Download Assembly of Mutant-Template Telomerase RNA into Catalytically

Research Article
Assembly of Mutant-Template Telomerase RNA into Catalytically
Active Telomerase Ribonucleoprotein That Can Act on
Telomeres Is Required for Apoptosis and Cell Cycle
Arrest in Human Cancer Cells
1,2
2
Amir Goldkorn and Elizabeth H. Blackburn
1
Department of Internal Medicine, Division of Hematology and Oncology and 2Department of Biochemistry and Biophysics,
University of California at San Francisco, San Francisco, California
Abstract
The telomerase ribonucleoprotein is a promising target for
cancer therapy, as it is highly active in many human
malignancies. A novel telomerase targeting approach combines short interfering RNA (siRNA) knockdown of endogenous human telomerase RNA (hTer) with expression of a
mutant-template hTer (MT-hTer). Such combination MT-hTer/
siRNA constructs induce a rapid DNA damage response,
telomere uncapping, and inhibition of cell proliferation in a
variety of human cancer cell lines. We tested which functional
aspects of the protein catalytic component of telomerase
[human telomerase reverse transcriptase (hTERT)] are required for these effects using human LOX melanoma cells
overexpressing various hTERTs of known properties. Within 3
days of MT-hTer/siRNA introduction, both growth inhibition
and DNA damage responses were significantly higher in the
setting of wild-type hTERT versus catalytically dead hTERT
or mutant hTERT that is catalytically competent but unable to
act on telomeres. These effects were not attenuated by siRNAinduced knockdown of the telomeric protein human Rap1 and
were additive with knockdown of the telomere-binding
protein TRF2. Hence, the effects of MT-hTer/siRNA require a
telomerase that is both catalytically competent to polymerize
DNA and able to act on telomeres in cells. (Cancer Res 2006;
66(11): 5763-71)
Introduction
Telomerase-based therapy provides a novel approach to the
treatment of many cancers. Telomeric DNA contains tandem
repetitive DNA sequences located at the ends of human chromosomes (1). The two essential functions of telomeres are protecting
chromosome ends (the ‘‘capping’’ function of telomeres) and facilitating their complete replication, usually involving the action of
telomerase, a ribonucleoprotein enzyme. Telomerase consists of
two core components: a reverse transcriptase protein [called
human telomerase reverse transcriptase (hTERT) in humans] and
human telomerase RNA (hTR or hTer in humans). hTer contains
a short template sequence used by hTERT to synthesize telomeric
DNA (2).
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Elizabeth H. Blackburn, Department of Biochemistry and
Biophysics, University of California at San Francisco, San Francisco, CA 94143-2200.
Phone: 415-476-4912; Fax: 415-514-2913; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-3782
www.aacrjournals.org
As a result of telomerase down-regulation in many normal
somatic cells, human chromosomes can lose up to 50 to 200
nucleotides of telomeric sequence per cell division (3). Such
shortening of telomeres has been proposed to be a mitotic clock
that monitors cell divisions; sufficiently short telomeres and lack
of telomerase may signal cellular senescence (3, 4). In contrast,
highly active telomerase is a common feature of a wide variety of
human cancers (5). Attempts to attenuate telomerase function in
cultured human cancer cells have led to telomere shortening (6)
and, eventually, to cellular apoptosis, thus limiting the cancer cell
growth in vitro (7, 8) and providing further evidence that
telomerase-dependent telomere maintenance promotes tumorigenesis. It was recently shown that depleting the endogenous wildtype hTer (wt-hTer) of cancer cells, with either short interfering
RNAs (siRNA) or ribozymes, leads to reduced telomerase activity,
growth inhibition, and reduction in metastatic progression in
mouse models. Intriguingly, these effects of simply reducing
telomerase RNA levels are accompanied by changes in the global
gene expression profile, but they do not involve bulk telomere
shortening or telomere uncapping nor do they elicit a DNA damage
response or require p53 (9, 10).
A second telomerase-based strategy against cancer cells exploits
the telomerase activation common in cancer cells, in effect turning
telomerase itself into a weapon against these cells. This was
accomplished by mutating the template of hTer and introducing
that mutant-template hTer (MT-hTer) gene into cancer cells and
mouse xenograft models (11, 12), where it resulted in cell killing
and slowing of tumor growth. The effects were specific to the
MT-hTer mutation and depended on telomerase expression but not
on p53 or pRb function. However, in contrast to the effects of
telomerase RNA depletion alone, MT-hTer expression induced a
DNA damage response and formation of DNA damage foci,
including DNA damage foci at telomeres (9, 13). Furthermore,
combining siRNA targeting endogenous hTer together with MThTer on the same construct had a synergistic inhibitory effect on
cell proliferation, consistent with more effective replacement of
endogenous wt-hTer by MT-hTer (12). The construct containing
both MT-hTer and siRNA against endogenous hTer will henceforth
be termed MT-hTer/siRNA.
The hypothesis for the toxic effects of MT-hTer expression was
that the MT-hTer complexed with hTERT to form a ribonucleoprotein that adds incorrect nucleotide repeats to the end of the
telomere (14), preventing binding of telomere-protective proteins.
However, whether it is the action of catalytically active telomerase
on telomeres that causes the DNA damage and inhibition of proliferation in human cancer cells has remained unproven. An
alternative mechanism of cell growth inhibition is suggested by
5763
Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
studies of telomerase in yeast, in which wt-hTer has a direct
telomere-protective function (15). This protection possibly involves
the known in vivo association of telomerase with telomeres during
G1 phase (16, 17), a cell cycle stage during which polymerization by
telomerase does not occur. These findings raised the possibility
that an aberrant telomerase, such as that containing MT-hTer,
may itself elicit a cellular response, for example, if it were unable to
perform a comparable protective function in human cells, thus
potentially contributing to the toxic effects of MT-hTer expression.
