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Vol. 63, Issue 3, 632-638, March 2003
Experimental Chemotherapy Laboratory, "Centro di Ricerca Sperimentale," Regina Elena Cancer Institute, Rome, Italy (A.B., C.G., S.A., B.B., D.D.B., G.Z.); Cellular Biotechnology and Hematology Department, University "La Sapienza", Rome, Italy (R.E., A.A.); and Oncology Department, "Mario Negri" Research Institute, Milan, Italy (M.D.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The aim of this study was to investigate the role of telomerase function on the chemosensitivity of melanoma cells. To this end, ecteinascidin-743 (ET-743) and cisplatin [cis-diamminedichloroplatinum(II) (CDDP)], two DNA-interacting drugs that invariably cause an arrest in the G2/M phase, and 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (LND), a mitochondria-targeting drug inducing a G1 block, were used. As experimental model, human melanoma clones showing reduced human telomerase reverse transcriptase (hTERT) expression and telomerase activity and characterized by telomere dysfunction were used. Reconstitution of telomerase activity by exogenous hTERT expression improved telomere function and reduced the sensitivity to CDDP and ET-743 without affecting LND susceptibility. The decreased sensitivity to CDDP and ET-743 was mainly caused by the ability of cells to recover from drug-induced damage, evaluated in terms of both chromosomal lesions and cell survival. The ability of hTERT-reconstituted cells to recover from drug-induced damage was attributable to the restoration of cell cycle progression. In fact, the cells without hTERT restoration remained for a prolonged time in the G2/M phase, and this cell cycle alteration made irreversible the drug-induced S-G2/M block and led to the activation of apoptotic program. On the contrary, the hTERT-reconstituted cells progressed quickly through the cell cycle, thus acquiring the capacity to recover from drug-induced block and to protect themselves from the G2/M phase-specific drug-triggered apoptosis.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Major
obstacles for anticancer chemotherapy are the cytotoxicity of
anticancer agents to normal cells and the occurrence of resistant tumor
cells to chemotherapeutic agents. Therefore, new chemotherapeutic
strategies, which could reduce the cytotoxicity of normal cells and
reverse chemoresistance of tumor cells, represent an important goal for
the development of selective cancer therapies. Telomerase activity has
been found in almost all tumors but not in adjacent normal cells (Shay
and Bacchetti, 1997
). The most prominent hypothesis is that the
maintenance of telomere stability is required for long-term
proliferation of tumors. Therefore, telomerase has become a target for
the development of new anticancer therapeutic agents.
The relationship between telomerase and the vulnerability to
drug-induced apoptosis is poorly understood. Antisense inhibition of
telomerase increases the susceptibility of glioblastoma cells to
CDDP-induced apoptosis (Kondo et al., 1998) and enhances apoptosis in
pheochromocytoma cells induced by a variety of stimuli (Fu et al.,
1999). Similarly, telomerase inhibition by ribozyme cleavage of
telomerase mRNA sensitizes breast epithelial cells to topoisomerase inhibitors through the activation of the apoptotic program (Ludwig et
al., 2001). These findings suggest a protective role of telomerase against drug-induced apoptotic cell death. Recently, a fast-increasing number of articles seem to indicate that the simple inhibition of
telomerase may not result in the anticancer effect (Bearss et al.,
2000). Therefore, although telomerase may not be a universal target for
cancer therapy, targeting the telomere maintenance mechanisms could be
important for future successful therapeutic anticancer strategies. In
this context, by using telomerase-deficient mouse, other studies have
demonstrated that telomere dysfunction, rather than telomerase per se,
is the principal determinant governing chemosensitivity against agents
that induce double-strand DNA breaks (Wong et al., 2000; Lee et al.,
2001).
The present study examines the impact of telomerase function on the
sensitivity of melanoma cells to ET-743, a novel marine natural
compound that shows a good activity against a variety of tumors
(Izbicka et al., 1998; Valoti et al., 1998; Villalona-Calero et al.,
2002); CDDP, one of the most effective and broadly used anticancer
drugs; and LND, a dichlorinated derivative of indazole-3-carboxylic acid, which plays a significant role in reversing or overcoming multidrug resistance (Citro et al., 1991; Silvestrini et al., 1992).
