Anti-apoptotic Role of Telomerase in Pheochromocytoma Cells*
Weiming
Fu,
James G.
Begley,
Michael W.
Killen, and
Mark P.
Mattson
From the Sanders Brown Research Center on Aging and the Department
of Anatomy & Neurobiology, University of Kentucky,
Lexington, Kentucky 40536
 |
ABSTRACT |
Telomerase is a protein-RNA enzyme complex
that adds a six-base DNA sequence (TTAGGG) to the ends of chromosomes
and thereby prevents their shortening. Reduced telomerase activity
is associated with cell differentiation and accelerated cellular
senescence, whereas increased telomerase activity is associated with
cell transformation and immortalization. Because many types of cancer have been associated with reduced apoptosis, whereas cell
differentiation and senescence have been associated with increased
apoptosis, we tested the hypothesis that telomerase activity is
mechanistically involved in the regulation of apoptosis. Levels of
telomerase activity in cultured pheochromocytoma cells decreased prior
to cell death in cells undergoing apoptosis. Treatment of cells with the oligodeoxynucleotide TTAGGG or with 3,3'-diethyloxadicarbocyanine, agents that inhibit telomerase activity in a
concentration-dependent manner, significantly enhanced
mitochondrial dysfunction and apoptosis induced by staurosporine,
Fe2+ (an oxidative insult), and amyloid
-peptide
(a cytotoxic peptide linked to neuronal apoptosis in Alzheimer's
disease). Overexpression of Bcl-2 and the caspase inhibitor zVAD-fmk
protected cells against apoptosis in the presence of telomerase
inhibitors, suggesting a site of action of telomerase prior to caspase
activation and mitochondrial dysfunction. Telomerase activity decreased
in cells during the process of nerve growth factor-induced
differentiation, and such differentiated cells exhibited increased
sensitivity to apoptosis. Our data establish a role for telomerase
in suppressing apoptotic signaling cascades and suggest a mechanism
whereby telomerase may suppress cellular senescence and promote tumor formation.
 |
INTRODUCTION |
Telomeres consist of repeats of the sequence TTAGGG/CCCTAA at the
ends of chromosomes. These DNA repeats are synthesized by enzymatic
activity associated with an RNA-protein complex called telomerase
(1-3). Telomerase activity decreases dramatically during the processes
of growth arrest and cell differentiation (4-6). Indeed, in most
somatic cells, telomerase activity is low or nonexistent, and telomere
length decreases with increasing cell divisions; telomere shortening
has therefore been proposed to play a role in cellular senescence
(7-9). The latter hypothesis recently gained strong support from
studies showing that the life span of normal human fibroblasts can be
extended if they are transfected with a vector encoding the telomerase
catalytic subunit (10). The mechanism whereby telomerase activity
suppresses cellular senescence has not been established.
Apoptosis is a stereotyped form of cell death that occurs in a variety
of physiological and pathological settings (11, 12). Apoptosis is
characterized by cell shrinkage, plasma membrane blebbing, and nuclear
chromatin condensation and DNA fragmentation. Biochemical features of
apoptosis include loss of plasma membrane phospholipid asymmetry,
activation of one or more cysteine proteases of the caspase family,
mitochondrial dysfunction, and release of factors from mitochondria
that induce nuclear destruction (13-15). During the aging process,
damaged senescent cells are eliminated by apoptosis (16). Abnormal
apoptosis is associated with many different disease states, including
cancers, in which apoptosis is suppressed (17), and neurodegenerative
disorders, in which apoptosis is enhanced (18). Because cell
immortalization is often associated with both increased telomerase
activity (19, 20) and increased resistance to apoptosis (21), we
performed experiments aimed at determining whether telomerase actively
modulates the cell death process.
 |
EXPERIMENTAL PROCEDURES |
PC12 Cell Cultures and Experimental Treatments--
Control
vector-transfected PC12 cells (PC12-V) and PC12 cells stably
overexpressing human Bcl-2 (PC12-Bcl2) were established using methods
described previously (22, 23). Cultures were maintained in plastic
culture flasks and subcultured onto polyethyleneimine-coated plastic
60-mm dishes for telomerase activity assays, 22-mm2
polyethyleneimine-coated glass coverslips for analyses of apoptosis and
mitochondrial transmembrane potential, or polyethyleneimine-coated 24-well plates for 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays. Cells were maintained in RPMI medium supplemented 10% with heat-inactivated horse serum and 5% with heat-inactivated fetal bovine serum (at 37 °C in a 5%
CO2 atmosphere). Immediately prior to experimental
treatment, the medium was replaced with RPMI medium containing 1%
fetal calf serum. 3,3'-Diethyloxadicarbocyanine (DODCB)1 (24) was purchased
from Sigma and was prepared as a 500× stock in dimethyl sulfoxide.
Synthetic oligodeoxynucleotide telomeric repeat DNA sequence
(5'-TTAGGG-3') and an oligonucleotide with a scrambled sequence
(5'-TGTGAG-3') were purchased from IDT (Coralville, IA) and were
prepared as 1 mM stocks in sterile deionized water. The
telomerase inhibitors were administered in a 1-h pretreatment and
remained in the medium during the rest of the experiment. Staurosporine
(STS) was purchased from Sigma and was prepared as a 500× stock in
dimethyl sulfoxide. Synthetic amyloid
-peptide 25-35 (A
) was
purchased from Bachem (Torrence, CA) and was prepared as a 1 mM stock in sterile deionized water 2 h prior to use.
