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Molecular and Cellular Biology, August 2000, p. 5690-5699, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence for a Telomere-Independent "Clock"
Limiting RAS Oncogene-Driven Proliferation of Human Thyroid
Epithelial Cells
C. J.
Jones,1
D.
Kipling,1
M.
Morris,1
P.
Hepburn,1
J.
Skinner,1
A.
Bounacer,1
F. S.
Wyllie,1
M.
Ivan,1
J.
Bartek,2
D.
Wynford-Thomas,1,* and
J. A.
Bond1
Cancer Research Campaign Laboratories,
Department of Pathology, University of Wales College of Medicine, Heath
Park, Cardiff CF14 4XN, United Kingdom,1 and
Institute of Cancer Biology, Danish Cancer Society, DK-2100
Copenhagen, Denmark2
Received 9 December 1999/Returned for modification 3 January
2000/Accepted 27 April 2000
 |
ABSTRACT |
An initiating role for RAS oncogene mutation in several
epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce
long-term proliferation in an epithelial cell in contrast to the
premature senescence observed in fibroblasts; and (ii) what is the
"clock" which eventually triggers spontaneous growth arrest even in
epithelial clones generated by mutant RAS. The early response to
RAS activation in thyroid epithelial cells showed two
features not seen in fibroblasts: (i) a marked decrease in expression
of the cyclin-dependent kinase inhibitor (CDKI) p27kip1 and
(ii) the absence of any induction of p21waf1. When
proliferation eventually ceased (after up to 20 population doublings)
this occurred despite undiminished expression of mutant RAS and was
tightly correlated with a return to the initial high level of
p27kip1 expression, together with the de novo appearance of
p16ink4a. Importantly, neither the CDKI changes nor the
proliferative life span of RAS-induced epithelial clones was altered by
induction of telomerase activity through forced expression of the
catalytic subunit, hTERT, at levels sufficient to immortalize human
fibroblasts. These data provide a basis for cell-type differences in
sensitivity to RAS-induced proliferation which may explain the
corresponding tumor-type specificity of RAS mutation. They also show
for the first time in a primary human cell model that a
telomere-independent mechanism can limit not only physiological but
also oncogene-driven proliferation, pointing therefore to a tumour
suppressor mechanism additional, or alternative, to the telomere clock.
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INTRODUCTION |
RAS mutation occurs at
high frequency in several human epithelial tumor types, notably those
of colon (8), pancreas (1), and thyroid (36,
58). Analyses of clinical samples indicate its involvement at
early (premalignant) stages and in pancreas and thyroid are consistent
with a role as the initiating molecular event. In the case of thyroid,
where a suitable cell culture model exists, this has been strongly
supported by the results of in vitro gene transfer experiments (7,
37). Whereas normal thyrocytes exhibit a very low proliferative
rate (as is also the case in the intact gland) and cease growing in
even optimal culture conditions after less than 3 population doublings
(PD), introduction of mutant RAS induces a dramatic proliferative
response, resulting in generation of clones whose final size (up to
107 cells) and well-differentiated phenotype are consistent
with those of a small benign thyroid tumor (adenoma) in vivo
(7).
This prolonged clonal expansion contrasts sharply with the more widely
studied effects of RAS mutation in primary fibroblasts (39, 55, 73), in which proliferation is sharply limited by
induction of a premature senescence state. Nevertheless, even in
thyrocytes, proliferation does not continue indefinitely and eventually
spontaneously ceases after 15 to 25 PD (7, 37), terminating
in a viable state of growth arrest, resembling replicative senescence. Spontaneous immortalization has never been observed despite many hundreds of gene transfer experiments in our laboratory.
In vivo, the vast majority of thyroid adenomas also appear to reach a
self-limiting quiescent end point. Such limitations in tumor growth are
often ascribed to insufficient ability to promote new blood
vessel formation and/or to invade surrounding tissues. Importantly,
however, the observation that a restriction similar to clonal expansion
occurs even in tissue culture indicates, on the contrary, that a
cell-intrinsic mechanism which is independent of tissue architecture
and is most likely to be based on number of elapsed cell divisions is responsible.
Interest in intrinsic proliferative life span barriers (PLBs) (4,
52, 69) has been heightened recently by the demonstration that
one such cell division "clock" is based on the progressive shortening of chromosome telomeres, which has been shown to trigger replicative senescence in a number of normal human cell types (3) by a pathway involving p53 (5, 21) and the
cyclin-dependent kinase inhibitor (CDKI) p21Waf1
(11). Here we have set out to examine the relationship
between this mechanism and that responsible for limiting the
proliferative response to activated RAS in thyroid epithelial cells. In
striking contrast to the fibroblast paradigm, the results point to a
telomere-independent clock operating through a distinctly different set
of CDKI changes, which may represent a novel class of PLB responsible
for arresting oncogene-induced tumor development in some types of human
epithelial cells at an early (premalignant) stage.
