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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 3–4 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 14–3-3 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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).



View larger version (27K):
[in this window]
[in a new window]
 
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.

 
H-Rasv12 Is Initially Mitogenic but Later Induces Programmed Cell Death
In the absence of TSH, H-Rasv12 increases cell growth (Fig. 2AGo), 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. 2BGo). Addition of doxycycline to the rtTACL3 control cell line does not alter cell growth (Fig. 2Go, A and B).



View larger version (27K):
[in this window]
[in a new window]
 
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.

 
By 3–6 days after the induction of H-Rasv12 expression in the presence of TSH, the Ras 25 and Ras 39 cells began detaching (Fig. 3AGo). These cells were found to be apoptotic as determined by DNA laddering (Fig. 3BGo). H-Rasv12-induced apoptosis was further verified by measuring cells with DNA strand breaks via the APO-BrdU assay (Table 1Go). The rate of apoptosis in response to acute expression of H-Rasv12 was concentration dependent (Fig. 3CGo).



View larger version (36K):
[in this window]
[in a new window]
 
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.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Apoptosis Is Induced by Acute Expression of H-Rasv12, MEK1S217E/S221E, or Racv12 before Cell Detachment

 
H-Rasv12 Activates Multiple MAP Kinase Superfamily Pathways in PCCL3 Cells
As shown in Fig. 4AGo, 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 1–147) fused to the activation domain of human Elk-1 (residues 307–428), 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. 4DGo).



View larger version (24K):
[in this window]
[in a new window]
 
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(1–147)]. 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 (1–147). 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.

 
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. 4Go, 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 1–223). 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. 4EGo).

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. 5BGo), inhibited doxycycline-induced apoptosis by about 50% in Ras 39 cells (Fig. 5AGo). 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, MEK1–55 and MEK1–65, express the MEK1S217E/S221E mutant protein in a doxycycline- dependent manner (Fig. 6AGo). After treatment with doxycycline, both clones activate the ERK pathway as analyzed through Western blotting utilizing phospho-antibodies for ERK1/ERK2 (Fig. 6AGo) and Elk-1 reporter assays (~5-fold, Fig. 6BGo). Six days after addition of doxycycline, both MEK1–55 and MEK1–65 cells become apoptotic as measured through cell detachment, DNA fragmentation (Fig. 6Go, C and D), and APO-BrdU assays (Table 1Go).



View larger version (23K):
[in this window]
[in a new window]
 
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.

 


View larger version (34K):
[in this window]
[in a new window]
 
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, MEK1–55, and MEK1–65 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 MEK1–55 cells with expression vectors for either Gal4-Elk1 alone, Gal4-Elk1 and MEK1, or Gal4 (1–147). 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, MEK1–55, and MEK1–65 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, MEK1–55, and MEK1–65 cell lines incubated with or without doxycycline for 6 days in H6 complete medium.

 
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 Rac1–88 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 Rac1–88 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. 7AGo).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 7. Acute Expression of Rac1v12 Induces Apoptosis

A, Rac1–88 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 Rac1–88 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 Rac1–88 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.

 
Acute activation of Rac1v12 induces apoptosis in thyroid cells (Fig. 7BGo) 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 Rac1–88 cells increased from approximately 11% in the absence of doxycycline to approximately 80% in its presence (Table 1Go).

In contrast to the MEK1 inhibitor, the p38MAPK specific inhibitor SB203580 did not block H-Rasv12-induced apoptosis (Fig. 8AGo) when used at a concentration (30 µM) that decreased H-Rasv12-induced p38MAP kinase activation to basal levels (Fig. 8BGo). These results suggest that the SAPK/JNK pathway, but not the p38MAPK pathway, mediates H-Rasv12-induced apoptosis in PCCL3 thyroid cells.



View larger version (29K):
[in this window]
[in a new window]
 
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.

 
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 1Go). 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. 9AGo, 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. 9BGo). 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. 9CGo). H89 was used at the minimal concentration verified to decrease cAMP response element-binding protein (CREB) phosphorylation to near basal levels (Fig. 9DGo). 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. 9CGo). 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).



View larger version (29K):
[in this window]
[in a new window]
 
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.

