Articles in PresS. Am J Physiol Heart Circ Physiol (October 2, 2009). doi:10.1152/ajpheart.00068.2009
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Doxorubicin Induces Senescence or Apoptosis in Rat Neonatal Cardiomyocytes by Regulating the
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Expression Levels of the Telomere Binding Factors 1 and 2
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(H-00068-2009R3)
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Paolo Spallarossa1a, Paola Altieri1a, Concetta Aloi1, Silvano Garibaldi1, Chiara Barisione1, Giorgio
Ghigliotti1, Giuseppina Fugazza2, Antonio Barsotti1, Claudio Brunelli1
1
Research Center of Cardiovascular Biology, Division of Cardiology, and 2Laboratory of
Cytogenetics, Department of Internal Medicine, University of Genova, Italy.
a
both authors contributed equally to this study
Running Head: TRF1, TRF2 and doxorubicin cardiotoxicity
Corresponding Author:
Paola Altieri
Research Center of Cardiovascular Biology, Division of Cardiology
University of Genova
Viale Benedetto XV, 6
16132-Genova
Italy
Tel.:+39 0103537990
Fax: +39 0103538655
E-mail: [email protected]
Copyright © 2009 by the American Physiological Society.
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ABSTRACT
Low or high doses of doxorubicin induce either senescence or apoptosis, respectively, in
cardiomyocytes. The mechanism by which different doses of doxorubicin may induce different
stress-response cellular programs is not well understood. A recent study showed that the level of
telomere dysfunction may induce senescence or apoptosis. We investigated the pathways to both
apoptosis and senescence in neonatal rat cardiomyocytes and in H9c2 cells exposed to a single
pulsed incubation with low or high doses of doxorubicin. High dose doxorubicin strongly reduces
TRF2 expression while enhancing TRF1 expression and determines early apoptosis. Low dose
doxorubicin induces down-regulation of both TRF2 and TRF1, it also increases the senescenceassociated-β-galactosidase activity, down-regulates the checkpoint kinase Chk2, induces
chromosomal abnormalities and alters the cell cycle. The involvement of TRF1 and TRF2 with
apoptosis and senescence was assessed by siRNA interference. The cells maintain telomere
dysfunction and a senescent phenotype over time, and undergo late death. The increase in the
phase>4N, and the presence of micronuclei and anaphase bridges indicate that cells die by mitotic
catastrophe. p38 modulates TRF2 expression, while JNK and cytoplasmic p53 regulate TRF1. Pretreatment with specific inhibitors of MAPKs and p53 may either attenuate the damage induced by
doxorubicin or shift the cellular response to stress from senescence to apoptosis.
In conclusion, various doses of doxorubicin induce differential regulation of TRF1 and TRF2
through p53 and MAPK, which is responsible for inducing either early apoptosis or senescence and
late death due to mitotic catastrophe.
Keywords: p53, MAPKs, anthracyclines.
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INTRODUCTION
The clinical use of anthracyclines in anti-cancer treatment is limited by their adverse cardiotoxic
effects which include cardiomyopathy and heart failure (22).
At high doses, cardiotoxicity often occurs within a few months, while low doses rarely cause
deterioration of ventricular function in the first year after therapy (63). However, there is now
increasing evidence that several years after therapy one out of three patients treated with low dose
anthracyclines develops hypokinetic cardiomyopathy (21). A number of mechanisms have been
proposed to explain anthracycline cardiotoxicity. Even though the most accredited hypothesis states
that anthracyclines induce myocyte loss through oxidative stress and apoptotic cell death (3, 51),
there is controversy whether apoptosis contributes to late onset cardiotoxicity induced by low-doses
of doxorubicin (4). Maejima Y et al, have recently shown that when cultured neonatal rat
cardiomyocytes are exposed to low concentrations of doxorubicin, the cells do not enter apoptotic
program, but exhibit a senescence-like phenotype (3).
The hallmark of cellular senescence is the cell cycle arrest that is accompanied by important
changes in many aspects of cell morphology (20, 42). Senescence is the result of changes in the
expression of many proteins that regulates cell cycle, cytoskeletal function and cellular
architecture, and cause impairment of cell functions, including the regenerative capacity (62, 31, 19,
58).
Studies performed on tumor cells indicate that low doses of doxorubicin, as well as several
anticancer agents, induce mitotic catastrophe, a phenomenon that is characterized by chromosomal
abnormalities and abnormal mitosis that leads to late cell death. It has also been shown that cells
which ultimately die of mitotic catastrophe initially show a senescence-like phenotype (18).
Therefore, the induction of senescence has been proposed as a novel mechanism of cardiotoxicity
induced by low doses of doxorubicin (35).
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A number of studies suggest that telomere dysfunction plays a main role in the stress-induced
senescence program and in apoptosis (30, 60). Telomeres are specialized, repetitive, non-coding
sequences of DNA bound by several proteins, including telomere binding factors 1 and 2 (TRF1
and TRF2), which play a crucial role in telomere biology and govern chromosomal stability. The
TRF1 multi protein complex regulates telomere length and specifically affects mitotic progression,
while TRF2 is critical for maintaining the telomere t-loop "end-capping" structure, whose function
is to prevent chromosome end-to-end fusion and chromosome abnormalities (54, 60).
