Contrasting effects of activating mutations of GαS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells

Abstract

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

Full text

Contrasting eects of activating mutations of GaS and the thyrotropin
receptor on proliferation and dierentiation of thyroid follicular cells
M Ludgate
1
, V Gire
1
, M Crisp
1
, R Ajjan
2
, A Weetman
2
, M Ivan
1
and D Wynford-Thomas*
,1
1
Cancer Research Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Cardi CF4 4XN,
UK;
2
Department of Medicine, University of Sheeld, Clinical Sciences Centre, Northern General Hospital, Sheeld, UK
The cyclic AMP pathway is a major regulator of
thyrocyte function and proliferation and, predictably,
its inappropriate activation is associated with a sub-set of
human thyroid tumours. Activating mutations are,
however, more common in the thyrotropin receptor
(TSHR) than in its downstream transducer, Gas. To
investigate whether this re¯ects an inherent dierence in
their oncogenic potency, we compared the eects of
retrovirally-transduced mutant (A623I) TSHR or
(Q227L) Gas (GSP), using the rat thyroid cell line
FRTL5 and primary human thyrocytes. In FRTL5,
expression of GSP or mutant (m) TSHR induced a 2 ± 3-
fold increase in basal levels of cAMP. This was
associated with TSH-independent proliferation (assessed
by both cell number and DNA synthesis) and function (as
shown by increased expression of thyroglobulin (Tg) and
the sodium/iodide symporter). In primary cultures,
expression of mTSHR, but not GSP, consistently
induced formation of colonies with epithelial morphology
and thyroglobulin expression, capable of 10 ± 15 popula-
tion doublings (PD) compared to less than three in
controls. Thus, while mTSHR and GSP exert similar
eects in FRTL5, use of primary cultures reveals a
major dierence in their ability to induce sustained
proliferation in normal human thyrocytes, and provides
the ®rst direct evidence that mTSHR is sucient to
initiate thyroid tumorigenesis.
Keywords: TSH receptor; G protein; cyclic AMP;
oncogene; thyroid
Introduction
In physiological conditions, growth and function of
thyroid follicular epithelial cells are regulated princi-
pally by binding of thyrotropin (TSH) to its receptor
(TSHR) which then activates adenylate cyclase and the
cAMP cascade via the hetero-trimeric transducer
protein Gs (Vassart and Dumont, 1992). In a variety
of inherited and acquired pathological states, thyroid
hyperfunction and proliferation are associated with
activating mutations in elements of this signal pathway,
notably TSHR itself (Van Sande et al., 1995; Russo et
al., 1996; Parma et al., 1997; Fuhrer et al., 1997) and
the asub-unit of the Gs protein (O'Sullivan et al.,
1991; Russo et al., 1995a,b; Parma et al., 1997; Fuhrer
et al., 1997).
To date, two mutations in Gas (GSP) have been
identi®ed, Q227L and R201H, which activate the
protein by `locking' it in its GTP-bound signalling
conformation. In contrast, ligand-independent activa-
tion of TSHR can occur through point mutation at
more than 20 dierent sites, the majority clustered in
its trans-membrane and intra-cellular domains (re-
viewed in Paschke et al., 1994). In thyroid neoplasia,
point mutations are found, as would be predicted,
mainly in the sub-set of benign tumours which display
both increased growth and hyperfunction, i.e. autono-
mously functioning `toxic adenomas' or `hot nodules'.
More rarely they can also occur in cancers (Said et al.,
1994; Russo et al., 1995a; Paschke and Ludgate 1997)
which have elevated cAMP levels but, due presumably
to loss of dierentiation, do not exhibit increased
thyroid hormone secretion.
Interestingly, however, in all thyroid tumour types
the prevalence of mutation of GSP appears to be much
lower than that of TSHR despite the fact that both
might be expected to have the same ability to activate
the cAMP pathway. In four series totalling 114 cases of
`hot nodule' (O'Sullivan et al., 1991; Russo et al.,
1995b, 1996; Parma et al., 1997) the frequency of GSP
mutation varied from 0% (Fuhrer et al., 1997) to 38%
(O'Sullivan et al., 1991) with a weighted mean of 14%.
In contrast, TSHR mutation varied from 20% (Russo
et al., 1996) to 82% (Parma et al., 1997) with a mean
of 47%. Since this is probably an underestimate, given
the greater potential for missing mutations in TSHR,
the latter must be at least 3 ± 4-fold more common than
mutation of GSP.
This dierence in prevalence may of course itself
merely re¯ect the greater range of functionally
signi®cant targets for mutation in TSHR (at least 20
codons) compared to GSP (only two). More interest-
ingly, though, it could alternatively be due to an
inherently greater biological potency of mTSHR as an
oncogene, which may result from its ability to activate
signal transduction pathways additional to cAMP; it is
already known for example that at least some mutants
stimulate phospholipase C and the inositol phosphate
cascade (Parma et al., 1995). This issue has not been
adequately addressed.