FeSO4 (Sigma) was prepared as a 1 mM stock in
water. In order to differentiate cells into a neuron-like phenotype,
the culture medium was replaced with RPMI medium containing 0.1%
bovine serum albumin and 50 ng/ml nerve growth factor (NGF).
Quantification of Apoptosis--
Following experimental
treatments, cells were fixed in 4% paraformaldehyde, and membranes
were permeabilized with 0.2% Triton X-100 and stained with the
fluorescent DNA-binding dye Hoechst 33342 as described previously (23).
Hoechst-stained cells were visualized and photographed under
epifluorescence illumination (340 nm excitation and 510 nm barrier
filter) using a × 60 oil immersion objective (200 cells/culture
were counted, and counts were made in at least four separate
cultures/treatment condition). Analyses were performed without
knowledge of the treatment history of the cultures. The percentage of
"apoptotic" cells (cells with condensed and fragmented DNA were
considered apoptotic) in each culture was determined. We
previously found that this method for assessment of apoptosis is
superior to the terminal transferase uridyl nick end-labeling assay
(which also detects necrotic cells) and that the death of PC12 cells
induced by STS, Fe2+, and A
(at the concentrations used
in the present study) can be completely blocked by caspase inhibitors
and macromolecular synthesis inhibitors (23, 32-34).
Telomerase Activity Assay--
Telomerase activity was measured
using a polymerase chain reaction-based telomeric repeat amplification
protocol (TRAP) as described previously (25, 26) using a kit from Oncor
(Gaithersburg, MD). Briefly, cells (105-106
cells/culture) were scraped in PBS, pelleted by centrifugation for 5 min at 400 × g, and resuspended in 200 µl of lysis
buffer that contained 0.5% CHAPS, 1 mM MgCl2,
1 mM EGTA, 0.1 mM benzamidine, 5 mM
-mercaptoethanol, 10% glycerol, and 10 mM Tris-HCl, pH
7.5. The lysate was incubated for 30 min at 4 °C and centrifuged at 12,000 × g for 20 min at 4 °C, and protein
concentration of the supernatant was determined using a BCA kit
(Pierce). Cell extract (1-200 ng of protein) was added to a reaction
mixture containing 10× TRAP buffer (15 mM
MgCl2, 630 mM KCl, 0.5% Tween 20, 10 mM EGTA, 0.1% bovine serum albumin, and 200 mM
Tris-HCl, pH 8.3), 50× dNTP mixture (2.5 mM each of dATP,
dTTP, dGTP, and dCTP), TS primer (5'-AATCCGTCGAGCAGAGTT-3'), TRAP
primer mixture (Oncor), and 5 units/µl Taq polymerase. The
mixture was incubated at room temperature for 20 min, followed by a
30-min incubation at 30 °C. The mixture was then subjected to 30 cycles of amplification (94 °C for 30 s, 53 °C for 45 s, and 72 °C for 60 s). Each reaction product was amplified in
the presence of a 36-base pair internal TRAP assay standard. Samples
were loaded on a 12.5% nondenaturing polyacrylamide gel, and DNA was
electrophoresed through the gel. Gels were stained with SyBr (Molecular
Probes), and images were captured using an AlphaImager. For
quantification of relative telomerase activity in each sample, the
total density of bands 1-10 of the characteristic ladder (band 1 being
the band immediately above the internal standard band) was quantified
using NIH Image software, and values are expressed relative to a
control value as indicated in the figure legends. TRAP assays were
performed on serial dilutions of extracts from untreated control PC12
cells in order to establish the linear response range of the assay and to thereby select appropriate protein concentrations for analyses of
extracts from cells subjected to experimental treatments. Control reactions included tubes lacking cell extract or containing cell extract treated with 200 µg/ml RNase.
Measurement of Mitochondrial Transmembrane Potential--
The
dye rhodamine 123 was used as a measure of mitochondrial transmembrane
potential using methods described previously (27, 28). Briefly,
cultures were incubated for 30 min in RPMI medium containing 5 µM rhodamine 123 and were then washed with Locke's buffer: NaCl, 154 mM; KCl, 5.6 mM;
CaCl2, 2.3 mM; MgCl2, 1 mM; NaHCO3, 3.6 mM; glucose, 5 mM; HEPES, 5 mM (pH 7.2). Cellular fluorescence
was imaged using a confocal laser scanning microscope with excitation
at 488 nm and emission at 510 nm; cells were selected randomly under
bright-field optics and then scanned with the laser. Levels of cellular
fluorescence were quantified from the fluorescence images using
ImageSpace software (Molecular Dynamics); the fluorescence measurements
were made in the entire cell body and are expressed as average pixel
intensity/cell body. Measurements were made in at least 40 cells/culture (analyses were performed without knowledge of treatment
history of the cultures).
 |
RESULTS |
Telomerase Inhibitors Enhance Apoptosis of Undifferentiated PC12
Cells--
Because tumor cells are generally more resistant to
apoptosis than are normal cells (21, 23) and because telomerase
activity is increased in tumor cells (19, 20), we determined whether inhibition of telomerase would modify the cell death process. In order
to determine an appropriate concentration of cell extract that would
allow reliable quantification of relative levels of telomerase activity
in PC12 cells, the basal level of telomerase activity was measured in
serial dilutions of PC12 cell extract (1-200 ng). An example of
such an assay is shown in Fig.