Furthermore, the finding that siRNA directed against endogenous
wt-hTer could alone inhibit cell growth (9) raised the possibility
that the synergism observed between MT-hTer and the antitelomerase RNA siRNA might reflect other cause(s) besides the
telomere uncapping predicted from incorporation of mutant
telomeric DNA repeats.
Here, we test and investigate the mechanism by which MT-hTer/
siRNA exerts its toxic effects. The experimental strategy was to
introduce a MT-hTer/siRNA construct into cells that were already
overexpressing one of three functional variants of the hTERT
protein. The phenotypic effects of MT-hTer/siRNA were then
determined in the setting of each hTERT variant. One hTERT
mutant tested was a catalytically inactive hTERT enzyme, the
‘‘dominant-negative’’ point mutant D868A hTERT (DN-hTERT).
The second hTERT mutant was N125A+T126A-hTERT, which
creates an enzyme that is catalytically active in vitro but fails to
elongate the telomeres in cells (18). The N125A+T126A mutation
is located in the so-called ‘‘dissociates activities of telomerase’’
(N-DAT) region, thought to be necessary for action of telomerase
on telomeres (19). Specifically, substitution of amino acids 122 to
127 in hTERT, which includes the two highly conserved residues
N125 and T126, has been shown previously to interfere with the
efficient utilization of telomeric G-rich DNA primer substrates by
reconstituted telomerase in vitro, although the ability to elongate
a nontelomeric DNA substrate remained the same as wild-type
(20). Hence, the N125 and T126 residues are thought to be involved
in the ‘‘anchor’’ site function of telomerase that increases the
affinity of telomerase for its telomeric DNA primer substrate to be
elongated.
The results of the experiments described here indicate that, to
exert its toxic effects, MT-hTer/siRNA needs to assemble into a
telomerase enzyme complex containing a hTERT that is both
catalytically competent and can act on telomeres in the cell and
that the potency of the rapid cell growth inhibition is not weakened
by depletion of telomeric proteins TRF2 or human Rap1 (hRap1).
Materials and Methods
Plasmid construction. The hTERT alleles were engineered as described
previously (18). Briefly, the N125A+T126A mutations at codons 125 and 126
and the D868A mutation at codon 868 were introduced using PCR-based
site-directed mutagenesis. All of the hTERT alleles were cloned into a
pBabe-puro retroviral expression vector. The MT-hTer/siRNA was engineered as described previously (12). Briefly, wt-hTer was PCR cloned from
human genomic DNA and subcloned into BlgII/SalI site in pIU1-T7 plasmid
(generously provided by Dr. Edouard Bertrand, University Montpellier,
Montpellier, France). MT-hTer was then generated by site-directed
mutagenesis to create an hTer with the templating domain sequence
5¶-UAUAUAUAUAA-3¶, AU5-MT-hTer (11, 12). The siRNA targeting wt-hTer
was engineered by PCR using a pTZ-U6 plasmid (generously provided by Dr.
John J. Rossi, Beckman Research Institute of the City of Hope, Duarte, CA)
using primers as described previously (12). The PCR product was subcloned
along with the MT-hTer into pHRCMVGFPWSin18 vector. siRNA targeting
TRF2 and hRap1, as well as a scrambled siRNA sequence that does not
Cancer Res 2006; 66: (11). June 1, 2006
affect mRNA levels or cell proliferation, was packaged using this same
lentiviral vector system with sequences as described previously (13).
Virus production. Recombinant lentiviruses were generated by transfecting pMD.G and pCMVDR8.91 plasmids (generously provided by
Dr. Didier Trono, University of Geneva, Geneva, Switzerland; ref. 21), along
with the MT-hTer/siRNA plasmid, into 293T cells with LipofectAMINE 2000
(Invitrogen, Carlsbad, CA). Recombinant retroviruses were generated by
transfecting hTERT pBabe-puro plasmids into Phoenix packaging cell line
with LipofectAMINE 2000. For both lentiviral and retroviral transfections,
virus-containing medium was harvested 48 and 72 hours after transfection
and filtered through 0.45-Am filters.
Cell culture and infection. LOX melanoma cells were seeded in six-well
tissue culture plates at 2 104 per well in duplicate or triplicate on the
evening before infection. Infection with pBabe-puro retrovirus containing
WT-hTERT or DN-hTERT or N125A+T126A-hTERT was achieved by
incubation with virus-containing culture medium supplemented with
8 Ag/mL polybrene for 8 hours. After 8-hour infection, fresh culture
medium was added, and the cells were maintained in culture for 2 days. At
the end of day 2, puromycin-containing culture medium was added, and the
cells were maintained in culture for 4 more days. At the end of 4 days of
puromycin selection (day 6 after hTERT infection), cells were reseeded at
2 104 per well in duplicate. On the following day (day 7 after hTERT
infection), cells were infected with lentiviral MT-hTERT/siRNA constructs at
>95% efficiency of infection as indicated by green fluorescent protein (GFP)
expressed from the same lentiviral vector. Lentiviral siRNA constructs
targeting TRF2 or hRap1 were infected in a similar fashion. Cells were
counted at 3, 6, and 9 days after lentiviral infection using a hemocytometer
under direct illumination microscopy.
Apoptosis measurement by fluorescence-activated cell sorting. After
cells were trypsinized and counted at each time point, 1 105 cells were
washed and resuspended in PBS and then stained with Annexinallophycocyanin conjugate (Invitrogen) per manufacturer’s protocol. Cells
were also stained with 1 Ag/mL propidium iodide (Invitrogen) as a dead
cell counterstain. Stained cells were fluorescence sorted on a FACSAria cell
sorter (Becton Dickinson, San Jose, CA).
Cell cycle analysis by fluorescence-activated cell sorting. Immediately before trypsinizing and counting the cells at each time point, a subset
of cells was treated with 10 Amol/L BrdUrd (Sigma, St. Louis, MO) for
1 hour. Trypsinized BrdUrd-treated cells were washed in PBS and fixed with
70% ethanol. Cells were stained overnight using anti-BrdUrd Alexa Fluor
647 monoclonal antibody (Invitrogen) per manufacturer’s protocol and then
fluorescence sorted on a FACSCalibur cell sorter (Becton Dickinson).