Most signalling studies in this ®eld have been
performed, for ease of experimental manipulation, by
expression of mutant genes in non-thyroid rodent cell
lines (Parma et al., 1993; Paschke et al., 1994) which
are inappropriate for assessing their relative prolifero-
genic eects on thyroid cells. Even where cells of
thyroid origin have been used, this has nearly always
been the rat thyrocyte cell line FRTL5. In the case of
GSP, expression of the Q227L mutant conferred
complete TSH independence for DNA synthesis
*Correspondence: D Wynford-Thomas
Received 21 September 1998; revised 11 March 1999; accepted 11
March 1999
Oncogene (1999) 18, 4798 ± 4807
ã
1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc

(Muca and Vallar, 1994) but, interestingly, required the
addition of forskolin to achieve the same increase in
cell number as induced by TSH in control cells. Similar
results were obtained using the PDE inhibitor,
rolipram (Nemoz et al., 1995). TSH-independent
proliferation has also been seen with mTSHR, the
extent varying in one study with the nature of the
mutant (Porcellini et al., 1997), although it was not
clear whether even the most potent construct (T632I)
reached the level of TSH stimulated controls. In
another study, comparing the M453T mutant with
GSP, only the mutant receptor was able to induce
clones with anchorage-independence and tumorigeni-
city in nude mice (Fournes et al., 1998), providing a
®rst hint that mTSHR may be a more potent
oncogene.
Published FRTL5 studies all suer from limitations
however. Some were performed on pools of clones
(Porcellini et al., 1997) which, given the heterogeneity
of the response expected in gene transfer experiments,
can lead to information being lost due either to over-
growth of more vigorously growing clones or by a
dilution eect from clones with low expression of the
transgene. Others involved selection of individual
clones based on their ability to overgrow the back-
ground culture in the absence of TSH (Fournes et al.,
1998), which introduces a strong bias for the most
proliferative phenotype and risks masking dierences
in potency between GSP and TSHR. Furthermore,
most previous studies have examined only eects on
proliferation rather than dierentiated thyrocyte
functions. Finally, in FRTL5, TSH has little eect on
phospholipase C unless highly unphysiological concen-
trations of hormone are used (Vassart and Dumont,
1992), indicating that these cells may not be a good
model system to investigate phenotypic responses
dependent on this pathway.
Primary human cells are obviously the ideal
experimental model to test the ability of a putative
oncogene to initiate tumour development and we have
successfully employed retrovirus vectors to demon-
strate this for two other thyroid oncogenes, RAS and
RET (Bond et al., 1994). However, in a recent study
(Ivan et al., 1997) using a retroviral vector expressing
Q227L GSP, we found that despite performing
infections in a variety of conditions in which the
positive control ± mutant RAS ± generated large
numbers of colonies, expression of GSP in human
thyrocytes in primary monolayer cultures induced only
morphological responses (cytoskeletal changes and
cellular hypertrophy), with very little proliferation.
These in vitro ®ndings are in general in agreement with
in vivo studies in mice transgenic for the R201H GSP
mutant (Michiels et al., 1994) under the control of the
bovine thyroglobulin promoter. These mice developed
only late-onset sporadic thyroid nodules, rather than
the expected generalized hyperplasia, suggesting either
that only a tiny sub-population of follicular cells are
sensitive to this growth stimulus, or more likely that
there is a need for a second oncogenic event.
Taken together these data suggest that GSP
mutation alone may be insucient to account for the
formation of a thyroid tumour and that the observed
predominance of TSHR mutations may indeed re¯ect
its greater biological potency. The purposes of this
study were therefore to compare in more detail the
eects of GSP and mutant TSHR on growth and
function in FRTL5 cells, using a phenotypically neutral
method of clonal selection, and furthermore, to
compare their biological eects on human primary
thyrocytes.
Results
Eect of GSP, wild-type and mutant TSHR expression
on FRTL5 cells
Selection of clones following retroviral infection Mono-
layers of FRTL-5 (SB5 sub-clone; Burns et al., 1992)
were infected with retroviral vectors encoding: (i)
mutant GSP (rat Q227L); (ii) mutant TSHR (human
A623I); (iii) wild-type (wt) TSHR or (iv) neo-only
(control) genes. From each infection, several hundred
G418-resistant clones were obtained, from which six
were picked at random for preliminary analysis (data
not shown). The GSP clones were clearly very
heterogeneous in behaviour. In the absence of TSH,
two had growth rates comparable to that of neo
controls in the presence of TSH, while the remaining
four showed varying degrees of TSH dependence, two
of which also displayed increased cellular size (area)
and altered cytoplasmic morphology. Three clones
were selected to represent this range of phenotypes:
gsp6 (TSH-independent); gsp1 (partially TSH-depen-
dent); and gsp8 (partially TSH-dependent plus
morphological change). The six mTSHR clones were
similarly heterogeneous, and again, three representative
clones were selected for detailed study: MT1 (TSH-
independent); MT2 (partially TSH-dependent) and
MT3 (partially TSH-dependent with morphological
change). The six wtTSHR clones were much more
homogeneous. None proliferated in the absence of
TSH and there were no morphological changes. Three
were therefore selected at random: WT1, WT4 and
WT6 (in some experiments only the last two were
included). The neo-only clones were also homogeneous
and so to limit the total number of clones which
needed to be analysed in later experiments, these were
pooled to form a single control population (neo).