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MATERIALS AND METHODS |
Cells and culture conditions.
Primary monolayer cultures of
follicular epithelial cells (>99% epithelial as judged by cytokeratin
immunostaining) were prepared from surgical samples of normal thyroid
tissue by protease digestion and mechanical disaggregation
(66) and maintained in a 2:1:1 mixture of Dulbecco's
modified Eagle's medium, Ham's F-12 medium, and MCDB104 (all from
Life Technologies, Paisley, United Kingdom) (7) supplemented
with 10% fetal calf serum (Imperial Laboratories, London, United
Kingdom). Normal human diploid fibroblasts (HCA2 cells, kindly provided
by James Smith, Houston, Texas) were grown in Dulbecco's modified
Eagle's medium (Life Technologies) supplemented with 10% fetal calf
serum (Imperial Laboratories). Senescence occurred at an estimated PD
level of 65 to 70.
Retroviral vectors.
Replication-defective amphotropic
retroviral vectors encoding the Val-12 mutant of human H-RAS
(psi-CRIP-DOEJ) together with the neo gene for selection in
G418 and the vector-only control (psi-CRIP-neo) were used as previously
described (7). To allow dual selection, we constructed
retrovirus vectors for hTERT and HPV E7 based on pBABEpuro
(46), which confers resistance to puromycin. pBABEpuro-hTERT
has been described recently (68); pBABEpuro-E7 was
constructed by PCR synthesis of the E7 open reading frame together with
a consensus upstream Kozak sequence and appropriate restriction sites
to allow ligation into the BamHI and EcoRI sites of pBABEpuro.
Retroviral gene transfer.
Primary thyroid epithelial cells
were plated at ~5 × 105 per 60-mm-diameter dish and
infected 2 days later with retrovirus-containing medium from
near-confluent producer cells, containing 8 µg of Polybrene per ml
(7). Three days later, cells were passaged and maintained in
medium with or without G418 (400 µg/ml) or puromycin (2.5 µg/ml) as indicated.
Assessment of DNA synthesis by BrdU incorporation.
Cells
were labeled by incubation in 10 µM bromodeoxyuridine (BrdU) for
1 h, following which nuclear incorporation was detected by
immunoperoxidase immunocytochemistry as previously described (4). The proportion of labeled nuclei (labeling index
[LI]) was determined from a count of >500 cells per datum point.
Analysis of mutant RAS expression by reverse
transcription-PCR.
Poly(A)+ RNA was extracted from
normal monolayers or from pooled colonies generated by retroviral
vector DOEJ, using a Micro-FastTrack mRNA isolation kit (Invitrogen
Corp., San Diego, Calif.). A partial H-ras cDNA was synthesized using a
reverse transcription-PCR kit (Perkin-Elmer Cetus, Norwalk, Conn.) with
primer 5'-TGGACGAATACGACCCCACT-3', located downstream of
codon 12. This was then amplified using this primer together with
upstream primer 5'-CTGAGGAGCGATGACGGAAT-3' in a PCR mixture
consisting of 30 cycles of 1-min denaturation at 94°C, 1-min
annealing at 60°C, and 1-min extension at 72°C, with an additional
4-min extension after the final cycle. A single 95-bp product was seen
on gel electrophoresis. This was then sequenced using an ABI-Prism
cycle sequencing kit (PE-Applied Biosystems) with the downstream primer
described above.
Detection of SA
-Gal activity.
Endogenous
senescence-associated mammalian -galactosidase activity (SA-Gal)
(15) was assessed histochemically (7) using X-Gal
substrate
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside).
Immunocytochemical analysis.
For p16ink4a,
monolayers were fixed in acetone-methanol, 1:1 (10 min at 
20°C),
and a standard indirect immunoperoxidase procedure applied, using mouse
monoclonal antibody DCS-50 (43) (Oncogene Research
Products), followed by peroxidase-conjugated rabbit anti-mouse immunoglobulin (Ig) (Dako). A similar procedure was followed for H-RAS
using monoclonal Y13-259 followed by swine-anti-rat Ig-peroxidase (Dako) and for the corresponding rat control antibody (antipolyoma large T).
For p21waf1, p27kip1, and cyclin D1, cultures
were fixed in 4% paraformaldehyde (10 min; or 4 min in the case of
cyclin D1) and then pretreated with 50 mM glycine (10 min), 0.2%
Triton X-100 (10 min), and 0.3% H2O2 (3 min),
and nonspecific binding was blocked with 2% horse serum (30 min).
