© 2004 Landes Bioscience. Do Not Distribute
by different methods has been shown to lead not only to progressive telomere shortening
and ensuing cell death,11but also to telomere length-independent apoptosis induction.12
Etoposide has been shown to generate topoisomerase DNA cleavable sites in telomeres
in vitro and in vivo.13Contradictory data exists on the interaction between etoposide and
telomerase. Upregulation of telomerase activity was observed in the human leukemic cell
line HL60,14,15and a number of pancreatic tumor cell lines after etoposide treatment.16
However a decrease in telomerase activity was observed in hepatocarcinomas and other
leukemic cell lines,17and no change in levels of telomerase in haematopoietic,18and
nasopharyngeal carcinoma cells.19There is evidence that inhibition of telomerase sensi-
tizes cells to topoisomerase II poisons including etoposide,20,21and that overexpression of
the catalytic subunit of telomerase (hTERT) in normal cells led to an increased resistance
against etoposide.20,22However, others could not confirm a telomerase dependency of the
[Cell Cycle 3:9, 1169-1176; September 2004]; ©2004 Landes Bioscience
Thomas von Zglinicki1,*
1Henry Wellcome Biogerontology Laboratory; 2Northern Institute for Cancer
Research; Newcastle University; Newcastle upon Tyne UK
*Correspondance to: Thomas von Zglinicki; Henry Wellcome Biogerontology
Laboratory; Newcastle General Hospital; Newcastle upon Tyne NE4 6BE; Tel.:
+44.191.256.3310; Fax +44.191.256.3445; Email: firstname.lastname@example.org
Received 06/21/04; Accepted 07/13/04
Previously published online as a Cell CycleE-publication:
telomeres, telomerase, DNA damage, etoposide,
apoptosis, growth arrest, tumor
fluorescence-detected alkaline DNA
hTERT human telomerase reverse transcriptase
growth inhibitory concentration (50%
telomere repeat amplification protocol
This work was supported by a Cancer Research
UK stipend to J.J., by a Newcastle Healthcare
Charity research grant to G.S. and by Research into
Ageing programme grant 252 to T.v.Z.
The Role of Telomeres in Etoposide Induced Tumor Cell Death
Etoposide, a topoisomerase II poison is used in the treatment of a number of solid
tumors. Contradictory data exist on the role of the telomere/telomerase complex in etopo-
side induced apoptosis. Therefore we examined the effects of etoposide treatment in the
neuroblastoma cell line SHSY5Y, with very short telomeres and the acute lymphoblastic T
cell line 1301, which displays extremely long telomeres. Both short-term and continuous
exposure to the drug were examined. Etoposide induced widespread DNA damage
followed by DNA damage foci formation and ultimately growth arrest and apoptosis in
a concentration-dependent manner. However, length of telomeres and of single stranded
telomeric G rich overhangs did not change significantly under the treatments in any cell
line. There was no significant induction of single-strand breaks in the G-rich strand of
telomeres. Telomerase activity was transiently upregulated under low concentrations of
etoposide, while high concentrations resulted in decreased telomerase activity only after
onset of apoptosis. Telomerase overexpression protected against etoposide induced
apoptosis in fibroblasts. The data suggest that telomeres are not major signal transducers
towards growth arrest or apoptosis after etoposide treatment. However, upregulation of
telomerase might be part of an attempted adaptative response, which protects cells by a
mechanism that might be independent of telomere length maintenance.
Topoisomerase II enzymes,1break double strands of DNA and induce topological
changes in DNA.2Etoposide, a topoisomerase II poison, stabilizes the cleavable complex
formation converting topoisomerase into physiological toxins that introduce high levels of
transient protein associated breaks in the genome of treated cells.3Levels of topoisomerase
II are generally elevated in cells that are undergoing rapid proliferation and due to the
mechanism of drug action, the higher the physiological concentration of topoisomerase II,
the more lethal the poison becomes.