Eects on proliferation
Cell number (Figure 1a and b; Table 1) Control (neo)
cells in the absence of TSH showed a consistent initial
reduction of *40% in cell number at day 2 (due
presumably to failure of some cells to attach).
Thereafter there was a slow recovery, returning by
day 7 to the number seeded. In the presence of TSH
(5 mU/ml) there was no initial cell loss, and near-
exponential growth leading to a 6 ±7-fold increase by
day 7.
In the absence of TSH, the wtTSHR clones behaved
almost identically to the neo population. In contrast,
both the GSP and mTSHR clones all showed some
degree of TSH-independence. Although all but two
(gsp6 and 8) suered the initial cell loss, this was
followed by a period of exponential growth with
doubling times similar to that of TSH-stimulated neo
cells (with the exception of gsp8). Cell numbers
attained by day 7 were therefore intermediate between
those seen in neo controls in the absence and presence
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4799

of TSH. The highest values, displayed by clones gsp6
and MT1, were not signi®cantly lower than that of neo
plus TSH.
In the presence of TSH (Figure 1b), the initial loss in
cell number was less marked but interestingly nearly all
GSP, wt and mTSHR clones still showed lower
numbers than neo controls at day 2. Thereafter,
though, all clones grew at rates similar to or greater
than neo, and reached larger ®nal cell numbers at day
7, the highest values being seen in the three mTSHR
clones.
DNA synthesis (Table 1) In the absence of TSH,
*30% of the control (neo) cells incorporated BrdU
during a 24 h labelling period corresponding to days
3 ± 4 of the growth curves. This was surprising given
that no increase in cell number occurred during this
time and presumably re¯ects either ongoing cell death
or DNA synthesis without cell division (endoredupli-
cation). In the presence of TSH, the BrdU LI increased
to *70%.
In the absence of TSH, clones expressing wt TSHR
had an LI very close to that of the neo control. In
contrast, all the GSP and mTSHR clones showed much
higher values, in two cases (gsp6 and MT1), reaching
levels not signi®cantly lower than that seen in neo in
the presence of TSH.
Eect on tissue-speci®c dierentiation
Morphology In the absence of TSH, control neo cells
exhibited a ¯attened morphology which was restored to
Figure 1 (a) Growth curves of FRTL5 (SB5) clones in the absence of TSH. Individual GSP, wtTSHR (WT) or mTSHR (MT)
clones were seeded at 5610
4
per dish and counted on days 2, 4, 7 and 11. For comparison, results with the pooled neo population
are also shown in the presence or absence of TSH (5 mU/ml); (b) Growth curves in the presence of TSH (legend as for (a)). Note
logarithmic ordinate scale; s.e. values omitted for clarity
a b
Table 1 Eects of gsp, wt and mTSHR expression on proliferation and function of FRTL5 (SB5) cells compared to the eect of TSH
stimulation
Proliferation Function
Clone
a
TSH cAMP
b,c
DT
d
(h)
Cell no
e
Day 2
Cell No
e
Day 7
BrdU
f
LI Tg
g
NIS
h
7IBMX
NIS
h
+IBMX
Neo
Neo
7
+
100
230+8
70
43
64+5.8
132+12
114+8.2
614+76
31+2.0
72+2.5
38+0.8
51+1.5
4.0+0.12
304+9.2
320+13
120+2.9
WT1
WT4
WT6
7
7
7
160+8
80+9
110+10
ND
92
46
ND
58+6.4
42+7.0
ND
110+19
100+6.6
29+1.5
30+2.0
28+2.0
ND
62+2.5
59+2.0
4.0+0.3
1.0+0.12
1.0+0.12
130+3.5
12+1.2
60+3.5
MT1
MT2
MT3
7
7
7
285+8
230+14
330+13
30
48
38
58+7.4
36+5.4
44+2.8
456+30
154+11
232+20
70+4.5
51+3.0
50+3.0
73+3.5
66+3.0
70+4.0
6.0+0.12
29+2.3
13+1.7
60+2.9
230+9.2
310+9.2
gsp1
gsp6
gsp8
7
7
220+9
300+17
240+13
45
35
114
56+9.4
96+2.0
142+2.4
212+24
472+40
276+46
47+3.5
65+5.5
50+4.0
48+8.0
70+6.0
63+5.0
15+1.2
140+10
16+0.58
200+4.6
17+2.9
160+4.0
a
`Neo' are pooled clones.
b
All results are expressed as mean of three or four replicates+s.e.
c
Per cent of basal level in neo controls.
d
Doubling
time calculated from steepest part of growth curve.
e
Per cent of number seeded at day 0.
f
Per cent of nuclei labelled after 24 h incubation with
BrdU.
g
Per cent of cells positive by immuno¯uorescence.
h
Ratio of NIS: b-actin transcript abundance (610
2
)
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4800

the normal, more cuboidal, shape within 24 ± 48 h of
addition of TSH. In the absence of TSH, the wtTSHR
clones closely resembled the neo controls, but GSP
clones were less ¯attened and the mTSHR clones
resembled TSH-stimulated controls.