Anti-p21 (Clone 6B6; Cambridge Bioscience, Cambridge, United Kingdom),
anti-p27 (Transduction Laboratories), or anti-cyclin D1 (DCS-6; Santa
Cruz) mouse monoclonal antibodies were applied followed by the
mouse-specific avidin-biotin-peroxidase (ABC) system (Novocastra).
For cyclin D3, cells were fixed in 2% paraformaldehyde and then
permeabilized in methanol followed by 0.1% Triton X-100 as
described
(
14). Detection was by the ABC method using monoclonal
antibody DCS-22 clone E8 (
2) as the primary
antibody.
For all antigens, sites of antibody binding were visualized by the
deposition of brown polymer following incubation in
diaminobenzidine-hydrogen
peroxide
solution.
Immunoblotting.
Cells were lysed for 5 min at 4°C by 1%
NP-40 in 150 mM NaCl, 50 mM Tris (pH 8.0), and 5 mM EDTA buffer, which
contained 1 mM phenylmethylsulfonyl fluoride and 0.01 mg each of
aprotinin and leupeptin per ml. Protein samples (30 µg) were
separated by sodium dodecyl sulfate-12% polyacrylamide gel
electrophoresis and electroblotted to Transblot polyvinylidene
difluoride membrane (Bio-Rad Labs, Hemel Hempstead, United Kingdom).
Anti-p16 antibody (as above) was applied, followed by goat anti-mouse
Ig-peroxidase conjugate and visualization by the ECL detection system
(Amersham, Little Chalfont, United Kingdom). The filter was stained
with India ink, and quantitation of the specific signal and the amount of protein loaded was performed using a Bio-Rad imaging densitometer running Molecular Analyst software.
TRF length analysis.
Genomic DNA (1 µg), prepared as
previously described (30), was digested with 10 U each of
RsaI and HinfI and separated on 0.5% agarose
Tris-borate-EDTA gels. Gels were denatured with 1.5 M NaCl-0.5 M NaOH
(15 min), neutralized with 1.5 M NaCl-0.5 M Tris, pH 8.0 (10 min), and
then dried onto 3MM paper (Whatman) for 1 h at room temperature
followed by 30 min at 60°C. Gels were then removed from the paper and
hybridized at 37°C overnight in 5× SSC (1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate)-5× Denhardt's-0.5 mM pyrophosphate-10 mM
Na2HPO4 with a single-stranded DNA probe
(CCCTAA)3 (500 ng/gel), which was end-labeled
with [
-32P]ATP (3,000 Ci/mmol) by 10 U of T4
polynucleotide kinase (Amersham). After washing with three changes of
0.1× SSC at 37°C and two at room temperature, gels were wrapped in
Saran and signals were detected using a STORM phosphorimager (Amersham
Pharmacia Biotech) from which mean terminal restriction fragment (TRF)
length was calculated (35) using Molecular Analyst software
(Bio-Rad).
Telomere repeat amplification protocol (TRAP assay).
Cells
(107 per 185 µl) were lysed in hypotonic-detergent buffer
(1 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 0.5%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS),
10% glycerol) for 30 min on ice followed by centrifugation at
100,000 × g for 30 min at 4°C. Extracts (3,000 cell
equivalents) were assayed with or without pretreatment for 10 min at
85°C (to abolish telomerase activity).
Telomerase activity was assayed according to the standard TRAP protocol
(
32) except that the wax barrier was avoided and
Taq polymerase and the second primer, CX, were added to
reaction
mixtures prewarmed to 92°C following elongation of the TS
primer.
Telomerase products were resolved in 10% polyacrylamide gels
and
visualized by Sybr Gold staining and fluoroimaging, again using
a
STORM system. Extracts of cell line 293 were used as a positive
control
(
10). The incorporation of an internal standard
(
67)
demonstrated that there were no inhibitors of PCR
detectable with
the quantities of protein
used.
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RESULTS |
Induction of thyrocyte proliferation by mutant RAS correlates with
reduced nuclear p27kip1 expression.
Normal human
thyroid epithelial cells in primary culture were stably transduced with
an amphotropic retroviral vector encoding mutant (V12) H-RAS. As
described previously (7), this results in the generation of
rapidly growing colonies (approximately 50 per dish of 105
cells infected) which are clearly distinguishable from the surrounding uninfected monolayer by 7 to 10 days after infection. Normal
(uninfected) thyroid cells cease proliferating after <3 PD and, even
prior to this, exhibit a very low proliferative rate, which accounts for the low yield of transduced cells compared to that seen for example
with fibroblast cell lines.
Using immunocytochemistry to permit analysis of small cell numbers and
to reveal changes in subcellular distribution, we initially
examined
the expression of several cell cycle regulators which
have been
implicated in RAS-induced proliferation in other models,
namely cyclin
D1, p21
waf1, and p27
kip1. We also included
cyclin D3, which has been shown to play a particularly
important role
in normal thyroid epithelial cells (
14).
