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Departments of Surgery (M.A.Z., M.S., Y.G., Y.T., W.C.D.), Pathology (W.H.W.), and Medicine (M.A.L.), Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; National Institute of Diabetes, Digestive, and Kidney Diseases (L.D.K.), National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Martha A. Zeiger, M.D., F.A.C.S., 600 North Wolfe Street, Carnegie 681, Department of Surgery, Division of Surgical Oncology and Endocrine Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-8611.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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s have been documented in subsets of thyroid
neoplasms. To test the oncogenic potential of activated Gs in
transgenic mice, we used the cholera toxin A1 subunit that
constitutively activates Gs and used the rat thyroglobulin gene
promoter for targeting this transgene (TGCT) to thyroid follicular
cells. Three (M1392, F1358, and F1286) of six founders identified were
able to transmit the transgene to their offspring and thyroid glands
from these mice contained elevated levels of cAMP. Concentrations of
serum thyroxine were elevated as early as 2 months of age (M 1392 and F
1286). F1358 mice were euthyroid until 8 months of age, at which time
they developed hyperthyroidism. All three TGCT lines developed thyroid
hyperplasia independent of their thyroxine levels. DNA image analysis
of thyroid follicular cells from both the hyper and euthyroid mice
showed that DNA index and "S+G2/M" phase were increased compared
with normal, changes similar to that seen in poor prognosis human
carcinomas. These data suggest that the Gs-adenylyl cyclase-cAMP
pathway has an important role in thyroid hyperplasia and the transgenic
mouse models reported herein will allow further examination of the role
of this pathway in thyroid oncogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s and G/G11) of the
adenylyl cyclase (AC) and phospholipase C (PLC) signaling pathways,
respectively (1, 2, 3). Several studies suggest that TSH is involved not
only in normal physiological growth but also in thyroid tumorigenesis
(4, 5). Indirect evidence implicating TSH in the development of thyroid
neoplasia includes work by Jemec in 1980 that showed thyroid hormone
administration and hypophysectomy prevented thyroid hyperplasia and
neoplasia in rats given the goitrogen, 1-methyl-2-mercapto-imidazol
(MMI) (6). Clinical evidence includes: 1) the observation that there is
an increased incidence of follicular and anaplastic carcinoma in areas
with concomitant endemic goiters (7, 8); and 2) that suppression of TSH
by administration of thyroid hormone decreases the local recurrence
rate as well as the rate of distant metastases in patients with thyroid
cancer (9, 10). Relevant to our study, activating mutations in both
Gs and the TSH receptor, which increase cAMP, have been documented
in hyperfunctioning adenomas and differentiated thyroid carcinomas
(11, 12, 13, 14, 15, 16), further supporting the involvement of the TSH-Gs-AC-cAMP
pathway in thyroid oncogenesis.
Two transgenic mouse models that examine the oncogenic potential of
constitutively elevated cAMP in the thyroid have been developed; one in
which Gs with an activating mutation
(GsR201H) is expressed (17), and one in
which the A2 adenosine receptor is overexpressed (18). Transgenic mice
expressing GsR201H develop
hyperfunctioning thyroid adenomas that are associated with elevated
cAMP levels, increased radioactive iodine uptake, as well as elevated
serum triiodothyronine and thyroxine levels. Transgenic mice
overexpressing the A2 adenosine receptor develop thyroid hyperplasia
and hyperthyroidism in association with a constitutively activated cAMP
cascade (18).
Because the GsR201H mouse model still expresses
endogenous, nonactivated Gs, and because signaling pathways
activated by the A2 adenosine receptor may exist that heretofore have
not been described, we chose to create a transgenic mouse model in
which, theoretically, Gs molecules are more specifically and more
completely activated. To accomplish this, we used the cholera toxin A1
subunit (CT) and placed it under the control of the thyroid-specific
thyroglobulin gene promoter (TG). Three transgenic mouse lines were
created that developed thyroid hyperplasia. Mice from two of these
lines were hyperthyroid as early as two month of age, and mice from the
third line were euthyroid until 8 months of age, at which time they
become hyperthyroid. The DNA index and proliferation fraction were
elevated in all three lines and aneuploidy, measured by DNA cytometry,
was similar to that seen in poor prognosis human carcinomas (19, 20, 21, 22).