During the period of initial selection in G418, it was
noticed that several groups of cells became enlarged
and ¯attened and displayed ®brillar changes in the
cytoplasm reminiscent of `stress ®bres'. As selection
proceeded, these changes disappeared in most clones,
but remained a prominent feature in gsp8 and MT3
(Figure 2).
Basal cAMP The basal level of cAMP production in
control neo cells in the absence of TSH was
*30 fmoles/mg protein. Only one of the wtTSHR
clones (WT1) had a signi®cantly higher level (160% of
control; P50.01) (Table 1). In contrast, all of the
GSP and mTSHR clones had basal levels ranging
from 220 ± 330% of the neo control and similar or
greater than those seen in neo cells in the presence of
TSH.
Thyroglobulin expression In the absence of TSH,
weak cytoplasmic immuno¯uorescence was observed
with anti-Tg antibody in *40% of control neo cells,
which increased to *50% in the presence of TSH,
accompanied by a marked increase in intensity (Figure
3). In contrast, in all TSHR or GSP expressing clones,
even in the absence of TSH, the percentage of Tg-
positive cells was similar to or higher than that seen in
controls in the presence of TSH (Table 1). Surprisingly,
this also included the two wtTSHR clones, whose
cAMP levels were not signi®cantly greater than the
control values. The highest percentage of positive cells
however was observed in the GSP and mTSHR clones,
which also showed more intense ¯uorescence than the
wtTSHR cells (Figure 3).
Expression of the Na
+
/I
7
symporter gene (Figure
4) In the absence of TSH, transcripts were often
below the limit of detection by RT ± PCR in control
neo and wtTSHR clones. In contrast, detectable levels
were present in all GSP and mTSHR clones. The
highest levels were seen in MT2 and gsp6, the latter
approaching that of TSH-stimulated neo controls
(Table 1). Results in the presence of the PDE
inhibitor, IBMX, were largely as expected with an
increase in the NIS transcript in the majority of cases
(Figure 4b; Table 1). The exceptions were the two
conditions showing highest levels in the absence of
IBMX, i.e. gsp6 and neo plus TSH, in which a
paradoxical reduction of NIS transcript levels was
observed.
Eects of GSP, wt and mutant TSHR expression on
primary human thyrocytes
Primary thyrocytes in monolayer culture were infected
with retroviral vectors either in 10% FCS or using a
reduced-serum medium (1% FCS, supplemented with
insulin and TSH) in an attempt to avoid the potential
de-dierentiating eect of serum on thyroid cells. In
both cases, a well-characterized retroviral vector
encoding mutant H-RAS (Bond et al., 1994) was used
as a positive control.
Colony formation in 10% FCS (Figure 5) In 10%
FCS, the H-RAS vector generated approximately 20
well-dierentiated epithelial colonies per dish of
5610
5
cells infected using thyrocytes from three out
of four fresh and three out of eight stored samples
(from 12 independent subjects). Most colonies were
able to proliferate for at least 20 PD. The neo-only
vector as expected failed to generate any colonies,
consistent with the very limited proliferative capacity
of normal human thyrocytes (Wynford-Thomas,
1993). GSP also failed to induce any colony
formation in 12 out of the 13 thyrocyte preparations
infected and in the remaining case very few colonies
were obtained.
In contrast, the mTSHR vector generated 10 ± 15
colonies of epithelial morphology per dish from four
out of ®ve fresh and two out of eight stored thyrocyte
samples. Typically, colonies grew for several weeks and
reached a maximum size of 1000 ± 30 000 cells,
equivalent to 10 ± 15 PD (assuming no cell death).
Cessation of growth was associated with alterations in
cellular morphology, characterized by spreading,
¯attening and appearance of cytoplasmic stress ®bres
with occasional pseudopodia (Figure 5). Similar results
were obtained when thyrocytes derived from the same
thyroids were infected with the wtTSHR vector,
although with a lower yield (2 ± 5 colonies per dish).
Figure 2 Phase contrast micrographs showing altered cellular morphology in FRTL5 clone gsp8 (2) compared to neo controls in
the presence of TSH (1). (6100)
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4801

It must be emphasized that these dierences in yield
of colonies are not explicable by dierences in the titre
of the corresponding vectors, at least as far as can be
assessed using epithelial cell lines (FRTL5 and A431)
as targets.
