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Molecular and Cellular Biology, June 2001, p. 3862-3875, Vol. 21, No. 12
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.12.3862-3875.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Coexistence of Alternative Lengthening of Telomeres
and Telomerase in hTERT-Transfected GM847 Cells
Kilian
Perrem,
Lorel M.
Colgin,
Axel A.
Neumann,
Thomas R.
Yeager, and
Roger R.
Reddel*
Cancer Research Group, Children's Medical
Research Institute, Westmead, Sydney, New South Wales 2145, Australia
Received 1 November 2000/Returned for modification 7 December
2000/Accepted 26 March 2001
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ABSTRACT |
It has been shown previously that some immortalized human cells
maintain their telomeres in the absence of significant levels of
telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of
very heterogeneous length, ranging from very short to very long. Here
we report the effect of telomerase expression in the ALT cell
line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells
resulted in lengthening of the shortest telomeres; this is the first
evidence that expression of hTERT in ALT cells can induce
telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells
after more than 100 population doublings. Very long telomeres and
ALT-associated promyelocytic leukemia (PML) bodies continued to be
generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when
the GM847 cell line was fused with two different
telomerase-positive tumor cell lines, the ALT phenotype was
repressed in each case. These hybrid cells were telomerase
positive, and the telomeres decreased in length, very rapidly at first
and then at the rate seen in telomerase-negative normal cells.
Additionally, ALT-associated PML bodies disappeared. After the
telomeres had shortened sufficiently, they were maintained at a stable
length by telomerase. Together these data indicate that the
telomerase-positive cells contain a factor that represses the
ALT mechanism but that this factor is unlikely to be
telomerase. Further, the transfection data indicate that ALT
and telomerase can coexist in the same cells.
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INTRODUCTION |
Telomeres are specialized structures
consisting, in human cells, of TTAGGG repeat sequences (4,
33) which, together with specific binding proteins
(5), form caps at the ends of chromosomes that are
essential for chromosome stability (11, 17). Telomeres shorten with each round of cell division (1, 2, 18, 21, 28,
34) due, at least in part, to the "end replication problem" (26, 44). It is hypothesized that critically shortened
telomeres can trigger growth arrest and senescence (19)
and that this is a key factor in cellular aging and control of cell
division potential (reviewed in reference 36). There may
be additional factors, however, that also act as a mitotic clock and
cause a permanent exit from the cell cycle (37). All
immortalized human cell lines and most tumors that have been studied to
date have an active telomere maintenance mechanism, which strongly
suggests that prevention of telomere shortening is necessary for the
unlimited proliferative potential of these cells (9).
The telomere maintenance mechanism in most immortalized cells and tumor
cells utilizes the ribonucleoprotein enzyme telomerase, which
compensates for sequential telomere shortening at each cell division by
catalyzing the addition of repeat sequences (15). Telomerase is either absent or present at low levels in most normal human somatic cells (3, 24). Previous studies, however,
have shown that a number of in vitro immortalized human cell lines (8), tumor-derived cell lines, and tumors (6)
maintain their telomeres in the absence of detectable
telomerase activity by one or more mechanisms, referred to as
alternative lengthening of telomeres (ALT) (9). Telomere
lengths in cells utilizing ALT range in size from almost undetectable
to greater than 50 kb; this phenotype is not present in these cells
prior to immortalization (8). Recently it has been shown
that ALT involves telomere-telomere recombination (14).
In this study we demonstrate that induction of telomerase
activity in the ALT cell line GM847, by expression of the
telomerase catalytic subunit hTERT, significantly lengthens the
shortest telomeres. This is the first report demonstrating such an
effect by exogenous expression of hTERT in ALT cells and suggests that sufficient levels of other components of the telomerase complex required for telomere lengthening are present in these cells. Very long
heterogeneous telomeres and other hallmarks of ALT are still present in
these cells, suggesting that ALT is not repressed by and is able to
coexist with telomerase. To determine the effect of endogenous
telomerase on GM847 telomeres, we analyzed
telomerase-positive somatic cell hybrids generated by fusion of
GM847 with the telomerase-positive cell lines HT-1080 and T24.
Previously it was reported that there was continuing telomere
shortening in these hybrid cells despite the presence of
telomerase activity and that ALT was repressed (35). We now show that the telomeres in the
hybrids eventually reach a stable length and undergo no further
shortening at later population doublings (PDs). The data from the hTERT
transfection experiments indicate that telomerase is unlikely
to cause the complete repression of ALT that was seen in the somatic
cell hybrids. In addition, telomerase did not prevent
shortening of long telomeres in the hybrid cells. These findings have
implications for our understanding of how telomere maintenance
mechanisms are controlled in human cells.
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MATERIALS AND METHODS |
Plasmid expression vectors.
A cDNA insert encoding the human
telomerase catalytic subunit hTERT (23) was
subcloned into the mammalian expression vector pCIneo (Promega,
Madison, Wis.). The construct was verified by DNA sequence analysis. A
pIRESneo construct (Clontech, Palo Alto, Calif.) containing a
dominant-negative hTERT insert (3-1) was kindly provided by Murray O. Robinson, Amgen Corporation, and has been described previously
(20).