Normal thyrocytes, after 7 days in culture, expressed readily
detectable levels of cyclin D1, D3, (not shown), and p21 in
the
majority of nuclei (Fig.
1a); in
particular nearly 100% of
cells exhibited strong nuclear
immunostaining for p27 (Fig.
1a).
As expected, the BrdU LI was
extremely low (<1%) and nearly all
cells were positive for SA
-Gal,
which has been widely reported
as a marker of replicative senescence in
other cell types (
15).
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FIG. 1.
Expression of cell cycle regulators during the life span
of thyroid epithelial clones induced to proliferate by mutant RAS. (a)
Representative photomicrographs of normal thyroid epithelial cells (N)
and colonies induced by mutant RAS at an early rapidly proliferating
stage (RAS-E) and at a late stage at the end of their proliferative
life span (RAS-L). Expression of p21waf1,
p27kip1, and p16ink4a and incorporation of BrdU
were analyzed by immunocytochemistry (positivity indicated by brown
peroxidase reaction product); the senescence-associated marker
SA-Gal was assessed by X-Gal histochemistry (blue reaction product).
In some panels, nuclei are lightly counterstained with hematoxylin to
aid visualization. Note a reduction in nuclear p27 correlating with
high BrdU labeling in RAS-E colonies and the increase in p16 in RAS-L
colonies (magnification ×25). (b and c) Quantitative analyses of
normal epithelial cells and colonies expressing mutant RAS at early (1 to 2 weeks), middle (2 to 3 weeks), and late (>5 weeks) stages showing
an inverse correlation between a proportion of nuclei containing
detectable p27 ( ) and a proportion in cell cycle S phase as shown by
BrdU incorporation ( ) and a direct correlation between p27 and
SA-Gal expression ( ) note less-marked changes in proportions of
nuclei containing detectable p21 ( ) and cyclin D1 ( ). Results are
presented as means of >300 cells per datum point ± standard
errors of the mean (error bars). (d) Western blot-ECL analysis of
p16ink4a expression (upper panel) in normal thyroid
epithelial cells (N), RAS-induced colonies at an intermediate stage of
their life span (RAS MID), and end-stage colonies (RAS L). The lower
panel shows part of an India ink-stained filter to assess equality of
protein loading. Figures show integrated optical density values
obtained by scanning densitometry from which the relative increase in
p16 signal corrected for total protein between RAS MID and RAS L is
calculated at 20-fold.
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In colonies induced to proliferate by mutant RAS at the earliest time
point analyzable (7 to 10 days) BrdU LI had increased
to 36%,
accompanied by a fall in the SA
-Gal index to 7%. This
proliferative
response was associated with a slight reduction
in the proportion of
cells expressing detectable nuclear p21,
which failed to reach
statistical significance, and an increase
in the proportion expressing
cyclin D1 from ~70% to nearly 100%
(Fig.
1a and c). Cyclin D3
expression remained essentially unchanged
at this (and subsequent) time
point at between 50 and 60% of nuclei
(not shown). The major change
observed however was a marked fall
in p27 expression, which was
detectable in only 16% of cells (nuclei)
expressing mutant RAS,
compared to 95% in the surrounding normal
monolayer (Fig.
1a and
b).
To control for the possibility that the reduction in p27 content in
early RAS-induced colonies might merely be a secondary
consequence of
the much higher proportion of proliferating cells,
we also examined an
analogous model in which primary thyrocytes
are induced to proliferate
by simian virus 40 T following infection
with the retroviral vector
psi-CRIP-SVU19 (
6). Despite similar
proliferation rates, and
at similar sizes, colonies generated
in this way failed to show any
reduction in p27 expression (data
not
shown).
Spontaneous cessation of RAS-induced thyrocyte proliferation
correlates with reexpression of nuclear p27 and de novo expression of
p16ink4a.
RAS-induced growth ceased by 5 weeks
postinfection at a final colony size varying from 104 to
106 cells. Colonies were analyzed as above at this and at
intermediate time points postinfection. Growth arrest was associated
with, and explicable by, a decline in BrdU LI, which eventually
returned to normal levels (~1%), together with a corresponding
restoration of SA-Gal expression and a flattened, senescence-like
morphology (Fig. 1a).
The changes in p21 and cyclin D1 expression seen in early RAS colonies
reverted to near-normal in terminally arrested colonies;
however, the
time course showed an imperfect correlation with
BrdU LI at
intermediate time points (Fig.
1c). In contrast, p27
expression showed
a tight inverse correlation throughout with
BrdU LI and a direct
correlation with SA
-Gal (Fig.
1b).