Immunofluorescence microscopy. Cells were grown on glass coverslips
for 3 days, fixed with 2% formaldehyde in PBS, and permeabilized with 0.2%
Triton X-100 in PBS. Immunostaining was done using primary antibodies
against 53BP1 (BD Transduction Laboratories, San Jose, CA) followed by
secondary antibody conjugated with Alexa Fluor 568 (Invitrogen). DNA was
visualized with 0.2 Ag/mL 4¶,6-diamidino-2-phenylindole (DAPI). All images
were analyzed using a Nikon (Kawasaki, Japan) Eclipse E600 microscope
with 60 or 100 objective.
Results
MT-hTer alone causes early cell growth inhibition, apoptosis,
and cell cycle arrest. Before the experiments combining expression of MT-hTer/siRNA and hTERT variants (see Materials and
Methods; Fig. 1), separate experiments were done to characterize
and control for the timing and magnitude of growth inhibition
contributed by each individual construct. First, LOX melanoma
cells (a telomerase-positive human cancer cell line) were infected
separately with lentiviral vectors expressing either the MT-hTer
or the siRNA directed at wt-hTer (Fig. 1A and B). Both constructs
contained a GFP marker, and infection efficiency was >95% as
quantified by percentage of cells with GFP fluorescence. MThTer expression and knockdown of wt-hTer by the siRNA were
confirmed by Northern and reverse transcription-PCR analyses
5764
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Mutant-Template Telomerase Mechanism
Figure 1. Telomerase mutants and
experimental scheme. A, sequence of
previously described MT-hTer and its
predicted copied DNA sequence (12). B,
sequence of previously described siRNA
targeting endogenous wt-hTer (12)
showing the targeted wt-hTER
sequence (red rectangle ) containing
the 11-nucleotide templating domain
(underlined ). C, map of the previously
described evolutionarily conserved
domains of hTERT (36, 37) showing the
positions of the different mutations made in
the hTERT protein and their corresponding
phenotypes: TRAP catalytic activity,
rescue from senescence, and effect
on telomere length (18). Proposed role
of each of the hTERT mutants at the
telomere. D, experimental scheme
outlining the sequential infections and
subsequent analyses.
(Supplementary Figs. S1 and S2). Growth analysis revealed that
although both constructs exerted an inhibitory effect, MT-hTer
produced marked growth inhibition in this cell line almost
immediately after its introduction, readily measurable by day 3
and statistically significant by day 6 (P = 0.02; Fig. 2A). In contrast, in
this experiment, siRNA directed against wt-hTer only began to
inhibit cell growth after f1 week from its introduction; although
slowing of cell proliferation was detected by day 9, in these
experiments, the difference by day 9 had not achieved statistical
significance.
To characterize the mechanism of growth inhibition induced by
MT-hTer expression, at each time point, cells were quantitatively
assessed for apoptosis and cell cycle arrest using Annexin V and
BrdUrd staining, respectively (Fig. 2B and C). In the cells
overexpressing MT-hTer, a 3-fold increase in apoptosis was
apparent by day 3 and remained statistically significant (P <
0.05) throughout the experiment (Fig. 2B). At day 3, MT-hTer
expression also led to a 28% relative reduction in S-phase fraction
( from 46% to 33% in S phase) and a concomitant increase in
G2 fraction, suggestive of a G2 arrest (Fig. 2C). The reduction in
S-phase fraction induced by MT-hTer was less pronounced at
the later time points possibly because the BrdUrd analysis was
done on unselected populations, which over time became enriched
for cells that were not expressing the growth-inhibitory MT-hTer.
In contrast to the early effects of MT-hTer, no significant increase
in apoptosis or cell cycle arrest was detectable in cells overexpressing just the siRNA against wt-hTer for up to 9 days. Notably,
control LOX cells infected with empty vector only showed a level
of baseline apoptosis. This phenomenon was not attributable to
the lentiviral infection, as we observed similar levels of baseline
apoptosis in uninfected control LOX cells using the same Annexin
V assay (data not shown). Thus, we attribute this finding to the
baseline characteristic of the assay in this cell line.
MT-hTer/siRNA inhibits proliferation in the setting of WThTERT but is minimally effective in the setting of catalytically
inactive hTERT or a DAT mutant hTERT. Having characterized
www.aacrjournals.org
the growth inhibition effects of each individual construct, we
proceeded to investigate the mechanism of action of MT-hTer/
siRNA. The MT-hTer/siRNA was introduced into LOX cells, in
which either WT-hTERT or one of two mutant hTERT alleles of
known properties (18) was ectopically overexpressed (Fig. 1C).
WT-hTERT was used as a control for ectopic overexpression of the
catalytic subunit of telomerase, hTERT. The first hTERT mutant
was a catalytically inactive hTERT enzyme, the DN-hTERT. The
second hTERT mutant was N125A+T126A-hTERT, which creates a
DAT mutant enzyme that is catalytically active in vitro but fails to
elongate the telomeres (18, 20).
To ensure that the effects of the MT-hTer/siRNA construct
could be compared in cells that were growing at equal rates, the
short-term effect on LOX cell growth of overexpressing each of
the hTERT alleles alone was characterized. LOX cells were
infected separately with retroviral vectors expressing WT-hTERT,
DN-hTERT, or N125A+T126A-hTERT. Equivalent levels of expression for each of the hTERT variants were confirmed by Northern
analysis (Supplementary Fig. S3). Previously published work has
shown that the D868A (DN) and N125A+T126A (DAT) mutations
do not affect the ability of hTERT to assemble with hTer (22, 23)
and that ectopic expression of the various hTERT alleles produces
telomerase ribonucleoprotein with the expected enzymatic
activity (18, 19, 22). This was confirmed in the present experiments using LOX cells by telomeric repeat amplification protocol
(TRAP) assays, showing preserved telomerase activity in the
WT-hTERT and N125A+T126A-hTERT mutants and diminished
activity (80-85% reduction) in the DN-hTERT mutant (Supplementary Fig. S4). After the LOX cells were retrovirally infected
with the hTERT variants, growth analysis of the puromycinselected cell populations revealed no effect on proliferation for up
to 14 days by any of the hTERT alleles (Fig. 3). Thus, although
overexpression of the mutant hTERTs eventually leads to telomere
shortening and has long-term effects on cell growth (12, 18), there
were no growth rate effects seen in the duration of the present
experiments.