Transfection of GM847 cells with plasmid vectors.
GM847DM
cells were seeded at a density of 106 cells/10-cm dish and
incubated overnight. Cells were transfected the following day with 5 µg of plasmid DNA using 30 µl of Fugene-6 transfection reagent
(Roche, Basel, Switzerland) and 1 ml of OPTI-MEM reduced serum medium
(Life Technologies, Rockville, Md.) for 2 h at 37°C, after which
time Dulbecco's modified Eagle's medium plus 10% fetal bovine serum
was added. Cells were harvested 18 to 24 h later with trypsin-EDTA
and seeded at a concentration of 104 cells/10-cm dish in
medium containing 300 µg of Geneticin (G418 sulfate; Roche) per ml.
Individual colonies were isolated after 2 weeks of selection, using
4-mm sterile, trypsin-soaked filter paper discs, and then were passaged
continuously in medium containing Geneticin.
Cell culture and photomicrography.
All cells used in this
study were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 50 µg of gentamicin per
ml and placed at 37°C in a 5% CO2 humidified incubator.
Cells were photographed at a magnification of ×15 on a phase-contrast microscope.
TRAP assay.
Cell lysates were prepared using the
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)
detergent lysis method, and 2 µg of total protein was used in each
assay. The protein concentration of lysates was measured using the
Bio-Rad Protein Assay kit (Hercules, Calif.). The PCR-based telomere
repeat amplification protocol (TRAP) assay for telomerase
activity was performed essentially as described earlier
(24). Amplification products were separated on a 10%
nondenaturing polyacrylamide gel, stained with SYBR-green I (Molecular
Probes, Eugene, Oreg.) and visualized using a Storm 860 imager
(Molecular Dynamics, Sunnyvale, Calif.). A 150-bp rat myogenin cDNA
fragment containing TRAP primer flanking sequences was added to a
number of reactions as a PCR internal control and has been previously
described (46). The intensity of this internal control
band is inversely proportional to the level of telomerase activity.
Terminal restriction fragment (TRF) analysis.
Genomic DNA
was prepared from the parental and hybrid cultures, and 40 µg was
digested with restriction enzymes HinfI and RsaI,
which cut throughout the genome but not within telomeres. Quantitated
samples (1 µg) were loaded onto a 1% agarose gel in 0.5×
Tris-borate-EDTA (TBE) buffer and separated using a CHEF-DR II
pulsed-field electrophoresis apparatus (Bio-Rad) in recirculating 0.5×
TBE buffer at 14°C with a ramped pulse speed of 1 to 6 s at 200 V for 14 h. The gels were dried, denatured, hybridized to a
[
-32P]dATP 5'-end labeled telomeric oligonucleotide
probe, (TTAGGG)3, and exposed to Kodak XAR film at 
80°C
for 18 h.
FISH analysis and immunocytochemistry.
Chromosome
preparations from colcemid (Roche)-arrested cells were obtained
according to standard cytogenetic methods. Fluorescence in situ
hybridization (FISH) with a Cy3-conjugated telomere-specific peptide-nucleic acid probe (PE Biosystems, Framingham, Mass.) was
essentially performed according to the guidelines of Lansdorp et al.
(25).
Briefly, slides were treated with RNase A (100 µg/ml in 2× SSC [1×
SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) at 37°C for 60 min,
rinsed in 2× SSC, equilibrated in 10 mM HCl (pH 2.0), and digested
with pepsin (0.01% [wt/vol] in 10 mM HCl) at 37°C for 10 min. This
was followed by three washes with phosphate-buffered saline (PBS) and
postfixation in 1% formaldehyde-PBS for 10 min at 25°C. Dehydrated
slides were denatured and probed with Cy3-conjugated (C3TA2)3 peptide-nucleic acid (0.6 µg/ml in 70% formamide, 1% blocking reagent [Roche], and 10 mM
Tris, pH 7.2) at 80°C for 3 min on a heating block, followed by
hybridization at 25°C for 2 h. After hybridization, slides were
washed at 25°C with 70% formamide-10 mM Tris (pH 7.2) and 0.05 M
Tris-0.15 M NaCl (pH 7.5) containing 0.05% Tween 20. Slides
were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) (0.6 µg/ml; Sigma, St. Louis, Mo.) and mounted with
antifade-mounting medium (2.33% [wt/vol] DABCO [Sigma] in 90%
glycerol-20 mM Tris, pH 8.0). Metaphases were evaluated on a Leica
DMLB fluorescence microscope with appropriate filter sets, and DAPI and
Cy3 images were captured on a cooled CCD camera (SPOT2; Diagnostic
Instruments, Sterling Heights, Mich.), merged, and further processed
for illustrative purposes with Adobe Photoshop 6.0.