Given its established role in senescence in other cell types, we also
examined expression of the CDK inhibitor p16
ink4a. There
was a clear increase from undetectable levels in normal
colonies to
readily detectable levels in end-stage RAS colonies
(Fig.
1a); however,
the rather diffuse, predominantly cytoplasmic,
pattern of
immunostaining obtained with antibody DCS-50 was difficult
to quantify
microscopically. In this case, therefore, sufficient
cells were
collected to perform a limited Western blot analysis
(Fig.
1d) which
confirmed the absence of signal in normal cells
and a 20-fold increase
between proliferating (early) and arrested
(end-stage) RAS
colonies.
The possibility that the above changes were due simply to decreased
mutant RAS expression in late-stage colonies, for example
through
methylation of the retroviral promoter, was excluded by
analysis at
both protein (Fig.
2a to d) and mRNA
(Fig.
2e) levels,
which confirmed the persistent overexpression of the
mutant compared
to the endogenous wild-type sequence in both early- and
late-stage
colonies.
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FIG. 2.
Persistence of mutant RAS expression in late-stage
RAS-induced colonies. (a to c) Immunocytochemical analysis of H-RAS
protein using anti-ras antibody Y13-259 in normal thyroid cells (a);
early, proliferating colonies induced by mutant H-RAS (b); and
late-stage, growth-arrested colonies (c). (d) Late-stage colony
immunostained with a species-matched antibody to an irrelevant antigen
(polyomavirus large T) as negative control. Note that although antibody
Y13-259 detects both mutant and wild-type RAS, comparison of panels b
and c with normal (uninfected) thyroid cells (a) indicates that nearly
all the immunostaining observed here can be attributed to the mutant
protein (magnification, ×75). (e) Sequence (antisense) of cDNA
surrounding codon 12 of H-RAS derived by RT-PCR of mRNA from normal
(N), pooled early-stage (E), or late-stage (L) mutant RAS-induced
colonies. The point mutation (arrowed) appears as a C A transversion
at position 2 of codon 12, equivalent to GT in the coding sequence.
Note that only mutant mRNA is detectable in both E and L, consistent
with a much higher expression of the vector-encoded mutant RAS in both
cases compared to the endogenous wild-type gene.
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Cessation of RAS-induced thyrocyte proliferation is not dependent
on telomere erosion.
Mean telomere length, measured as TRF size
after HinfI/RsaI digestion of genomic DNA, varied
from 9 to 11 kbp in different isolates of human thyrocytes at the end
of their normal proliferative life span (Fig.
3).
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FIG. 3.
Analysis of telomere length and telomerase activity in
thyroid cells expressing mutant RAS. (a) TRF analysis of
HinfI/RsaI-digested genomic DNA from normal
thyrocytes (N), and pools of early (proliferating) (RAS E) and
late-stage (RAS L) RAS colonies derived from two separate human thyroid
samples (Thy.1 and Thy.2). Mean TRF values were calculated from
densitometry data as described in text. (b) TRF analysis of normal (N)
and late-stage (RAS L) RAS colonies derived from a third thyroid sample
(Thy.3), together with colonies expressing both mutant RAS and the
catalytic subunit of human telomerase (RAS + hTERT). Senescent
human fibroblasts (strain HCA2) are shown for comparison (HDF SEN). (c)
Telomerase activity assessed by TRAP assay in normal cells (N), in
pools of early colonies expressing mutant RAS (RAS E) and late-stage
colonies expressing mutant RAS alone (RAS L) or with hTERT (RAS L + hTERT). Each sample is analyzed with (+) or without () prior heat
treatment (85°C for 10 min). The immortal human epithelial cell line
293 is included as a positive control. The arrow indicates the position
of the internal telomerase amplification standard as a control for
nonspecific PCR inhibition.
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Analysis of DNA from pooled RAS-induced colonies in comparison to the
normal cells from which they were derived showed only
variable,
low-magnitude TRF shortening. In end-stage colonies
this varied in
different experiments from ~1.4 kbp down to unresolvable
differences
(Fig.
3a and b). Furthermore, the final mean TRF value
(typically ~10
kbp) was always much larger than that observed
in normal human
fibroblasts at the end of their replicative life
span (~7 kb in our
hands) (Fig.
3b).
To address the possibility that normal thyrocytes, unlike fibroblasts
and most other primary cultures, might circumvent telomere
erosion
through physiological expression of telomerase, we assayed
telomerase
activity in lysates using the well-established in vitro
TRAP assay
(
32). No activity could be detected in any normal
sample,
nor in nearly all lysates of pooled RAS colonies at both
early and late
stages (Fig.
3c). Only one sample (Thy.1 RAS-E
[Fig.
3c]) out of more
than 30 analyzed was found to be positive,
and this only at extremely
weak levels (<1% of the positive control
293 cell
line).