5765
Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
The requirements for MT-hTer/siRNA activity were tested with
a serial infection experiment (Fig. 1D). First, retroviral vectors
containing each of the hTERT alleles were introduced into LOX
cells and subjected to puromycin selection as above. Then,
lentiviral vector containing MT-hTer/siRNA and GFP, or an empty
control vector containing GFP only, was introduced into those
same LOX cells at high efficiency as quantified by percentage of
cells with GFP fluorescence. The DNA damage and cell growth
responses to the MT-hTer/siRNA were thus quantified in the
settings of overexpressed WT-hTERT or overexpressed mutant
Figure 2. MT-hTer causes early cell growth inhibition, apoptosis, and cell cycle arrest. A, growth curves of LOX cells infected with lentiviral vectors expressing
MT-hTer and a GFP marker or siRNA against wt-hTer and a GFP marker. Two days after infection, cells were sorted by fluorescence-activated cell sorting (FACS), and
GFP-positive cells were reseeded. Cell counts were obtained at 3, 6, and 9 days after reseeding. Points, mean of duplicate infections. B, sample FACS analysis
of Annexin V and propidium iodide staining in MT-hTer-treated cells compared with vector control-treated cells. Increased proportion of MT-hTer-treated cells
undergoing apoptosis relative to siRNA-treated or vector control-treated cells at each time point. C, sample FACS analysis of BrdUrd and propidium iodide staining in
MT-hTer-treated cells compared with vector control-treated cells. Decreased proportion of MT-hTer-treated cells in S phase and increased proportion in G2 phase
relative to siRNA-treated or vector control-treated cells at each time point.
Cancer Res 2006; 66: (11). June 1, 2006
5766
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Mutant-Template Telomerase Mechanism
Figure 3. Individual hTERT alleles exert no short-term effect on LOX cell
proliferation. Growth curves of LOX cells infected with retroviral vectors
containing WT-hTERT, DN-hTERT, or N125A+T126A-hTERT. Two days after
infection, cells were subjected to puromycin selection for 4 days. Then, cells
were reseeded and cell counts were obtained over the following 14 days.
Points, mean cell counts of triplicate infections conducted in parallel.
hTERTs. All infections were done in duplicate, and the experiment
was repeated with similar results.
LOX cell proliferation was quantified using hemocytometry on
days 3, 6, and 9 after MT-hTer/siRNA was introduced (Fig. 4A and
B). By day 3, cell proliferation was significantly inhibited (P = 0.001)
in the setting of WT-hTERT expression. In contrast, cells expressing
DN-hTERT or N125A+T126A-hTERT were not significantly affected
by MT-hTERT/siRNA at days 3 or 6, although, on day 9, MT-hTer/
siRNA-induced growth inhibition in the DN-hTERT cells became
statistically significant. Overall, on days 3, 6, and 9, MT-hTer/siRNA
induced progressively greater growth inhibition,f3-, 13-, and
19-fold, respectively, in the setting of WT-hTERT expression. In
contrast, cells expressing DN-hTERT or N125A+T126A-hTERT
experienced no growth inhibition at day 3 after introduction of
MT-hTer/siRNA, only 1.5-fold inhibition at day 6 (not statistically
significant), and f3.5-fold at day 9 [showing statistical significance
(P = 0.03) for DN-hTERT only; Fig. 4B and C].
MT-hTer/siRNA induces DNA damage foci in the setting of
WT-hTERT but elicits significantly fewer such foci in the
presence of catalytically inactive hTERT or a DAT mutant
hTERT. It was previously shown that the growth inhibition
induced by two different MT-hTer/siRNA constructs, the AU5
construct used in the present experiments and 47A MT-hTer, was
associated with the appearance of DNA damage foci in treated
cells (9, 13). For 47A, >80% of the foci colocalized at telomeres; in
the AU5-MT-hTer cells, a lower percentage of foci were telomeric
(9, 13).3 To quantify DNA damage focus formation in the AU5-MThTer/siRNA-treated cells, at each time point (days 3, 6, and 9), in
parallel to the cell counts described above, a subset of cells was
grown on glass coverslips and examined microscopically following
fixation and staining with antibody against 53BP1, which localizes
to DNA damage foci (13). In the LOX melanoma cells expressing
MT-hTer/siRNA in the setting of WT-hTERT (Fig. 5A), large
numbers of bright, prominent 53BP1 foci appeared, consistent
3
Additional data to be submitted elsewhere.
www.aacrjournals.org
with DNA damage foci as shown previously (9, 13). Costaining
with DAPI (Fig. 5A, blue) showed that these foci localized to the
nuclei, and GFP positivity (Fig. 5A, green) indicated that the cells
expressed the MT-hTer/siRNA. Cells overexpressing MT-hTer/
siRNA in the setting of WT-hTERT also displayed typical
morphologic changes characterized by overall enlargement and
rounding of cell bodies, relative loss of cytoplasmic projections,
nuclear enlargement and multilobulation, cytoplasmic vacuolization, and occasional presence of anaphase bridges (Fig. 5A).