Quantitative histogram analysis of fluorescence intensities was
performed with ImagePro Plus 4.0 software (MediaCybernetics,
Silver
Spring, Md.) on 24-bit RGB images captured with exposure
times of 0.5, 1.0, 1.5, and 2.0 s with no gamma adjustment. Image
bitmap pixel
values ranged from 0 (black) to 255 (full scale)
following a linear
function of a measured intensity with increasing
exposure time.
Typically, four intensity values were recorded
for the maximum
intensities of the short (p) and the long (q)
arm of the Y chromosome
with the four different exposure times.
The ratio of p arm intensity to
q arm intensity was plotted as
a bar chart on a logarithmic
scale.
ALT-associated promyelocytic leukemia (PML) bodies (APBs)
(
47) were detected and visualized either by telomere FISH
to interphase
nuclei or by immunostaining methanol-fixed cells on
chamber slides
with a mouse monoclonal hTRF2 antibody (Upstate
Biotechnology,
Lake Placid, N.Y.) for 60 min at 25°C followed by a
rabbit anti-mouse
fluorescein isothiocyanate secondary antibody for 30 min. Images
were captured as described
above.
Somatic cell hybridization.
A universal hybridizer ALT cell
line, GM847DM (double mutant, simian virus 40 [SV40]-immortalized
human fibroblasts), was obtained from O. Pereira-Smith, Baylor College
of Medicine, Houston, Tex. HT-1080 (fibrosarcoma) and T24 (bladder
carcinoma) telomerase-positive cell lines were obtained from
the American Type Culture Collection (Manassas, Va.). Cell fusion was
performed as previously described (35).
Estimation of telomere shortening rate.
TRF gel lanes were
scanned by a computing densitometer (Molecular Dynamics). Discrete
telomere band sizes were calculated by plotting the relative position
on a linear curve generated by regression analysis of molecular weight
marker positions. Rates of shortening were calculated by reduction in
band sizes over specific PD ranges.
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RESULTS |
Exogenous expression of telomerase in GM847 cells lengthens
the shortest telomeres.
We transiently expressed the
telomerase catalytic subunit hTERT in the ALT cell line GM847
and found, using TRAP, that this reconstituted telomerase
activity which was absent in vector control and untransfected cells
(Fig. 1a). This finding was consistent with other reports showing activation of telomerase in GM847
cells by both transient (45) and stable (13)
transfection of hTERT alone. To assess the impact of exogenous
telomerase activity upon ALT, we generated 10 stable
clones of GM847 cells by transfection of hTERT (GM847/hTERT) and found
8 out of 10 to be telomerase positive (Fig. 1b). The
GM847/hTERT stable clones and vector control clones were
analyzed, at the earliest time points possible, by TRF to measure
telomere lengths. Telomere lengths of each clone, however, appeared
heterogeneous and characteristic of ALT whether or not
telomerase activity was detectable (data not shown). Thus, by
TRF analysis the ALT telomere phenotype appeared to be unaffected by
exogenous telomerase activity in GM847 cells.
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FIG. 1.
TRAP analysis of GM847 cells containing both transient
and stable expression of hTERT. An internal control (rat myogenin) for
the PCR is indicated by an arrow. LB, lysis buffer negative control.
(a) Transient transfection of exogenous hTERT into GM847 cells
(GM847/hTERT) is compared to untransfected GM847 or pCIneo vector
controls. (b) Stable clones of GM847 transfected with hTERT.
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Twelve subclones were generated by limiting dilution of two
telomerase-positive clones at late passage, GM847/hTERT-3 (PD
115) and GM847/hTERT-6 (PD 143). TRAP analysis showed that all
of the
subclones were telomerase positive (data not shown). The
telomere lengths of GM847/hTERT-3 and -6 clones and their subclones
were then compared with those of untransfected GM847 cells by
telomere
FISH with a fluorochrome-labeled telomere probe (Fig.
2). GM847 cells show heterogeneous
telomere FISH signals ranging
from undetectable to very large (Fig.
2a), and this pattern was
apparent in 50 out of 50 metaphase spreads
(Table
1). There were
similar numbers of
undetectable telomeres present in each of the
nuclei examined amounting
to approximately 6% of the total (Table
1). This telomere FISH pattern
is consistent with the telomere
length heterogeneity in GM847 cells
documented by TRF analysis
(
8,
35).
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FIG. 2.
Telomere length analysis by FISH on individual metaphase
chromosomes using a peptide-nucleic acid telomere probe. Examples of
very intense telomere signals (corresponding to very long telomeres)
are indicated by arrows with filled heads and chromosome ends with
undetectable or almost undetectable telomere signals are indicated by
arrows. (a) Untransfected GM847 cells showing widely varying signal
intensities indicative of ALT telomere length heterogeneity. (b) A
metaphase from the GM847/hTERT-3 parental mass culture that has
ALT-like heterogeneous telomere lengths. (c and d) GM847/hTERT-3
parental cell metaphases showing a detectable FISH signal at each
chromosome. GM847/hTERT-3 subclone 9 (e) and GM847/hTERT-6 subclone 11 (f) also show detectable FISH signals at each chromosome end.