We believe that our results both confirm and expand upon previous
studies that implicate the Gs-AC-cAMP pathway in thyroid neoplasia
(17, 18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The animal protocols (MO93M028 and MO96M164) have been approved by the Animal Care and Use Committee at Johns Hopkins University.
Transgenic mice, Southern analysis and PCR
We have previously detailed the creation of a functional pUC
plasmid containing the thyroglobulin gene promoter (TG), the cholera
toxin A1 subunit (CT), and polyadenylation signal sequence derived from
the human GH (hGH) gene (23, 24, 25, 26). Although the 3'-untranslated region
contains the entire hGH and polyadenylation signal sequence, hGH is not
expressed, because there is no promoter region for the hGH in this
transgene. In addition, hGH was not detected in the conditioned medium
of FRTL-5 cells transfected with this construct (24, 25, 26) nor in cell
suspensions from thyroid glands of the transgenic mice (data not
shown). The TGCT transgene was excised from pUC by digestion with
KpnI and EcoRI enzymes (Fig. 1
)
and microinjected into the pronucleus of single cell B6/C3 F1 mouse
embryos at the Johns Hopkins University Transgenic Core Facility (27).
The injected cells were then implanted into the oviduct of CD-1
pseudo-pregnant mice. Identification of transgenic founder mice was
done by PCR and Southern analysis of genomic DNA that was extracted
from tail biopsies of 3-week-old pups (28). Five-millimeter tail
biopsies were taken from each mouse, and DNA was extracted overnight at
37 C in the following buffer: 1% SDS, 50 mM Tris-HCl, pH
8, 20 mM NaCl, 1 mM EDTA, and proteinase K
(final concentration 1 mg/ml; Life Technologies, Gaithersburg, MD). The
quality of tail DNA was assessed by testing the ability to amplify the
gene for mouse milk protein (MWAP) (28). One microliter of a 1/10
dilution of this crude DNA solution was used for PCR amplification of
the TGCT minigene using the following protocol: 1 µl template was
mixed with 20 pmol of each primer, 200 µM each dNTP, 10
mM Tris-HCl, 1.5 mM MgCl2, 50
mM KCl, and 2.5 U Taq DNA polymerase in a volume
of 100 µl, and was amplified for 35 cycles consisting of denaturation
at 94 C for 1 min, annealing at 54 C for 1 min, and extension at 72 C
for 2 min. Final extension was continued for 7 min. The PCR products
were then electrophoresed on a 2% agarose gel in Tris-Borate-EDTA
buffer [100 mM Tris-HCl, pH 8.3, 100 mM boric
acid, and 2 mM EDTA (Biofluids, Inc., Rockville, MD)] and
stained with ethidium bromide. HaeIII-digested 
x174 RF
DNA (New England Biolabs) was used to determine size of PCR products.
For Southern blot analysis, genomic DNA was extracted from the crude
DNA preparations of mouse tail by phenol/chloroform extraction and
precipitated with isopropanol. 10 µg DNA in TE buffer (10
mM Tris-HCl, pH 7.5, and 0.1 mM EDTA) was
digested with DNA endonuclease, including BamHI and
EcoRI (New England Biolabs), size-fractionated on a 0.8%
agarose gel containing 1 µg/ml ethidium bromide, transferred to nylon
membranes (Schleicher & Schuell, Keene, NH) and probed with the
[32P]-labeled 3'-untranslated region of the TGCT gene
(Fig. 1). HindIII-digested 
page DNA (New England
Biolabs) was used to determine size of digested genomic DNA.