It is believed that telomeric dysfunction beyond a certain limit triggers a DNA damage response
mediated by p53, a tumor suppressor protein, and by mitogen-activated protein kinases (MAPKs)
(53, 23).
MAPKs are highly conserved serine/threonine kinases that are activated in response to a wide
variety of stimuli and play a role in numerous cell functions including survival, growth and
proliferation (59, 68, 67). Both p53 and MAPKs are involved in doxorubicin-induced cardiotoxicity
(32, 52). The signal transduction pathways of doxorubicin-induced senescence and the mechanism
by which different doses of the same stressing agent may induce either apoptosis or senescence and
mitotic catastrophe are still poorly understood.
In the present study we investigated the cellular response to stress that occurs in rat neonatal
cardiomyocytes exposed to a pulsed incubation with apoptotic and sub-apoptotic concentrations of
doxorubicin. Our results confirm that sub-apoptotic doses of doxorubicin induce senescence and,
for the first time, we demonstrate the following: (a) cardiomyocytes with a senescence-like
phenotype undergo late cardiac death by mitotic catastrophe; (b) various doses of doxorubicin
induce a differential regulation of TRF1 and TRF2 that is associated with the induction of either
apoptosis or senescence; (c) p53 and MAPK activation is not only downstream to telomere
dysfunction, as suggested by previous literature, but it is also a doxorubicin-induced signal that
affects TRF1 and TRF2 expression; (d) increases in p53 expression levels, as well as the
cytoplasmic accumulation of p53, which are the result of low-doses of doxorubicin, down-regulate
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TRF1 and play an active role in inducing senescence, while the inhibition of p53 results in a
transition of the cellular response to doxorubicin treatment from senescence to apoptosis.
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MATERIALS AND METHODS
All materials, unless otherwise indicated, were supplied by Sigma-Aldrich (Poole,UK).
Cell and culture conditions
Ventricular myocytes from 2-day-old Sprague-Dawley rats were purified and cultured as described
(2). H9c2 rat heart-derived embryonic myocytes (American Type Culture Collection) were cultured
as previously described (52). Cells were always used at less than 70% of confluence.
Cells were pre-incubated for 1 hour with or without the ERK1/2 pathway inhibitor PD98059 (50
μmol/L) (Calbiochem), the JNK inhibitor SP600125 (20 μmol/L), the p38 MAPK inhibitor
SB203580 (3 μmol/L), and with the p53 inhibitor Pifithrin-alpha (PFT) (5 μmol/L) (32). They were
then incubated with or without different doses of doxorubicin for 3 hours (14) and analyzed at the
time indicated for each experiment. In order to evaluate MAPK phosphorylation, cells were
incubated with various doses of doxorubicin for 20 minutes. Since both PFT and the MAPK
inhibitors were dissolved in 0.1% dimethyl sulphoxide (DMSO), an equivalent amount of vehicle
was added to both the control and to the drug-treated samples when the experiments were
performed with these inhibitors.
RT-PCR
RT-PCR was performed using the previously described procedure (56). The quantity of mRNA was
normalized for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Amplification of the cDNA by polymerase chain reaction (PCR) was performed using the primers
shown in Table 1. The PCR products were quantified by a gel documentation system with analysis
software (Syngene).
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Immunoblotting
Immunoblotting was performed using the previously described procedure (52). After various
treatments, cells were processed to determine the levels of TRF2 (clone 4S794.15, Imgenex), TRF1
(C-19, Santa Cruz Biotechnology), p53 (clone C6A5, BioVision), α-Tubulin (B-7, Santa Cruz
Biotechnology), actin (I-19, Santa Cruz Biotechnology), the phosphorylated MAPK p-p38 (D-8,
Santa Cruz Biotechnology), p-ERK (E-4, Santa Cruz Biotechnology), p-JNK (G-7, Santa Cruz
Biotechnology). The quantity of protein was normalized for GAPDH (0411, Santa Cruz
Biotechnology).
SA-b-gal activity (Senescence-associated -β-Galactosidase Staining)
Cells were stained for β-gal activity as described by Dimri et al (12). The number of SA-b-gal
positive cells was determined in 100 randomly chosen low-power fields (x100) and expressed as a
percentage of all counted cells (36).
Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining
Cells were labeled with Annexin V–FITC and propidium iodide (A/PI), (51) and 100 randomly
selected fields were counted using a fluorescence microscope. The number of stained cells was
normalized to the total number of cells as counted by phase contrast microscopy of the same field.
Images from independent fields were captured and processed using Metamorph software (Universal
Imaging; Downingtown, PA).
Trypan Blue Exclusion
Trypan blue exclusion was used to determine the viability of cardiomyocytes. After treatments as
described above, cells were trypsinized and incubated with 0.4% trypan blue dye for 2 min and
were observed with the use of a hemocytometer under a light microscope. Cells that were able to
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exclude the stain were considered viable and the percentage of non-blue cells over total cell number
was used as an index of viability.