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Analysis of GM847/hTERT-3 cells, however, revealed detectable telomere
FISH signals at almost every chromosome end (Fig.
2c
and d) in the
majority of nuclei at both early and late passage.
This suggested that
the shortest telomeres had been lengthened
in the majority of cells
within the population. Chromosome ends
without a detectable telomere
FISH signal were present in only
12 out of 50 GM847/hTERT-3 metaphase
nuclei at an early passage
and 13 out of 50 nuclei at a later passage
(Table
1). Approximately
one quarter of the GM847/hTERT-3 metaphases
examined, therefore,
still had the very heterogeneous telomere FISH
signals characteristic
of untransfected GM847 cells (Fig.
2b), and this
subpopulation
persisted with passage in culture (Table
1). Although no
telomerase-negative
subclones of GM847/hTERT-3 were detected,
it is possible that
hTERT expression had been downregulated in a
proportion of cells
and this resulted in retention of, or reversion to,
a characteristic
ALT telomere
phenotype.
Similar results were obtained for the GM847/hTERT-6 clone, in which
most of the short telomeres had also been lengthened.
Approximately one
quarter (23 to 30%) of the GM847/hTERT-6 clone
cells contained a
subpopulation of cells with at least one undetectable
telomere signal
(Table
1). However, it is likely that even in
this subpopulation some
of the very short telomeres had been lengthened
by telomerase
activity, because the number of undetectable telomeres
was reduced by
much more than one quarter compared to that of
the untransfected GM847
cells. It is noteworthy that some very
large signals were still evident
in nuclei that had no undetectable
telomere signals (Fig.
2). This
indicated that although the number
of very short telomeres was reduced
in these clones, very long
telomeres were still
present.
FISH analysis of GM847/hTERT-3 subclone 9 (Fig.
2e) and GM847/hTERT-6
subclone 11 (Fig.
2f) revealed detectable telomeric
signals on every
chromosome examined in 49 out of 50 and 50 out
of 50 metaphases,
respectively (Table
1). It is interesting that
although cells with
undetectable telomeres were present within
the GM847/hTERT-3 and -6 populations and persisted at later passages,
this phenotype was very
rare in the GM847/hTERT-3 and -6 subclones.
A reason for this outcome
may be that cells within the GM847/hTERT
populations that have
lengthened the shortest telomeres might
have a selective growth
advantage during cloning by limiting
dilution.
These FISH data therefore show that the shortest telomeres have been
lengthened in most of the GM847 cells that express exogenous
hTERT. The
effect was most noticeable after subcloning, and this
may have been due
to the additional population doublings that
the cells undergo during
the subcloning process, during which
time telomere lengthening by
telomerase would have progressed.
Every metaphase of
untransfected GM847 cells that was examined,
without exception, had
several chromosome ends with an undetectable
telomere FISH signal
(Table
1). This indicates that the lengthening
of the short telomeres
seen in GM847/hTERT cells is due to telomerase
rather than
random selection of putative preexisting cells within
the untransfected
GM847 population that happened to have no short
telomeres. The data
were confirmed by FISH examination of additional
GM847/hTERT
clones, which showed in five of five clones that the
shortest
telomeres had clearly been lengthened; this phenotype
was detected in
zero of five GM847 untransfected subclones and
in zero of two vector
control clones (data not
shown).
The ALT mechanism remains active in GM847/hTERT cells.
FISH
analysis of early- and late-passage GM847/hTERT-3 and GM847/hTERT-6
cells and subclones derived from them showed that these cells retained
the very large telomere FISH signals characteristic of ALT cells (Fig.
2). This suggested that ALT was still active in these cells, but to
confirm that this was the case, additional telomere FISH analyses were
performed on GM847/hTERT-3 subclones 6 and 9 and GM847/hTERT-6
subclones 2 and 11 that had been obtained by limiting dilution at
late-passage levels (PD 115 and PD 143, respectively). Very large FISH
signals corresponding to long telomeres were detectable in every
metaphase examined (Fig. 3), indicating that ALT telomere maintenance was still operating in these cells. Further cytogenetic analysis of metaphase nuclei within each subclone revealed a striking heterogeneity in the chromosomal location of the
long telomeres (Fig. 4). This pattern was
also seen in untransfected GM847 cells but not in
telomerase-positive cells (data not shown). This is
consistent with previous data indicating that GM847 cells maintain
their telomeres by telomere-telomere recombination and copy switching
(14). As the subclones were derived from single cells,
this heterogeneity must have been generated subsequent to subcloning,
indicating that ALT was still active for more than 115 or 143 PD (in
GM847/hTERT-3 or GM847/hTERT-6 cells, respectively) after initial
transfection with hTERT.
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FIG. 3.
Telomere FISH analysis, using a peptide-nucleic acid
telomere probe, on individual metaphase nuclei from subclones of
GM847/hTERT-3 and GM847/hTERT-6 (all at PD 25 after subcloning of
late-passage cells). Examples of very intense telomere signals
(corresponding to very long telomeres) are indicated by arrows.