CDDP and ET-743 are DNA-interacting drugs that cause an arrest in the
G2/M phase (Sorenson and Eastman, 1988;
Takebayashi et al., 2001a; Gajate et al., 2002), whereas LND is a
mitochondria-targeting drug inducing a G1 block
(Del Bufalo et al., 1996). Unlike CDDP and LND, the mechanism of action
of ET-743 is not yet fully elucidated. It binds to guanines at the N2
position in the minor groove (Pommier et al., 1996), and its cytotoxic
effect and cell cycle perturbation (Erba et al., 2001) seem related to
an alteration of the transcription regulation that occurs in a
promoter-dependent fashion (Jin et al., 2000; Minuzzo et al., 2000).
Moreover, cells with mismatch repair defects, which are tolerant to
some alkylating agents and to CDDP, are sensitive to ET-743, whereas
cells deficient in transcription-coupled nucleotide excision repair,
which are very sensitive to CDDP, are resistant to ET-743 (Damia et
al., 1996; Takebayashi et al., 2001b).
We demonstrate here that the reconstitution of telomerase activity, by exogenous hTERT expression, improved telomere function and decreased sensitivity to CDDP and ET-743 without affecting LND susceptibility. The reduced drug sensitivity was caused by the ability of hTERT to modify cell-cycle progression, enabling the cells to recover from drug-induced G2/M block and consequently protecting them from apoptosis.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cells and Culture Conditions.
MAS51 and MAS53 c-Myc
low-expressing clones were previously obtained by transfecting the M14
human melanoma line with an expression vector carrying the exon 2 + exon 3 of c-myc cDNA cloned in antisense orientation (Biroccio et al.,
2001). The c-Myc low-expressing clones were then infected with
retroviruses encoding hTERT (+hTERT cells) or the gene for the
puromycin resistance only (
hTERT cells) and used after 14 and 35 population doublings (PD), corresponding to the second and fifth
culture passages, respectively, after infection (Biroccio et al.,
2002).
hTERT and +hTERT cells were grown at 37°C (5%
CO2/95% air atmosphere) in RPMI 1640 medium
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, 2 mM L-glutamin, and antibiotics and containing neomycin (0.8 mg/ml; Invitrogen) and puromycin (0.5 µg/ml; Sigma, Milan, Italy).
Treatments and Clonogenic Assay.
Clinical grade CDDP,
ET-743, and LND were obtained from Pharmacia (Milan, Italy), PharmaMar
(Tres Cantos, Madrid, Spain), and Angelini (Rome, Italy), respectively.
Drug dilutions were freshly prepared before each experiment. Cells were
seeded in 60-mm Petri dishes (Nunc-Mascia Brunelli, Milan, Italy) at a
density of 2 × 105 cells/dish. Cells were
exposed to different doses of CDDP (ranging from 0.3 to 16 µM) for
2 h, ET-743 (ranging from 1 to 50 nM) for 1 h, and LND
(ranging from 0.07 to 0.6 mM) for 24 h, and the analysis was
performed at the end of treatments. The doses of CDDP and ET-743 that
inhibit cell survival by approximately 50%
(IC50) in all the cellular lines were used as
equitoxic doses. In particular, we used the doses of 7 µM CDDP and 20 nM of ET-743 for the treatment of M14 and +hTERT cells and 1.7 µM
CDDP and 1 nM ET-743 for hTERT cells. To evaluate cell colony-forming
ability, aliquots of cell suspension from each sample were seeded into
60-mm Petri dishes with complete medium and incubated for 10 to 12 days. Colonies were stained with 2% methylene blue in 95% ethanol and
counted (1 colony 
50 cells). Surviving fractions were
calculated as the ratio of absolute survival of the treated sample to
the absolute survival of the control sample. All of the experiments
were repeated four times in triplicate.
Western Blot.
Western blot and detection were performed as
reported previously (Biroccio et al., 2001). Briefly, 40 µg of total
proteins were loaded on denaturing SDS-polyacrylamide gel
electrophoresis. Immunodetection of the c-Myc protein was performed
using a 1:1000 dilution of the anti-c-myc monoclonal antibody clone
9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). To determine the
amount of protein transferred onto nitrocellulose membrane, 
-actin
was used as control. The relative amounts of the transferred proteins were quantified by scanning the autoradiographic films with a gel
densitometer scanner (Bio-Rad, Milan, Italy) and normalized to the
related -actin amounts.
