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1B-Adrenergic Receptor Transgene Promotes Malignant Transformation of Thyroid Follicular Cells1
IRIBHN (C.L., J.D., G.V., M.P.) and the Medical Genetics Department (G.V.), Free University of Brussels Campus Erasme; and the Histology Laboratory, University of Louvain Medical School (J.-F.D.), Brussels, Belgium; and the Departments of Medicine and Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center (S.C., R.L.), Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Dr. Catherine Ledent, IRIBHN, Free University of Brussels Campus Erasme, 808 route de Lennik, B-1070 Brussels, Belgium.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1B adrenergic receptor, which is known to couple to both
cascades in transfected cell lines. The expression of the transgene
resulted, as expected, in the activation of phospholipase C and
adenylyl cyclase, as demonstrated by the direct measurement of
IP3 and cAMP in thyroid tissue. The phenotype resulting
from this dual stimulation included growth stimulation, hyperfunction,
cell degeneracy attributed to the overproduction of free radicals, and
the development of malignant nodules invading the capsule, muscles, and
blood vessels. Differentiated metastases were found occasionally in old
animals. The development of malignant lesions was more frequent and of
earlier onset than in our previous Tg-A2aR model, in which
only the cAMP cascade was stimulated. These observations demonstrate
that the cAMP and IP3/Ca2+/DAG cascades can
cooperate in vivo toward the development of thyroid
follicular cell malignancies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-adrenergic agonists (10, 11, 12).
Stimulation of this cascade by the TSH receptor at high TSH
concentrations has also been demonstrated in the human thyrocyte (13),
but not in the dog thyrocyte (7). Stimulation of the inositol
1,4,5-triphosphate (IP3)/Ca2+/DAG cascade has
been reported to promote H2O2 production
(through the stimulation of the ill defined
H2O2-generating system), iodide organification,
and thyroid hormone synthesis (9, 14, 15). The third cascade
stimulating proliferation is the tyrosine
kinase/ras/mitogen-activated protein kinase pathway. Factors
known to regulate this pathway in thyroid cells include insulin-like
growth factor I, epidermal growth factor (EGF), and hepatocyte growth
factor (3, 16, 17). Although EGF and hepatocyte growth factor promote
proliferation and induce dedifferentiation of the thyroid cell,
insulin-like growth factor I and insulin have a permissive effect on
the mitogenic actions of TSH, EGF, and phorbol esters (6).
We have previously generated transgenic mouse lines expressing the
adenosine A2a receptor under the control of the thyroid
cell-specific thyroglobulin promoter (18). Presumably through the
continuous release of adenosine by thyroid tissue, the adenosine
receptor expressed in thyroid cells acts as a constitutive activator of
the cAMP cascade, leading to the development of a toxic
hyperfunctioning goiter. This transgenic model reproduces the
pathogenesis and phenotype of autonomous hyperfunctional adenomas and
nonautoimmune familial hyperthyroidism secondary to the permanent
activation of adenylyl cyclase by constitutively active TSH receptor
mutants (19, 20). A similar, although milder, phenotype was obtained in
mice expressing in their thyroid a constitutively activated
Gs, the G protein that activates adenylyl cyclase
(21).