Finally we asked whether stable induction of telomerase activity
through forced expression of the catalytic sub-unit of telomerase,
hTERT, would, as in fibroblasts and several other cell types
(
3),
lead to an extension of replicative life span in
RAS-induced colonies.
Thyrocytes were infected with the V12H-RAS-neo
vector as before,
together with an in-house amphotropic vector
(pBABEpuro-hTERT)
(
68) encoding hTERT together with
puromycin resistance, or with
the pBABEpuro vector without
hTERT as a control. After selection
in G418 plus puromycin, a similar
yield of clones was obtained
with or without hTERT
expression.
Randomly selected groups of 20 colonies expressing mutant RAS alone and
20 expressing RAS and hTERT were followed up in detail.
No differences
in growth rate or morphology were seen (not shown),
and both sets of
colonies ceased proliferating after a similar
time (4 to 5 weeks).
Based on final colony cell counts (assuming
no cell death), the mean
numbers of PD undergone in the two groups
were calculated as 13 (range,
9 to 15) and 12 (range, 9 to 14)
respectively.
The phenotype of end-stage colonies expressing hTERT plus mutant RAS
with respect to morphology, BrdU incorporation, SA
-Gal
staining, and
expression of p21 and p27 was indistinguishable
from that of the
corresponding colonies generated by mutant RAS
alone (compare Fig.
4 with Fig.
1).
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FIG. 4.
Phenotype of end-stage thyroid epithelial colonies
induced by coexpression of mutant RAS plus the catalytic subunit of
telomerase, hTERT. Representative photomicrographs showing
phase-contrast morphology (a), BrdU incorporation (all nuclei negative
in this field) (b) and expression of p21waf1 (c),
p27kip1 (d), and SA-Gal (e) as assessed as in Fig. 1. (b
to d) These panels are lightly counterstained with hematoxilin.
Magnification, ×50.
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The TRAP assay was performed on pooled RAS and hTERT colonies to check
that the lack of effect on life span was not due to
ineffective
expression. A high level of activity was observed
(Fig.
3c), comparable
to that obtained in fibroblasts whose life
span was successfully
extended by infection with our hTERT vector
(
68).
Infection of normal thyrocytes with the pBABEpuro-hTERT
vector alone failed to generate any colonies, either with or
without
puromycin
selection.
These data indicate that terminal growth arrest occurring both in
normal thyrocytes and in those expressing mutant RAS is
regulated by a
mechanism which comes into operation at mean telomere
lengths well
above those associated with senescence in fibroblasts
and furthermore,
unlike the latter, is not abrogated by forced
expression of
telomerase.
Expression of human papillomavirus (HPV) E7 fails to extend
proliferative life span of thyrocyte colonies induced by mutant
RAS.
The correlation between spontaneous growth arrest in
end-stage RAS-induced colonies and the increased expression of CDKIs p16 and p21 suggests that further tumor progression would require abrogation of one or both of these inhibitory controls. This is consistent with analyses of clinical samples (17) which have shown reduced expression of p27 in carcinomas compared to adenomas (although without evidence for mutation). Inactivating genomic abnormalities have also been reported at the p16 locus, and we have
observed promoter methylation or gene deletion in the majority of
thyroid cancer cell lines and in up to 25% of well-differentiated thyroid cancers (27, 29, 70).
We therefore predicted that experimental abrogation of p16 and/or p27
function in thyrocytes expressing mutant RAS should
confer a further
extension of proliferative life span. Attempts
to achieve this directly
by anti-sense p27 or p16 expression have
so far proven problematic; we
therefore employed HPV E7 which
is known to directly antagonise members
of the p27 family of CDKIs
and to block their effect downstream by
inactivating
Rb.
Thyrocytes were coinfected with the V12H RAS-neo vector together with
either our pBABEpuro vector encoding E7, or pBABEpuro
alone.
Contrary to prediction, no significant difference in the
final size of
colonies was observed as a result of E7 expression,
which was confirmed
by immunocytochemical analysis (not shown).
There was, however, a
marked difference in the final fate of the
E7-expressing colonies in
that instead of reaching a viable quiescent
end point they continued to
proliferate, but with increasing cell
death (Fig.
5) which finally resulted in colony
degeneration.
A terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick
end labeling (TUNEL) assay (not shown) confirmed
that end-stage
E7/RAS colonies were undergoing apoptosis. A similar
outcome was
seen if the E7 vector was replaced with one encoding both
E7 and
E6, indicating that, as observed previously (
4),
apoptosis
in thyrocytes is p53 independent.
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FIG. 5.