Colony formation in 1% FCS In 1% FCS colonies
were obtained from two out of ®ve freshly
disaggregated thyrocytes with a similar yield to that
seen with 10% FCS, i.e. approximately 20 H-RAS,
10 mTSHR and two wtTSHR colonies per dish of
5610
5
infected. Proliferative capacity however was
greatly reduced, being limited to no more than ®ve
PD, even in the RAS colonies. In the case of TSHR
this was accompanied by an early onset of
morphological alterations including cellular ¯attening
and cytoskeletal changes. (Similar results were
obtained irrespective of the inclusion of TSH at 0.03
or 5 mU/ml up to 1 day following retroviral
infection.) Infection of stored follicles in 1% FCS
was unsuccessful in all but one case due to poor cell
attachment.
Expression of thyroglobulin To con®rm the cellular
identity of the colonies obtained from primary culture,
expression of Tg was analysed by immuno¯uorescence.
In all colonies obtained with wt or mTSHR
cytoplasmic positivity was observed in the majority of
cells (Figure 6). As observed with FRTL5, the intensity
of immunostaining was greater with mTSHR than with
wtTSHR as was the percentage of positive cells
(74+4.5% and 51+4% respectively).
Figure 3 Thyroglobulin expression in FRTL5 detected by immuno¯uorescence (rhodamine label). All in the absence of TSH unless
otherwise indicated. (1) negative control (neo with ®rst antibody omitted); (2) neo; (3) neo+TSH; (4) clone WT 4; (5) clone MT1;
(6) clone gsp6. (6150)
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4802

Discussion
Activated GSP and TSHR both confer TSH-independent
growth and function on FRTL5 cells
As expected, in FRTL5 (SB5) cells, expression of either
activated Gas or TSHR was able to stimulate cAMP
production and thyroglobulin expression, to levels
similar to that induced by TSH. For Tg, a signi®cant
increase was also induced by over-expression of
wtTSHR, re¯ecting its limited constitutive activity
(Van Sande et al., 1995; Parma et al., 1993; Duprez
et al., 1994) but interestingly this was not associated
with any increase in cAMP. We also investigated
expression of another recently cloned thyroid-speci®c
gene ± the Na
+
/I
7
symporter (Dai et al., 1996) ±
although the non-availability of antibodies restricted
this to assessment of transcript abundance. Increased
expression was seen in some GSP and mTSHR clones
but to a more limited extent than for Tg, remaining
well below the TSH-stimulated value in all but one
clone. Since the corresponding cAMP levels were
higher than those of TSH-stimulated controls this
indicates the need for additional signal pathways.
Furthermore, the inability of mTSHR to mimic
extracellular TSH stimulation (the only example of
this in our study) points to the involvement of
pathway(s) whose stimulation by ligand binding must
be quantitatively or qualitatively dierent from that
following mutational activation. Nevertheless, NIS
transcript levels approaching that induced by TSH
could be achieved by inclusion of IBMX, implying that
cAMP can be sucient at supra-physiological levels.
Interestingly though, the paradoxical fall in NIS on
addition of IBMX in conditions where it was already
highly expressed (gsp6 and neo plus TSH) indicates
that the optimal level of cAMP may be quite critical.
Excessive levels are perhaps inhibitory through
activation of ICER, an alternatively spliced form of
the CREM transcription factor, which has already
been demonstrated in the FRTL5 cell line (Lalli and
Sassonecorsi, 1995).
GSP and mTSHR (but not wtTSHR) also conferred
TSH-independent proliferation, as assessed both in
terms of DNA synthesis and increase in cell number.
Large variations were observed within each group, only
one GSP and one mTSHR clone approaching complete
TSH-independence. It is unlikely that this relates to
dierences in expression of the mutant genes since (in
contrast to one previous study (Porcellini et al., 1997))
there was no consistent relationship to cyclic AMP
levels. Expression was not measured directly here
because basal cAMP was considered a more mean-
ingful biological endpoint, and the only way to
Figure 5 Phase contrast micrographs of human primary
thyrocytes expressing mutant H-RAS or mTSHR in 10% FCS.
(1) Typical epithelial colony induced by mutant RAS; (2) typical
early mTSHR-induced epithelial colony; (3) mTSHR colony
following several weeks in culture, showing cellular enlargement,
¯attening and prominent cytoskeletal striations. (6200)
Figure 4 Duplex RT ± PCR analysis of sodium/iodide symporter
(NIS) and b-actin transcripts in representative FRTL5 clones
expressing mutant GSP, mutant TSHR or wild-type TSHR, in the
absence (a) or presence (b)of2m
MIBMX (all in the absence of
TSH). A pooled control population (neo) in the presence or
absence of TSH is shown for comparison. Note that the ®gure
shows just one of three replicate analyses, and therefore does not
match exactly the means shown in Table 1
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4803

eectively compare between GSP and mTSHR eects.
The important ®nding for this study is that despite the
inter-clonal variation, there was no signi®cant differ-
ence in the mean cAMP level or TSH-independent
proliferative response between the GSP and TSHR
clones, the respective basal cAMP levels (% of neo
control) being 281+29% and 253+24%, with
corresponding BrdU LI values of 57+5.6% and
54+6.5% (data derived from Table 1).