With the aim of investigating the effect of the concomitant stimulation
of both the cAMP and IP3/Ca2+/DAG pathways on
thyroid function and proliferation in vivo, we generated
transgenic mouse lines expressing a mutant of the
1B-adrenergic receptor that activates both cascades
(22). This mutant was previously reported to be constitutively active
in transfected cell lines (23) and in heart muscle cells of transgenic
mice (24). As expected from the stimulation of the cAMP pathway,
transgenics expressing the mutant 1B-receptor partially
reproduced the phenotype of the mice expressing the
A2a-adenosine receptor. Nevertheless, marked differences
were found in the function of the gland, the evolution of the
phenotype, and the frequency and timing of malignant tumor development,
all changes attributed to the stimulation of phospholipase C.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1B-adrenergic mutant receptor
hybrid gene (Tg-1BAR)1B-adrenergic receptor complementary DNA (cDNA; -14 to
+1745 relative to the start codon) was cloned into the polylinker of
pSG5 (Stratagene, La Jolla, CA). A StuI-SalI
restriction fragment of pSG5-1B comprising the second
intron of the rabbit ß-globin gene, the 1B cDNA, and
the polyadenylation signal was further cloned in pBluescript
SK+, downstream of a bovine thyroglobulin gene promoter
fragment (-2036 to +9 bp relative to the CAP site). Transgenic mice were generated as previously described (25). All animals were anesthetized with either avertin or ether before surgical procedures. Animals were kept at the central housing facility of the Free University of Brussels Medical School, and all procedures involving them were made in accordance with the regulations and guidelines of the Belgian State and European Union after approval by the local ethical committee. The linearized construct (1–2 pl of a 2.5 µg/ml solution) was microinjected into the pronuclei of fertilized eggs from a C57BL/6J x DBA/2J F2 cross. Screening of transgenic animals was performed by Southern blotting of DNA extracted from tail biopsies and hybridization with a bovine thyroglobulin gene promoter probe.
Hormonal, functional, and second messenger assays
Blood samples were obtained by cardiac or orbital puncture under
anesthesia. Total T4 levels were assayed on sera by
standard RIA (T4 Coat-a-Count, Clinical Assays, Cambridge,
MA).
Iodide uptake was measured by counting whole thyroid glands 4 h after an ip injection of [125I]NaI (15 µCi). Organification was assayed by trichloroacetic acid (5%, wt/vol) precipitation of thyroid homogenates as previously described (26). Protein-bound radioiodine was expressed as a percentage of the total iodide uptake.
For cAMP measurements, thyroid glands were collected under anesthesia and immediately immersed in boiling water. After 5 min, tissues were homogenized in a glass homogenizer, and insoluble material was removed by centrifugation. The soluble fraction was lyophilized and redissolved in water. cAMP was assayed by RIA following the kit manufacturer’s instructions (cAMP125 assay system, RPA 509, Amersham, Aylesbury, UK).
For inositol phosphate determinations, thyroid glands were collected under anesthesia and flash-frozen in liquid nitrogen. They were thawed in 1 ml ice-cold 5% perchloric acid and homogenized. After centrifugation, supernatants were titrated to pH 7.5 with a solution containing 60 mM HEPES, 1.5 M KOH, and Universal Indicator. Precipitated KClO4 was removed by centrifugation. Samples were purified using Amprep SAX minicolumns (RPN 1908, Amersham), and IP3 was measured following the kit manufacturer’s instructions (D-myo-inositol 1,4,5-triphosphate 3H assay system, TRK 1000, Amersham).
Binding assays
Crude 40,000 x g membrane fractions were
prepared from thyroid glands as previously described (27) and
resuspended in binding buffer [50 mM Tris-HCl (pH 7.4), 5
mM EDTA, and 150 mM NaCl]. Protein content
estimation was carried out using the Lowry assay, as modified by
Peterson (28). Binding assays were performed on 10–100 µg membrane
proteins, using the 1-specific antagonist
2-{ß-(hydroxy-3-[125I]iodophenyl)ethylaminomethyl}-tetralone
(2200 Ci/mmol; New England Nuclear, Hertfordshire, UK) as tracer at a
saturating concentration (250 pM; reported
Kd, 30–50 pM) (29). Nonspecific
binding was determined in the presence of a large excess (50
µM) of prazosin (Sigma Chemical Co., St. Louis, MO).
Incubations were performed for 1 h at 25 C in 250 µl binding
buffer. Bound ligand was separated by filtration on GF-C membranes
(Whatman, Maidstone, UK) and counted.