Representative images are shown. (a and b) GM847/hTERT-3 subclone 6; (c
and d) GM847/hTERT-3 subclone 9; (e and f) GM847/hTERT-6 subclone 2; (g
and h) GM847/hTERT-6 subclone 11.
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FIG. 4.
Detection of long telomeres on specific chromosomes in
metaphase spreads of GM847/hTERT subclones. Footnote a, ten metaphase
nuclei were examined for each subclone. Numbers represent the number of
instances of long telomeres on the same chromosome within a single
metaphase. Footnote b, chromosomes that could not be identified exactly
were assigned to groups based on their size and arm index. Group number
(chromosomes): A (1 through 3); B (4 and 5); C (6 through 12, X); D (13 through 15); E (16 through 18); F (19 and 20); G (21, 22, Y).
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We then compared the telomere FISH signals of the p and q arms of the Y
chromosome, which was present in single copy in subclones
of
GM847/hTERT-3 and GM847/hTERT-6. It was found that within each
GM847/hTERT subclone there was dramatic variation in the ratio
of these
signals, consistent with the GM847 untransfected control,
which was not
seen in the telomerase-positive cell line, GM639
(SV40-immortalized skin fibroblasts) (Fig.
5). This marked fluctuation
in p:q ratio
must have occurred subsequent to subcloning, consistent
with the
presence of continuing ALT activity in these cells.
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FIG. 5.
Ratio of p arm to q arm telomere lengths on the Y
chromosome from 20 metaphase spreads of each GM847/hTERT subclone
indicated, including a GM847 (ALT) and GM639
(telomerase-positive) control. Each bar represents the ratio
for an individual metaphase. The ratio was calculated using the Y
chromosome mean telomere fluorescence signal intensities from captured
images of each metaphase nucleus at exposure times of 0.5, 1.0, 1.5, and 2.0 s.
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We examined the GM847/hTERT cells for the presence of APBs. APBs are
PML bodies containing low-molecular-weight telomeric
DNA and
telomere-associated proteins and are present in all ALT
cell lines
examined to date (including GM847) but not in
telomerase-positive
or mortal cells (
47), and they
can be readily detected in interphase
nuclei from metaphase
preparations by telomere FISH. In exponentially
growing cultures of ALT
cells, APBs are present in approximately
5% of the population. Their
occurrence has a close temporal correlation
with activation of ALT in
cell lines immortalized in vitro (
47).
They are thus an
excellent marker of ALT activity, although the
role of APBs in the
mechanism of ALT is unknown. APBs were detectable
by telomere FISH at
all PD levels examined for GM847/hTERT-3 and
GM847/hTERT-6 cells and in
subclones of these cells (Fig.
6).
The
proportion of cells with detectable APBs was maintained within
the
range characteristic of untransfected GM847 cells (Table
2)
(
47). These data suggest
that ALT can still operate in the presence
of exogenous hTERT, and it
coexists with telomerase activity as
a telomere maintenance
mechanism in GM847/hTERT cells.
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FIG. 6.
Detection of APBs in interphase nuclei of GM847/hTERT
cells by telomere FISH. Representative images are shown (magnification,
×100). (a) Untransfected GM847; (b) GM847/hTERT-3 (PD 42); (c)
GM847/hTERT-3 (PD 138); (d) GM847/hTERT-3 subclone 9 (PD 25); (e)
GM847/hTERT-6 (PD 150); (f) GM847/hTERT-6 subclone 11 (PD 25).
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Endogenous telomerase is required for the immortal
phenotype in somatic cell hybrids of ALT cells and
telomerase-positive cells.
The coexistence of
telomerase and ALT was further investigated by utilizing
somatic cell hybrids of ALT and telomerase-positive cells. We
previously generated such hybrids by fusion of GM847 cells with both
HT-1080 and T24 telomerase-positive cells and showed that the
resulting hybrid clones were telomerase positive (35). Investigation of telomere dynamics in these cells
revealed an initial rapid deletion of large telomeric tracts,
indicating repression of ALT, followed by a normal rate of telomere
shortening which suggested that telomerase was unable to
maintain the telomeres (35). DNA fingerprinting and
immunostaining for SV40 T-antigen expression at both early and late PDs
indicated that the clones were indeed hybrids, containing genetic
material from both parental cells (data not shown). A number of these
hybrid clones were continuously passaged to determine the effect of
endogenous telomerase on the ALT phenotype.
Four hybrid clones, GM847 × HT-1080 (G/HT) clones E and K and
GM847 × T24 (G/T) clones G and L, were passaged for approximately
300 PDs with no occurrence of growth arrest or senescence. These
cells
were positive in the TRAP assay at each PD level tested,
indicating
that they retained in vitro telomerase activity throughout
this
period of growth (Fig.
7a). To
determine whether telomerase
was actually required for
continuing growth of the hybrid cells,
we transfected clones G/HT K and
G/T L with a dominant-negative
hTERT (dn hTERT) expression plasmid.