For each hTERT variant (WT-hTERT, DN-hTERT, or
N125A+T126A-hTERT) infected with MT-hTer/siRNA or vector
control, we counted 100 cells in duplicate and scored each cell as
‘‘53BP1 positive’’ (z1 53BP1 foci) or negative (Fig. 5B). LOX cells
expressing WT-hTERT had significantly more 53BP1 DNA damage
foci than those expressing DN-hTERT or N125A+T126A-hTERT: on
day 3, 42% of WT-hTERT cells that received MT-hTer/siRNA
contained one or more distinct 53BP1 foci compared with 20% to
22% of the DN-hTERT and N125A+T126A-hTERT cells that received
MT-hTer/siRNA (P = 0.002 and 0.01, respectively) and 15% to 20% of
any hTERT cells that received vector control. In a repeat
experiment, by day 3, 20% of WT-hTERT cells that received MThTer/siRNA contained multiple (z4) 53BP1 foci, compared with
only 2% to 4% in the DN-hTERT and N125A+T126A-hTERT cells
that received MT-hTer/siRNA (data not shown). Hence, in the
setting of WT-hTERT, the production of DNA damage foci induced
by MT-hTer/siRNA correlated with growth inhibition.
MT-hTer inhibition of proliferation is not attenuated by
knockdown of other telomere-associated proteins. The mechanism by which the addition of mutant template to telomeres
inhibits cell growth was investigated further, first by knocking
down the telomeric protein TRF2, which binds telomeric DNA
sequence specifically and is involved in normal telomere end
protection and length regulation (24, 25). The hairpin siRNA used
was the same siRNA construct recently validated in our laboratory
to knock down TRF2 protein levels in the same LOX cell line used
in the present work (13). This siRNA construct was expressed in
the LOX cells using the same lentiviral infection protocol described
previously (13). TRF2 knockdown after siRNA expression in
LOX cells was confirmed by Western immunoblot (Supplementary
Fig. S5A). Three days after infection with the anti-TRF2 siRNA, a
second infection with a lentiviral construct containing just the
MT-hTer was done. Consistent with successful knockdown of TRF2,
a significant reduction in cell proliferation, to f5 105 down from
f9.5 105 in control samples treated in parallel with scrambled
siRNA, was observed (Fig. 6A, blue columns). Expression of the
MT-hTer on top of this TRF2 knockdown further inhibited cell
growth down to 2.8 105 cells (P = 0.02; Fig. 6A, maroon columns).
A similar experiment was done in which the levels of the protein
hRap1 were knocked down. hRap1 is recruited by TRF2 to the
telomere, where it acts as a negative regulator of telomere length
(26). The present knockdown experiments were done again using
the same hairpin siRNAs recently validated in our laboratory to
knock down hRap1 in the same LOX cell line (13), and knockdown
was confirmed by Western immunoblot (Supplementary Fig. 5SB).
In contrast to the above findings with siRNAs targeting TRF2,
the short-term knockdown of hRap1 alone did not significantly
reduce cell proliferation (Fig. 6B, blue columns). Expression of the
MT-hTer in these cells caused significant growth inhibition as in
the control cells that received the scrambled siRNA (P = 0.04;
Fig. 6B, maroon columns). Therefore, depleting hRap1 did not
diminish the effect of the MT-hTer on cell growth.
5767
Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
Discussion
Figure 4. MT-hTer/siRNA inhibits proliferation in the setting of WT-hTERT
but is minimally effective in the setting of catalytically inactive hTERT or a
DAT mutant hTERT. A, growth curves of LOX cells after infection with
retroviral vectors expressing WT-hTERT, DN-hTERT, or N125A+T126AhTERT followed by infection with lentivirus expressing either MT-hTer/siRNA
and GFP or control vector with GFP only. B, relative cell counts at
3, 6, and 9 days after infection with MT-hTer/siRNA or vector control.
Columns, mean of duplicate samples. C, fold reduction in cell count induced
by MT-hTer/siRNA relative to vector control in LOX cells expressing each of
the hTERT alleles.
Cancer Res 2006; 66: (11). June 1, 2006
A telomerase-targeting construct has previously been developed
that is highly toxic to a variety of cancer cell lines, inducing
profound growth inhibition, DNA damage, and apoptosis over a
time course of days (12). This construct was designed to take
advantage of a two-pronged approach, siRNA that knocks down
endogenous wt-hTer coupled with a MT-hTer. The results reported
here are consistent with the interpretation that the effects of MThTer/siRNA require mutant telomeric DNA synthesis on telomeres.
These results provide the most direct evidence to date that the
mechanism by which MT-hTer/siRNA exerts its toxic effects on
human cancer cells is to combine with endogenous hTERT,
forming a catalytically active telomerase that is capable of adding
incorrect DNA repeats to the telomeres.
We showed this mechanism of action of MT-hTer/siRNA by a
novel approach using telomerase enzyme (hTERT) variants with
previously characterized properties. We began with the following
hypothesis. Two conditions must both be met in order for the
MT-hTer-hTERT ribonucleoprotein to exert its toxic effects by
the proposed mechanism (adding incorrect nucleotides to the
telomere): (a) the hTERT must be enzymatically active and (b) the
hTERT must be able to act on the telomeres in vivo. We tested
this hypothesis by ectopically overexpressing three hTERT alleles
before treatment with the MT-hTer/siRNA construct: functional WT-hTERT, enzymatically inactive DN-hTERT, and the DAT
N125A+T126A-hTERT. Then, we quantified the effects of MT-hTer/
siRNA in each of these intracellular hTERT milieus.
Before the serial infection experiments, we quantified the
individual effects on LOX cell proliferation contributed by each
of the component constructs comprising MT-hTer/siRNA. When
tested separately, the MT-hTer produced cell cycle arrest,
apoptosis, and consequent growth inhibition significantly more
rapidly and intensely than the siRNA directed against wt-hTer.
The independent growth-inhibitory effects of siRNA directed at
wt-hTer were not significant over the time course of the present
experiments. These results suggest that, in this cell line, the earliest
phase of the cancer cell inhibition by MT-hTer/siRNA is primarily
dependent on the mutant template, likely through its previously
shown ability to induce a DNA damage response (12). Notably,
recent evidence also indicates that siRNA directed at wt-hTer
functions via additional pathways (9). Ongoing studies in our
laboratory aim to separate and better define the respective
contributions of MT-hTer and siRNA against wt-hTer to the
phenotypes of DNA damage and growth inhibition.