Northern blotting procedure
Polyadenylated RNA was isolated using the FastTrack kit
(Invitrogen, San Diego, CA). After glyoxal denaturation, RNA samples
(15 µg/lane) were fractionated on a 1% agarose gel in 10
mM phosphate buffer (pH 7.0) and transferred to nylon
membranes (Pall Biodyne A, Glen Cove, NY) as previously described (30).
DNA probes were -32P labeled by random priming (31).
After hybridization and washing, the filters were autoradiographed
using Amersham ß-max films.
Histological and immunohistological procedures
For light microscopy, tissues were fixed by immersion for
24 h in Bouin’s solution and embedded in paraffin by standard
procedures. Six-micron sections were stained with hematoxylin and
eosin.
For determination of the proliferation index, bromodeoxyuridine (BrdU) was injected ip (0.05 mg/g BW) 1 h before death. Thyroids were fixed in 70% ethanol-30% acetic acid, embedded in paraffin, and cut at 6-µm intervals. BrdU was detected by immunochemistry (32), using a mouse monoclonal anti-BrdU antibody (Becton Dickinson, Rutherford, NJ), a sheep biotinylated antimouse Ig serum (Amersham), and a streptavidin-biotinylated horseradish peroxidase complex (Amersham).
For semithin sections and electron microscopy, thyroids were fixed by immersion for 2 h in 2.5% glutaraldehyde (Taab, Reading, UK) in 0.1 M cacodylate buffer, dehydrated in an ethanol series of increasing strength, and embedded in LX112 (Ladd Research Industries, Burlington, VT). Sections (0.5 µm thick) were cut and stained with 1% toluidine blue for light microscopy. Ultrathin sections (30–40 nm) were cut and mounted on copper grids, stained with uranyl acetate and lead citrate, and examined on a Philips EM 301 electron microscope.
Statistical analysis
All presented data represent the mean and SD.
Statistical analysis was performed by using the Graphpad Instat
software. Student’s t test was used to determine the
statistical significance of the differences observed among groups for
the various parameters. When SDs were significantly
different between groups (as determined by the F test), therefore
preventing the use of Student’s t test, logarithmic
transformation of the data was made before statistical analysis, or
Welch’s alternate t test (33) was applied instead.
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Results |
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1B-receptor1B-adrenergic
receptor cDNA under control of the bovine thyroglobulin gene promoter
(Fig. 1
). Stable transgenic lines were established by
backcrossing the founder animals (DBA/2J x C57BL/6J F2 cross)
with the C57BL/6J parental line. The bovine thyroglobulin gene promoter
was shown previously in a number of transgenic models to be strictly
specific for the thyroid follicular cell (18, 26, 34, 35, 36). Expression
of the transgene was verified by Northern blotting, using an
1B-adrenergic receptor probe. 1B
transcripts could not be detected in the thyroid of control animals
(not shown) or in that of other transgenic lines displaying
undifferentiated (Tg-AgT) (26) or highly differentiated
hyperfunctioning goiters (Tg-A2aR; Fig. 2
(18). Abundant transcripts were found in the thyroid of transgenic
animals (Fig. 2). Binding assays were also performed to detect the
presence of 1B-binding sites in thyroid membranes of the
transgenic mice. Using the specific 1-adrenergic
receptor antagonist
2-{ß-(hydroxy-3-[125I]iodophenyl)ethylaminomethyl}-tetralone
as ligand, a small number of binding sites was found on wild-type mouse
thyroid membranes (mean ± SD, 9 ± 2 fmol/mg
membrane proteins). On membranes prepared from the thyroid of
transgenic animals (5 months old, line 51), 1-binding
sites were increased about 10-fold (mean ± SD,
115 ± 9 fmol/mg membrane proteins), demonstrating the functional
expression of the transgene (not shown). In these conditions, the
nonspecific binding was 8 ± 1 (mean ± SD)
fmol/mg membrane proteins. The direct measurement of IP3
and cAMP in thyroid tissue obtained from mice belonging to line 51
confirmed the expected dual stimulation of the
IP3/Ca2+/DAG and cAMP cascades (Fig. 3).