Human telomeres are specialized DNA-protein structures localized at the ends of
chromosomes. They are composed of tandemly repeated hexanucleotides TTAGGG,
bound to specific proteins. Their shortening with each round of DNA replication is caused
by different mechanisms, one of these being their sensitivity to DNA damage. Thus,
telomere shortening can greatly be accelerated or decelerated by controlling oxidative stress
within the cells.4Telomeres end in single stranded overhangs of the G-rich strand, which
appear to be essential for telomeric higher order structure,5and possibly for the generation
of DNA damage signals from telomeres.6-8In the vast majority of cancer cells, telomere
shortening is counteracted by telomerase, a reverse transcriptase that uses an RNA tem-
plate to add telomeric repeats onto the ends of chromosomes.9,10Inhibition of telomerase
sensitivity of various tumor cells to etoposide.23,24
Given these inconsistent results, we decided to examine whether
there is a role for the telomere/telomerase complex in etoposide
induced cell death. Neither telomere length, telomere strand break
frequency, or length of G-rich telomeric single-stranded overhangs
changed in etoposide-treated cells before onset of apoptosis. We
conclude that telomeres are not directly involved in the signalling
pathway to etoposide-induced tumor cell growth arrest or apoptosis.
However, telomerase was transiently upregulated in response to
etoposide treatment in SHSY5Y cells and overexpression of telomerase
protected human cells against etoposide-induced apoptosis, probably
by an indirect mechanism.
MATERIALS AND METHODS
Cell Culture and Drug Solutions. The neuroblastoma cell line
SHSY5Y,25was obtained from ATCC, USA and acute lymphoblastic T cells
1301 were from Dako, Denmark. Cells were grown in RPMI medium plus
10% foetal calf serum at 37˚C under air plus 5% CO2. MRC5 human
fibroblasts were obtained from the ECACC and grown in DMEM. MRC-5
fibroblasts at PD 33 were telomerized using retroviral supernatant from
ΦNX cells transfected with the retroviral vector pLCP-hTERT (Clontech)
containing the gene for the human reverse transcriptase (hTERT) under
control of the CMV promoter and selected with 0.75 µg/ml puromycin.
Lifespan of MRC-hTERT was greatly extended for at least 120 PD.
Telomerase activity (analysed in a TRAP assay) was comparably high to tumor
cells, telomeres (measured in southern blot) were stably maintained at
around 9 kb. Etoposide was dissolved in methanol prior to each experiment
and diluted into medium. All reagents were acquired from Sigma-Aldrich
Sulphorhodamine B (SRB) Assay. Cells were seeded into multiple
96 well plates and incubated for 24 hours before treatment with varying
concentrations of etoposide for four hours. Daily a plate would be fixed
with 50% trichloroacetic acid up to 6 days after treatment. All plates were
then stained with 0.4% SRB in 1% acetic acid for 30 mins, washed with 10
mM Tris pH 10.5 for 10 mins and read on a spectrophometric plate reader
at 570 nm.
Cell Counting and Apoptosis. After appropriate etoposide treatment,
non adherent and adherent cells were collected in RPMI plus 10% FCS,
counted using a haemocytometer (VWR International, Leicester, UK) and
stained with 10 µg/ml propidium iodide for 15 mins at 4˚C to determine
the number of cells with intact plasma membrane. The cells were immedi-
ately used for apoptosis analysis in a PAS-PPCS flow cytometer (Partec
GmbH, Munster, Germany). Apoptosis was assessed as the fraction of small,
granular apoptotic cells that are distinguishable from viable cells by their
lower forward and higher sideward light scatter.26
Immunofluoresence. Cells were grown onto coverslips, treated with
etoposide, fixed with 2% paraformaldehyde in PBS for 10 mins and
permeabilized in PBG (0.2% cold water fish skin gelatine, 0.5 % BSA in
PBS) with 0.5 % Triton X-100 for 45 mins at room temperature. Cells were
incubated with anti phosphor histone H2A.X (Upstate, Cat#05-636) in
PBG with 0.5% Triton X-100 and washed. Alexa fluor 594 goat anti mouse
IgG (Molecular probes Cat# A11005) was used as a secondary antibody.
Cells were stained with DAPI and imaged in a Zeiss epifluorescence micro-
Fluorescence Detected Alkaline DNA Unwinding Assay (FADU).