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cAMP production
Thyroid glands were removed from mice euthanized with 5–10 mg
pentobarbital (Abbott Laboratories, North Chicago, IL). Each thyroid
gland was incubated at 37 C for 1 h in 300 µl NaCl-free HBSS
containing 0.4% BSA and 0.5 mM isobutyl-methylxanthine
(25). After the addition of 1 ml 5% perchloric acid, samples were
homogenized and centrifuged to remove protein debris (1, 25).
Supernatants were recovered and neutralized with 5 M KOH
and recentrifuged to remove insoluble salts. Total cAMP from one
aliquot of the supernatant was measured by RIA (DuPont-New England
Nuclear, Boston, MA) (1, 25). Diphenylamine solution was added to the
pellets to measure DNA (25, 30). Values are reported as pmol cAMP/µg
DNA.
Histology
Thyroids glands were fixed in 10% formalin and embedded in
paraffin. Five-micrometer sections were stained with hematoxylin-eosin
stain and slides were reviewed by a single pathologist (WHW) in a
blinded fashion.
Image cytometry
Five-micrometer sections from formalin-fixed and
paraffin-embedded tissue were deparaffinized. Touch preparations from
fresh thyroid tissues were also used. They were Feulgen-stained (Becton
Dickinson, Elmhurst, IL) by hydrolysis in 4 N HCl at 28 C
for 1 h. Sections were then incubated in CAS DNA staining reagent
(Becton Dickinson) for 1 h at room temperature in the dark, rinsed
in 0.25% sulfite water and 0.5 N HCl three times for 5 min
each, air dried for 3 h, and mounted with Accu Mount 60 (Baxter
Healthcare Co., McGaw Park, IL) (31).
Feulgen-stained thyroid cells were analyzed using the computerized CAS 200 Image Cytometer (Becton Dickinson) with a 40x achromatic lens. 500 nuclei per slide were measured. Data were collected on total DNA, DNA index, nuclear size, nuclear shape and 20 separate Marcovian-Nuclear texture features. DNA histograms were analyzed with the CAS Cell Measurement program version 3.0 (Becton Dickinson). Ploidy value was determined as the major peak value of DNA Index histogram. The fraction of cells in S-phase and G2/M phases of cell cycle was estimated by standard modeling software.
Statistical analysis
All assays were performed in duplicate. Differences between
groups were evaluated by ANOVA. The statistical significance of the
differences was determined by Student’s t test or
Cochran-Cox’s test, when the variation in the data were uniform or not
uniform, respectively. Values were considered significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and Southern blot analysis (Fig. 3) of DNA from offspring. Two founder mice (M1363 and
F1394) were infertile and died at 1 and 5 months, respectively. M1382
was unable to transmit transgene DNA. Only three (M1392, F1286, and
F1358) of the six founders survived, were able to transmit the
transgene to their offspring, and form the basis of this report.
Southern blot analysis showed that these three founders carried between
1 and 3 copies of the transgene (Fig. 3). Because the B6/C3 mouse
strain exhibit spontaneous thyroid abnormalities, including thyroid
neoplasia in 2.3% of mice between 90 and 110 weeks of age (32), we
cross-bred the TGCT founders with FVB/N mice (33), a strain that
exhibits no thyroid abnormalities (34).
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). Serum levels of TSH were undetectable in these
hyperthyroxinemic mice, confirming hyperthyroidism (data not shown).
Mouse TSH from control mice between 2 and 4 months of age was 17.8
± 8.9 ng/ml (n = 14). Mice from the transgenic line F1358 were
euthyroid (T4, 6.0 ± 1.9 µg/dl, n = 15) for 4
months (Fig. 4), but some of the mice developed
hyperthyroxinemia after 8 months. The average thyroxine levels were
significantly lower in younger mice (
8 months, 6.4 ± 1.9
µg/dl, n = 17) than in older mice (
8 months, 10.0 ± 4.7
µg/dl, n = 13) (Fig. 5). Half the offspring from the
hyperthyroid lines (M1392 and F1286) were treated empirically with
0.05% methimazole (MMI) and 5 µg/ml thyroxine in their drinking
water. Females from of these lines were infertile unless treated with
MMI and thyroxine.