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Morphological analysis of the transgenic model
Animals of the six transgenic lines were killed at ages ranging
from birth to 24 months, and macroscopic or microscopic abnormalities
were searched for in the thyroid and other organs. Primary lesions were
restricted to the thyroid, as expected from the properties of the
thyroglobulin gene promoter directing the expression of the transgene.
The morphological changes, when present, were grossly similar in all
transgenic lines, but the timing of their appearance, and the extent
and frequency of alterations were variable from one line to another.
Structural abnormalities occurred earliest in line 51; the evolution
was somewhat slower in line 57 and was much delayed in line 66. In the
other three lines (no. 23, 36, and 40), the morphological alterations
were moderate and of late appearance. This evolution was correlated
with the serum thyroid hormone levels; lines 51 and 57 were the two
lines that exhibited severe hyperthyroidism from birth, whereas animals
from line 66 became hyperthyroid at later stages (Fig. 3
). The
evolution was also correlated with the abundance of transgene-derived
transcripts in the thyroid as determined by Northern blotting;
transcripts were more abundant in line 51 than in line 57 or 66 (data
not shown). We focused most of the analysis on the transgenic lines
displaying the strongest phenotype. Functional assays were performed
essentially on animals from line 51, histological data were collected
mostly from animals from lines 51, 57, and 66. We will first describe
the morphological changes observed in line 51. The variation in
phenotypic development in the other lines will be described later.
The thyroids from mice of line 51 were increased in size from the first
weeks of life, and some reached a weight of 130 mg by 12 months
(control, 2 mg). The enlargement was regular and symmetrical (Fig. 4, A and B), and the general organization into follicles
was preserved. The structure of the gland and the cell morphology were,
nevertheless, altered. Normal thyroid tissue consists of round or ovoid
follicles of relatively regular size and shape, lined with a cuboidal
epithelium. In transgenic animals expressing the mutated
1B-adrenergic receptor, clusters of densely packed cells
forming narrow or branched follicular-like structures (Fig. 4C) and
irregularly shaped follicles with numerous papillary infoldings (Fig. 4D) were frequent. Large follicles with weakly eosin- or periodic
acid-Schiff-stained colloid were also found. Abundant cell debris,
macrophages, monocytes, and neutrophils were present in the colloid.
Follicles were lined by flattened epithelial cells (Fig. 4F) or by
cylindrical or cuboidal cells (Fig. 4D). The cells sometimes displayed
a pseudostratified organization, with nuclei found at all levels of the
epithelium (Fig. 4E). Nuclei were round and regularly shaped, with a
normal chromatin organization (Fig. 4E). Necrotic cells with condensed
nuclei were frequently found in the epithelium or lumen (Fig. 4F). The
apical region of the cells was often protruding into the lumen and was
vacuolar, presenting an "empty" aspect (Fig. 4E). Ultrastructural
analysis attributed this clear aspect to the presence of large
cytoplasmic vesicles containing electron-lucent material (Fig. 5A) or markedly dilated cisternae of the rough
endoplasmic reticulum (Fig. 5B) that sometimes occupied most of the
cell volume. Electron-dense inclusions with homogeneous or
heterogeneous contents were frequent (Fig. 5, A and B). Mitoses (Fig. 5H) were found with a relatively high frequency (they are extremely
rare in the thyroid of adult controls). Bromodeoxyuridine incorporation
confirmed the higher proliferation rate of thyroid cells in transgenic
animals compared to controls (Fig. 3). Vascularization of the thyroid
was normal or moderately increased compared to that in control
animals.