1A, and values of relative
telomerase activity are shown in Fig. 1B. Essentially no
bands were detected in TRAP assay samples lacking cell extract, and
treatment of the cell extract with RNase prior to TRAP assay completely
eliminated telomerase activity, demonstrating specificity of the assay
(Fig. 1C). Based upon these results, we used 20-100 ng of
PC12 cell extract for subsequent assays. A comparison of relative
levels of telomerase activity in HeLa cells (known to exhibit high
telomerase activity) and PC12 cells indicated that PC12 cells have a
quite high basal level of telomerase activity (Fig. 1C). We
next examined the effects of two different putative telomerase
inhibitors on telomerase activity in PC12 cells. One inhibitor was the
six-base oligonucleotide TTAGGG, which corresponds to the sequence that
telomerase adds to telomeres; this oligonucleotide was previously shown
to be effective in suppressing telomerase activity in various cultured
cell lines (29-31). The second inhibitor was the compound DODCB, an
agent previously shown to bind selectively to dimeric hairpin
quadruplexes (24) and to inhibit telomerase activity.2 As expected, both
TTAGGG and DODCB caused decreases in telomerase activity in PC12 cell
homogenates (Fig. 1, C and D). A scrambled control oligonucleotide (TGTGAG) had no effect on telomerase activity (data not shown). The IC50 values for DODCB and TTAGGG were
approximately 2 and 4 µM, respectively (Fig.
1D). Maximal inhibition of telomerase activity was observed
with concentrations of DODCB and TTAGGG in the range of 10-50
µM. DODCB and TTAGGG, at concentrations effective in
suppressing telomerase activity (1-10 µM DODCB and 2-20
µM TTAGGG), did not affect the TRAP assay when added
immediately prior to the polymerase chain reaction step (data not
shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of telomerase inhibition by
DODCB and TTAGGG in cultured PC12 cells. A, TRAP assay
showing basal levels of telomerase activity in serially diluted
extracts of PC12 cells containing the indicated amounts of protein.
IS, internal TRAP assay standard. B, plot of
relative telomerase activity versus PC12 extract amount (the
value for 1 ng of extract was set at 1.0). C, TRAP assay of
PC12 cell extracts from cultures treated with increasing concentrations
of DODCB. Extract, from an assay performed in the absence of
cell extract. HeLa, 20 ng HeLa cell extract. Lanes marked
DODCB concentration contained 20 ng of extract from PC12
cells that had been treated for 12 h with the indicated
concentrations of DODCB. RNase, 20 ng of extract from
untreated PC12 cells that had been pretreated with 200 µg/ml RNase
prior to the TRAP assay. D, plots of relative telomerase
activity in PC12 cell extracts from cultures that had been treated for
20 h with the indicated concentrations of either DODCB or TTAGGG
(the value for untreated cultures was set at 10). Each reaction
contained 20 ng of cell extract.
|
|
In order to determine whether telomerase activity influenced the
vulnerability of PC12 cells to apoptosis, we employed STS, A
, and
Fe2+, three agents known to induce apoptosis in PC12 cells
(23, 32). Cultures were pretreated with TTAGGG, TGTGAG, or DODCB prior
to exposure to STS, A
, or Fe2+. Basal levels of
apoptosis were approximately 4-5% in vehicle-treated control cultures
and were not significantly affected by TTAGGG or DODCB at
concentrations (10-20 µM) that inhibited telomerase activity by over 80% (Figs. 1D and
2A), although there was a
trend toward an increased basal level of apoptosis in cultures exposed to the telomerase inhibitors (Fig. 2A). Apoptosis induced by
each insult (STS, A
, and Fe2+) was significantly
enhanced in cultures treated TTAGGG and DODCB but not in cultures
treated with scrambled DNA (Fig. 2, A and B).
These results suggested an anti-apoptotic role for telomerase activity.
Alterations in mitochondria, including membrane depolarization and
release of apoptotic factors, occur prior to nuclear alterations in a
variety of cells undergoing apoptosis (13). We previously showed that
STS, A
, and Fe2+ can each induce such mitochondrial
alterations in PC12 cells (23, 33, 34). As expected, STS, A
, and
Fe2+ each caused mitochondrial membrane depolarization
measured as a decrease in levels of rhodamine 123 fluorescence (Fig.
3). The magnitude of the decrease
in mitochondrial membrane potential was significantly greater in PC12
cells treated with TTAGGG or DODCB than in cells treated with vehicle
or scrambled DNA (Fig. 3), suggesting that the anti-apoptotic action of
telomerase occurs at a step prior to mitochondrial alterations.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 2.
Telomerase inhibitors enhance apoptosis
induced by staurosporine, amyloid
-peptide, and Fe2+ in PC12 cells.
A, cultures of PC12 cells were pretreated for 1 h with
saline (vehicle), 20 µM telomeric repeat DNA (TTAGGG), 20 µM scrambled DNA (TGTGAG), or 10 µM DODCB.
Cultures were then exposed for 24 h to 0.2% dimethyl sulfoxide
(control), 1 µM STS, 20 µM A ,
or 10 µM Fe2+. The percentage of cells
exhibiting apoptotic nuclei in each culture was calculated, and values
are the mean and S.E. of determinations made in four separate cultures.
Values for vehicle-treated cells exposed to STS, A , and
Fe2+ were significantly greater than the corresponding
control value (p < 0.01). Values for cells treated
with TTAGGG or DODCB and then exposed to STS, A , or Fe2+
were significantly greater than corresponding values for cultures
treated with vehicle or TGTGAG (p < 0.01). Analysis of
variance with Scheffe's post hoc tests was used. B, PC12
cells were exposed for 20 h to 0.2% dimethyl sulfoxide
(Control), 1 µM STS, or 20 µM
TTAGGG (1 h pretreatment) plus 1 µM STS. Cells were then
either fixed and stained with the DNA-binding dye Hoechst 33342 (top row) or were photographed under phase-contrast optics
(bottom row). Note that STS induced nuclear chromatin
condensation/fragmentation and cell shrinkage and that both of these
manifestations of apoptosis were exacerbated in cells cotreated with
TTAGGG.
|
|

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 3.