Cytogenetic Analysis.
To obtain chromosome
preparation, cells, in the log phase of growth, were incubated with 0.1 µg/ml of colcemid for 1 h and trypsinized, and then they were
incubated with hypotonic 0.075 M KCl for 20 min, fixed with methanol to
acetic acid (3:1, v/v), dropped onto frosted microscope slides, and
air-dried overnight. Chromosome aberration frequency was evaluated in
at least 50 Giemsa-stained metaphases from two simultaneously grown
cultures for each line and each treatment (7 µM of CDDP for 2 h
and 20 nM ET-743 for 1 h). For all of the experiments, metaphase
preparations of the different cells were performed simultaneously under
the same conditions. The 
2 test was used for
statistical analysis.
Bromodeoxyuridine Labeling.
Progression of cells
through the cell-cycle phase was analyzed by flow cytometry (BD
Biosciences, San Jose, CA) using bromodeoxyuridine (BrdU; BD
Biosciences) incorporation, as described previously (Biroccio et al.,
2001). Briefly, cells were pulsed with BrdU (10 µM for 15 min)
24 h after the end of CDDP and ET-743 treatments. At the end of
BrdU pulse and at 4 h intervals after the pulse, the cells were
fixed and the DNA was denatured. Cells were then incubated with 2 µg/ml of mouse anti-BrdU (clone BMC 9318, Roche Diagnostics,
Indianapolis, IN) for 30 min at room temperature, and the BrdU-positive
cells were revealed with fluorescein isothiocyanate-conjugated anti-mouse monoclonal antibody (1:20; DAKO, Glostrup, Denmark). To
evaluate the percentage of cells in each phase of the cell cycle after
propidium iodide staining (1 µg/ml), the fraction of BrdU-positive
cells was divided into three regions by their DNA content
(G1, S, and G2/M). The
three compartments were chosen for each cell line at the end of the
pulse and were left unchanged during the following intervals of the analysis.
Detection of Apoptosis. Apoptotic cells were detected by using the FlowTACS in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-based Apoptosis Detection Kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instruction. Briefly, 2 × 106 cells were collected 48 h after the treatments with equitoxic doses of CDDP and ET-743, fixed with 3.7% formaldehyde/phosphate-buffered saline, washed with phosphate-buffered saline, and permeabilized with 100 µl of Cytonin (Trevigen, Gaithersburg, MD) for 30 min at room temperature. The fragmented DNA was revealed by incubating the samples with the labeling reaction mix for 1 h at 37°C, and the fluorescein isothiocyanate-labeled cells were then stained with 50 µg/ml propidium iodide solution containing 750 µg/ml RNAase for 30 min at room temperature and in the dark. The flow-cytometric analysis was performed using FACSCalibur (BD Biosciences).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reconstitution of hTERT Decreases CDDP and ET-743 Sensitivity.
In this study, an experimental model obtained previously by our group
was used (Biroccio et al., 2002). Briefly, we first generated
M14-derived c-Myc low-expressing clones (MAS51 and MAS53), characterized by the reduction of hTERT expression, telomerase activity, and telomere shortening compared with the parental line (Biroccio et al., 2001). Second, telomerase activity was restored in
the c-Myc low-expressing clones (Biroccio et al., 2002) by infection
with amphotropic viruses, encoding either hTERT (+hTERT cells) or the
puromycin resistance only (hTERT cells).
hTERT cells after 35 PD
(MAS51/V35, MAS53/V35)
after infection were used to study the role of telomerase function on
drug sensitivity. Figure 1 shows the
survival curves of the different clones exposed to increasing doses of
CDDP and ET-743, two DNA-interacting drugs that invariably cause an
arrest in G2/M phase, and LND, a
mitochondria-targeting drug, inducing a G1 block
(Del Bufalo et al., 1996). No difference in cell survival was observed
between hTERT and +hTERT cells treated with LND. On the contrary, the
survival curves of the hTERT cells treated with CDDP and ET-743
showed an exponential decrease in cell survival with the increasing of
doses and, at the highest drug concentrations, the curves decreased in
the third decade, reaching values of approximately 0.1%.