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, C and B, and 6A). The mean size and number of
the nodules increased with the age of the animals, and their higher
proliferation rate was confirmed by BrdU incorporation (not shown). The
nodules were usually homogeneous and displayed various architectural
and differentiation patterns. They were generally more dense than the
surrounding tissue, and the structural organization included
follicular, microfollicular, papillary, trabecular, and solid masses,
with a higher frequency of papillary structures. The cell morphology
was sometimes similar to that of the surrounding thyroid gland, but
most of the nodules completely lost their clear cell aspect. In large
nodules, foci of cells were found, with large nuclei adopting
morphological criteria that characterize human papillary carcinomas
(ground glass aspect, pseudoinclusions; Fig. 6B). In a few cases, all
morphological criteria of thyroid follicular cell differentiation were
lost; spindle cell foci were found, without formation of identifiable
follicles (Fig. 6C). The nodules were never encapsulated. They
frequently acquired structural characteristics that are considered
malignant criteria for human thyroid tumors. In these cases, the
capsule surrounding the gland became markedly thickened, irregular, and
frequently invaded by the growing nodule (Fig. 6A); local invasion also
involved muscular tissue. Some of the nodules appeared highly
vascularized, to the point that areas of the gland were transformed
into vascular lakes filled with digitations of thyroid tissue (Fig. 6D). When this feature was encountered, invasion of blood vessels,
vascular emboli in large capsular or pericapsular veins (Fig. 6E), as
well as differentiated lung metastases (Fig. 6F) were frequently
observed. The overall frequency of lung metastases was estimated to be
20% in animals over 12 months of age.
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). In animals
more than 15 months of age, the largest goiters were found in mice
derived from line 66, and these goiters resulted generally from a
single large nodule per lobe. Some of the most malignant phenotypes
were also found in these mice (Fig. 6, C, D, and E), and the
circulating T4 levels generally correlated with the size of
the goiter. For the three other transgenic lines, mild alterations were
found in old animals, including a slight increase in thyroid size and
heterogeneity in follicle size and shape (not shown). Small papillary
nodules were occasionally found. None of these animals was
hyperthyroid.
Functional and proliferative status of the thyroid gland
Thyroid hormone levels reflected the functional activity of the
gland; they were elevated in most animals from lines 51 and 57 (Fig. 3). In line 51, young mice were hyperthyroid, but older mice with large
nodules were characterized by a wider range of T4
measurements, presumably resulting from the functional properties of
the individual nodules. As a result, the mean T4 level in
line 51 exhibited only a mild increase with age (Fig. 3). In line 57,
young animals were hyperthyroid as well, and mean T4 levels
increased strongly with age and the development of secondary nodules
(Fig. 3). In line 66, T4 levels were normal during the
first months of age and increased significantly in older animals
bearing large nodules (Fig. 3), suggesting that in this line, most
nodules were indeed functional.
Iodide uptake and organification were estimated after ip injection of
[125I]NaI in animals (1, 2, 6, and 13 months) belonging
to line 51. Uptake and organification were increased compared to those
in controls, in proportion to the weight of the gland (Fig. 3). The
efficiency of iodine incorporation into iodoproteins (protein-bound
radioiodine) was similar in transgenic and control animals at all ages
(Fig. 3).
The proliferation rate was assayed by measuring the percentage of cells
labeled after a single ip injection of BrdU. This measurement was made
on animals from line 51 at different ages (Fig. 3). Compared with
control animals, which show very little thyroid cell turnover after the
first few weeks of life, the labeling index of transgenic thyroids was
maintained at a higher level throughout life, although it decreased
progressively.
The expression level of thyroid-specific genes was
investigated by Northern blotting in the thyroids of mice transgenic
for the mutant 1B-adrenergic receptor and was compared
to that in other transgenic lines characterized by dedifferentiation
(simian virus 40 large T antigen expression, Tg-TAg) or
hyperfunctioning (A2a-adenosine receptor expression,
Tg-A2aR) of the thyroid cell (Fig. 2). Controls were tested
in parallel, but the amount of messenger RNA obtained was much lower
than that in the transgenic lines, and the results are not shown in the
figure. Thyroglobulin, thyroperoxidase, and TSH receptor transcripts
were all present in the thyroid of Tg-1BAR mice,
although they were less abundant than in Tg-A2aR mice.