 
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. 10Go, 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. 10Go, A, B, and D). Similarly, Rac1v12-induced apoptosis is TSH dependent, as displayed by the decrease in viability measured by MTT assays (Fig. 10C).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 10. MEK1S217E/S221E and Rac1v12-Induced Apoptosis Is Dependent on TSH

rtTA7 and MEK1–55 (panel A) or MEK1–65 (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, Rac1–88 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, MEK1–55, and MEK1–65 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.

 
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. 11Go, A–C). TSH did not modulate the ability of doxycycline to induce H-Rasv12 in these cell lines (not shown).



View larger version (46K):
[in this window]
[in a new window]
 
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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 4–6 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 4–6 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{gamma}. 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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). Coon’s 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 Coon’s 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 10–3 vector downstream of the heptamerized tetO and a minimal CMV promoter. The pUHG 10–3 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 pUHD172–1neo, which directs high level expression of rtTA. The rTTACl3 cell line was created by cotransfecting rtTA7 cells with the pUHG 10–3 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 2–3 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 (3–6 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 manufacturer’s 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. Back

Received for publication April 19, 2000. Accepted for publication August 9, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bos JL 1988 The ras gene family and human carcinogenesis. Mutat Res 195:255–271[Medline]
  2. McCormick F, Wittinghofer A 1996 Interactions between Ras proteins and their effectors. Curr Opin Biotechnol 7:449–456[CrossRef][Medline]
  3. Lemoine NR, Mayall ES, Wyllie FS, Williams ED, Goyns M, Stringer B, Wynford-Thomas D 1989 High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis. Oncogene 4:159–164[Medline]
  4. Namba H, Rubin SA, Fagin JA 1990 Point mutations of ras oncogenes are an early event in thyroid tumorigenesis. Mol Endocrinol 4:1474–1479[Abstract]
  5. Downward J 1998 Ras signalling and apoptosis. Curr Opin Genet Dev 8:49–54[CrossRef][Medline]
  6. Fukasawa K, Vande WG 1997 Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability. Mol Cell Biol 17:506–518[Abstract]
  7. Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J, Evan G 1997 Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385:544–548[CrossRef][Medline]
  8. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME 1995 Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331[Abstract]
  9. Minden A, Lin A, Claret FX, Abo A, Karin M 1995 Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81:1147–1157[Medline]
  10. Brenner B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F, Gulbins E 1997 Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem 272:22173–22181[Abstract/Free Full Text]
  11. Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, Sanchez I 1997 Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA 94:3302–3307[Abstract/Free Full Text]
  12. Gulbins E, Bissonnette R, Mahboubi A, Martin S, Nishioka W, Brunner T, Baier G, Baier-Bitterlich G, Byrd C, Lang F 1995 FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway. Immunity 2:341–351[Medline]
  13. Kawasaki H, Morooka T, Shimohama S, Kimura J, Hirano T, Gotoh Y, Nishida E 1997 Activation and involvement of p38 mitogen-activated protein kinase in glutamate- induced apoptosis in rat cerebellar granule cells. J Biol Chem 272:18518–18521[Abstract/Free Full Text]
  14. Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J 1997 Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J 16:2783–2793[Abstract/Free Full Text]
  15. Marte BM, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J 1997 R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways. Curr Biol 7:63–70[Medline]
  16. Yao R, Cooper GM 1995 Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267:2003–2006[Medline]
  17. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME 1997 Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241[Medline]