Similar TSH-independent stimulation of cAMP and
proliferation have been reported separately both for
GSP (Muca and Vallar, 1994) and a range of TSHR
mutants (Porcellini et al., 1997) but few functional
parameters were examined. The only previous study to
attempt a direct comparison between GSP and TSHR
clones obtained from the same parent population
(Fournes et al., 1998) concluded that GSP exerted a
weaker eect than mTSHR (M453T) in terms of cAMP
stimulation and doubling time in monolayer. However
the former result was obtained in the presence of
IBMX, and the latter may have been in¯uenced by the
method of clonal selection (TSH-independent prolifera-
tion rather than `neutral' selection in G418).
On the basis of behaviour of FRTL5 cells in
monolayer culture, the concensus from both the
present and previous work is therefore that there is
little or no evidence for dierence in the biological
potencies of activated GSP and TSHR on which to
explain their dierent mutational frequencies in thyroid
tumours.
GSP and mTSHR exert very dierent eects on normal
human thyrocytes
In contrast to the above, our data on primary cells
reveals a massive dierence in the ability of mTSHR
and GSP to induce sustained proliferation in normal
thyrocytes. Unlike the FRTL5 line, these cells are
normally capable of only a few cell divisions in culture,
even in the presence of TSH (Dumont et al., 1992;
Wynford-Thomas, 1993). Whereas expression of GSP
nearly always failed to stimulate proliferation, expres-
sion of mTSHR consistently generated colonies of Tg-
positive epithelial cells which were able to proliferate
for up to 15 PD Interestingly, although fewer in
number, similar colonies could also be obtained by
over-expression of the wtTSHR. Presumably, as was
seen with Tg expression in FRTL5, this re¯ects its
weak but signi®cant constitutive activity (Van Sande et
al., 1995) although, if so, it implies that the normal
cells are more responsive than FRTL5, since no TSH-
independent growth was observed with wtTSHR in the
latter. Unfortunately, in the primary culture model, it
was not possible to further investigate this by
measurement of receptor expression or cAMP levels.
Constitutive TSHR activity provides a signal not
mimicked by GSP or physiological TSH stimulation
The two major questions arising from the results with
primary thyrocytes are: ®rstly, why GSP does not
produce an eect equivalent to that of mTSHR and
secondly, why is the latter so much more potent as a
growth stimulus than stimulation by extracellular TSH.
One potentially artefactual explanation for the
ineectiveness of GSP is that forced expression of a
mutant Gas sub-unit does not mimic exactly the
naturally occurring mutation in the endogenous gene
because it does not lead to a corresponding release of
free b/gsub-units. The latter can act in their own right
as signal transducers, interacting for example with PI-
3-kinase and tyrosine kinases (Van Biesen et al., 1996),
and could theoretically be required for the mitogenic
response in primary cells. However, this would not
explain the lack of sustained proliferation following
administration of agents, notably cholera toxin
(Wynford-Thomas, 1993) which activate endogenous
Gs and closely mimic the eect of Gas mutation.
It seems most likely therefore that the essential signals
missing in the case of GSP are derived from a pathway
which diverges upstream of Gs, i.e. at the receptor itself.
It is known that TSHR activates pathways additional to
cAMP, notably the phospho-inositide pathway (via Gq
and phospholipase-Cb) (Vassart and Dumont, 1992)
and at least in one cell line model (Kupperman et al.,
1993) there is evidence that cross-talk with RAS is
involved in the mitogenic response. What is puzzling
though is how expression of mTSHR can lead to
sustained proliferation, whereas stimulation of normal
thyrocytes by extra-cellular TSH does not lead to more
than two or three PD either in vitro (Wynford-Thomas,
1993) or in the intact animal (Wynford-Thomas et al.,
Figure 6 Thyroglobulin expression in colonies of human thyrocytes derived by infection with vectors expressing: (1) wtTSHR; and
(2) mTSHR. Rhodamine immuno¯uorescence (6150)
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4804

1982). One plausible explanation is that constitutive,
ligand-independent, receptor signalling may not invoke
receptor downregulation which is a well recognized
response to ligand-mediated activation (Rapoport et al.,
1982; Lalli and Sassonecorsi, 1995). (Use of a
heterologous promoter to drive TSHR in this and
previous studies would, of course, circumvent the
transcriptional component of down-regulation (Lalli
and Sassonecorsi, 1995) but would not prevent receptor-
G protein uncoupling (Rapoport et al., 1982)).
Although in FRTL5, down-regulation is also known
to occur (Lalli and Sassonecorsi, 1995), its negative
in¯uence must be of insucient magnitude to prevent a
sustained proliferative response to TSH. Intriguingly
though, dierential sensitivity of ligand-dependent and
-independent stimulation to downregulatory control
could provide an explanation for the observation
(Fournes et al., 1998) that anchorage-independent
opposed to monolayer growth of FRTL5 was
strikingly stimulated by mTSHR, but not by TSH-
stimulation of the wt receptor (nor by GSP).