Reconstitution of hTERT decreased sensitivity to CDDP and ET-743 at all
the doses of drugs used. The behavior of the survival curves was
biphasic, characterized by a shoulder region followed by an exponential phase at the highest doses, even though the surviving fraction remained
between the first and the second decade. The degree of sensitivity to
both drugs was strictly associated with the PD of the +hTERT cells. In
fact, at 7 µM CDDP, the surviving fraction of the +hTERT cells was
approximately 20% after 14 PD and 60% after 35 PD. Similarly, at 20 nM ET-743, the surviving fraction of the +hTERT cells was approximately
15% after 14 PD and 50% after 35 PD. Moreover, after 35 PD, the
sensitivity to both drugs was superimposable to that of the M14
parental line. To exclude the possibility that the decreased CDDP and
ET-743 sensitivity observed in +hTERT cells with the increasing PD was
caused by a reactivation of c-Myc expression, Western blot analysis was performed. As shown in Fig. 2, no
difference in c-Myc protein expression was evident between hTERT and
+hTERT cells, as well as between +hTERT cells at different PD.
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), to
explain the difference in drug sensitivity, telomere status was
analyzed. Data reported in Table 1 show
that hTERT cells had a high frequency of telomeric fusions with a mean value, between MAS51/V35 and
MAS53/V35, of 1.24. A significant decrease in
telomeric fusion frequency appeared in +hTERT cells already after 14 PD
and became more evident after 35 PD (p < 0.001) compared with hTERT cells. The mean values of telomeric fusion frequency of the two +hTERT 14 and 35 PD were 0.79 and 0.23, respectively. A representative metaphase of hTERT
(MAS51/V35) and +hTERT after 14 (MAS51/T14) and 35 (MAS51/T35) PD, showing the maximum number of
telomeric fusions per metaphase observed (four, three, and two,
respectively), is shown in Fig. 3.
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Reconstitution of hTERT Enables the Cells to Recover from CDDP and
ET-743-Induced Damage.
We next analyzed the ability of cells to
recover from CDDP and ET-743-induced damage. The experiments were
performed by using hTERT (MAS51/V35) and +hTERT
(MAS51/T35) cells after 35 PD after infection
treated with equitoxic doses of both drugs, chosen from the
dose-response curves (see Fig. 1). The doses of 7 µM CDDP for +hTERT
and M14 cells and 1.7 µM for hTERT cells that reduced cell survival
by approximately 50% (IC50) were used. Figure
4 shows that immediately after the end of
CDDP treatment, a similar behavior between the survival curves of
hTERT and +hTERT was observed. However, a different ability to
recover from CDDP damage was evident. In fact, at 96 h, the
surviving fraction of hTERT cells was approximately 50%. On the
contrary, +hTERT cells showed a surviving fraction of approximately
100%, indicating that these cells were able to completely recover from
CDDP-induced damage. The doses of ET-743, corresponding to
IC50, were 20 nM for the +hTERT and M14 cells and
1 nM for the hTERT cells. After a decrease of cell survival by
approximately 70%, a plateau phase in the hTERT cells was observed,
indicating that they were unable to recover from the ET-743-induced
damage. On the contrary, the survival fraction of +hTERT cells reached
approximately 80% of cell survival at 96 h. The survival curves
of the +hTERT cells treated with both drugs were superimposable to
those of M14 cells.
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hTERT and in +hTERT cells. In addition, both hTERT and +hTERT
cells treated with CDDP or ET-743 exhibited various chromosome
aberrations such as aspecific chromatid and chromosome breaks and
exchange figures involving two or more chromosomes. During the time
after the treatment, the rate of induced chromosome damage
significantly (p < 0.01) decreased in +hTERT cells,
whereas no significant difference was observed in hTERT cells. The
results obtained when using the MAS51 clone, with and without hTERT
reconstitution, were similar to those obtained with the MAS53 clone
(data not shown).
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Reconstitution of hTERT Enables the Cells to Recover from CDDP and
ET-743-Induced G2/M Block.