Cell immunofluorescence
TRF1, TRF2 and p53 expression were all documented by immunofluorescence as described by
Verzola et al (61). Cardiomyocytes were grown on chamber slides to subconfluence and incubated
for three hours in the presence or absence of doxorubicin in cells that had been pre-treated with or
without MAPK inhibitors, after which they were analyzed. Subsequently the cells were washed
with cold PBS and fixed in 2% paraformaldehyde for 10 minutes. After an overnight incubation
with anti-TRF1, anti-TRF2 or anti-p53 antibodies at 4°C, cells were washed extensively with PBS
and exposed to biotinylated conjugated secondary antibody (Vector) for 30 minutes. After being
washed, cells were incubated with FITC-streptavidin (Sigma) for 30 minutes. Slides were observed
under a fluorescence microscope. Images from independent fields were captured and processed
using the Metamorph software.
Flow cytometric analysis
Nuclear DNA content: Trypsinized and floating cells were pooled, washed twice with PBS and
resuspended in 400 μl of hypotonic solution labeled with propidium iodide, (5 μg/ml PI, 0.1% w/v
Na citrate, 0.1%Triton X-100 in sterile water). Cells were incubated on ice for 30 min until DNA
content analysis.
Propidium Iodide labelling: Trypsinized and floating cells were pooled, washed twice with PBS,
and suspended in PI solution.
All test cells were monitored by Fluorescence Activated Cell Sorting (Becton-Dickinson) and data
were analyzed using CellQuest software (Becton Dickinson).
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Chromosome analysis
Twenty-four hours after treatment with doxorubicin, cells were exposed to Colcemid (0.04 μg/ml)
for 90 minutes at 37°C and to hypotonic treatment (0.075 mol/L KCl) for 15 minutes at room
temperature. Cells were fixed in a methanol and acetic acid (3:1 by volume) mixture for 15 minutes
and then washed three times in the fixative. The slides were air-dried and stained with Giemsa for
analysis.
F-actin detection
Cardiomyocytes growing on slides were fixed, permeabilized and labelled simultaneously in PBS
containing 50μg/ml lysopalmitoyphospatidylecholine, 3.7 % formaldehyde and 5 units/ml of
fluorescent phallotoxin (A-12379alexaTM488 phalloidin, Molecular probes, Inc). Cells were rapidly
washed three times with PBS and were viewed by fluorescent microscopy (38). In order to quantify
the fluorescence and to measure cells area, image analysis was performed by the Leica Q500 MC
Image Analysis System (Leica, Cambridge, UK). Three hundred cells were randomly analyzed for
each sample, and the optical density of the signals was quantitated by a computer. The video image
was generated by a CCD Camera connected through a frame grabber to a computer. Single images
were digitized.for image analysis at 256 grey levels. Imported data were quantitatively analyzed by
Q500MC Software-Qwin (Leica, Cambridge, UK). The single cells were randomly selected by the
operators by using the cursor and then positive areas were automatically estimated. Constant optical
threshold and filter combination were used.
TRF1 and TRF2 siRNA transfection
ON-TARGET plus SMARTpool short interfering RNAs (siRNA), for silencing the
expression of target genes TRF1 and TRF2 and ON-TARGETplus Non-Targeting Pool as a control
were purchased from Dharmacon. All transfections were carried out using the manufacturer’s
protocol with DharmaFECT1 transfection reagent (Dharmacon). Briefly, cardiomyocytes were
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trypsinized, counted, and plated at a density of 104 cells/cm2. After 24 hours, cells were transfected
with 10, 50, or 100 nmol/l of SMARTpool siRNA or control siRNA using DharmaFECT1 reagent
and analyzed after 24, 48 or 72 hours with A/PI staining, SA-b-gal activity and Western blot
analysis.
Statistical analysis
Data are reported as mean ± standard error (SE) of four independent experiments. Statistical
analysis was performed by one-way ANOVA followed by Bonferroni post-hoc test and Wilcoxon
signed rank test when appropriate.
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RESULTS
Low and high doses of doxorubicin have different effects on plasma membrane integrity and Bax-
α/Bcl-2 protein ratio
Twenty-four and 48 hours after having been exposed to doxorubicin for 3 hours, annexin
V/propidium iodide double staining revealed the typical pattern of early-stage apoptosis [A(+)/ PI()] and late-stage apoptosis [A(+)/PI(+)] only in cells that had been exposed to 1 μmol/L doxorubicin
(Figures 1A and 1B). Following treatment with 0.1 μmol/L doxorubicin, we observed that the
percentage of A(+) cells was lower, and that 86% of the A(+) cells were PI(+) at 24 hours,
suggesting that the integrity of the plasma membrane was precociously lost. As a further apoptosis
index we measured the bax-bcl2 ratio which increased 3-fold upon treatment with 1 μmol/L
doxorubicin, while it remained unchanged in cells treated with 0.1 μmol/L doxorubixcin (Figure
1C). Enhancement of the ratio indicates that cells underwent apoptosis. Thus, these results suggest
that 0.1 μmol/L doxorubicin induces non apoptotic cardiac damage.
Low doses of doxorubicin induce abnormal mitosis associated with a senescent-like phenotype and
alterations of cytoskeletal protein levels.
Senescent cells can be identified by the expression of enzymatic SA-b-gal activity at pH 6.0 and by
an enlarged, flattened phenotype (12). Forty-eight hours after having been exposed to doxorubicin
for 3 hours, SA-b-gal activity expression was 34%, 20%, and 8% in cells treated with 0.1, 0.05, and
0.01 μmol/L , respectively, while it remained unchanged in cells treated with higher doses (Figure
2A). Cells treated with 0.1 μmol/L doxorubicin showed enlarged volume, flattened morphology,
and the appearance of vacuolated cells (Figure 2B).