Single and double strand breaks were measured using the semi automated
FADU assay.27Briefly cells were treated with etoposide for four hours,
washed in PBS and lysed in 0.25 M meso-inositol, 1M MgCl2, 10 mM
Na2P04/NaH2PO4, pH 7.2. The lysed cells were transferred onto a 96-well
plate (replicates of 8–10), kept at 4˚C, and denaturation buffer (9 M urea,
10 mM NaOH, 25 mM CDTA (trans-1,2-diaminocyclohexane-N,N,N’N’-
tetraacetic acid), 0.1% SDS) was added, followed by an alkali solution
(200 mM NaOH, 40% denaturation buffer) at 37˚C for 90 mins. DNA
was stained with the intercalating dye SYBR Green (diluted 1:25,000 in 13
mM NaOH) and fluorescence was measured in a fluorescence reader
Spectrafluor Plus (Tecan, Crailsheim, Germany) at λex 492 nm and λem
520 nm. The fluorescence intensity is inversely correlated to the number of
DNA strand breaks present at the time of lysis. DNA damage percentage
was calculated as 100x (P0- Px) / P0, with P0: fluorescence intensity of an
untreated control sample and Px: fluorescence intensity of the treated cell
Cell Cycle. Non adherent and adherent treated cells were collected,
washed in PBS and fixed in 70% ice cold ethanol for 1 hour. Cells were
washed in PBS, resuspended in DAPI stain/PBS and analysed in a PAS-
PPCS flow cytometer using UV excitation (Partec GmbH, Munster,
BrdU Incorporation. Cells were treated with etoposide for 48 hours and
incubated with 20 µM 2-bromo-5-deoxuridine (BrdU) for 1 hour. Cells
were then fixed in 70% ice cold ethanol, washed in PBS plus 0.5% bovine
serum albumin (BSA) and denatured in 2M HCl in PBS plus 0.5% BSA at
room temperature for 20 mins. After neutralization for 2 mins in 0.1 M
sodium borate pH 8.5 cells were stained with FITC-AntiBrdU (Pharmingen
Cat#33284X, 20 µl of antibody in 30 µl of dilution buffer) or FITC-IgG
control (Pharmingen Cat# 35404X) for 30 mins. Cellular DNA was stained
with 10 µg/ml propidium iodide for 30 mins and samples were analysed in
PAS-PPCS flow cytometer (Partec GmbH, Munster, Germany) with FL1 in
log4 scale and FL3 in linear scale.
Telomere and Overhang Lengths. Cells were embedded in 0.65% low
melting agarose plugs at a density of 107cells/ml before treatment with
proteinase K.28DNA was completely digested by Hinf1 (60 U per plug;
Roche) at 37˚C. Plugs were analysed in a 1% agarose gel by pulsed field gel
electrophoresis (Biorad) at 3 V/cm for SHSY5Y cells and 5.5 V/cm for
1301 cells for 17 hours with a switching time of 2 to 10 in 0.5 x TBE. To
measure telomere restriction fragment lengths in SHSY5Y cells, the gel was
dried at room temperature, denatured (1.5 M NaCl, 0.5 M NaOH) for 30
mins, neutralized (1.5 M Nacl, 0.5 M Tris-HCl, pH 7.4) for 30 mins and
in-gel hybridized with 32P-γ-ATP (TTAGGG)4at 43˚C for 16 hours. For
the 1301 cells, gels were southern blotted to Hybond-N+ membranes and
hybridized with 32P-γ-ATP (TTAGGG)4at 43˚C for 16 hours. All gels/
blots were washed 4 times in 0.2 x SSC/0.2 x SSC & 0.1% SDS at 43˚C for
30 mins each and then exposed to a phosphoimager screen overnight. Signals
were visualized using a phosphoimager (Storm 820, Molecular Dynamics,
Amersham). Average fragment length per lane was calculated as weighted
mean of the optical density using the AIDA densitometry software (Raytek,
The length of single-stranded terminal overhangs in telomeres was
measured by in-gel hybridization of telomeric probes onto nondenatured
DNA as described,28,29with the following modifications: In-gel hybridiza-
tion with 32P-γ-ATP (CCCTAA)4was performed on non denatured gels at
37°C for 16 hours. Gels were then washed as for telomeres though at 37˚C
and visualized. Under these conditions, only single-stranded G-rich DNA is
available as target for hybridization. Afterwards, DNA was denatured and
gels were hybridized as above to obtain the total telomeric signal. To test for
unspecific degradation of DNA, gels were rehybridized with the minisatellite
Single Strand Breaks in the G Rich Strand of Telomeres. DNA was
digested overnight at 37˚C with Hinf1. Digested DNA was preincubated
for 2 hours in 1x alkaline buffer (50 mM NaOH, 1 mM EDTA) and then
electrophoresed at 26 V for 20 hours for SHSY5Y cells and 40 V for 24
hours for 1301 cells in 0.7% agarose in alkaline buffer. Gels were then neu-
tralized (1.5 M Nacl, 0.5 M Tris-HCl, pH 7.4) for 1 hour and dried at room
temperature. Gels were hybridized with 32P-γ-ATP (AATCCC)4at 43˚C
for 16 hours and washed 4 times in 0.2 x SSC at 43˚C for 30 mins each. They
were then exposed to a phosphoimager screen overnight. Signals were visual-
ized using a phosphoimager (Storm 820, Molecular Dynamics, Amersham).