In separate experiments, the WT-hTERT-overexpressing, DNhTERT-overexpressing, and N125A+T126A-hTERT-overexpressing
LOX cells all grew similarly for the short periods (10-14 days)
monitored after selection. Because overexpression of each of the
hTERT constructs in LOX cells did not significantly alter the shortterm LOX growth phenotype, we were able to quantify any cell
growth differences arising from subsequent expression of MThTer/siRNA against a constant cell proliferation rate. Hence, the
differential effects observed when MT-hTer interacted with each of
the hTERT alleles can be attributed to the differences in those
predefined functional capacities of hTERTs rather than to some
other inherent differences between the hTERT alleles. This was a
critical validation, especially in light of recent publications showing
a significant long-term effect on cell activation produced by TERT
expression alone in some systems (27, 28).
When we proceeded to introduce the combined MT-hTer/siRNA
construct into cells expressing the hTERT alleles, as early as 3 days
5768
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Mutant-Template Telomerase Mechanism
after introduction of MT-hTer/siRNA, there was a marked
inhibition of proliferation in the setting of WT-hTERT expression
(>2-fold reduction in cell number) but no growth inhibition in cells
expressing the DN-hTERT or N125A+T126A-hTERT alleles. These
growth effects correlated with the results of 53BP1 staining, which
showed that the proportion of cells sustaining any demonstrable
DNA damage as a result of MT-hTer/siRNA expression was f2to 3-fold higher in the WT-hTERT-expressing cells than in the
mutant hTERT-expressing cells. Similarly, the proportion of cells
with multiple (z4) foci of DNA damage was f6-fold higher in the
WT-hTERT-expressing cells than in the mutant hTERT-expressing
cells.
These data show that MT-hTer/siRNA causes DNA damage and
growth inhibition in a manner dependent on the action of WThTERT, with MT-hTer/siRNA losing effectiveness if the introduced
hTERT is enzymatically inactive (DN-hTERT). Similarly, MT-hTer/
siRNA was ineffective in the setting of a DAT hTERT allele that has
lost the ability to elongate telomeres in vivo (N125A+T126AhTERT). Previous publications have shown that mutations in the
DAT region render hTERT specifically unable to act on telomeric
primers (20, 29) and that in vivo activity could be restored to the
DAT mutant through fusion to TRF2, which localizes to the
telomere (30). Thus, the relative absence of MT-hTer/siRNA
growth-inhibitory effects in the setting of the N125A+T126AhTERT mutant, which has an impaired capacity to act on
telomeres, signifies that the MT-hTer/siRNA toxic effects most
likely occur at the telomere. This is consistent with previous
findings that DNA damage foci induced by MT-hTer expression
colocalize at telomeres, with up to 80% telomeric colocalization
observed with some constructs (9, 13).3 Therefore, we conclude
that MT-hTer must complex with enzymatically active hTERT and
act on telomeres to exert its effects. The most likely explanation for
these results is that MT-hTer/siRNA does in fact work through
addition of incorrect nucleotides to the telomere and a DNA
damage response, leading to inhibition of growth.
Although MT-hTERT/siRNA was significantly more toxic to the
cells expressing WT-hTERT, later in the time course, we also noted
a modest growth-inhibitory trend in the cells expressing DNhTERT and N125A+T126A-hTERT, which was not statistically
significant, except with DN-hTERT at day 9. We speculate that,
occasionally, the N125A+T126A-hTERT can act on telomeres or
that endogenous hTERT, although expected generally to be
outcompeted by the overexpressed mutant forms, assembled with
the MT-hTer. This possibility is born out by the TRAP assay analysis
of the hTERT variants (Supplementary Fig. S4), which showed that
telomerase activity in the DN-hTERT-expressing cells, though
diminished by 80% to 85% relative to parental controls, was still
present. Another contributing factor may be that the siRNA
portion of the construct may begin to exert its independent
growth-inhibitory effects by day 9 as suggested by the growth
results shown in Fig. 2A and in previous work (9, 12). Any such
late inhibitory effects of the siRNA are expected to be more
readily discernable in the cells that expressed DN-hTERT or
N125A+T126A-hTERT because, unlike the WT-hTERT-expressing
cells, the DN-hTERT-expressing and N125A+T126A-hTERTexpressing cells were relatively unaffected by the earlier, more
potent growth inhibition caused by the MT-hTer portion of the
construct.
We tested whether the effects of MT-hTer/siRNA were mediated
by the telomere-interacting proteins hRap1 and TRF2. hRap1
does not bind normal telomeric DNA directly; nevertheless, its
www.aacrjournals.org
Figure 5. MT-hTer/siRNA induces DNA damage foci in the setting of
WT-hTERT but elicits significantly fewer such foci in the presence of
catalytically inactive hTERT or a DAT mutant hTERT. A, sample
immunofluorescence micrographs of 53BP1-negative cells, 53BP1-positive
cells, and cells with anaphase bridges. LOX melanoma cells were infected
with retroviral vectors expressing each of the hTERT alleles (WT, DN, or
N125A+T126A) followed by 4-day puromycin selection. At the end of day 4 of
puromycin selection, cells were passaged onto glass coverslips at 2 104 per
coverslip and infected the following morning with lentivirus expressing MT-hTer/
siRNA and a GFP marker or vector control and a GFP marker. Cells were
maintained in culture for 3 days and then fixed and stained with 53BP1 (red )
antibody. Blue, DNA was visualized with 0.2 Ag/mL DAPI; green, GFP was
also visualized using the appropriate wavelength filters. A microscope
(Nikon Eclipse E600) with 60 and 100 objectives and a CoolSNAP fx
charge-coupled device camera and software (Roper Scientific, Tucson, AZ)
were used to visualize the images, which were subsequently color coded and
merged using Adobe Photoshop. B, percentage of 53BP1-positive LOX cells
after treatment with retroviral vectors expressing each of the hTERT alleles
followed by 3-day infection with lentivirus expressing MT-hTer/siRNA and a
GFP marker or vector control and a GFP marker. 53BP1 foci in 100 cells
were counted after immunofluorescence analysis and rated as 53BP1 positive
(z1 53BP1 foci) or negative. Columns, mean of duplicate experiments.