Some cells also contained several nuclei of unequal size, including micronuclei (Figure 2B), while
nuclei staining documented the presence of anaphase bridges (Figure 2C).
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Cells treated with 1 μmol/L doxorubicin exhibited condensed nuclei, which are characteristic of
apoptotic cells, (Figures 2B ). Cell viability, determined by trypan blue exclusion analysis, was
maintained for the first four days after exposure to 0.1 μmol/L doxorubicin, while it decreased in a
dose-dependent manner after exposure to 1 and 10μM doxorubicin (Figure 2D)
We then examined how pulsed incubation with doxorubicin affects cytoskeletal remodeling.
Phalloidin staining showed that at a dose of 0.1 μmol/L, doxorubicin increases cell size and both the
length and the density of the cytoplasmic actin fibers, while at a dose of 1 μmol/L it leads to the
disruption of the actin fibers (Figure 2E). Western blot analysis showed a 300% increase in actin
levels and a 50% reduction in alfa tubulin levels in cells incubated with 0.1 μmol/L doxorubicin
(Figure 2F).
Flow cytometric analysis was used to assess changes in DNA content. At 48 hours, cells treated
with 1 μmol/L doxorubicin demonstrated an increase in the sub-G1 phase and a decrease in the
G2/M phase, while cells treated with 0.1 μmol/L doxorubicin showed an increase in the S-phase and
in the hyperploid (>4N DNA) cell population (Figure 3A). In doxorubicin 0.1 μmol/L treated cells,
we also observed both a down-regulation of the expression of the DNA damage-induced checkpoint
kinase Chk2, which normally prevents the induction of mitosis, and polyploid metaphases with
several chromosomal abnormalities including dicentric chromosome, end-to-end fusion, and ring
and string configuration (Figure 3B and 3C). These findings indicate the presence of mitotic
catastrophe.
Doxorubicin regulates the expression of TRF1 and TRF2
Doxorubicin at 0.1 μmol/L decreased protein and mRNA levels of TRF1 and TRF2, while at 1
μmol/L reduced TRF2 , and enhanced TRF1 expressions (Figures 4A and 4B). These results were
confirmed by fluorescence analysis (Figure 4C).
To analyze the influence of TRF1 and TRF2 levels on cellular response, we selectively silenced
TRF1 or TRF2 using the siRNA transfection technique. By performing transfection with various
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doses of siRNA targeting TRF2, we obtained a dose dependent down-regulation of the TRF2
protein levels (Figure 5A). The 10 nmol/l siRNA induced 18% knockdown of TRF2 protein that
was associated with senescent morphology changes, including SA-b-gal positivity, marked increase
in cell size, flattened morphology and early PI positivity, while the 100 nmol/l siRNA induced a
55% knockdown of the TRF2 protein that was associated with apoptosis. siRNA at a dose of 50
nmol/l produced a 35% knockdown of TRF2 protein that was associated with the presence of both
senescent and apoptotic cells (Figure 5C), which is likely the result of the various levels of TRF2
silencing in each cell. By transfection with 100 nmol/l of siRNA targeting TRF1, we obtained a
60% knockdown of the TRF1 protein (Figure 5B). As shown in Figure 5C, the transfection with
siRNA targeting TRF1 did not alter the cells.
Doxorubicin regulates TRF1 and TRF2 expression through MAPK and p53 mediated pathways
We observed that 0.1, 1, and 10 μmol/L doxorubicin induced dose-dependent phosphorylation of
p38 and JNK, while ERK 1/2 was only activated by 10 μmol/L doxorubicin. We found no changes
in total MAPK protein levels (Figure 6A). Using specific inhibitors, we analyzed the role played by
p38, JNK, and p53 in mediating the effect of doxorubicin on TRF1 and TRF2 expression (Figure
6B). Pre-treatment with the p38 inhibitor, SB203580, attenuated the down-regulation of TRF2
induced by both 0.1 and 1 μmol/L doxorubicin, but did not modify the effects of doxorubicin on the
TRF1 expression. Conversely, pre-treatment with the JNK inhibitor, SP600125, did not influence
the effects of doxorubicin on the expression of TRF2, but blunted both the 1 μmol/L doxorubicininduced TRF1 up-regulation, and the 0.1 μmol/L doxorubicin-induced TRF1 down-regulation. Pretreatment with the p53 inhibitor, PFT, did not influence the effects of doxorubicin on the expression
of TRF2, but did modulate the effects on TRF1 expression. PFT abolished the down-regulation of
TRF1 in cells incubated with 0.1 μmol/L doxorubicin and increased the TRF1 up-regulation in cells
incubated with 1 μmol/L doxorubicin.