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cAMP levels
Thyroids from M1392, F1286, and F1358 mice that had not been
treated with MMI and thyroxine were examined. Basal levels of cAMP were
significantly elevated in all three lines: M1392 (1.65 ± 0.59
pmol/µg DNA, n = 3), F1286 (1.05 ± 0.82 pmol/µg DNA,
n = 3), and F1358 (0.96 ± 0.92 pmol/µg DNA, n = 8)
compared with control (0.08 ± 0.03 pmol/µg DNA, n = 6)
(Fig. 6). cAMP levels in thyroids from M1392 and F1286
mice were elevated as early as 2 months of age. In contrast, cAMP
levels in thyroids from the euthyroid F1358 mice increased in an
age-dependent manner after 3 months (Fig. 7). There was
no correlation between cAMP and T4 levels (data not
shown).
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) and
hyperplasia histologically (Fig. 9). More severe thyroid
hyperplasia was seen in the hyperthyroid M1392 and F1286 lines than in
the euthyroid F1358 line at the same age (2–4 months of age). One
striking feature seen microscopically was the papillary pattern
associated with intense mitotic activity, despite the absence of
infiltration, psammoma bodies or the cytological features that are
associated with papillary cancer (Fig. 9).
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DNA image cytometry
Three thyroids from each of the M1392 and F1358 lines
were examined by DNA image cytometry. All exhibited both an increase in
their DNA index and "S + G2/M" phase (Table 1),
consistent with the development of aneuploidy and an increase in
mitosis. The most dramatic changes were seen in one animal (M143) from
line M1392 whose thyroid had three aneuploid peaks (Fig. 10). These changes in DNA cytometry were similar to
that seen in poor prognosis human carcinomas (19, 20, 21, 22).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s (gsp) in up to 69% of well differentiated thyroid
carcinomas. Similarly, Zou et al. have demonstrated p53
mutations in both well differentiated and anaplastic thyroid carcinomas
(37). There also exists clinical and pathological evidence that well
differentiated carcinomas may progress to anaplastic carcinomas (38, 39). Two studies (39, 40) have demonstrated the presence of well
differentiated thyroid carcinoma in up to 50% of anaplastic carcinomas
on pathological sections. Additional supporting evidence that thyroid
tumors may arise via a series of mutational events includes the fact
that these tumors are of monoclonal origin (41, 42, 43) and therefore
likely result from multiple and complimentary mutational events (44),
although the possibility that a single hit may cause thyroid neoplasm,
cannot be excluded (45).
Activating mutations of the TSH receptor and Gs lead to elevated
cAMP levels and have been postulated to represent early events
associated with the development of thyroid neoplasia. In this study, we
created transgenic mice in which CT expression results in elevated
cAMP, hyperthyroxinemia, thyromegaly and thyroid hyperplasia. Although
there was no direct evidence by Northern analysis or Western blotting
that the CT was expressed, the data from PCR and Southern analysis
suggest correct integration of CT, and the documented cAMP elevation
indicates that the CT gene was indeed translated.
In addition to the current study, two previous transgenic models that
examined the Gs-AC-cAMP pathway (17, 18) lend credence to the
proposed hypothesis. In contrast to transgenic mice expressing an
activated form of Gs (GsR201H) that develop nodular
thyroid hyperplasia (17), TGCT mouse lines develop diffuse hyperplasia
and at an earlier age (2 months vs. 8 months, respectively).