To evaluate whether the
inability to recover the drug damage elicited by the hTERT cells was
related to alterations in cell-cycle progression, BrdU incorporation
assay was performed. Figure 6 shows the
bivariate DNA/BrdU distribution, performed at different times after 15 min of BrdU labeling, in hTERT (MAS51/V35),
+hTERT (MAS51/T35), and M14 cells untreated or
treated with CDDP and ET-743 at equitoxic doses. It is evident that
both drugs caused a similar block in the S-G2/M
phase of cell cycle 24 h after the end of treatments (0 h),
regardless of hTERT expression. However, a significant difference among
the lines was observed during the progression through the cell-cycle
phases. In fact, although the +hTERT cells were able to recover from
the CDDP and ET-743-induced block to the same extent as M14 cells, a
strong perturbation of the cell cycle was still evident in the hTERT
cells 16 h after labeling. This effect was mainly caused by a
different progression through the cell cycle between hTERT and +hTERT
cells. In fact, the analysis of the percentage of BrdU-positive cells
in the different phases of cell cycle (Fig.
7) demonstrated that hTERT cells
remained in the G2/M phase for a prolonged time
with respect to +hTERT or M14 cells, thus causing a delay in the
repopulation of the G1 and S phases. As a
consequence, the CDDP and ET-743-induced G2/M
block was irreversible in the hTERT cells, in which approximately 80% of BrdU-positive cells were still arrested in
G2/M. On the contrary, the restored progression
into cell cycle in the +hTERT cells permitted them to overcome the
drug-induced G2/M block, as observed in the M14
parental line.
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hTERT cells led them to activate
the apoptotic program, a TUNEL assay was carried out by flow cytometry,
in which the fragmented DNA is simultaneously analyzed with DNA
content. Two-parameter flow-cytometry analysis (Fig.
8) demonstrated that the hTERT cells
treated with both drugs gave a TUNEL-positive signal in the
G2/M-phase region (approximately 20%), whereas
no apoptotic cells were detected in either the +hTERT or M14 cells. Therefore, it follows that CDDP and ET-743 treatments induced DNA
fragmentation in the G2/M phase in the hTERT
cells only, suggesting that the prolonged permanence in
G2/M of the hTERT cells was responsible for the
CDDP and ET-743 sensitivity.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We report that the reconstitution of telomerase activity was
able to modify the functional status of telomeres. In fact, the +hTERT
cells showed a number of total end-to-end fusions significantly lower
than those of the hTERT cells. The improved telomere function gradually occurred with the increase of population doublings. The
restored telomere function rendered the cells less sensitive to CDDP
and ET-743 without affecting LND susceptibility. In particular, hTERT
cells containing dysfunctional telomeres were more sensitive to CDDP
and ET-743 than were +hTERT cells possessing functional telomeres.
Moreover, we demonstrated that telomere dysfunction, rather than
telomerase activity, determined the chemosensitivity. In fact, by using
+hTERT cells at two different population doublings showing the same
level of telomerase activity (Biroccio et al., 2002) but different
telomere status, we demonstrated that the degree of chemosensitivity
strictly depended on the severity of telomere dysfunction. Our results
are in agreement with the data reported by DePinho et al.,
demonstrating that telomere dysfunction alters the radio- and
chemotherapeutic profile of cells derived from telomerase RNA-null mice
(Wong et al., 2000; Lee et al., 2001). On the other hand, attenuation
of telomerase activity without telomere shortening does not increase
sensitivity to anticancer agents (Folini et al., 2000).
The decreased sensitivity to CDDP and ET-743 after reactivation
of telomerase function was caused by the ability of cells to recover
from drug-induced damage in terms of cell survival. Similarly, the
persistence of structural chromosomal lesions was evident only in
hTERT cells treated with both drugs, whereas these aberrations were
largely resolved in +hTERT cells. The ability of the +hTERT cells to
recover from drug-induced damage was attributable to the restored cell
cycle progression. In fact, although the hTERT cells remained for a
prolonged time in the G2/M phase with a delay in
the repopulation of the G1 and S phases, hTERT
reconstitution restored normal cell cycle. The delayed progression of
the G2/M phase, observed in hTERT cells,
rendered irreversible the S-G2/M block induced by
both drugs. In fact, at equitoxic doses, both drugs caused a similar
arrest of the S-G2/M phases, which is recovered only in +hTERT cells, although a strong perturbation of the cell cycle
was evident in the cells without hTERT reconstitution. Our data are
strengthened by the use of the G1-blocking drug
LND, because the susceptibility to this compound was similar in all the
cell lines regardless of hTERT expression and telomere dysfunction. The
decreased drug sensitivity was mainly caused by telomere dysfunction rather than by telomerase activity per se, because, as we demonstrated previously, the mutant biologically inactive hTERT, which is
catalytically active but unable to maintain telomeres, was unable to
restore cell cycle (Biroccio et al., 2002).