5769
Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Cancer Research
Figure 6. MT-hTer inhibition of
proliferation is not attenuated by
knockdown of other telomere-associated
proteins. Lentiviral vectors expressing
control scrambled siRNA or siRNA
targeting TRF2 (A) or hRap1 (B ) were
introduced into LOX cells at high efficiency
as confirmed by GFP marker expression.
This was followed by a second infection
with lentiviral vectors containing MT-hTer
and GFP or vector control and GFP. Cell
counts 3 days after the second infection.
A and B, columns, results of two separate
experiments done in duplicate.
involvement at the telomere is substantiated by a growing body of
research. In budding yeast, Rap1 binds telomeric DNA directly and
acts as a negative regulator of telomere length (31, 32). The
function of Rap1 seems to be conserved in its mammalian
orthologue, hRap1, which is recruited by TRF2 to the telomere,
where it acts as a negative regulator of telomere length (26).
The present experiments with hRap1 sought to define its role in the
growth inhibition generated by MT-hTer. MT-hTer expression
produced the same degree of growth inhibition in hRap1
knockdown cells as in control cells. Hence, hRap1 depletion does
not reduce the effect of MT-hTer in inducing growth inhibition.
TRF2 directly binds telomeric DNA sequence specifically and
also recruits hRap1 to the telomeres (24, 25). It has been proposed
that incorporation of aberrant telomeric repeats may disrupt the
normal binding of TRF2 to the telomeric DNA (11, 33). Disruption
of TRF2 binding, in turn, may lead to telomere ‘‘uncapping’’ and a
DNA damage response as suggested by one set of experiments
in HeLa cancer cells, where expression of a dominant-negative
TRF2 allele that lacked the telomeric DNA binding domain and
depleted TRF2 from telomeres led to increased apoptosis in a p53dependent manner (34). In the present experiments, TRF2
knockdown alone produced the expected growth inhibition
phenotype in LOX cells as described previously (13). Notably,
expression of MT-hTer in these cells after TRF2 had been knocked
down led to a further reduction in cell proliferation rate. Relative
to vector control, MT-hTer expression produced a degree of
growth inhibition comparable to that seen when the MT-hTer was
expressed with no TRF2 knockdown. Therefore, the relative
absence of TRF2 seems to act in an additive fashion with MThTer. Interestingly, previous work in our laboratory has shown
that the apoptotic response generated by MT-hTer in HCT116
References
1. Telomeres. Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press; 2006.
2. Greider CW, Blackburn EH. A telomeric sequence in
the RNA of Tetrahymena telomerase required for
telomere repeat synthesis. Nature 1989;337:331–7.
Cancer Res 2006; 66: (11). June 1, 2006
cancer cells or a transformed fibroblast cell line is not dependent
on p53 (12). Hence, TRF2 depletion and MT-hTer effects may
involve different pathways.
In summary, the current studies offer insight into a phenomenon that has been reported repeatedly (11, 12, 35), that interference with telomerase elicits an acute apoptotic response that
cannot be explained by the traditional role ascribed to telomerase
(prevention of gradual telomere shortening with serial cell
divisions). Here, we show that DNA damage, cellular apoptosis,
and inhibition of cancer cell proliferation indeed occur rapidly
when telomerase function is disrupted by introduction of MT-hTer
but only when that MT-hTer incorporates into a catalytically
active telomerase capable of normal action on telomeres in vivo.
These findings, together with the additive effects of growth
inhibition caused by knockdown of the telomere-interacting
proteins hRap1 and TRF2, suggest that MT-hTer exerts its toxicity
by altering critical interactions at the telomere. Thus, our work
begins to elucidate not only the manner by which a novel agent,
MT-hTer, inhibits cancer cell proliferation but also the underlying
delicate balance that makes telomerase interference a promising
therapeutic approach.
Acknowledgments
Received 10/19/2005; revised 3/7/2006; accepted 4/4/2006.
Grant support: NIH Basic Research in Hematology and Oncology T32 grant
DK007636 (A. Goldkorn) and NIH/National Cancer Institute grant CA96840 and
Bernard Osher Foundation (E.H. Blackburn).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Jue Lin and Dana Smith for critical reading of the article, Maura DevlinClancy for helping with the preparation and editing of the article, and Brad Stohr for
helpful input on experiment design.
3. Allsopp RC, Vaziri H, Patterson C, et al. Telomere
length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A 1992;89:10114–8.
4. Blackburn EH. Telomere states and cell fates. Nature
2000;408:53–6.
5. Shay JW, Bacchetti S. A survey of telomerase activity in
human cancer. Eur J Cancer 1997;33:787–91.
5770
6. Strahl C, Blackburn EH. Effects of reverse transcriptase inhibitors on telomere length and telomerase
activity in two immortalized human cell lines. Mol Cell
Biol 1996;16:53–65.
7. Hahn WC, Stewart SA, Brooks MW, et al. Inhibition of
telomerase limits the growth of human cancer cells. Nat
Med 1999;5:1164–70.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Mutant-Template Telomerase Mechanism
8. Zhang X, Mar V, Zhou W, Harrington L, Robinson MO.
Telomere shortening and apoptosis in telomeraseinhibited human tumor cells. Genes Dev 1999;13:2388–99.
9. Li S, Crothers J, Haqq CM, Blackburn EH. Cellular and
gene expression responses involved in the rapid growth
inhibition of human cancer cells by RNA interferencemediated depletion of telomerase RNA. J Biol Chem
2005;280:23709–17.
