequent. Microinjection of activated Ras protein into human thyroid cells
sensitized the cells to apoptosis after treatment with
PI3K inhibitors (15). Therefore, acute expression of
Ras renders human cells more susceptible to apoptosis. Nonetheless, human thyroid cells exhibit a far
lower apoptotic index than rodent cells. Given the
more stable karyotype in human vs. rodent cells, the
ability of Ras to stimulate proliferation in human cells
may be secondary to the more intact genomic surveillance mechanisms in these cells.
Ras-transformed rodent thyroid cells are readily isolated; therefore, compensatory changes that allow the
survival of Ras-expressing cells must occur at a high
frequency. Our findings revealed very different regulation of cyclin D1 by Ras in acute vs. chronic settings.
Acute Ras expression markedly decreased cyclin D1
expression, whereas stable cell lines selected to survive Ras expression exhibited increased D1 protein
levels compared with parental cells. The differential
effects of acute vs. chronic Ras expression on cyclin
D1 were selective in that expression of cyclins D2 and
D3 were not up-regulated in Ras-transformed cells.
This finding is especially interesting based on a report
in Rat-1 fibroblasts where Ras stimulated apoptosis as
a consequence of increased turnover of cyclin D1
protein (26). Increased D1 expression in Ras-transformed cells may be integral to their survival. Interestingly, cyclin D1 is overexpressed in thyroid tumors
(27–34). Given the pleiotropic effects of Ras, it is likely
that multiple events, perhaps including up-regulation
of cyclin D1, contribute to the survival of Ras-expressing thyroid cells. Analysis of these changes might gen-
456 Mol Endocrinol, March 2003, 17(3):450–459
Cheng et al. • Ras Stimulates Apoptosis
Fig. 6. Ras Effects on Cell Cycle Progression in the Presence of 3H
WRT cells were transferred to basal medium for 24 h, infected with Ras or LacZ virus (m.o.i. 10) overnight in basal medium,
and subsequently incubated in basal medium for a further 24 h. 3H was then added, and cells harvested 6, 20, 24, and 30 h later.
A representative FACS analysis is presented. Two experiments were performed with similar results.
erate strategies with which the apoptotic program
could be reactivated in Ras-transformed cells.
In summary, our findings highlight the existence of
an apoptotic program activated by Ras in thyroid cells
and provide a molecular explanation for the enhanced
sensitivity of Ras-transformed cells to apoptosis. Our
findings are the first to suggest a correlation between
Ras effects on cell cycle progression and apoptosis in
thyroid cells and to invoke a role for cyclin D1 in the
survival of Ras-transformed thyroid cells.
MATERIALS AND METHODS
Reagents
The following antibodies were used: Ras (OP41; Oncogene
Research, Boston, MA), activated MAPK (9101S; Cell Signal-
ing, Beverly, MA), Fas ligand (F37720; Transduction Laboratories, Lexington, KY), cyclin B (610219; PharMingen, San
Diego, CA), cyclin D2 (AHF0112; BioSource International,
Camarillo, CA), ␤-galactosidase (55976; Cappel Laboratories, Durham, NC); and from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA): p21cip1 (SC-397), p27kip1 (SC-527), cyclin A
(SC-596), cyclin D1 (SC-450), cyclin D3 (SC-16), cyclin E
(SC-481), and actin (SC-1615).
Cell Culture
WRT, FRTL-5 (purchased from ATCC, Manassas, VA) and
PC-Cl3 cells (kindly provided by Dr. J. Fagin, University of
Cincinnati, Cincinnati, OH) were cultured in Coon’s modified
Ham’s F12 medium supplemented with bovine thyrotropin (1
mU/ml), insulin (10 ␮g/ml), transferrin (5 ␮g/ml), and 5%
donor calf serum (referred to as 3H). Cells were rendered
quiescent by incubation in Coon’s modified Ham’s F12 medium supplemented with 0.2% fatty-acid free BSA (basal
medium) for 72 h. In some studies, cells were treated with
thyrotropin-deficient 3H medium (referred to as 2H).
Cheng et al. • Ras Stimulates Apoptosis
Mol Endocrinol, March 2003, 17(3):450–459
457
Fig. 7. Ras Effects on the Expression of Cell Cycle Regulators in 3H
WRT cells in 3H were infected with Ras for the times
indicated, floating and adherent cells collected, and cell lysates prepared and analyzed by Western blotting with the
indicated antibodies.