Different promoters (bovine TG in the Gs model vs. rat TG in the
TGCT model) may account for this finding. The difference between these
two phenotypes may also be a result of the number of activated Gs
molecules or the extent to which it is activated: the TGCT transgene
activates all endogenous Gs protein in thyroid cells (26), whereas
in GsR201H transgenic mice, only a subset of Gs are
activated. Although the complete activation of Gs may not be
required to develop thyroid neoplasia, comparison of the degree of
activation may still be relevant. Likewise, the A2 adenosine receptor
transgenic model is associated with elevated cAMP levels (18), but the
A2 adenosine receptor pathway may be associated with activation of
other signal transduction pathways heretofore not described. In our
model, there was no concomitant stimulation of the PLC pathway (data
not shown). In this respect, our model may therefore provide a more
complete and more specific activation of Gs than the previous
models.
The basis for the different phenotypes exhibited by the 3 TGCT lines
remains unclear but may be explained by the different cAMP levels.
Thyroid glands from two lines (M1392 and F1286) showed high levels of
basal cAMP and hyperthyroidism as early as two month of age. In
contrast, mice from F1358 were euthyroid until 8 months of age with
correspondingly lower cAMP levels. The cAMP levels in F1358 thyroids
increased after 3 months and the mice developed hyperthyroidism after 8
months. Although there was no correlation between cAMP levels in the
thyroid glands and blood thyroxine level in F1358, cAMP level itself
may explain the variations in phenotype. Although cAMP elevation can
mimic TSH-induced iodide uptake in vivo (46, 47), its
elevation may require a longer time to cause hyperthyroidism. In
addition, activated Gs mutations are reported in nonfunctioning as
well as functioning thyroid tumors (11, 12, 13, 14), suggesting that elevated
levels of cAMP may not always be associated with increased hormone
synthesis. Further examination of protein kinase A and other distal
components of the cAMP pathway in younger euthyroid F1358 mice may
provide insight into this phenomenon.
We have previously reported that FRTL-5 cells permanently transfected with the TGCT transgene have markedly elevated levels of cAMP and undergo malignant transformation when implanted into nude mice (26). The transgenic mice in this study developed thyroid hyperplasia but showed no evidence of neoplasia. This discrepancy may reflect a difference in host immune cell recognition phenomena with rat thyroid follicular cells transfected with the TGCT minigene able to undergo unchecked malignant transformation in nude mice. Alternatively, although FRTL-5 cells maintain normal thyroid cell characteristics, including TSH-dependent growth and iodide uptake (48, 49), it is reasonable to presume that these immortalized cells already harbor another mutation(s), such that with the introduction of TGCT, the cells experienced an additional hit that confers a malignant phenotype (44). In support of this concept, a recent clinical report suggests (50) that elevation of cAMP alone may be insufficient to induce thyroid neoplasia. Our data confirm this hypothesis.
Both the histology and the DNA cytometry present in thyroid glands from our TGCT model are, however, consistent with early steps seen in thyroid neoplasia. This is supported by the fact that the aneuploidy peaks demonstrated in some of the TGCT thyroid glands are seen only in poorly differentiated human carcinomas (19, 20, 21, 22). There were no definitive features of papillary carcinoma seen in our model and yet, intense mitotic activity was observed, a feature associated with thyroid neoplasia and not hyperplasia (19, 20, 21, 22). With MMI and thyroxine treatment to prevent premature death from hyperthyroidism, thyroid malignancy may still develop over time in these transgenic lines.
In summary, we have both confirmed and extended the hypothesis that
Gs-AC-cAMP pathway is involved in thyroid hyperplasia. Although our
TGCT model of thyroid hyperplasia and hyperthyroxinemia suggests that
stimulation of the cAMP signaling pathway is insufficient to promote
malignant thyroid growth in mice, further studies are still needed to
fully understand the underlying mechanism of thyroid oncogenesis.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
Received January 30, 1997.
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) and F1286 (
)
show increased cAMP production as early as 2–3 months of age in
comparison with control (
) show a
slightly higher cAMP production within 100 days, and the cAMP
production increased in an age-dependent manner.