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We then analyzed whether pre-treatment with these inhibitors influenced the effects of doxorubicin
on the number of SA-b-gal and annexin positive cells (Figures 6C and 6D). We observed that in
cells exposed to 1 μmol/L doxorubicin, pre-treatment with SB203580 drastically reduced the
number of A(+) cells, and specifically the number of cells in late apoptosis characterized by
A(+)/PI(+) double positivity, while pre-treatment with SP600125 or PFT did not produce any
effects. When we used 0.1 μmol/L doxorubicin, we found that pre-treatment with SB203580,
SP600125, and PFT reduced the number of SA-b-gal cells from 34% to 7%, 18%, and 16%
respectively. Pre-treatment with SB203580 decreased the number of A(+)/PI(+) cells, while pretreatment with SP600125 and PFT not only increased the number of damaged cells, but it induced
the appearance of the A(+)/PI(-) cell population. These results indicate that when doxorubicin is
used at sub-apoptotic doses, pre-treatment with SP600125 or PFT switches the cell response to the
stress from senescence to apoptosis. Since pre-treatment with PFT and SP600125 on cells treated
with 0.1 μmol/L doxorubicin produced similar effects on TRF1 expression such senescence like
phenotype, and A(+)/PI(-) staining, this prompted us to investigate the relationship between p53
and JNK. We observed that the 0.1 μmol/L doxorubicin-induced expression of p53 is JNK
dependent (Figure 7A). Fluorescence microscopy confirmed these results and showed that p53
accumulation occurs in the cytoplasm and not in the nucleus where it is considered a marker of p53
mediated apoptosis (Figure 7B). This result further confirms that the 0.1 μmol/L doxorubicin dose
is not apoptotic.
Taken together, these data prove that TRF1 and TRF2 are at the center of the pathways that lead to
senescence or apoptosis in response to doxorubicin. Figure 8 shows the putative signaling pathways
involved in doxorubicin-induced senescence or apoptosis.
The effects of a single pulsed incubation with low dose doxorubicin are still detectable 21 days later
Neonatal rat cardiomyocytes cannot be cultured at length, therefore, in an attempt to examine the
long-term effects (21 days) of a 3 hour pulsed exposure to low dose doxorubicin, we used H9c2
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cardiomyocytes. We repeated all the experiments described in the previous paragraphs and obtained
the same results that we had found with neonatal rat cardiomyocytes. FACS analysis showed that
the percentage of PI(+) cell population increased from 8% to 58% at day 6, and decreased to 53% at
day 15 and to 24% at day 21, a percentage that was 3 times the basal value (Figure 9A). Both cell
size (Figures 9A and 9B) and the percentage of SA-b-gal positive cells (Figure 9C) increased
following doxorubicin treatment and peaked at day 6, still remaining above the basal values at day
21. TRF1 and TRF 2 protein expression remarkably decreased at day 6 and was still lower than the
basal value at day 21 (Figure 9D). No significant differences were observed in cells incubated
without doxorubicin. These experiments show that brief exposure to low dose doxorubicin induces
cell damage that lasts over time.
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DISCUSSION
Different doses of doxorubicin induce senescence or apoptosis through different modulation of
TRF1 and TRF2 expression levels.
This study, which investigates the physiological bases of doxorubicin-induced senescence, was
conceived moving from two lines of evidence. The first arises from the study by Eom et al (18) who
found that chronic exposure of hepatoma cells to high doses of doxorubicin induced apoptosis,
while lower doses induced senescence and late death by mitotic catastrophe, and from the study by
Maejima et al who were the first to demonstrate that low doses of doxorubicin induced senescence
in neonatal cardiomyocytes (35). The second line of evidence is represented by the study of Lechel
et al. who demonstrated that low and high levels of TRF2 inhibition are respectively associated with
senescence and apoptosis (30). The data of the present study indicate that different doses of
doxorubicin affect the expression of the telomeric binding proteins TRF1 and TRF2 differently, and
that these, in turn, decide the type of response to the stress, i.e., senescence or apoptosis. Our data
also show that TRF2 plays a prominent role in deciding the type of cellular response. By using the
siRNA technique we confirmed that high levels of TRF2 down-regulation induce apoptosis, low
levels of TRF2 down-regulation induce senescence, and we also found that the selective
knockdown of TRF1 induces neither senescence nor apoptosis. These results are in agreement with
a number of studies which found that a dominant negative allele of hTRF1 does not affect the
growth and viability of a variety of primary and transformed human cells (60, 25, 26, 13)
We documented that if doxorubicin induces high levels of TRF2 down-regulation, apoptosis occurs
regardless of the level of TRF1 expression. Conversely, if the stress induces a moderate level of
TRF2 down regulation, as it does with doxorubicin 0.1, TRF1 is crucial for deciding cell response.
A consensual reduction of TRF1 expression induces senescence, otherwise apoptosis occurs.
This finding is in line with previous studies which demonstrated that in particular cellular
conditions, TRF1 can affect cell cycle progression and is an important signal for cell survival. Lu et
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al observed that TRF1 inhibition suppresses the ability of mitotic kinase NIMA to induce premature
mitotic entry and apoptosis (34). Kishi et al found that TRF1 over-expression can induce apoptosis
in cells with short telomeres (27) but not in those containing long telomeres. The similarity between
our and Kishi's data may be better appreciated keeping in mind that short telomeres and TRF2
down-regulation have similar effects on cell biology (24).
Doxorubicin-induced senescent cardiomyocytes show morphologically flattened and enlarged cell
shapes associated with cytoskeleton remodeling that is characterized by a significant increase in the
level of actin protein with well visible polymerized F-actin filaments crossing the whole cell, and
by a decrease in tubulin levels. Cytoskeletal components are key regulators of cellular architecture.