Telomerase inhibitors exacerbate
mitochondrial membrane depolarization induced by apoptotic insults in
PC12 cells. Cultures of PC12 cells were pretreated for 1 h
with saline (vehicle), 20 µM telomeric repeat
DNA (TTAGGG), 20 µM scrambled DNA (TGTGAG), or 10 µM DODCB. Cultures were then exposed for 6 h to
0.2% dimethyl sulfoxide (control), 1 µM STS,
20 µM A , or 10 µM Fe2+.
Levels of rhodamine 123 fluorescence were quantified, and values are
the mean and S.E. of determinations made in four separate cultures.
Values for vehicle-treated cells exposed to STS, A , and
Fe2+ were significantly less than the corresponding control
value (p < 0.01). Values for cells treated with TTAGGG
or DODCB and then exposed to STS, A , or Fe2+ were
significantly less than corresponding values for cultures treated with
STS, A , or Fe2+ alone (p < 0.02).
Analysis of variance with Scheffe's post hoc tests was used.
|
|
Telomerase Activity Decreases in Cells Undergoing
Apoptosis--
We next determined whether levels of telomerase
activity changed in PC12 cells undergoing apoptosis. STS, A
, and
Fe2+ each caused a time-dependent increase in
the number of PC12 cells exhibiting apoptotic nuclei beginning 8 h
following treatment and progressing through 24 h (Fig.
4A). Levels of telomerase
activity were reduced within 4-8 h of treatment with each apoptotic
insult and continued to decrease through 16 h (Fig.
4B).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Telomerase activity is decreased in cells
undergoing apoptosis. A, PC12 cell cultures were
exposed for the indicated time periods to vehicle (0.2% dimethyl
sulfoxide) (control), 1 µM STS, 20 µM A , or 10 µM Fe2+, and
levels of apoptosis were quantified. Values are the mean and S.E. of
determinations made in four separate cultures. B, PC12 cell
cultures were exposed for the indicated time periods to vehicle (0.2%
dimethyl sulfoxide), 1 µM STS, 20 µM A ,
or 10 µM Fe2+, and levels of telomerase
activity in cell extracts (100 ng of extract) were quantified. Values
are expressed as a percentage of the telomerase activity level in
untreated control cultures and represent the mean and S.E. of
determinations made in three cultures.
|
|
Bcl-2 and a Caspase Inhibitor Protect against the
Apoptosis-enhancing Actions of Telomerase Inhibitors--
Expression
of the proto-oncogene bcl-2 is correlated with resistance of
many types of tumor cells to apoptosis, and overexpression of Bcl-2 in
cultured cells confers resistance to apoptosis induced by an array of
agents, including STS, A
, and Fe2+ (35). We previously
generated lines of PC12 cells stably overexpressing Bcl-2 and
demonstrated their resistance to apoptosis induced by a variety of
insults (23, 32). In agreement with a recent study (41), we found that
levels of telomerase activity were approximately 2-fold greater in PC12
cells overexpressing Bcl-2 (PC12-Bcl2) compared with vector-transfected
control PC12 cells (PC12-V) (levels were 194 ± 8% of the control
level; n = 3 cultures). We next quantified levels of
apoptosis in PC12-V and PC12-Bcl2 cells following exposure to
telomerase inhibitors (TTAGGG and DODCB) in combination with apoptotic
insults (STS, A
, and Fe2+). PC12 cells overexpressing
Bcl-2 were very resistant to apoptosis induced by each insult in the
presence of telomerase inhibitors (Fig.
5A), as well as in the absence
of telomerase inhibitors (data not shown; see Refs. 23 and 32). These
results indicate that the apoptosis-enhancing action of telomerase
inhibitors occurs at a stage of the apoptotic cascade prior to that at
which Bcl-2 exerts its anti-apoptotic action.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
Bcl-2 and the caspase inhibitor zVAD-fmk
protect PC12 cells against apoptosis in the presence of telomerase
inhibitors. A, vector-transfected PC12 cells (PC12-V)
and PC12 cells overexpressing Bcl-2 (PC12-Bcl2) were pretreated for
1 h with saline (Control), 10 µM DODCB,
or 20 µM TTAGGG. Cultures were then exposed for 24 h
to 0.2% dimethyl sulfoxide (Control), 1 µM
STS, 20 µM A , or 10 µM Fe2+
(Fe). The percentage of cells with apoptotic nuclei in each
culture was quantified, and values are the mean and S.E. of
determinations made in four separate cultures. Each value for PC12-Bcl2
cells exposed to an apoptotic insult was significantly less than the
corresponding value for PC12-V cells (p < 0.01).
B, PC12 cell cultures were pretreated for 1 h with
saline (Control), 100 µM zVAD-fmk, 100 µM zVAD-fmk + 10 µM DODCB, or 100 µM zVAD-fmk + 20 µM TTAGGG. Cultures were
then exposed for 24 h to 0.2% dimethyl sulfoxide
(Vehicle), 1 µM STS, 20 µM A ,
or 10 µM Fe2+. The percentage of cells with
apoptotic nuclei in each culture was calculated, and values are the
mean and S.E. of determinations made in four separate cultures. The
control value is significantly greater than each of the other three
values for cultures exposed to STS, A , and Fe2+
(p < 0.005).
|
|
Members of the caspase family of cysteine proteases play important
roles in effecting apoptotic cell death in most, if not all, types of
mammalian cells (36, 37). In order to determine whether caspase
activation played a role in the apoptosis-enhancing actions of
telomerase inhibitors, we employed the broad-spectrum irreversible
caspase inhibitor zVAD-fmk (38). Apoptosis induced by STS, A
, and
Fe2+ was prevented by zVAD-fmk (Fig. 5B).
zVAD-fmk also prevented apoptosis in PC12 cells exposed to combinations
of TTAGGG or DODCB and each apoptotic insult (Fig. 5B).