Compared to controls, there was an increase in thyroperoxidase and TSH
receptor transcripts and a decrease in thyroglobulin transcripts (not
shown). The three differentiation markers were undetectable in Tg-TAg
mice, as previously described (26).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Constitutive activity of G protein-coupled receptors was first
described (23) for an 1B-adrenergic receptor mutated in
the third cytoplasmic loop (Arg288Lys,
Lys290His, and Ala293Leu). This mutant receptor
was characterized by a marked increase in affinity for agonists (but
not antagonists) and by a basal coupling to Gq in
the absence of agonists (23). Expression in NIH-3T3 cells demonstrated
the transforming potential of the mutant (29). This mutant was recently
used to generate transgenic mice under the control of the -myosin
heavy chain, leading to heart muscle-specific expression and cardiac
hypertrophy (24). The Ala293>Leu mutation has been shown
to contribute to the constitutive properties of this mutant receptor,
as any amino acid substitution at position 293 led to increased basal
activity (37). Although their main signaling pathway is through
phospholipase C, the 1B receptor, its mutants, as well
as other 1 receptor subtypes were shown to also activate
the cAMP pathway in tissues or transfected cell lines (22, 38, 39, 40)
(Cotecchia, S., unpublished observations) by direct (Gs
activation) and indirect mech-anisms.
1B mutant receptors, therefore, appeared as an ideal
means of constitutively activating phospholipase C and adenylyl cyclase
in transgenic mice. A hybrid gene was engineered by placing the mutant
receptor (23, 41) under the control of the bovine thyroglobulin gene
promoter. This Tg-1BAR construct was used to generate
six transgenic lines. The expression of the transgene was confirmed by
Northern blotting, and the presence of functional
1-adrenergic receptors in thyroid tissue was determined
by ligand binding assays. Direct measurements of cAMP and
IP3 levels in the thyroids of transgenic animals confirmed
the dual stimulation of adenylyl cyclase and phospholipase C by the
mutant receptor. The stimulation of the cAMP pathway, however, was
weaker than that in our previous Tg-A2aR model (18). This
was not unexpected from a mutant receptor whose main coupling is
through the phospholipase C pathway.
Mice belonging to the Tg-1BAR lines developed a
phenotype including hyperthyroidism and the development of thyroid
nodules, ultimately leading to malignant tumors. This phenotype,
however, was quite variable in its intensity and in the timing of its
appearance from one line to another. This variability was correlated to
the level of expression of the transgene. We reported previously, using
the bacterial chloramphenicol acetyltransferase gene as reporter gene,
that the expression from the thyroglobulin gene promoter in transgenic
mice is highly variable from line to line (34). Variability in the
expression level and in the severity of the phenotype was observed for
other transgenes expressed under the dependence of the same promoter
(18, 26, 36). Such variability is believed to be dependent on the site
of integration of the transgene in the mouse genome.
Animals from lines 51 and 57 were markedly hyperthyroid from birth,
whereas mice from line 66 developed hyperthyroidism only in association
with the growth of large nodules in their thyroid. The serum
T4 values of young animals (<5 months old) did not reach
the high levels that characterized Tg-A2aR mice (18), in
accordance with the lower stimulation of the cAMP cascade in the
Tg-1BAR model. Nevertheless, with the development of
nodules in older animals (>5 months old) from lines 57 and 66,
T4 values reached levels similar to those in the
Tg-A2aR model. The Tg-1BAR mice also
exhibited a high capacity for taking up iodine and incorporating it
into iodoproteins. As expected and similar to the situation in our
previous Tg-A2aR model, activation of the cAMP cascade by
the mutant 1B-adrenergic receptor resulted in
stimulation of thyroid function.
There were, however, marked differences between the present
Tg-1BAR mice and our previous Tg-A2aR model.
The thyroids from lines 51 and 57 were characterized by clear
follicular cells filled with large vacuoles resulting from organellar
degeneration and by a high frequency of cell necrosis. This was never
encountered in the Tg-A2aR model, in which the epithelium
lining the follicles was normal, and necrosis was exceptional.