10. Nosrati M, Li S, Bagheri S, et al. Antitumor activity of
systemically delivered ribozymes targeting murine
telomerase RNA. Clin Cancer Res 2004;10:4983–90.
11. Kim MM, Rivera MA, Botchkina IL, Shalaby R, Thor
AD, Blackburn EH. A low threshold level of expression of
mutant-template telomerase RNA inhibits human tumor
cell proliferation. Proc Natl Acad Sci U S A 2001;98:7982–7.
12. Li S, Rosenberg JE, Donjacour AA, et al. Rapid
inhibition of cancer cell growth induced by lentiviral
delivery and expression of mutant-template telomerase
RNA and anti-telomerase short-interfering RNA. Cancer
Res 2004;64:4833–40.
13. Xu L, Blackburn EH. Human Rif1 protein binds
aberrant telomeres and aligns along anaphase midzone
microtubules. J Cell Biol 2004;167:819–30.
14. Marusic L, Anton M, Tidy A, Wang P, Villeponteau B,
Bacchetti S. Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells
leads to altered telomeres that correlate with reduced
cell viability. Mol Cell Biol 1997;17:6394–401.
15. Chan SW, Blackburn EH. Telomerase and ATM/Tel1p
protect telomeres from nonhomologous end joining.
Mol Cell 2003;11:1379–87.
16. Smith CD, Smith DL, DeRisi JL, Blackburn EH.
Telomeric protein distributions and remodeling through
the cell cycle in Saccharomyces cerevisiae. Mol Biol Cell
2003;14:556–70.
www.aacrjournals.org
17. Taggart AK, Teng SC, Zakian VA. Est1p as a cell cycleregulated activator of telomere-bound telomerase.
Science 2002;297:1023–6.
18. Kim M, Xu L, Blackburn EH. Catalytically active
human telomerase mutants with allele-specific biological properties. Exp Cell Res 2003;288:277–87.
19. Armbruster BN, Banik SS, Guo C, Smith AC, Counter
CM. N-terminal domains of the human telomerase
catalytic subunit required for enzyme activity in vivo .
Mol Cell Biol 2001;21:7775–86.
20. Lee SR, Wong JM, Collins K. Human telomerase
reverse transcriptase motifs required for elongation of a
telomeric substrate. J Biol Chem 2003;278:52531–6.
21. Naldini L, Blomer U, Gallay P, et al. In vivo gene
delivery and stable transduction of nondividing cells by
a lentiviral vector. Science 1996;272:263–7.
22. Bachand F, Autexier C. Functional regions of human
telomerase reverse transcriptase and human telomerase
RNA required for telomerase activity and RNA-protein
interactions. Mol Cell Biol 2001;21:1888–97.
23. Cristofari G, Lingner J. The telomerase ribonucleoprotein particle. In: de Lange T, Lundblad V, Blackburn
EH, editors. Telomeres. 2nd ed. Cold Spring Harbor (NY):
Cold Spring Harbor Laboratory Press; 2006. p. 21–47.
24. Smogorzewska A, de Lange T. Regulation of telomerase by telomeric proteins. Annu Rev Biochem 2004;73:
177–208.
25. de Lange T. Protection of mammalian telomeres.
Oncogene 2002;21:532–40.
26. Li B, de Lange T. Rap1 affects the length and
heterogeneity of human telomeres. Mol Biol Cell 2003;
14:5060–8.
27. Sarin KY, Cheung P, Gilison D, et al. Conditional
telomerase induction causes proliferation of hair follicle
stem cells. Nature 2005;436:1048–52.
5771
28. Flores I, Cayuela ML, Blasco MA. Effects of
telomerase and telomere length on epidermal stem cell
behavior. Science 2005;309:1253–6.
29. Moriarty TJ, Ward RJ, Taboski MA, Autexier C. An
anchor site-type defect in human telomerase that
disrupts telomere length maintenance and cellular
immortalization. Mol Biol Cell 2005;16:3152–61.
30. Armbruster BN, Etheridge KT, Broccoli D, Counter
CM. Putative telomere-recruiting domain in the catalytic subunit of human telomerase. Mol Cell Biol 2003;
23:3237–46.
31. Hardy CF, Sussel L, Shore D. A RAP1-interacting
protein involved in transcriptional silencing and telomere length regulation. Genes Dev 1992;6:801–14.
32. Levy DL, Blackburn EH. Counting of Rif1p and Rif2p
on Saccharomyces cerevisiae telomeres regulates telomere length. Mol Cell Biol 2004;24:10857–67.
33. Guiducci C, Cerone MA, Bacchetti S. Expression of
mutant telomerase in immortal telomerase-negative
human cells results in cell cycle deregulation, nuclear
and chromosomal abnormalities, and rapid loss of
viability. Oncogene 2001;20:714–25.
34. Karlseder J, Broccoli D, Dai Y, Hardy S, de Lange T.
p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 1999;283:1321–5.
35. Takai H, Smogorzewska A, de Lange T. DNA
damage foci at dysfunctional telomeres. Curr Biol 2003;
13:1549–56.
36. Nakamura TM, Morin GB, Chapman KB, et al.
Telomerase catalytic subunit homologs from fission
yeast and human. Science 1997;277:955–9.
37. Xia J, Peng Y, Mian IS, Lue NF. Identification of
functionally important domains in the N-terminal
region of telomerase reverse transcriptase. Mol Cell Biol
2000;20:5196–207.
Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.
Assembly of Mutant-Template Telomerase RNA into
Catalytically Active Telomerase Ribonucleoprotein That Can
Act on Telomeres Is Required for Apoptosis and Cell Cycle
Arrest in Human Cancer Cells
Amir Goldkorn and Elizabeth H. Blackburn
Cancer Res 2006;66:5763-5771.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/66/11/5763
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2006/06/09/66.11.5763.DC1
This article cites 35 articles, 24 of which you can access for free at:
http://cancerres.aacrjournals.org/content/66/11/5763.full#ref-list-1
This article has been cited by 6 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/66/11/5763.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2006 American Association for Cancer
Research.