Collectively, the data suggest that the anti-apoptotic action of
telomerase is exerted at a relatively early stage in the cell death
process, prior to the points at which Bcl-2 and caspase inhibitors act.
Telomerase Activity Is Decreased, and Vulnerability to
Apoptosis Is Increased in Differentiated PC12 Cells--
Primary
differentiated neurons are known to be more vulnerable to apoptosis,
induced by a variety of insults, than are various neural tumor cell
lines (23). Because cellular differentiation is often associated with a
decrease in levels of telomerase activity (4-6, 39), we sought to
determine whether a relationship exists between vulnerability to
apoptosis and telomerase activity in PC12 cells. We first compared the
vulnerability of undifferentiated PC12 cells and PC12 cells that had
been differentiated into a neuron-like phenotype by treatment with NGF
to apoptosis induced by STS and A
. Differentiated PC12 cells were
significantly more vulnerable than undifferentiated PC12 cells to
apoptosis induced by STS and A
(Fig.
6).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Differentiation of PC12 cells is associated
with increased vulnerability to apoptosis. Undifferentiated PC12
cell cultures and cultures that had been differentiated into a
neuron-like phenotype by treatment for 10 days with NGF (see under
"Experimental Procedures") were exposed for 24 h to the
indicated concentrations of STS (A) or A (B).
The percentage of cells with apoptotic nuclei in each culture was
calculated, and values are the mean and S.E. of determinations made in
four separate cultures. Values for differentiated cells were
significantly greater than corresponding values for undifferentiated
cells with STS concentrations of 0.2 µM
(p < 0.02), 0.5 µM (p < 0.01), 1 µM (p < 0.01), and 2 µM (p < 0.02) and with A
concentrations of 5 µM (p < 0.05), 10 µM (p < 0.01), 20 µM
(p < 0.01), and 50 µM (p < 0.05). Analysis of variance with Scheffe's post hoc tests was
used.
|
|
Previous studies have shown that telomerase activity decreases upon
differentiation of NT2 teratocarcinoma cells into a neuron-like phenotype in response to treatment with retinoic acid (39). We obtained
similar results in PC12 cells following exposure to NGF, a treatment
that induces differentiation of PC12 cells into a neuron-like
phenotype. Following exposure to NGF, there was a progressive decrease
in telomerase activity, with levels declining to approximately 10% of
control levels by day 8 (Fig.
7A). In contrast to the case
of undifferentiated PC12 cells (Fig. 2), when differentiated PC12 cells
were exposed to apoptotic insults in the presence of the telomerase
inhibitors TTAGGG or DODCB, levels of apoptosis were not different
from levels in differentiated cells exposed to the insults in the
absence of telomerase inhibitors (Fig. 7B). These data
demonstrate a strong correlation between reduced telomerase activity
and increased vulnerability to apoptosis, and when taken together with
the data showing that telomerase inhibitors enhance apoptosis in
undifferentiated PC12 cells, they suggest a role for telomerase in
suppressing apoptosis.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Telomerase activity decreases in PC12 cells
during NGF-induced differentiation into a neuron-like phenotype, and
telomerase inhibitors do affect differentiated PC12 cells.
A, PC12 cells were treated with NGF for the indicated time
periods (see under "Experimental Procedures"), and relative levels
of telomerase activity in cell extracts (100 ng) were measured. Values
are expressed as a percentage of telomerase activity in control
cultures not treated with NGF and represent the mean and S.E. of
determinations made in three separate cultures. B, PC12
cells were treated with NGF for 10 days. Cultures were then pretreated
for 1 h with saline (vehicle), 20 µM telomeric
repeat DNA (TTAGGG), or 10 µM DODCB. Cultures were then
exposed for 24 h to 0.2% dimethyl sulfoxide (Control),
1 µM STS, 20 µM A , or 10 µM Fe2+. The percentage of cells exhibiting
apoptotic nuclei in each culture was calculated, and values are the
mean and S.E. of determinations made in four separate cultures. Each
value for cultures treated with STS, A , and Fe2+ was
significantly greater than the corresponding value in control cultures
(p < 0.001).
|
|
 |
DISCUSSION |
Several lines of evidence obtained in the present study
suggest that telomerase activity plays a role in cellular
resistance to apoptosis. First, inhibition of telomerase
activity with TTAGGG and DODCB was associated with increased
vulnerability of PC12 cells to apoptosis induced by three
different insults (STS, A
, and Fe2+). These
data are consistent with a very recent study showing that
glioblastoma cell lines with high levels of telomerase
activity exhibit reduced sensitivity to cisplatin-induced
apoptosis and that overexpression of telomerase antisense
increases the vulnerability of the resistant cell lines (39). Second,
apoptotic insults caused a relatively rapid decrease in telomerase
activity that preceded nuclear manifestations of apoptosis. A decrease
in telomerase activity following exposure of human testicular cells to
the DNA-damaging apoptotic agent cisplatin was recently documented
by Burger et al. (40). Third, stable overexpression of Bcl-2
resulted in an increase in telomerase activity and resistance to
apoptosis. The latter finding is consistent with a recent study showing
that overexpression of Bcl-2 in human cervical carcinoma (HeLa) cells resulted in increased telomerase activity (41). Fourth, we found that
telomerase activity was decreased upon differentiation of PC12 cells
and that such differentiated cells exhibited increased sensitivity to
apoptosis. Although further work will be required to establish the
mechanism responsible for the resistance of cells with high levels of
telomerase activity to apoptosis, the ability of sustained telomerase
activity to prevent cellular senescence (10) and of telomerase
inhibitors to enhance apoptosis (present study) strongly suggest a
central role for telomerase in modulation of cell death pathways.