Functionally, the enhanced actin fibers may construct a frame structure that is needed to support the
enlarged cells (45, 1). Cytoskeletal components are also involved in cellular processes such as cell
differentiation and cell cycle regulation, and are essential mediators of the cell signaling pathways
that regulate senescence (41, 42). The reduction of alpha-tubulin levels in senescent cells has been
viewed as a process that impairs cellular microtubule integrity, and may disrupt the normal mitotic
processes (56).
Some structural alterations of senescent cells are specific for the cell type. For example, while our
data and those of Alexander et al document an association between the senescent phenotype and
over expression of actin in cardiomyocytes and human osteosarcoma cells (1), Nishio et al
documented a reduction in the levels of actin protein in senescent fibroblasts. Differently from
neonatal cardiomyocytes that do not express vimentin (29, 70), senescent fibroblasts show an
extraordinary production of vimentin (42) that, by anchoring cytoplasmic p53, prevents nuclear
import of p53 and p53-dependent apoptosis (41).
We found that a pulsed, brief, incubation with doxorubicin 1 μmol/L induces apoptosis through a
p53-independent pathway. This result is in agreement with previous studies that employed similar
doses of doxorubicin (66), but contrasts with other studies which used higher concentrations of
doxorubicin and/or for longer amount of time, leading to the discovery that apoptosis occurs
18
through a p53-dependent pathway (50, 12). Accordingly, we think that the role of p53 in
doxorubicin induced apoptosis in cardiac muscle cells may vary depending on the concentration and
duration of treatment.
We also found that doxorubicin 0.1 μmol/L induces a senescent phenotype, accompanied by an
increase in the p53 levels that accumulate in the cytoplasm. Many studies have demonstrated that
cytoplasmic p53 may contribute to apoptosis through transcription-independent mechanisms by
amplifying the transcription-dependent apoptotic signals triggered by nuclear p53 (50). However,
there are also studies that suggest a protective role for cytoplasmic p53. In agreement with the
above mentioned study by Nishio et al, Qu et al also demonstrated that p53 cytoplasmic
accumulation induced by endoplasmic reticulum stress prevents p53 stabilization and p53-mediated
apoptosis upon DNA damage (46).
Nithipongvanitch et al found nuclear, cytoplasmic and mitochondrial accumulation of p53
following acute doxorubicin treatment, and demonstrated that the loss of mitochondrial p53 renders
mitochondrial DNA more susceptible to oxidative stress, thus suggesting that mitochondrial p53
accumulation may participate in mithocondrial DNA repair as a rapid adaptive response to
oxidative stress in cardiomyocytes (43).
Our data show that p53 plays an active, protective role. In fact, if the incubation of low prosenescent doses of doxorubicin was preceded by pre-treatment with PFT, the p53 inhibitor, then
cells underwent apoptosis rather than showing a senescence phenotype. This finding is in agreement
with a study by Elmore et al. who found that breast tumor cells which are acutely exposed to
doxorubicin exhibit an increase in p53 activity and a dramatic increase in beta-galactosidase. They
also found that inactivation of wild-type p53 results in a transition of the cellular response to
doxorubicin treatment from replicative senescence to delayed apoptosis (17).
Both literature as well as our own experimental data collectively suggest that the following
mechanisms may be involved in doxorubicin-induced senescence. Doxorubicin induces TRF2
down-regulation which causes telomere uncapping and ultimately cell death. At low doses,
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doxorubicin also induces TRF1 knockdown. While TRF1 inhibition does not play a crucial role in
unstressed cells, it is of great importance in cells treated with low doses of doxorubicin because it
prevents premature mitotic entry and apoptosis. p53 plays a key role in down-regulating TRF1 in
cells treated with low doses of doxorubicin. If p53 activity is inhibited by PFT, the exposure to low
doses of doxorubicin produces TRF1 over-expression, thus the cellular response program to
doxorubicin shifts from senescence to apoptosis. How p53 regulates TRF1 expression is an open
question. We hypothesize that p53 may function in the cytoplasm by relaying a negative regulator
of TRF1 transcription.
The present study shows that p53 and MAPK activation is a doxorubicin-induced signaling which
regulates TRF1 and TRF2 expression: p38 mediates the effects on TRF2, while JNK regulates
TRF1 through p53 activation at low doses of doxorubicin, and in a p53-independent manner at high
doses of the drug. Previous studies showed that p38 and p53 are senescence-executing molecules
that are activated by telomere dysfunction (23, 25). We do not believe that the previous
observations conflict with our present results since the point of MAPK and p53 activation can be
either upstream or downstream from telomere dysfunction. This is in agreement with the study by
Iwasa H et al., who found that p38 is activated by H2O2 and that p38 activation persists after H2O2
removal (23). It may be argued that doxorubicin directly induces MAPK and p53 activation, which
determines telomere dysfunction, which in turn increases their activation.