The cell cycle deregulation after alteration of telomere function
has been reported by other authors. In particular, the expression of
mutant telomerase in immortal telomerase-negative human cells results
in chromosome fusion and abnormal cell cycle, with the ratio between
the percentage of cells in G1 and
G2/M being decreased (Guiducci et al., 2001).
Similar cell cycle deregulation has been reported by altering the
expression of telomere function-regulating proteins, such as Pin2/TRF1
(Shen et al., 1997), or the new Pin2/TRF1-interacting protein PinX1
(Zhou and Lu, 2001).
However, even if an alteration in the cell cycle after telomere
dysfunction has already been described, it has not been correlated with
drug sensitivity. We demonstrated that the permanence in G2/M phase of CDDP- and ET-743-treated cells
without hTERT reconstitution led the cells to activate the apoptotic
program. Apoptosis was observed only in hTERT cells treated with both
drugs and, by using a two-parameter flow-cytometric analysis, we
demonstrated that apoptosis occurred at the G2/M
phase of cell cycle, strengthening the hypothesis that irreversible
G2/M block induced by treatment was responsible
for the CDDP and ET-743 sensitivity.
To the best of our knowledge, this is the first evidence demonstrating
that telomere dysfunction is involved in the susceptibility to CDDP and
ET-743 in melanoma cells. Therefore, on the basis of previous results
demonstrating the efficacy of telomerase inhibitors in cells with short
telomeres (Kelland, 2001) and recent evidence showing the antitumoral
activity of CDDP and ET-743 combination in several histotypes,
including melanoma (D'Incalci et al., 2002), a new approach consisting
of the use of telomerase inhibitors able to induce telomere
dysfunction, followed by CDDP/ET-743 treatment, could improve the
chemotherapeutic response of melanoma.
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Acknowledgments |
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We thank Adele Petricca for her helpful assistance in typing the manuscript and Paula Franke for language revision of this manuscript.
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Footnotes |
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Received August 5, 2002; Accepted November 18, 2002
This work was supported by grants from Italian Association for Cancer Research, Ministero della Salute (to A.B., G.Z., and M.D.), and Consiglio Nazionale delle Ricerche-Ministero dell'Istruzione, dell'Universitá, e della Ricerca. B.B. and S.A. are recipients of a fellowship from the Italian Foundation for Cancer Research.
Address correspondence to: G. Zupi, Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy. E-mail: zupi{at}ifo.it
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Abbreviations |
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CDDP, cisplatin
[cis-diamminedichloroplatinum(II)];
ET-743, ecteinascidin-743;
LND, lonidamine
[1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid];
hTERT, human
telomerase reverse transcriptase;
PD, population doubling;
BrdU, bromodeoxyuridine;
TUNEL, terminal deoxynucleotidyl transferase dUTP
nick-end labeling;
+hTERT, c-Myc low-expressing clones infected with
retroviruses encoding hTERT;
hTERT, c-Myc low-expressing clones
infected with the gene for the puromycin resistance only.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, MAS51/V35; 
, MAS53/V35), +hTERT (right)
cells after 14 (
, MAS51/T14; 
, MAS53/T14)
and 35 (
, MAS51/T35; 
, MAS53/T35) PD, and
M14 parental line (
; right) exposed for 2 h to different doses
of CDDP ranging from 0.3 to 16 µM (top), for 1 h to different
doses of ET-743 ranging from 1 to 50 nM (center), and for 24 h to
increasing doses of LND ranging from 0.07 to 0.6 mM (bottom). Surviving
fractions were calculated as the ratio of absolute survival of the
treated sample to the absolute survival of the control sample. The data
represent the mean and S.D. of four independent experiments.
), and breaks (