Previous studies have shown that the TTAGGG oligodeoxynucleotide is
effective in inhibiting telomerase activity in several different types
of cells in culture (29-31). Presumably, the TTAGGG DNA occupies the
activity of the telomerase enzyme complex and thereby prevents it from
adding bases to the ends of chromosomes. Reverse transcriptase
inhibitors, such as azidothymidine and carbovir, can block telomerase
activity and induce cell death in immortalized cell lines (42) and in
normal fibroblasts (43). We found that both TTAGGG and DODCB, a
compound previously shown to bind selectively to dimeric hairpin
quadruplexes (24), reduced telomerase activity in a
concentration-dependent manner. Concentrations of each
agent that were effective in suppressing telomerase activity by more than 80% were not toxic to PC12 cells but markedly increased apoptosis following exposure to STS and oxidative insults. When combined with
chemotherapeutic agents, such telomerase inhibitors may prove useful in
promoting death of cancer cells.
The established function of telomerase is to add the TTAGGG repeat to
the ends of chromosomes (1). The anti-apoptotic action of telomerase
suggested by our data could conceivably be related to telomere
elongation, if shortening of telomeres is an important event that
triggers apoptosis. The ability of exogenous TTAGGG to enhance
apoptosis is consistent with a role for the recognized DNA repeat
elongating function in the anti-apoptotic action of telomerase. Because
telomeres play a role in protecting DNA, it is possible that
telomerase could prevent DNA-damaging events that trigger apoptosis.
Indeed, DNA damage may be a critical event that triggers apoptosis in
response to a variety of stimuli (44). For example, apoptosis induced
by STS is associated with early DNA damage (45), and DNA damage is a
very early event in apoptosis induced by oxidative stress in human
bladder tumor cells (46). Consistent with a role for DNA damage being
an early and pivotal event in many apoptotic paradigms are data showing
that Bcl-2 overexpression (47-49) and caspase inhibition (48, 50) can prevent apoptosis induced by DNA-damaging agents. Our data showing that
Bcl-2 overexpression and caspase inhibitors protect PC12 cells against
the pro-apoptotic actions of telomerase inhibitors in three different
apoptotic paradigms are consistent with the latter studies and suggest
that telomerase suppresses an early event in the apoptotic cascade.
Our data suggest possible roles of telomerase in modulating apoptosis
that occurs in both physiological settings and pathological states.
Apoptosis occurs during the normal turnover of a variety of cell types
throughout the body. The relatively low level of telomerase activity in
many somatic cells that turn over (e.g. fibroblasts and
various epithelial cells) may be important in allowing the cells to
undergo apoptosis in response to appropriate environmental signals.
Abnormal growth of such tissues may occur in association with
dysregulated (increased) telomerase activity. In this regard,
inhibition of telomerase is increasingly recognized as an important
treatment strategy for many different kinds of cancer (51). The role of
telomerase in apoptosis of postmitotic cells that undergo apoptosis
should also be considered. We found that telomerase activity decreased
when PC12 cells were differentiated into a neuron-like phenotype.
Neurons and other postmitotic cells (e.g. cardiac myocytes)
that have negligible telomerase activity are known to be extremely
vulnerable to apoptosis induced by conditions such as ischemia or
exposure to oxidative stress (52, 53). It will be of considerable
interest to determine whether expression of telomerase in such cells
increases their resistance to apoptotic insults that are relevant to
the pathogenesis of both acute (e.g. stroke and traumatic
brain injury) and chronic (e.g. Alzheimer's and
Parkinson's diseases) neurodegenerative conditions (52, 54, 55).
 |
FOOTNOTES |
*
This research was supported by grants from the NIA and
NINDS, National Institutes of Health (to M. P. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: 211 Sanders Brown
Bldg., University of Kentucky, Lexington, KY 40536-0230. Tel.: 606-257-1412; Fax: 606-323-2866; E-mail:
mmattson{at}aging.coa.uky.edu.
2
W. Fu and M. P. Mattson, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
DODCB, 3,3'-diethyloxadicarbocyanine;
NGF, nerve growth factor;
STS, staurosporine;
A
, A
25-35;
Fe2+, FeSO4;
TRAP, telomeric repeat amplification protocol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
 |
REFERENCES |
-
Greider, C. W.,
and Blackburn, E. H.
(1989)
Nature
337,
331-337[CrossRef][Medline]
[Order article via Infotrieve]
-
Feng, J.,
Funk, W. D.,
Wang, S.-S.,
Weinrich, S. L.,
Avilion, A. A.,
Chiu, C.-P.,
Adams, R. R.,
Chang, E.,
Allsopp, R. C., Yu, J.,
Le, S.,
West, M. D.,
Harley, C. B.,
Andrews, W. H.,
Greider, C. W.,
and Villeponteau, B.
(1995)
Science
269,
1236-1241[Medline]
[Order article via Infotrieve]
-
Lingner, J.,
Hughes, T. R.,
Shevchenko, A.,
Mann, M.,
Lundblad, V.,
and Cech, T. R.