Cells with doxorubicin-induced senescent like phenotype may die of mitotic catastrophe
In an attempt to better understand the mechanism involved in doxorubicin-induced late cardiac
toxicity, we chose to expose cells to a pulsed, short term, incubation with doxorubicin because it
has been shown that prolonged treatment is lethal, even if the doses of the drug are low (18). We
observed that while 1 μmol/L doxorubicin led to rapid death through apoptosis, 0.1 μmol/L
doxorubicin induced a senescent-like phenotype and caused death through mitotic catastrophe of a
lower number of treated cells. In addition, we found that a relevant number of surviving myocytes
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had a senescent phenotype which was documented 21 days after treatment by the increase of cell
volume, by the presence of SA-b-gal activity, and by the down-regulation of TRF2 and TRF1
expression.
Mitotic catastrophe is a slower than apoptosis mode of death that results from DNA damage
coupled with altered functions of the various checkpoint mechanisms whose role is to arrest
progression into mitosis until repair (8). In the present study we observed the typical biochemical
and morphological features of mitotic catastrophe which include: the presence of multinucleated
cells with micronuclei and/or nuclei of unequal size (likely arising through abnormal mitosis),
anaphase bridges, changes in DNA content (>4N), evidence of chromosomal abnormalities (18, 30,
64). In agreement with Castedo et al., who found that the doxorubicin-induced inhibition of Chk2
facilitates the induction of mitotic catastrophe, we observed a down-regulation of the Chk2, a cell
cycle check point which governs the order and timing of cell cycle transition to ensure completion
of one cellular event prior to the commencement of another, and therefore acts as a negative
regulator of mitotic catastrophe (9).
It is well-known that polyploidy and chromosomal aberrations are the consequence of TRF2 downregulation. In fact, TRF2 down-regulation determines the molecular disassembly of the telomere
complex and causes the deprotection of chromosome ends which then become the substrate of
repair activities resulting in covalent fusion of two chromosomes and the appearance of anaphase
bridges which must be torn away to allow the mitosis to proceed. As a result, each daughter cell
will inherit a chromosome with a double strand break at one end that will be fused to another
uncapped end, perpetuating a cycle of breakage-fusion-bridge that may lead to cell death (10). If
cells do not escape from this cycle, cell death may occur even much later on and several mitoses
after the application of the stress (33).
Do telomere dysfunction and senescence play a role in doxorubicin-induced heart failure
cardiomyopathy?
21
Until a few years ago the prevailing belief was that the heart is a terminally differentiated organ
whose response to pathological loads or cell death can only be accomplished by hypertrophy of
differentiated cells (46).
In the past few years a number of studies have demonstrated that the heart contains a pool of stem
cells that are able to create new myocytes (5, 44, 37).
More recently, it has been shown that the heart is characterized by a heterogeneous population of
myocytes, and that small, immature, dividing myocytes can be found together with the old,
hypertrophied, non-replicating ones (10). There is currently increasing evidence of a slow, on-going
turnover of cardiomyocytes in the normal heart involving the death of cardiomyocytes and the
generation of new cardiomyocytes (19, 58, 7). A number of studies have demonstrated that the
heart contains a pool of resident progenitor cells (37), and a population of small, immature,
dividing, p16INK4a negative myocytes with long telomeres (48) that have been considered the link
between progenitor cells and more differentiated, non replicating cardiomyocytes. Conceptually,
heart failure may now be considered a myocyte-deficiency disease in which pump dysfunction
supervenes when cell loss is not compensated by hypertrophy of the existing myocytes and
regeneration of new myocytes. It must be kept in mind that we are already well aware of a model in
which the impaired function of regenerative cells contributes to myocardial dysfunction i.e., aging.
This is a process in which the impaired function of progenitor cells significantly contributes to the
dysregulation of endogenous repair mechanisms and explains the increased severity of
cardiovascular pathophysiology that is observed in the geriatric population (19, 57, 47, 15).
Neonatal rat cardiomyocytes share some characteristics with the population of small, immature,
replicating cells that are present in the adult heart, and which are reminiscent of a fetal/neonatal
phenotype (10). Thus, our experimental model may be considered a convenient indicator of what
might happen to these young and cardioregenerative cells when the heart is exposed to doxorubicin.
Current research in the field of cardioregenerative medicine is mainly focused on ways to stimulate
22
and strengthen the regenerative capacity of progenitor cells in response to stress. Future research
should also look into the possibility of protecting progenitor cells themselves from dangerous stress.
23
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32
FIGURE LEGENDS
Figure 1
Low dose doxorubicin induces non apoptotic cardiac damage. A, Representative photographs of
AnnexinV/propidium iodide staining of neonatal rat cardiomyocytes treated with two different
doses of doxorubicin. B, Percentage of AnnexinV(A) and/or propidium iodide (PI) positive cells at
the indicated doses and time points. (magnification, x200). C, mRNA Bax and Bcl2 expression six
hours after treatment with 0.1 or 1 μmol/L doxorubicin. Histograms illustrate the mRNA Bax/Bcl2
expression ratio. *:P<0.05 vs ct. ct=control, 0.1=0.1μmol/L doxorubicin, 1=1 μmol/L doxorubicin.
Figure 2
A, Quantitation of SA-b-gal activity in cardiomyocytes treated with different doses of doxorubicin.