This plasmid expresses an
hTERT protein which contains an amino acid
substitution within
the catalytic core and which has been
characterized previously
as an inhibitor of telomerase
activity. It has also been shown
to cause telomere shortening and can
induce apoptosis (
48) or
senescence (
10)
following transfection into telomerase-positive
human cell
lines.
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FIG. 7.
(a) TRAP analysis of G/T clones G and L and G/HT clones
E and K at the PD timepoints indicated. (b) TRAP analysis of hybrid
clones G/HT K (KDN) and G/T L (LDN) expressing dn hTERT. LB, lysis
buffer negative control. An internal control (rat myogenin) for the PCR
is indicated by an arrow.
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Fifteen colonies were isolated from the G/HT K (KDN) and 19 from the
G/T L (LDN) dn hTERT transfections. At early PDs, before
the colonies
had expanded sufficiently to permit harvesting of
cells for analysis, 3 KDN clones and 17 LDN clones ceased dividing,
with the cells displaying
the large, flat morphology characteristic
of senescence. Twelve KDN
clones and only two LDN clones underwent
sufficient PDs to allow
analysis of telomerase activity by TRAP
(Fig.
7b). KDN clones
3, 8, 10, and 11 showed complete inhibition
of telomerase
activity, with weak activity detectable in clones
2 and 9. All other
KDN clones that could be analyzed were telomerase
positive,
which was consistent with previous studies utilizing
this same dn hTERT
construct that reported the emergence of telomerase-positive
revertants (
10,
20). LDN clones 10 and 11 also showed
complete
inhibition of telomerase activity (Fig.
7b). TRF
analyses of a
subset of KDN and LDN clones showed telomere shortening
in the
absence of telomerase and stabilization or lengthening
in clones
that retained telomerase activity (data not shown).
The inhibition
of telomerase in these clones correlated with
the appearance of
a senescent phenotype. In contrast, the clones that
were telomerase
positive continued to divide, with no onset of
growth arrest.
Representative cell morphologies for both senescent and
proliferating
clones are shown in Fig.
8.
KDN 7 cells (Fig.
8a), which are telomerase
positive, divide
rapidly, grow at high densities, and have detectable
mitotic cells,
which is also characteristic of the other telomerase-positive
KDN clones. In contrast, KDN 10 and KDN 11 cells, which are
telomerase
inhibited, no longer divide, and many have become
enlarged (Fig.
8b and c), which was also apparent in LDN clones 10 and
11 (Fig.
8e and f). Most LDN clones stopped dividing at very early PD
levels
(when they had formed small colonies), and the few surviving
cells
also appeared enlarged and senescent (Fig.
8d). Telomerase is
therefore required for continued cell growth in these hybrids.
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|
FIG. 8.
Phase-contrast photomicrographs of dn hTERT transfected
subclones of G/HT hybrid clone K (KDN) and G/T hybrid clone L (LDN).
Magnification, ×15. Representative images are shown. (a) KDN clone 7;
(b) KDN clone 10; (c) KDN clone 11; (d) an LDN colony; (e) LDN clone
11; (f) LDN clone 10.
|
|
It has been reported previously that expression of dn hTERT in GM847
cells has no effect on proliferation or telomere lengths
of GM847 cells
(
16). Consistent with this finding, we recently
established stable clones of GM847 expressing dn hTERT, and there
was
no evidence of proliferation arrest at early PD levels (data
not
shown). These results support the conclusion that telomerase
is
the sole telomere maintenance mechanism in the hybrid
cells.
APBs are lost from the somatic cell hybrids.
As further
evidence that there was no ALT activity in the hybrid cells, we tested
each of the four hybrid clones for APBs. Hybrid clones at early and
late PDs were immunostained with anti-hTRF2 antibodies to visualize
APBs (Fig. 9). At the earlier PDs, in each case there were rare nuclei (1 out of 1,000 to 2,000) that had
detectable APBs (Fig. 9c, e, and g), but most had the punctate staining
pattern that is characteristic of telomerase-positive cells
(Fig. 9b). Thus, 0.05 to 0.1% of the hybrid cells had APBs at early
time points, which is at least a 30-fold reduction from the level in
untransfected GM847 cells (47). At later PDs there were no
APBs detected in any nuclei examined (Fig. 9d, f, and h). In contrast,
analysis of GM847/hTERT subclones showed no loss of APBs at later time
points (Table 2 and Fig. 6). These data provide further evidence that
ALT is fully repressed in the hybrid clones but is still functional in
GM847/hTERT clones.
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|
FIG. 9.
Detection of APBs by immunostaining with antibodies to
hTRF2. Representative cells (magnification, ×40) are shown. (a) GM847
ALT control. APBs are detectable in 1 to 5% of nuclei (arrow). (b)
GM639 (SV40-immortalized fibroblasts) telomerase-positive
control. No APBs are visible in any nucleus. (c through h) Somatic cell
hybrid clones at different PD levels. Left panels indicate rare
individual nuclei (arrow) at earlier PDs in which APBs could be
detected. Right panels are indicative of all nuclei examined at later
PDs for each hybrid in which only the punctate pattern of staining
characteristic of ALT-negative human cells (47) was seen.