(1997)
Science
276,
561-567[Abstract/Free Full Text]
-
Sharma, H. W.,
Sokoloski, J. A.,
Perez, J. R.,
Maltese, J. Y.,
Sartorelli, A. C.,
Stein, C. A.,
Nichols, G.,
Khaled, Z.,
Telang, N. T.,
and Narayanan, R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
12343-12346[Abstract]
-
Savoysky, E.,
Yoshida, K.,
Ohtomo, T.,
Yamaguchi, R.,
Akamatsu, K.,
Yamazaki, T.,
Yoshida, S.,
and Tsuchiya, M.
(1996)
Biochem. Biophys. Res. Commun.
226,
329-334[CrossRef][Medline]
[Order article via Infotrieve]
-
Reichman, T. W.,
Albanell, J.,
Wang, X.,
Moore, M. A. S.,
and Studzinski, G. P.
(1997)
J. Cell. Biochem.
67,
13-23[CrossRef][Medline]
[Order article via Infotrieve]
-
Cooke, H. G.,
and Smith, B. A.
(1986)
Cold Spring Harbor Symp. Quant. Biol.
51,
213-219[Medline]
[Order article via Infotrieve]
-
Harley, C. B.
(1991)
Mutat. Res.
256,
271-282[CrossRef][Medline]
[Order article via Infotrieve]
-
Kim, N. W.,
Piatyszek, M. A.,
Prowse, K. R.,
Harley, C. B.,
West, M. D.,
Ho, P. L. C.,
Coviello, G. M.,
Wright, W. E.,
Weinrich, S. L.,
and Shay, J. W.
(1994)
Science
266,
2011-2015[Medline]
[Order article via Infotrieve]
-
Bodnar, A. G.,
Ouellette, M.,
Frolkis, M.,
Holt, S. E.,
Chiu, C.-P.,
Morin, G. B.,
Harley, C. B.,
Shay, J. W.,
Lichtsteiner, S.,
and Wright, W. E.
(1998)
Science
279,
349-352[Abstract/Free Full Text]
-
Wyllie, A. H.,
Kerr, J. F. R.,
and Currie, A. R.
(1990)
Int. Rev. Cytol.
68,
251-306
-
Steller, H.
(1995)
Science
267,
1445-1449[Medline]
[Order article via Infotrieve]
-
Kroemer, G.,
Zamzami, N.,
and Susin, S. A.
(1997)
Immunol. Today
18,
44-51[CrossRef][Medline]
[Order article via Infotrieve]
-
Miller, D. K.
(1997)
Semin. Immunol.
9,
35-49[CrossRef][Medline]
[Order article via Infotrieve]
-
D'Mello, S. R.
(1998)
Curr. Top. Dev. Biol.
39,
187-213[Medline]
[Order article via Infotrieve]
-
Warner, H. R.
(1997)
Curr. Top. Cell Regul.
35,
107-121[Medline]
[Order article via Infotrieve]
-
Barrett, J. C.,
Annab, L. A.,
Alcorta, D.,
Preston, G.,
Vojta, P.,
and Yin, Y.
(1994)
Cold Spring Harb. Symp. Quant. Biol.
59,
411-418[Medline]
[Order article via Infotrieve]
-
Bredesen, D. E.
(1995)
Ann. Neurol.
38,
839-851[Medline]
[Order article via Infotrieve]
-
Greider, C. W., and Blackburn, E. H. (1996) Sci.
Am., February, 92-97
-
Rhyu, M. S.
(1995)
J. Natl. Cancer Inst.
87,
884-894[Abstract]
-
Hetts, S. W.
(1998)
J. Am. Med. Assoc.
279,
300-307[Abstract/Free Full Text]
-
Kane, D. J.,
Sarafian, T. A.,
Anton, R.,
Hahn, H.,
Gralla, E. B.,
Valentine, J. S.,
Ord, T.,
and Bredesen, D. E.
(1993)
Science
262,
1274-1277[Medline]
[Order article via Infotrieve]
-
Kruman, I.,
Bruce-Keller, A. J.,
Bredesen, D. E.,
Waeg, G.,
and Mattson, M. P.
(1997)
J. Neurosci.
17,
5089-5100[Abstract/Free Full Text]
-
Chen, W.,
Kuntz, I. D.,
and Shafer, R. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2635-2639[Abstract/Free Full Text]
-
Wright, W. E.,
Shay, J. W.,
and Piatyszek, M. A.
(1995)
Nucleic Acids Res.
23,
3794-3795[Medline]
[Order article via Infotrieve]
-
Kim, N. W.,
and Wu, F.
(1997)
Nucleic Acids Res.
25,
2595-2597[Abstract/Free Full Text]
-
Johnson, L. V.,
Walsh, M. L.,
Bokus, B. J.,
and Chen, L. B.
(1981)
J. Cell Biol.
88,
526-532[Abstract]
-
Mattson, M. P.,
Zhang, Y.,
and Bose, S.
(1993)
Exp. Neurol.
121,
1-13[CrossRef][Medline]
[Order article via Infotrieve]
-
Morin, G. B.
(1989)
Cell
59,
521-529[Medline]
[Order article via Infotrieve]
-
Zahler, A. M.,
Williamson, J. R.,
Chch, T.,
and Prescott, D. M.
(1991)
Nature
350,
718-720[CrossRef][Medline]
[Order article via Infotrieve]
-
Mata, J. E.,
Joshi, S. S.,
Palen, B.,
Pirruccello, S. J.,
Jackson, J. D.,
Elias, N.,
Page, T. J.,
Medlin, K. L.,
and Iversen, P. L.