TRAP Assay. Telomerase PCR ELISA (Roche) was used for analysis of
telomerase activity in SHSY5Y cells using 2 µg of protein according to the
THE ROLE OF TELOMERES IN ETOPOSIDE INDUCED TUMOR CELL DEATH
1170 Cell Cycle2004; Vol. 3 Issue 9
THE ROLE OF TELOMERES IN ETOPOSIDE INDUCED TUMOR CELL DEATH
Etoposide Induces S Phase Arrest and Apoptosis. Treatment of both cell
lines with etoposide induced arrest of net growth and cell loss in a concen-
tration-dependent manner. IC50values were measured by the sulphorho-
damine B (SRB) assay and/or cell counting for a bolus treatment which lasted
for 4 h (‘short treatment’) and a continuous treatment for at least 48 h
(Table 1). These two regimens, spanning concentration ranges from
0–350 µM for the short treatment and 0–5 µM for the continuous treat-
ment, were used in all subsequent experiments.
Short treatments with etoposide in concentrations of 100 µM and above
induced significant apoptosis in both SHSY5Y (Fig. 1A) and 1301 cells (Fig. 1B).
Continuous treatment with concentrations above 1 µM also induced sig-
nificant apoptosis in SHSY5Y (Fig. 1C) and less so in 1301 cells (Fig. 1D),
despite a significant inhibition of net growth (Table 1). In fact, the majority
of non-apoptotic cells were growth-arrested at 2 days after the onset of treat-
ments. Measurement of the cellular DNA content in SHSY5Y cells revealed
significant increases of the fraction of cells in S phase at 48 h after a short
etoposide treatment (Fig. 2A) and after continuous treatment (data not
shown). Neither of these cells incorporated BrdU (Fig. 2B). We conclude
that etoposide treatment induces S phase arrest and apoptosis with the
relative frequencies of these responses being dependent on both treatment
regime and cell type.
Etoposide Triggers a DNA Damage Response. FADU measurements
confirmed the expected concentration-dependent rise of DNA strand breaks
immediately after a 4 h treatment in both SHSY5Y cells (Fig. 3A) and 1301
cells (Fig. 3B), which correlates well with the observed frequencies of apop-
totic cells after short term treatment (Fig. 1A and B). We observed a better
strand break repair capacity of 1301 cells, resulting in significantly less
damage remaining after a further 4 h incubation (compare Fig. 3A and B),
which might be related to the ability of 1301 cells to redirect their response
to low etoposide concentrations from apoptosis towards growth arrest. The
DNA damage response was examined by staining with an antibody recog-
nizing the phosphorylated form of histone H2A.X (γ-H2A.X). γ-H2A.X is
a major component of DNA damage induced foci of proteins, which func-
tion as a signal transducer between DNA strand breaks and the cellular
Figure 1. Etoposide induced apoptosis in SHSY5Y (A & C) and 1301 cells (B & D). Frequency of apoptosis was measured for both short exposure (A & B)
and continuous treatment (C & D) by FACS. Data are mean +/- SEM from triplicate experiments. Etoposide concentrations are given in each figure.
GROWTH INHIBITORY CONCENTRATIONS (IC50)
FOR SHORT AND CONTINUOUS ETOPOSIDE TREATMENT
OF SHSY5Y CELLS AND 1301 CELLS (IN µM)
repair, growth arrest and apoptosis machinery.30DNA damage foci were
clearly evident in the majority of the cells even at 24 h after a short treatment
with as low as 15 µM etoposide (Fig. 3C).
Etoposide Treatment Does Not Influence Telomere Length. Telomere
shortening is a major mechanism to induce telomere uncapping and thus to
signal growth arrest and/or apoptosis.30-32To find out whether
etoposide-induced growth arrest and apoptosis was preceded or accompanied
by telomere shortening, telomere restriction fragment lengths were examined
by an in gel hybridization technique using all treatment regimes on both cell
lines. Average fragment lengths were calculated for each lane as weighted
mean of the optical density and are shown as white bars. Neither short
exposure (Fig. 4A) nor continuous treatment (Fig. 4B) of SHSY5Y cells had
any effect on telomere length. This was confirmed by quantitative evalua-
tion (Fig. 4C and data not shown). The same holds for the much longer
telomeres in 1301 cells (Fig. 4D and data not shown).