Coexpression of HPV E7 plus mutant RAS changes the
end-stage phenotype of thyroid epithelial clones to cell death rather
than growth arrest. Phase-contrast photomicrographs of typical
late-stage colonies generated either by mutant RAS plus HPV E7 (a) or
mutant RAS alone (b). Note the much more marked cell death and
detachment in panel a. Magnification, ×50.
|
|
| |
DISCUSSION |
Mechanisms underlying the sensitivity of thyroid epithelial cells
to RAS-induced proliferation.
The prolonged growth stimulation
induced by activated RAS in normal thyroid epithelial cells stands in
sharp contrast to the response observed in normal human fibroblasts, in
which proliferation is limited within a few PD by the onset of a
permanent growth arrest state, which, apart from being independent of
telomere erosion (65), closely resembles replicative
senescence mediated by both p14arf-p53 and
p16ink4a pathways (39, 55, 73).
This premature senescence phenotype, which we too have observed in
fibroblasts, has aroused great interest recently (
51)
as a
potential innate tumor suppressor mechanism and an explanation
for the
selective pressure for mutation of p53 and/or p16 in tumors
bearing
RAS mutations (
39,
73).
This concept does not fit well, however, with observations on
epithelial tumor models both in vivo (
18,
36,
58) and
in
vitro (
4,
37; this study), which indicate the
ability
of
RAS mutation to drive sustained proliferation for
at least
20 PD without apparent mutation of these tumor suppressor
pathways.
Furthermore, it is implausible, even on purely probabilistic
grounds,
that the few PD available before onset of permanent
RAS-induced
senescence would afford any reasonable chance of a cell
acquiring
the necessary tumor suppressor gene mutation(s) for further
clonal
expansion.
We suggest rather that premature senescence effectively renders tumor
induction by RAS impossible in those cell types, such
as fibroblasts,
in which it occurs and hence accounts for the
observed lack of
involvement of this oncogene family in those
(rare) tumors derived from
them, e.g., fibrosarcomas (
9). Conversely,
it is the absence
of this mechanism which confers on cells such
as thyrocytes their
susceptibility to RAS-induced tumorigenesis.
The pattern of expression
of CDKIs reported here provides an initial
insight into the underlying
differences in cell-cycle regulatory
pathways which may explain this
crucial context-dependent difference
in response to RAS
activation.
In fibroblasts (
55,
73), RAS-induced senescence is
associated with large increases in cellular content of two CDKIs
p16
and p21
the latter being driven by activation of p53, in turn,
probably resulting from increased levels of p14
arf
(
51). In thyrocytes, in contrast, there is no early
RAS-induced
increase in p21 or p16; on the contrary there is a dramatic
fall
in the level of another p21 family member, p27
KIP1,
which does not occur in fibroblasts (
73). The predicted
effect
of this is a release of inhibition of CDKs controlling
G
1/S transition,
either directly or, as suggested recently
(
12,
45,
57),
via a redistribution of p21 from CDK2-cyclin E
to CDK4-cyclin
D complexes, hence increasing CDK2 activity. It is
likely therefore
that the net level of CDKI activity is altered in
opposite directions
in thyrocytes and fibroblasts following RAS
activation.
p27 is expressed at high levels in normal thyrocytes both in vitro
(this study) and in the intact thyroid gland, in both human
(
17,
40,
60) and mouse (
13). Many studies suggest that
p27
plays a role in growth arrest accompanying differentiation
(
16,
49,
72), and p27-null mice exhibit hyperplasia of many
highly
differentiated cell populations, including endocrine glands
(
19,
33,
47) (although thyroid was not specifically studied).
Taken
together, this suggests that p27 is necessary for maintaining
the
normally very low proliferative rate of thyrocytes in vivo
and that its
loss is a plausible candidate mechanism for induction
of inappropriate
proliferation in these
cells.
RAS activation has been shown to induce destabilization of p27 in many
different experimental models, in most cases probably
through
ubiquitin-mediated degradation (
31,
38,
50,
59).
Although
other pathways such as RAF-mitogen-activated protein
kinase may be
necessary in some contexts (
31), the consensus
currently
favors rho as a key effector for this response (
25,
64). rho
is also an attractive candidate in our model, since
its degree of
activation has been previously reported in a fibroblast
model
(
48) to determine whether or not RAS induces p21 and hence
premature senescence. We speculate therefore that in thyrocytes
and
other epithelial cells susceptible to RAS-induced tumorigenesis,
in
contrast to refractory cell types such as fibroblasts, the
level of
activation of rho in response to RAS signalling is sufficient,
firstly,
to prevent induction of p21 by other RAS effector pathway(s)
and,
secondly, to induce destabilization of p27. In related work
(
22), we recently established the requirement for at least
two
effector pathways in RAS-induced thyrocyte
proliferation
mitogen-activated
protein kinase and phosphatidyl
inositol 3-kinase. The former
is a good candidate for the induction of
cyclin D1 by mutant RAS
(
25,
41), while the latter may be
responsible at least in
part for activation of rho (although we have
not yet excluded
a role for the Ral-GDS effector
pathway).