(c) G/HT K (PD 43); (d) G/HT K (PD 134); (e) G/T L (PD 58); (f) G/T L
(PD 148); (g) G/HT E (PD 23); (h) G/HT E (PD 62).
|
|
Endogenous telomerase eventually stabilizes and maintains
telomeres in the ALT-repressed hybrids.
The results from the dn
hTERT transfection experiment implied that telomerase activity
was important for the survival of the hybrid clones, presumably through
its role in telomere maintenance. This further implied that the
telomeres of these hybrids would eventually stop shortening. To test
this, TRF analysis of hybrid clones G/HT E and K and G/T G and L (Fig.
10) was performed. As observed
previously (35), the long telomeres contributed by GM847
cells were not maintained at early PDs and indeed were lost from clones
K, L, and G at a much faster rate than that observed in normal
telomerase-negative cells. In clone K, which lost the large
TRFs most rapidly, the telomere length then fluctuated around a mean
length below 8 kb, while clones E, G, and L entered a phase in which
the telomeres continued to shorten but at a less rapid rate (Fig. 10).
The telomere length dynamics in clone K suggest that there may be some
dysregulation of factors that stabilize telomeres which was not
apparent in the other clones (Fig. 10). Other studies have shown that
telomere length can vary in the presence of telomerase
(7, 22, 39) and that there may be fluctuation around a
mean telomere length (39).
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FIG. 10.
TRF analysis of hybrid clones G/HT E and K and G/T L
and G at the PD levels indicated. The analysis was performed using
pulsed-field gel electrophoresis as described in Materials and Methods.
Parental cell TRFs are shown in the left-hand panels.
|
|
The presence of discrete TRF bands allowed us to more accurately
measure the rate of shortening for clones E, G, and L (175,
67, and 44 bp/PD, respectively), and these were consistent with
the rates of
telomere shortening (50 to 200 bp/PD) that had been
previously reported
for mortal cells (
11,
18,
21). These
results therefore
show that telomerase did not maintain the long
telomeres on the
chromosomes contributed to the hybrids by the
ALT cells. The second
phase of shortening in three of the four
clones (G/HT E and G/T L and
G) occurred at a rate characteristic
of normal cells which have no
telomerase activity. At later PD
levels, however, the TRF
lengths stabilized, indicating telomere
maintenance by
telomerase.
| |
DISCUSSION |
We report that exogenous expression of hTERT in GM847 ALT cells
lengthens the shortest telomeres. Although transfection of hTERT into
ALT cells has been shown to induce in vitro TRAP activity (13,
45), it has not been demonstrated previously that this telomerase activity has any effect on the telomeres in vivo. In other circumstances it has been shown clearly that TRAP activity is not
synonymous with in vivo enzyme activity: hTERT that was modified at the
carboxyl terminus induced TRAP activity when expressed in
telomerase-negative mortal cells but was not able to maintain the telomeres (12). We have demonstrated in this study
that hTERT expression results in telomerase activity that acts
on some of the telomeres of GM847 cells, as detected by telomere FISH. This effect was most apparent after subcloning and continued passaging, most likely because the telomere lengthening was a gradual process that
did not occur equally in all cells within the population. Within the
untransfected GM847 population every metaphase nucleus examined had
chromosome ends without a detectable telomere FISH signal. This
indicates that the telomere lengthening was due to hTERT expression and
not due to random clonal selection of preexisting cells within the
population in which telomere lengthening had already occurred. As a
corollary, the data indicate that the GM847 cells express sufficient
levels of whatever factors, other than hTERT, are required for
telomerase-mediated lengthening of telomeres.
Although telomerase is active in the GM847/hTERT cells, these
cells retain hallmarks of ALT activity, that is, persistence of
heterogeneous telomeres and of APBs. Additionally, the very long
telomeres were present on different chromosomes in clonal populations
of late-passage GM847/hTERT cells, which is consistent with ALT
involving a telomere-telomere recombination mechanism (14). This indicates that telomerase and ALT are
both active in the GM847/hTERT cells. In contrast, when ALT was
repressed in somatic cell hybrids of GM847 with a
telomerase-positive cell line, the telomeres rapidly decreased
in length and the APBs disappeared. We previously found that some human
tumors exhibit both telomere maintenance mechanisms (6).
The present lack of assays suitable for analysis of individual cells
within tumors means that it is currently not possible to determine
whether these tumors contain subpopulations with one or other telomere
maintenance mechanisms, or whether there are tumor cells utilizing more
than one mechanism. The results reported here do not answer this
question regarding tumors, but they do show that it is possible for
more than one mechanism to be active in the same cell.
It has been demonstrated previously in yeast that cells lacking
telomerase utilize a recombination pathway to maintain
telomeres and continue proliferating (29, 32). It has been
suggested that this mechanism in yeast, which is dependent on RAD52,
may be a general alternative pathway for telomere maintenance in
eukaryotes (32). Some telomerase-negative
Saccharomyces cerevisiae cells survive by a mechanism that
closely resembles that in human ALT cells: type II survivors have
telomeres with extremely heterogeneous lengths (29, 42).