Yoon et al.13showed that the topoisomerase II cleavage site is 5’
TTAGG*G 3’ when etoposide was present. It was also suggested that not
only telomere shortening, but also an abundance of DNA single-strand
breaks in telomeres could lead to opening of the t-loop and uncapping of
telomeres.33Thus, we next examined the G-rich telomeric strand separately
using denaturing gel electrophoresis. There was no evidence of single
stranded DNA breaks on the telomeric G-rich strand after either continuous
treatment of SHSY5Y (Fig. 4E) or short exposure treatment of 1301 cells
Etoposide Does Not Influence the Length of Telomeric Single Stranded
G-Rich Overhangs. Telomeric single stranded overhangs have been impli-
cated in telomere structural maintenance,5and generation of a DNA
damage/growth arrest response.6,7,34In order to test whether etoposide
treatment might interfere with the integrity of the overhangs, we assessed
overhang length by measuring the relative hybridization signal intensity of
overhangs alone and whole telomeres.29Neither in continuously exposed
SHSY5Y cells (Fig. 5A) nor in 1301 cells after short treatment (Fig. 5B)
could any significant change of the relative overhang length be observed.
We conclude that etoposide treatment even at high concentrations does not
induce degradation of the G-rich telomeric overhang.
Telomerase Activity Decreases Only after Onset of Apoptosis.
Telomerase activity was measured by the semiquantitative TRAP ELISA in
SHSY5Y cells under a continuous etoposide treatment at various time
points and concentrations (Fig. 6). Increased telomerase activity was found
in cells treated for 48 h with low
concentrations of etoposide, which
was significant for the treatment
with 0.25 µM. At the same time, a
significant decrease in telomerase
activity became apparent after treat-
ment with 5 µM etoposide, the
highest concentration tested. 72 h
of treatment with etoposide in con-
THE ROLE OF TELOMERES IN ETOPOSIDE INDUCED TUMOR CELL DEATH
Figure 3. DNA damage, repair and
response in etoposide-treated cells.
DNA strand breaks were measured
by FADU after a 4 h etoposide treat-
ment (open bars) and after further 4
h recovery time allowing for repair
(filled bars) in SHSY5Y (A) and
1301 cells (B). Data are mean ±
SEM from six parallel measure-
ments. Significant repair effects are
denoted by an asterisk (p < 0.05,
paired t-test). (C) SHSY5Y cells were
fixed at 24 h after a 4 h treatment
with 15 µM etoposide and stained
as indicated. The merged image
shows strong foci formation in the
majority of cells.
Figure 2. Cell cycle analysis of etoposide-treated SHSY5Y cells. (A) S/G1
phase ratios were measured in DNA histograms from DAPI-stained cells at
the indicated times after short exposure treatment. Data are mean ± SEM
from triplicate experiments. Etoposide concentrations are indicated as in
Figure 1. (B) Scattergrams of cells treated for 48 h with either 3 µM etoposide
(right) or untreated (left). FITC-labeled anti-BrdU fluorescence is measured in
Fl1, and DNA content is measured in Fl3. Cell cycle phase positions G1, S,
and G2/M are indicated at the x-axis. Etoposide-treated cells do not incor-
1172Cell Cycle 2004; Vol. 3 Issue 9
THE ROLE OF TELOMERES IN ETOPOSIDE INDUCED TUMOR CELL DEATH
centrations at or above 1 µM significantly decreased telomerase activity as
well (Fig. 6). At these time points and concentrations more than 50% of the
cells are in apoptosis (Fig. 1C). This suggests that telomerase is first activated
in response to the cytotoxic treatment, and that the later decrease in telom-
erase activity is a consequence of etoposide-induced apoptosis rather than a
Telomerase Protects Human Fibroblasts from Apoptosis after
Continuous Etoposide Treatment. To examine whether telomerase expres-
sion could modify cellular responses to etoposide treatment, we stably
transfected normal diploid human fibroblasts (MRC5) with hTERT. These
cells display high telomerase activity and show greatly increased lifespan,30
but the expression of hTERT elongates telomeres only slightly and does not
change basal rates of apoptosis or growth arrest. We compared responses to
etoposide treatment between parental MRC5 and MRC5-hTERT cells.