How rho destabilizes p27 is still unclear. While some studies point to
a direct action (
38), others (
25) suggest that
it
is secondary to activation of CDK2-cyclin E (by other means),
leading
to increased CDK2-mediated phosphorylation of p27, which
is known to be
a key modification targeting it for ubiquitin-mediated
degradation
(
56,
61,
62). In other words, p27 degradation
may be both
upstream and downstream of CDK2 activation, thus creating
a positive
feedback loop which greatly complicates attempts to
establish
cause-effect
relationships.
Clearly, further work will be needed to establish the precise role of
p27 degradation in RAS-induced proliferation in our
model.
Nevertheless, the data presented here already open up a
route to
identifying the molecular determinants of susceptibility
to RAS-induced
tumorigenesis, which in turn may provide novel
therapeutic
targets.
Mechanisms limiting RAS-induced clonal expression: an antioncogenic
PLB not mediated by a telomere clock.
Proliferation of thyroid
epithelial cells induced by mutant RAS ceases spontaneously
after 15 to 25 PD despite continued expression of the activated
oncogene. This growth arrest is correlated with, and potentially
mediated by, increases in two CDKIs, p27 and p16. Although these may
act redundantly, recent insights into cell cycle control (12,
57) suggest a simpler, sequential model driven solely by
induction of p16 expression after a given number of PD. In addition to
its direct inhibitory action on CDK4 and CDK6, it is now known that an
increase in p16 can also lead to displacement of p21 from CDK4 or CDK6
to CDK2-cyclin E complexes, resulting indirectly in reduced activity of
the latter, an effect which may be further enhanced by formation of
inactive complexes between CDK2 and displaced cyclin D1
(44). Loss of CDK2 activity will reduce the phosphorylation
of p27 and hence lead to an increase in its steady-state level, an
effect which should be amplified by the feedback loop (56)
which results from further inhibition of CDK2. On this model,
therefore, the stabilization of p27, though triggered in the first
instance by the increase in p16, may nevertheless be necessary to
ensure sufficient inhibition of CDKs for complete growth arrest.
The above model is consistent with analyses of human thyroid cancers
(
17,
42,
60,
63) and derived cell lines (
27,
29),
which frequently show loss of p16 and/or decreased p27
expression in
cancers compared to normal or benign epithelia,
suggesting that loss of
either CDKI may permit escape from the
PLB limiting initial clonal
expansion driven by RAS. Unfortunately,
we have not as yet been able to
test this prediction experimentally
by direct manipulation of p27 or
p16. A more central abrogation
of this TSG pathway using HPV E7 was of
limited value, since although
appearing to relieve proliferative
arrest, net growth was offset
by increasing cell
death.
A key finding in the current work is the apparent independence of the
above-mentioned PLB on telomere erosion, as shown most
conclusively by
the failure of experimentally induced telomerase
activity to extend
life span despite stabilization of telomere
length. We must, therefore,
postulate the existence of a separate
cell division counting mechanism,
which acts at least in part
via induction of p16. Such a clock also
seems to operate in controlling
physiological (growth factor induced)
proliferative life span
in a variety of situations, including primary
fibroblasts in rodents
(
54) and, moreover, several specific
types of human epithelium,
including mammary epithelial cells (
20,
34), keratinocytes
(
34) and uroepithelial cells
(
53,
71) which undergo senescence
after a similar range of
PD (10 to 30 PD). The originality of
our observation is that it
demonstrates a crucial role for a p16-dependent,
telomere-independent
PLB in limiting not only physiological but
also oncogene-driven
proliferation.
In all of these situations the major question which remains is the link
between replicative age (elapsed divisions) and induction
of p16
expression. Demethylation of promoter sequences clearly
plays a part in
this (
20,
26) and there is long-standing evidence
for
reduction in DNA methylation during replicative ageing (
24);
the mechanistic details of such a clock, however, and the role
of
potential
trans-acting factors such as BMI-1 (
28)
remain
obscure. Given its potential importance as a natural tumor
suppressor
mechanism alternative to telomere erosion, this promises to
be
an area of major basic and therapeutic
significance.
| |
ACKNOWLEDGMENTS |
We thank the Cancer Research Campaign and the Medical Research
Council for grant support.
We also thank Michèle Haughton for primary cells and Theresa King
for manuscript and graphics preparation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cancer Research
Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom. Phone: 44 (029) 2074 2700. Fax: 44 (029) 2074 2704. E-mail:
KingTD{at}Cardiff.ac.uk.
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Molecular and Cellular Biology, August 2000, p. 5690-5699, Vol. 20, No. 15
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Abstract
Introduction