(1997)
Toxicol. Appl. Pharmacol.
144,
189-197[CrossRef][Medline]
[Order article via Infotrieve]
-
Guo, Q.,
Sopher, B. L.,
Pham, D. G.,
Furukawa, K.,
Robinson, N.,
Martin, G. M.,
and Mattson, M. P.
(1997)
J. Neurosci.
17,
4212-4222[Abstract/Free Full Text]
-
Kruman, I.,
Guo, Q.,
and Mattson, M. P.
(1997)
J. Neurosci. Res.
51,
293-308[CrossRef]
-
Keller, J. N.,
Kindy, M. S.,
Holtsberg, F. W.,
St Clair, D. K.,
Yen, H.-C.,
Germeyer, A.,
Steiner, S. M.,
Bruce-Keller, A. J.,
Hutchins, J. B.,
and Mattson, M. P.
(1998)
J. Neurosci.
18,
687-697[Abstract/Free Full Text]
-
Chao, D. T.,
and Korsmeyer, S. J.
(1998)
Annu. Rev. Immunol.
16,
395-419[CrossRef][Medline]
[Order article via Infotrieve]
-
Nicholson, D.,
and Thornbery, N.
(1997)
Trends. Biochem. Sci.
22,
299-306[CrossRef][Medline]
[Order article via Infotrieve]
-
Kidd, V. J.
(1998)
Annu. Rev. Physiol.
60,
533-573[CrossRef][Medline]
[Order article via Infotrieve]
-
Susin, S. A.,
Zamzami, N.,
Castedo, M.,
Daugas, E.,
Wang, H. G.,
Geley, S.,
Fassy, F.,
Reed, J. C.,
and Kroemer, G.
(1997)
J. Exp. Med.
186,
25-37[Abstract/Free Full Text]
-
Kondo, Y.,
Kondo, S.,
Tanaka, Y.,
Haqqi, T.,
Barna, B. P.,
and Cowell, J. K.
(1998)
Oncogene
16,
2243-2248[CrossRef][Medline]
[Order article via Infotrieve]
-
Burger, A. M.,
Double, J. A.,
and Newell, D. R.
(1997)
Eur. J. Cancer
33,
638-644[CrossRef][Medline]
[Order article via Infotrieve]
-
Mandel, M.,
and Kumar, R.
(1997)
J. Biol. Chem.
272,
14183-14187[Abstract/Free Full Text]
-
Strahl, C.,
and Blackburn, E. H.
(1996)
Mol. Cell. Biol.
16,
53-65[Abstract]
-
Yegorov, Y. E.,
Chernov, D. N.,
Akimov, S. S.,
Bolsheva, N. L.,
Krayevsky, A. A.,
and Zelenin, A. V.
(1996)
FEBS Lett.
389,
115-118[CrossRef][Medline]
[Order article via Infotrieve]
-
Martin, D. S.,
Stolfi, R. L.,
and Colofiore, J. R.
(1997)
Cancer Invest.
15,
372-381[Medline]
[Order article via Infotrieve]
-
MacManus, J. P.,
Rasquinha, I.,
Walker, T.,
and Chakravarthy, B.
(1996)
Hum. Cell
9,
197-204[Medline]
[Order article via Infotrieve]
-
Higuchi, Y.,
and Matsukawa, S.
(1997)
Free Radical Biol. Med.
23,
90-99[CrossRef][Medline]
[Order article via Infotrieve]
-
Weller, M.,
Malipiero, U.,
Aguzzi, A.,
Reed, J. C.,
and Fontana, A.
(1995)
J. Clin. Invest.
95,
2633-2643[Medline]
[Order article via Infotrieve]
-
Takeda, M.,
Kobayashi, M.,
Shirato, I.,
Osaki, T.,
and Endou, H.
(1997)
Arch. Toxicol.
71,
612-621[CrossRef][Medline]
[Order article via Infotrieve]
-
Miyake, H.,
Hanada, N.,
Nakamura, H.,
Kagawa, S.,
Fujiwara, T.,
Hara, I.,
Eto, H.,
Gohji, K.,
Arakawa, S.,
Kamidono, S.,
and Saya, H.
(1998)
Oncogene
16,
933-943[CrossRef][Medline]
[Order article via Infotrieve]
-
Antoku, K.,
Liu, Z.,
and Johnson, D. E.
(1997)
Leukemia
11,
1665-1672[CrossRef][Medline]
[Order article via Infotrieve]
-
Sharma, S.,
Raymond, E.,
Soda, H.,
Sun, D.,
Hilsenbeck, S. G.,
Sharma, A.,
Izbicka, E.,
Windle, B.,
and Von Hoff, D. D.
(1997)
Ann. Oncol.
8,
1063-1074[Abstract]
-
Linnik, M. D.,
Zobrist, R. H.,
and Hatfield, M. D.
(1993)
Stroke
24,
2002-2008[Abstract]
-
Sabbah, H. N.,
and Sharov, V. G.
(1998)
Prog. Cardiovasc. Dis.
40,
549-562[Medline]
[Order article via Infotrieve]
-
Guo, Q.,
Fu, W.,
Xie, J.,
Luo, H.,
Sells, S. F.,
Geddes, J. W.,
Bondada, V.,
Rangnekar, V.,
and Mattson, M. P.
(1998)
Nat. Med.
4,
957-962[Medline]
[Order article via Infotrieve]
-
Stern, G.
(1996)
Adv. Neurol.
69,
101-107[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.