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Molecular Endocrinology 17 (3): 450-459
Copyright © 2003 by The Endocrine Society

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

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: meinkoth{at}pharm.med.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NORMAL 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 impairs 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-expressing cells, those in which apoptosis is actively circumvented, survive in the form of thyroid tumors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1BGo). Infection with a virus containing the Escherichia coli lacZ gene resulted in time-dependent ß-galactosidase expression.



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  		Figure 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 µg 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 ß-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 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.

 
Expression of activated Ras stimulated a time-dependent increase in hypodiploid DNA content (Fig. 1CGo). 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. 1DGo), 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. 2AGo) and stimulated cell death to a similar extent and with similar kinetics (data not shown) in 3H and 2H as in basal medium (Fig. 2BGo).



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  		Figure 2. Ras Stimulates Apoptosis in the Presence of Thyrotropin and Other Growth Factors

A, Cell lysates (25 µg) 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.

 
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. 3Go). 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.



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  		Figure 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 µg 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.

 
Apoptosis Is Correlated with Aberrant Cell Cycle Progression
The magnitude of the apoptotic response was initially surprising in that WRT cells survive microinjection of Ras protein, where it stimulates DNA synthesis in assays typically measured at 48 h post injection (7). Therefore, we examined if infection with Ras virus-stimulated 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. 4AGo, and the results of several experiments summarized in Fig. 4BGo. 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. 4CGo). 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.



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  		Figure 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.

 
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. 5Go). 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.



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  		Figure 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.

 
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. 6Go). Cells were analyzed at times corresponding to early G1 (6 h), S (20–24 h), and G2/M (30 h) phase in 3H-treated parental cells. As expected, mock- and LacZ-infected 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. 6Go); 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.



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  		Figure 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.

 
Because the previous studies were performed after infection of growth-arrested cells, we also analyzed Ras effects in cycling cells (Fig. 7Go). 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 cell cycle progression are not due to infection of growth-arrested cells or a consequence of hormone or growth factor deprivation.



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  		Figure 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.

 
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. 8Go). 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 mock-infected cells (acute Ras in Fig. 8AGo). In cells selected to survive stable Ras expression (chronic Ras), p27 protein levels were also significantly reduced compared 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. 8BGo). Although D1 protein levels were markedly decreased upon acute expression of activated Ras, they were increased in Ras-transformed 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. 8CGo). 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.



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  		Figure 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 Ras-transformed 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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, 13, 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).

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 progression 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 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 {alpha} 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. 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, 28, 29, 30, 31, 32, 33, 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 generate 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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
The following antibodies were used: Ras (OP41; Oncogene Research, Boston, MA), activated MAPK (9101S; Cell Signaling, Beverly, MA), Fas ligand (F37720; Transduction Laboratories, Lexington, KY), cyclin B (610219; PharMingen, San Diego, CA), cyclin D2 (AHF0112; BioSource International, Camarillo, CA), ß-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 µg/ml), transferrin (5 µg/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).

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 µg 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).

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).

DNA Synthesis
DNA synthesis was assessed by BrdU incorporation as described in (37). Cells plated on glass coverslips were labeled 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.


   FOOTNOTES
 
This work was supported by Public Health Service Grant DK-55757 from NIDDK.

Abbreviations: 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.

Received for publication October 3, 2002. Accepted for publication November 25, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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