Both strains are significantly less sensitive to etoposide than either SHSY5Y
or 1301 cells. Treatment with 250 µM etoposide produced a strong DNA
damage response in all cells, whether they were telomerase-negative or
positive. Levels of γ-H2A.X staining are at saturation in essentially all cells
Figure 4. Telomere restriction fragment lengths after etoposide treatment. Etoposide concentrations (in µM) and times after onset of treatment (in h) are indi-
cated on top of the figures. White bars indicate average telomere length. The positions of size markers for SHSY5Y (23.1, 4.36, 2.32, 2.03 kbp) and
1301 cells (194, 97, 48.5, 23.1 kbp) are indicated. M = marker (A) SHSY5Y cells, short treatment. (B) SHSY5Y cells, continuous treatment. (C) Average
telomere lengths in SHSY5Y cells after short treatment. Gels were normalized to a standard and average fragment lengths of at least four experiments were
calculated. Data are mean ± SEM. (D) 1301 cells, short treatment. (E) Denaturing gel, SHSY5Y cells, continuous treatment. Only the G-rich telomeric strand
is detected by using a C-rich probe. (F) Denaturing gel, 1301 cells, short treatment. The G-rich telomeric strand is detected.
following short exposure to etoposide (Fig. 7A). Etoposide treatment with
concentrations between 50 and 250 µM for 48 h induced significant apop-
tosis in parental MRC5 fibroblasts. However, hTERT-expressing fibroblasts
were efficiently protected from apoptosis (Fig. 7B) and significantly
increased apoptosis levels were only found after treatments with doses
higher than 250 µM (data not shown). A similar protective effect of hTERT
overexpression against the cytotoxicity of etoposide and other topoisomerase
II poisons had been shown before in other human cell strains.20,22
There are several lines of evidence to suggest that telomere/telom-
erase complexes could be important signal transduction intermedi-
ates in drug-induced cell growth arrest and apoptosis. First, telomeres
are specifically sensitive to DNA damage induced by UV,35oxidative
stress,4,36and possibly, chemotherapeutic drugs including etopo-
side.13 Second, dysfunctional telomeres trigger growth arrest and/or
apoptosis via telomerespecific induction of DNA damage foci, also
termed senescence-associated DNA damage foci.30Third, inhibition
of telomerase sensitizes mice cells,37and human cells,20,21towards
cytotoxic drugs including etoposide. Accordingly, overexpression of
the catalytic subunit of telomerase increased the resistance of cells
against etoposide.20,22Fourth, etoposide treatment of tumor cells
has been claimed to shorten telomeres,13or to modify telomerase
expression and/or activity.38
However, a number of published results are contradictory. Some
data, both in mice,37and in human cells,23suggest that it is telomere
length rather than telomerase activity that modifies the sensitivity of
cells to anticancer drugs. In contrast to that, arguments for a telomere
length-independent protective action of telomerase per se were found
in a number of different systems.12,20,39Other authors, however, did
not detect any effect of telomerase inhibition on the sensitivity of
tumor cells to etoposide.23,24
The present study shows that etoposide induced widespread
DNA strand breaks and a classical DNA damage response as indi-
cated by DNA damage foci formation. This was followed by a
combination of growth arrest and apoptosis in a concentration- and
cell strain-dependent manner.
To establish the role of telomeres in this process, we measured not
only telomere length, but also length of single stranded telomeric G-
rich overhangs and the frequency of single-strand breaks in the G-rich
strand of telomeres. Since the importance of a telomere-dependent
THE ROLE OF TELOMERES IN ETOPOSIDE INDUCED TUMOR CELL DEATH
Figure 7 (Above). Effects of hTERT expression on the sensitivity of human
MRC5 fibroblasts to etoposide. (A) Cells were fixed immediately after a
250 µM 3 h treatment with etoposide. Control shows a merged image
(DAPI/γ-H2A.X) from untreated MRC5-hTERT. All etoposide-treated cells
showed intense γ-H2A.X staining. (B) Frequency of apoptotic MRC5 and
MRC5-hTERT fibroblasts after 48 h treatment with etoposide in the indicated
concentrations. Data are mean ± SEM from 3 experiments.