TSHR activation as an initiator of thyroid tumorigenesis
Irrespective of the above issues, our data show for the
®rst time that mTSHR is capable of driving clonal
expansion of normal human thyrocytes, providing
direct experimental support for its role as an initiating
event in thyroid tumorigenesis, analogous to that
already demonstrated by us for two other oncogenes
± RAS and RET (Bond et al., 1994). As with the latter,
proliferation eventually ceased, although after fewer
PD, following which cells enter a state of growth arrest
with morphological changes reminiscent of replicative
senescence (Wynford-Thomas, 1997a). The mechanism
of this intrinsic limitation of clonal expansion is
currently unknown although preliminary data suggest
that, at least in the case of RAS, increased expression of
the cyclin/CDK inhibitor p16
INK4a
plays a role (Bond JA
-unpublished). Comparison of mTSHR colonies in 1%
versus 10% FCS shows that the timing of this lifespan
checkpoint can be modulated by the growth factor
environment. Although the colony sizes observed in
vitro would probably not be sucient to account for a
clinically-evident tumour, this raises the important
possibility that the environment in the intact organ
may permit signi®cantly more PD to be achieved.
Clearly though, further tumour development beyond
the initial self-limiting clone must be dependent on
mutation of additional growth-regulatory genes, such
as p16
ink4a
(Wynford-Thomas, 1997a,b). If, as suggested
by our data, TSHR mutation usually generates smaller
clones in vivo than does RAS, the chance of such a
second event will be correspondingly smaller. This may
be one explanation for the relative rarity of tumours
initiated by TSHR mutation compared to RAS (when
all tumour types are considered together), despite the
much larger number of target sites for activating
mutations in the TSHR gene.
Materials and methods
Production of retroviral vectors
The rat Q227L GSP and H-RAS amphotropic retroviral
vectors driven by a MoMuLV LTR have been described
previously (Bond et al., 1994; Ivan et al., 1997). To construct
TSHR vectors, cDNAs for the entire coding sequences of the
wild-type (wt) and A623I mutant receptor (kindly provided
by Dr G Vassart, IRIBHN, Brussels) were subcloned into
plasmid pLNSX, which utilizes the SV40 early promoter for
the gene of interest. (Attempts to clone mTSHR cDNA into
LTR-driven vectors were, for unknown reasons, unsuccessful.
However, previous comparison of the two promoters driving
SV40T expression in primary thyrocytes did not reveal any
detectable dierences in strength ± Bond JA, unpublished.)
The resulting constructs, and the pLNSX control were
transfected into the OE ecotropic packaging line and
retroviral supernatants from pooled G418 resistant clones
subsequently used to infect the amphotropic packaging line,
Ccrip (Danos and Mulligan 1988). Supernatants from a panel
of G418 resistant Ccrip producer clones were evaluated using
the human epithelial cell line A431 as a recipient. Viral titre
was assessed in terms of the yield of G418 resistant colonies
and, in the case of the TSHR vectors, stable expression was
determined by measurement of speci®c [
125
I]TSH binding
(Costagliola et al., 1994). The optimum producer clones, all
with titres 410
6
c.f.u./ml, were designated MLWT1 and
MLAI1 for the wt and A623I mutant TSHR respectively, and
MLSX1 for the LNSX neo-only control.
Cells and culture conditions
The SB5 subclone (Burns et al., 1992) of FRTL5, selected for
its TSH dependency as previously described, which are
referred to as `FRTL5' throughout. Cells were routinely
cultured in a 2 : 1 : 1 mixture of DMEM, Ham's F12 and
MCDB 104 supplemented with 5% calf serum (Life
Technologies, Paisley, UK), 5 mU/ml bovine TSH (Sigma;
UK), 10 mg/ml insulin, 10
78
Mhydrocortisone, 45 mg/ml
ascorbic acid and 5 mg/ml transferrin (`5H medium'). For
experiments in basal conditions, cells were ®rst maintained in
the same medium but lacking TSH (`4H medium') for at least
4 days.
Human primary thyrocytes were obtained by protease
digestion of histologically-normal background tissue obtained
from thyroidectomies performed for solitary nodules as
previously described (Williams et al., 1988). Monolayer
cultures were maintained in a 2 : 1 : 1 mixture of DMEM,
Ham's F12 and MCDB104 with 1% or 10% FCS (Life
Technologies, Paisley, UK). In 1% FCS cultures, 10 mg/ml
insulin and 5 mU/ml or 0.3 mU/ml bovine TSH were also
included. Primary cultures from 13 dierent thyroids were
used. Five were freshly disaggregated; in the remainder
follicles had been stored frozen in DMSO prior to use.
Retroviral infection
Forty-eight hours after seeding in 5H medium, FRTL5 cells
were pretreated for 1 h with 8 mg/ml polybrene and then
exposed to retroviral supernatants for 2 h before refeeding
with fresh medium. Cultures were passaged the following
day, and after a further 24 h G418 selection commenced. Six
G418-resistant clones from each GSP, wtTSHR and mTSHR
infection were isolated, at random, using cloning rings.