  18. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G 1997 Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687–689[Abstract/Free Full Text]
  19. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ 1996 Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14–3-3 not BCL-X(L). Cell 87:619–628[Medline]
  20. Francis-Lang H, Zannini M, De Felice M, Berlingieri MT, Fusco A, Di Lauro R 1992 Multiple mechanisms of interference between transformation and differentiation in thyroid cells. Mol Cell Biol 12:5793–5800[Abstract]
  21. Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G, Rapp U, Ashworth A, Marshall CJ, Cowley S 1994 Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J 13:1610–1619[Abstract]
  22. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS 1995 The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81:1137–1146[Medline]
  23. Rak J, Mitsuhashi Y, Erdos V, Huang SN, Filmus J, Kerbel RS 1995 Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression. J Cell Biol 131:1587–1598[Abstract]
  24. Kinoshita T, Shirouzu M, Kamiya A, Hashimoto K, Yokoyama S, Miyajima A 1997 Raf/MAPK and rapamycin-sensitive pathways mediate the anti-apoptotic function of p21Ras in IL-3-dependent hematopoietic cells. Oncogene 15:619–627[CrossRef][Medline]
  25. Chen CY, Liou J, Forman LW, Faller DV 1998 Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras. Cell Death Differ 5:984–995[CrossRef][Medline]
  26. Fukasawa K, Rulong S, Resau J, Pinto dS, Woude GF 1995 Overexpression of mos oncogene product in Swiss 3T3 cells induces apoptosis preferentially during S-phase. Oncogene 10:1–8[Medline]
  27. Adler V, Pincus MR, Brandt-Rauf PW, Ronai Z 1995 Complexes of p21RAS with JUN N-terminal kinase and JUN proteins. Proc Natl Acad Sci USA 92:10585–10589[Abstract]
  28. Qiu RG, Chen J, Kirn D, McCormick F, Symons M 1995 An essential role for Rac in Ras transformation. Nature 374:457–459[CrossRef][Medline]
  29. Russell M, Lange-Carter CA, Johnson GL 1995 Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1). J Biol Chem 270:11757–11760[Abstract/Free Full Text]
  30. Chen YR, Wang X, Templeton D, Davis RJ, Tan TH 1996 The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and {gamma} radiation. Duration of JNK activation may determine cell death and proliferation. J Biol Chem 271:31929–31936[Abstract/Free Full Text]
  31. Wang TH, Wang HS, Ichijo H, Giannakakou P, Foster JS, Fojo T, Wimalasena J 1998 Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J Biol Chem 273:4928–4936[Abstract/Free Full Text]
  32. Saavedra HI, Knauf JA, Shirokawa JM, Wang JW, Ouyang B, Elisei R, Stambrook PJ, Fagin JA 2000 The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene 19:3948–3954[CrossRef][Medline]
  33. Cowley S, Paterson H, Kemp P, Marshall CJ 1994 Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77:841–852[Medline]
  34. Kupperman E, Wen W, Meinkoth JL 1993 Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase. Mol Cell Biol 13:4477–4484[Abstract]
  35. Miller MJ, Rioux L, Prendergast GV, Cannon S, White MA, Meinkoth JL 1998 Differential effects of protein kinase A on Ras effector pathways. Mol Cell Biol 18:3718–3726[Abstract/Free Full Text]
  36. Roger PP, Reuse S, Servais P, Van Heuverswyn B, Dumont JE 1986 Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture. Cancer Res 46:898–906[Abstract]
  37. Hafner S, Adler HS, Mischak H, Janosch P, Heidecker G, Wolfman A, Pippig S, Lohse M, Ueffing M, Kolch W 1994 Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol 14:6696–6703[Abstract]
  38. Bond J, Dawson T, Lemoine N, Wynford-Thomas D 1992 Effect of serum growth factors and phorbol ester on growth and survival of human thyroid epithelial cells expressing mutant ras. Mol Carcinog 5:129–135[Medline]
  39. Parma J, Van Sande J, Swillens S, Tonacchera M, Dumont J, Vassart G 1995 Somatic mutations causing constitutive activity of the thyrotropin receptor are the major cause of hyperfunctioning thyroid adenomas: identification of additional mutations activating both the cyclic adenosine 3',5'-monophosphate and inositol phosphate-Ca2+ cascades. Mol Endocrinol 9:725–733[Abstract]
  40. Gossen M, Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551[Abstract]
  41. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H 1995 Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766–1769[Medline]
  42. Knauf JA, Elisei R, Mochly-Rosen D, Liron T, Chen XN, Gonsky R, Korenberg JR, Fagin JA 1999 Involvement of protein kinase C{epsilon} (PKC{epsilon}) in thyroid cell death. A truncated chimeric PKC{epsilon} cloned from a thyroid cancer cell line protects thyroid cells from apoptosis. J Biol Chem 274:23414–23425[Abstract/Free Full Text]