Figure 5 (Above). Telomeric G-rich overhangs in SHSY5Y and 1301 cells
remain intact after exposure to etoposide. The overhang length is measured as
the ratio of hybridization intensities to the overhang alone vs the whole
telomere. (A) SHSY5Y cells after a continuous treatment. Data are mean
± SEM from 4 experiments. (B) 1301 cells after a short exposure treatment.
Data are mean ± SEM from 5 experiments.
Figure 6 (Above). Telomerase activity in SHSY5Y cells after continuous
etoposide treatment. Relative telomerase activity was measured using a
telomerase PCR ELISA. Data are mean +/- SEM from at least four indepen-
dent experiments. For each experiment, activities measured in untreated con-
trol samples were set as 100%. Statistically significant differences towards
untreated controls are marked by an asterisk (p < 0.05, ANOVA).
1174 Cell Cycle 2004; Vol. 3 Issue 9
signal transduction pathway, if it existed at all, might be different in
cells with long and short telomeres, we examined one cell line with
very short (SHSY5Y, average telomere length about 3.2 kbp) and
one with extremely long (1301, average telomere length about 80 kbp)
telomeres. None of the parameters mentioned above changed signif-
icantly in either cell line under the treatments despite induction of
growth arrest and/or apoptosis. This is not in accordance with Yoon
et al.13who show a slight shortening of telomere lengths in HeLa
cells after treatment with etoposide. Whether this discrepancy to our
results might be cell line-specific, is not clear. However, our data
clearly establish that measurable telomeric damage, might it be
shortening, accumulation of single strand breaks or deterioration of
the G-rich overhang, is not necessary as a signal transduction inter-
mediate in etoposide-induced tumor cell growth arrest and apoptosis.
We next examined telomerase activity levels in SHSY5Y cells after
continuous exposure to etoposide. It had been shown that treatment
with etoposide can transiently upregulate telomerase activity in some
tumor cells.14-16Under our conditions of continuous treatment, we
found a transient increase of telomerase activity after 48 h, which
was significant under the lowest etoposide concentration. High
concentrations of etoposide did not induce any immediate change in
telomerase activity. However, they resulted in a decrease of telom-
erase activity with increasing concentration and time of treatment,
which occurred after the massive onset of apoptosis. These results are
in accordance with others.18Thus, downregulation of telomerase is
a consequence, not a possible cause of etoposide-induced apoptosis
or growth arrest. However, the possibility remains that telomerase is
upregulated also in response to higher etoposide concentrations, but
that this is masked by a parallel induction of apoptosis. Thus, upreg-
ulation of telomerase might be part of a compensatory response of
tumor cells to cytotoxic treatments. In fact, upregulation of telom-
erase has been demonstrated in response to etoposide,14-16and to a
wide variety of other cytotoxic agents including oxidative stress.7
This is consistent with the idea of telomerase acting as a “survival
Telomerase could promote survival either by compensating for
telomeric damage or by some telomere-independent mechanism. Our
present data cannot discriminate between these two possibilities: The
constancy of telomere length under etoposide might be either due to
negligible damage in this specific structure, or due to damage compen-
sation by the action of telomerase. There are arguments in favor of a
telomere-independent mechanism, however. Firstly, an overexpression
of telomerase has been shown to induce the expression of a number
of DNA damage response and repair genes,39which are known to
act in a telomere-independent fashion. Second, if maintenance of
telomere length and/or structure by telomerase were important for
the resistance of tumor cells to cytotoxins, one should expect that
telomerase knock-down would result in compromised telomeres,
followed by early induction of apoptosis or growth arrest. In fact,
telomerase knock-down induces apoptosis in a variety of tumor cells
even under mild stress conditions. However, this occurs without any
change in telomere length.12,20
In conclusion, our data show that SHSY5Y and 1301 cells
respond to etoposide treatment by a combination of S phase arrest
and apoptosis. While a transient activation of telomerase is evident
under some conditions, which might be part of an attempted survival
response, we find no indication that telomeres and/or telomeric
damage play any preferential role as a signal transducer towards
apoptosis and/or growth arrest in etoposide-treated tumor cells.
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superhelical turns into DNA. Proc Nat Acad Sci USA 1976; 73:3872-6.
2. Wang JC. DNA topoisomerases. Ann Rev Biochem 1996; 65:635-92.
3. Kaufmann SH. Cell death induced by topoisomerase targeted drugs: More questions than
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