The type II mechanism in yeast and ALT in human cells both involve
recombination (14, 41). The data presented here, however,
suggest that the ALT mechanism in human cells may differ somewhat from
that in yeast. In yeast type II survivors, telomere lengthening only
occurs on very short telomeres (41), and it has been shown
that reconstitution of telomerase activity in S. cerevisiae inhibits this ALT-like mechanism and returns the
telomeres to wild-type lengths over a number of PDs (42).
In GM847/hTERT cells, however, the hallmarks of ALT activity persist
even when the shortest telomeres have been lengthened by telomerase.
We also report that in somatic cell hybrid clones, generated by fusion
of GM847 cells with each of two telomerase-positive cell lines
(ALT × telomerase), ALT is repressed and the telomeres are maintained
by endogenous telomerase activity. Features of ALT disappeared,
i.e., the abnormally long telomeres (35) and APBs.
Transfection of the ALT × telomerase hybrid cells with dn hTERT
demonstrated that their continued survival was dependent on
telomerase activity, which is consistent with the other data indicating that ALT was repressed. The factor(s) responsible for ALT
repression in these hybrids has yet to be identified, but the lack of
ALT repression in the hTERT-transfected GM847 cells also suggests that
it is unlikely to be telomerase. It is possible that
telomerase activity induced by overexpression of hTERT in ALT
cells is not entirely comparable to the endogenous telomerase activity in the hybrids. Further, we cannot completely exclude the
possibility that ALT repression in the hybrids is mediated via
telomerase, maybe indirectly, in concert with some other factor which is lacking in the GM847/hTERT cells and which is contributed along with telomerase activity to the hybrid by the
telomerase-positive cells. However, it has been previously
shown that ALT is repressed in ALT × normal somatic cell hybrids
(35); in these hybrids repression of ALT was clearly not
due to telomerase, as neither the normal cells nor the hybrids
expressed any telomerase activity.
Interestingly, in both the ALT × telomerase-positive hybrids
and in the ALT × normal fibroblast hybrids (35) there was
an initial rapid loss of telomeric tracts, suggesting that an active mechanism was involved. Rapid telomere loss has also been reported in
yeast, where cleavage of long telomeres to a wild-type size could occur
within a single cell division (27).
Candidate ALT repressors include telomere-associated proteins
contributed to the hybrids by the telomerase cells. It is very possible that more than one repressor of ALT exists in these cells, which would greatly reduce the probability of ALT revertants if telomerase activity was abrogated. Previous studies have shown that human telomere binding proteins hTRF1 (38, 43) and
hTRF2 (38) play a direct role in regulation of telomere
elongation by telomerase and prevent overextension of telomere
tracts. In yeast it has been shown that the telomere binding protein
Rap1p tightly regulates telomere lengths (31) and that
Rap1p binding proteins, Rif1 and Rif2, inhibit both telomerase
and type II recombination mechanisms (41). While it
remains entirely possible that proteins that regulate both
telomerase and ALT in human cells will be found, our somatic
cell hybrid data show that ALT and telomerase can be controlled separately.
Telomere shortening occurs at normal rates in the hybrid cells despite
the presence of telomerase, but this is eventually followed at
later PDs by telomere length stabilization at lengths characteristic of
telomerase-positive cells. This indicates, consistent with our
findings in GM847/hTERT cells, that short telomeres are lengthened by
telomerase. However, unlike ALT, there appears to be a control
mechanism in telomerase-positive cells that prevents maintenance by telomerase of telomeres above a certain length. There is strong evidence for the existence of such a regulatory mechanism in yeast (30, 31). Studies on
telomerase-positive human immortalized cells have also found
evidence for telomere length control and have suggested the existence
of an equilibrium mean length for individual telomeres, above which
shortening can occur in the presence of telomerase activity
(39). In some cases, telomeres can be maintained by
telomerase at subsenescent lengths (11, 40, 49).
The data presented here provide new insights into the regulation of
telomerase and ALT in human cells and also suggest some possible differences between ALT and the mechanism in yeast type II
survivors. In view of the current interest in developing anticancer therapeutics directed against telomerase, it will be of
particular interest to determine whether ALT and telomerase
sometimes coexist in individual tumor cells. An understanding of the
mechanisms whereby ALT is normally repressed may also identify useful
therapeutic targets.
| |
ACKNOWLEDGMENTS |
We thank Peter Rowe for his comments on the manuscript and Murray
Robinson, Amgen Corporation, for providing the dominant-negative hTERT construct.
These studies were supported by a project grant from the National
Health and Medical Research Council of Australia and the Carcinogenesis
Fellowship of the New South Wales Cancer Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Children's
Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia. Phone: 61 2 9687 2800. Fax: 61 2 9687 2120. E-mail:
rreddel{at}cmri.usyd.edu.au.
| |
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Molecular and Cellular Biology, June 2001, p. 3862-3875, Vol. 21, No. 12
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.12.3862-3875.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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