Pooled G418 resistant clones derived from the MLSX1
infected cells were used as a control neo-only population.
Primary human thyrocytes were similarly infected with
retroviral supernatants 48 h after plating, and G418 selection
started after a further 48 h. For experiments performed in
1% FCS, cultures were maintained in the presence of insulin
throughout, but TSH (0.3 or 5 mU/ml) was included only up
to the day following retroviral infection.
Proliferation assays (FRTL5 only)
Growth curves 5610
4
cells were seeded as multiple
replicates in 24 well plates in 4H or 5H medium. Triplicate
GSP and TSHR mutants in thyrocyte function and proliferation
MLudgateet al
4805

wells were trypsinized and counted at 2, 4 and 7 days and
also at 11 days for cultures in 4H. Results are expressed as
the mean of triplicates+standard error (s.e.).
DNA synthesis 5610
4
cells were seeded in 35 mm
dishes, in 4H or 5H medium. Seventy-two hours later
10 mMBrdU was added for a further 24 h, following
which monolayers were ®xed in 3.7% formaldehyde
(15 min), permeabilized (0.2% Triton-X100) and immu-
nostained using a mouse monoclonal anti-BrdU primary
antibody (Boehringer-Mannheim), in the presence of
10 U/ml DNAse followed by ¯uorescein-conjugated goat
anti-mouse IgG (Southern Biotechnology). The percen-
tage of labelled nuclei (LI) was determined from a count
of 4300 per dish and results expressed as means of
quadruplicate dishes+s.e.
cAMP assay
Five610
4
cells were seeded in 24 well plates in 4H medium.
72 ± 96 h later, the medium was replaced with Ham's F12
containing 1% BSA with varying concentrations of bovine
TSH. After 4 h incubation cells were extracted in HCl, and
extracts evaporated to dryness before resuspension in buer
for cAMP determination using a commercial radio-
immunoassay (Amersham, UK). Dierences in growth rate
of the clones were corrected by measuring total cellular
protein using a Bradford assay. Experiments were
performed in quadruplicate and results expressed as a
percentage (+s.e.) of the level in control cells in 4H
medium.
Detection of thyroglobulin by immunocytochemistry
Monolayers were ®xed with methanol (10 min, at 7208C),
permeabilized with 0.1% Triton-6100 in PBS (15 min), and
non-speci®c antibody binding blocked with 10% FCS in PBS
(30 min). Cells were then incubated with a rabbit polyclonal
anti-human thyroglobulin antibody (Dako, Carpinteria CA,
USA) at a 1 : 500 dilution for 1 h, followed by rhodamine-
conjugated goat anti-rabbit IgG (Southern Biotechnology)
(1 h). The percentage of cells containing immunodetectable
cytoplasmic Tg was scored for at least 300 cells per dish and
results expressed as mean+s.e. of quadruplicate dishes (or
colonies in the case of primary cells).
Semi-quantitative RT ± PCR analysis of NIS expression
Cells were grown to con¯uence in 6-well plates, in 4H or 5H
medium+IBMX. RNA was extracted (Chomczynski and
Sacchi, 1987) and reverse-transcribed using an oligo-dT
primer (Ajjan et al., 1998). PCR was performed in a 50 ml
reaction containing 2.5 units Taq polymerase (Promega),
0.1 mMof each dNTP, and 1.5 ml of cDNA, in a buer
consisting of 1 mMMgCl
2
,10mMTris HCl, 0.01% gelatin,
50 mMKCl, 0.1% Tween and 0.1% NP40. Ampli®cation was
performed using 30 cycles of 948C, 1 min; 558C, 1 min; and
728C, 1 min. The NIS primers were designed using the
published rat sequence (Dai et al., 1996) as follows: forward:
5'-CTG CGA CTC TCC CAC TGA-3'and reverse: 5'-CGC
AGC TCT AGG TAC TGG TA-3'. In addition, the
following b-actin primers were used as an internal standard:
forward: 5'-GTG GGG CGC CCC AGG CACCA-3'and
reverse: 5'-CTC CTT AAT GTC ACG CAC GAT TTC-3'.
Control reactions omitting the reverse transcription step were
also performed to exclude the possibility of genomic
contamination. Ten ml of the ampli®ed product were
separated by agarose gel electrophoresis, stained with
ethidium bromide and scanned into a Bio-Rad gel
documentation system for quanti®cation of band intensity.
Results are expressed as the ratio of NIS to b-actin signals
and expressed as the mean+s.e. of triplicate determinations.
Statistics
The signi®cance of dierences between means was assessed by
Student's t-test.
Acknowledgements
We are grateful to the Medical Research Council for grant
support, to Brahms Diagnostics and to Dr Gilbert Vassart
(Brussels) for supply of reagents, to Michelle Haughton for
technical support and to Theresa King for manuscript
preparation.
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