Adenoviruses
Adenoviruses encoding H-RasL61 and the E. coli LacZ gene
were kindly provided by Dr. J. Nevins (HHMI, Duke University)
and Dr. A. Zeleznik (Department of of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA), respectively.
Viruses were propagated by standard techniques in the 293
packaging cell line and titrated by endpoint dilution. Cells
were infected in basal medium at a multiplicity of infection
(infectious units/cell) ranging from 2.5–15. After infection,
fresh medium was added, and the cells incubated for various
times in basal, 2H or 3H medium.
Western Blotting
Whole cell lysates were prepared and 20–60 ␮g of total cell
protein analyzed as described previously (35). The antibodies
used for Western blotting and their sources are indicated
under reagents. Equal protein loading was confirmed in all
experiments.
FACS Analysis
Floating and trypsinized adherent cells were collected by
centrifugation, fixed in 70% EtOH in PBS at 4 C for 30 min,
stained with propidium iodide (0.1 mg/ml) in 0.1% Triton
X-100, 0.1 mM EDTA containing 100 U/ml ribonuclease A in
PBS for 30 min and subjected to FACS analysis. FACS analysis was performed by the Wistar Institute cytometry facility
(Philadelphia, PA) on an EPICS XL flow cytometer (Coulter
Corporation, Hialeah, FL).
Fig. 8. Expression of Cyclin D1 Is Differentially Regulated by
Acute vs. Chronic Ras Expression
A, Western blot showing expression of p21 and p27 in
lysates prepared from mock-infected WRT cells and WRT
cells infected with Ras (acute Ras) for 2 d in 3H medium.
Lysates from Ras-transformed WRT cells (11) after 48 h in 3H
(chronic Ras) were analyzed in parallel. B, Lysates prepared
from mock and Ras-infected WRT cells (acute Ras) in 3H
were analyzed by western blotting for cyclin D1 expression at
2 d post infection. Lysates prepared from WRT and Rastransformed WRT cells (chronic Ras) after 2 d in basal or 3H
medium were also analyzed. C, Lysates prepared from WRT
and Ras-transformed (chronic Ras) WRT cells after 2 d in
basal medium were analyzed for expression of cyclins D1,
D2, and D3 by Western blotting. Ras expression was similar
in the acutely infected and stably transfected cells.
Assays of Apoptosis
Hypodiploid DNA content was assessed by FACS analysis as
described above. For DNA laddering, DNA was isolated and
analyzed as described previously (13, 36).
with BrdU for 24-h intervals, fixed and stained for BrdU
incorporation, and the percentage of cells with replicated
DNA was scored. Over 200 cells on duplicate coverslips were
scored blinded for each condition.
DNA Synthesis
Acknowledgments
DNA synthesis was assessed by BrdU incorporation as described in (37). Cells plated on glass coverslips were labeled
Received October 3, 2002. Accepted November 25, 2002.
458 Mol Endocrinol, March 2003, 17(3):450–459
Address all correspondence and requests for reprints to:
Judy L. Meinkoth, Department of Pharmacology, Room 164
John Morgan Building, University of Pennsylvania School of
Medicine, 3620 Hamilton Walk, Philadelphia, Pennsylvania
19104-6084. E-mail: [email protected].
This work was supported by Public Health Service Grant
DK-55757 from NIDDK.
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Twenty-Second Annual
University of Kentucky
Symposium in Reproductive Sciences
May 16-17, 2003
Lexington, Kentucky
This annual symposium of the Reproductive Sciences Forum at the University of Kentucky
brings faculty, clinicians, and trainees in the field of reproductive sciences together to
exchange information and ideas with each other and with recognized leaders of the
reproductive sciences research community. The program consists of plenary lectures, a
poster session, meet-the-professor luncheon, and dinner.
For registration information visit our website at
www.mc.uky.edu/obg/Forum/Forum.htm
or contact:
Lothar Jennes, Ph.D.
Department of Anatomy & Neurobiology
University of Kentucky
800 Rose St.
Lexington, KY 40536
(859) 257-1093
[email protected]
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