Such degeneration of follicular cells has been shown to occur in
situations characterized by an excessive production of free radicals
(42). This aspect of the phenotype can be attributed to the stimulation
of the IP3/Ca2+/DAG cascade, in agreement with
the in vitro demonstration that phospholipase C stimulates
the H2O2 generation system and iodine
organification in thyroid cells (9, 43). Cell degeneracy was prominent
in line 51, where cell necrosis appeared to balance proliferation;
despite a high index of bromodeoxyuridine incorporation, the goiters
did not grow as large as in other lines (no. 57 and 66). In line 51,
many nodules that ultimately grew to a large size had often lost their
clear cell morphology, probably as a consequence of a selection
process. In support of this view, the T4 level in animals
from line 51 did not correlate with the size of the gland, suggesting
that most of the large nodules were poorly active. In animals from line
57, cell degeneracy was less pronounced, and the selection against
functional activity was reduced, as T4 levels increased
readily with the development of nodules. Clear cells were not observed
in young animals from the other lines in agreement with their overall
milder phenotype.
Another marked difference with the Tg-A2aR model was the rapid development of nodules, and their frequent evolution toward malignancy. Multiple nodules developed in lines 51 and 57, whereas animals from line 66 developed late, but very aggressive, functional nodules. Vascular invasion and lung metastases were relatively frequent in old animals. The higher malignancy of this model compared to that of the Tg-A2aR mice is presumably due to concomitant stimulation of phospholipase C and adenylyl cyclase. It has been shown in vitro that the DAG/protein kinase C arm of the cascade, if stimulated long enough, can promote the growth and dedifferentiation of thyroid cells (6). We demonstrate here in vivo that chronic phospholipase C stimulation can cooperate with the cAMP pathway toward proliferation and transformation, without affecting the differentiation of thyroid cells.
In conclusion, this work demonstrates that the permanent activation of both phospholipase C and adenylyl cyclase in the thyroid follicular cell of transgenic mice promotes as phenotypic characteristics stimulation of function, induction of cell degeneration attributed to the overproduction of free radicals, stimulation of proliferation, and tumor growth. Signs of malignancy, including vascular invasion, appear more frequently and earlier than in previous transgenic models. The present mouse lines constitute the most aggressive transgenic model of differentiated thyroid tumors to date, as the result of the expression of a single transgene. This model also demonstrates that membrane receptors coupled to phospholipase C may cooperate with the simultaneous stimulation of the cAMP cascade and contribute to the development of thyroid follicular cell malignancies. The possible relevance of our transgenic model for human cancer will require a search for genetic events (such as mutations in receptor genes) resulting in constitutive stimulation of the phospholipase C pathway in thyroid tumors.
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Acknowledgments |
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Footnotes |
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2 Chercheur Qualifié of the Fonds National de la Recherche
Scientifique of Belgium.
3 Present address: Institut de Pharmacologie et de Toxicologie,
Université de Lausanne, Bugnon 27, 1005 Lausanne,
Switzerland.
Received May 29, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-adrenergic receptors in the inhibitory
effect of catecholamines on the thyrotropin-induced release of
thyroxine by the mouse thyroid. Endocrinology 110:51–54[Abstract]
1A-
and 1B-adrenergic receptors mediate the effect of
norepinephrine on cytosolic calcium levels in rat PC C13 thyroid cells:
thyrotropin modulation of alpha 1B-linked response via a adenosine
3',5'-monophosphate-protein kinase-A-dependent pathway. Endocrinology 134:424–431[Abstract]
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M. D. Ringel, M. Saji, W. F. Schwindinger, D. Segev, M. A. Zeiger, and M. A. Levine Absence of Activating Mutations of the Genes Encoding the {alpha}-Subunits of G11 and Gq in Thyroid Neoplasia J. Clin. Endocrinol. Metab., February 1, 1998; 83(2): 554 - 559. [Abstract] [Full Text]
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