Representative pictures are shown (magnification, x200). B, Representative pictures illustrating the
morphological changes and SA-b-gal activity in cardiomyocytes treated with 0.1 and 1 μmol/L
doxorubicin. Cells were counterstained with Giemsa solution (magnification, x400). C,
Representative picture illustrating anaphase bridges in cardiomyocytes treated with 0.1 μmol/L
doxorubicin and stained with DAPI (magnification, x1000). D, Bar graph showing cell viability
determined by trypan blue exclusion analysis after exposure to 0.1 ,1 and 10 μmol doxorubicin. E,
Representative pictures illustrating phalloidin staining for F-actin filaments in cardiomyocytes
treated with 0.1 and 1 μmol/L doxorubicin (magnification, x1000). Bar graphs showing changes in
F-actin density and cell size in cardiomyocytes treated with 0.1 or 1 μmol/L doxorubicin. F,
Western blot analysis showing the levels of actin and alfa tubulin in cells incubated with 0.1 μmol/L
doxorubicin *:P<0.05 vs ct. *: P<0.05 vs ct; ^: P< 0.05 vs day 0; §: P< 0.05 vs day 1; ‡: P< 0.05 vs
vs day 2; #: P< 0.05vs day 3. ct=control. 10, 1, 0.1, 0.05, 0.01=10, 1, 0.1, 0.05 and 0.01μmol/L
doxorubicin, respectively.
33
Figure 3
A, Changes in DNA content following treatment with 0.1 or 1 μmol/L doxorubicin. Cells were
cultured for 24h after treatment, followed by ethanol permeabilization and propidium staining for
cell cycle analysis. B, mRNA Chk2 expression evaluated 6 h after doxorubicin treatment. C,
Metaphase spread in cardiomyocytes analyzed 24 hours after treatment with 0.1 μmol/L
doxorubicin. Giemsa-staining shows chromosomal abnormalities and endoreduplications
(magnification, x1000). ct=control, 0.1=0.1μmol/L doxorubicin, 1=1 μmol/L doxorubicin *P<0.05
vs ct; § P< 0.05 vs 1μmol/L.
Figure 4
Dose dependent effects of doxorubicin on the expression of TRF1 and TRF2 as analyzed by A,
Western blot; B, RT-PCR analysis; and C, immunofluorescence (magnification, x200).
Immunofluorescence and Western blot analysis were performed 24 hours after treatment. RT-PCR
analysis was performed 6 hours after treatment.. ct=control, 0.1=0.1μmol/L doxorubicin, 1=1
μmol/L doxorubicin *:P<0.05 vs ct.
Figure 5
Effects of the silencing of the TRF1 and TRF2 genes using specific siRNAs. Cardiomyocytes were
transfected with 100, 50, 10 nmol/l siRNA-targeting TRF2, or with 100nmol/l of siRNA-targeting
TRF1. Western blot analysis at 72 hours of TRF2, A, and TRF1 B, expression levels. Cells not
transfected (No Trfx) were used as the endogenous control for transfection. C, representative
pictures illustrating from left to right, Sa-b-gal activity (magnification, x200), Sa-b-gal
activity/counterstaining with Giemsa (magnification, x400), and AnnexinV(A) and/or propidium
iodide (PI) staining (magnification, x200) in cells analyzed after 48 hours of treatment. Bar graph
34
showing D, percertage of Sa-b-gal positive cells, and E, Percentage of AnnexinV(A) and/or
propidium iodide (PI) positive cells. *: P<0.05 vs ct.
Figure 6
A, The effects of exposure to various doses of doxorubicin on MAPKs. Total cell lysates were
analyzed after 20 minutes of exposure to doxorubicin by Western blot using antibodies specific for
phosphorylated and total forms of MAPK. The bar graph shows values for phospho MAPK
normalized to the amount of total enzyme and expressed as fold increase above control value which
was set set at 100. B, C, and D. Effects of SB203580 (SB), SP600125 (SP), and PFT on TRF1 and
TRF2 protein expression levels (panel B), percentage of SA-b-gal active positive cells (panel C)
and percentage of AnnexinV(A) and/or propidium iodide (PI) positive cells (panel D). Western blot
analysis was performed 24 hours after treatment. SA-b-gal activity and A/PI staining were assessed
48 hours after treatment. ct=control, 0.1=0.1μmol/L doxorubicin, 1=1 μmol/L doxorubicin. *:
P<0.05 vs ct; §: P< 0.05 vs 0.1 μmol/L; ‡: P< 0.05 vs 1 μmol/L; #: P< 0.05 vs SB.
Figure 7
Effects of 0.1μmol/L doxorubicin on p53 protein expression levels evaluated by Western blot
(panel A) and immunofluorescence (panel B) after pre-treatment with or without SB203580 (SB) or
SP600125 (SP). Analyses were performed 24 hours after treatment. ct=control, 0.1=0.1μmol/L
doxorubicin. *:P<0.05 vs ct; §: P< 0.05 vs 0.1 μmol/L.
Figure 8
Proposed signaling pathways involved in apoptosis and senescence induced by doxorubicin in
cardiac myocytes.
Figure 9
35
Time response analysis of the effects of a single treatment of 0.1 μM doxorubicin on H9c2 cells. A,
Representative FACS dot plots of PI staining. B, Representative FACS histograms of cell size.
Grey = day 0, red = day 6, blue = day 15; green = day 21. Table shows median and coefficient of
variation of the same experiment. C, Percent of SA-b-gal positive cells. D, Western blot analysis of
TRF1 and TRF2 expression. *:P<0.05 vs day 0; §: P< 0.05 vs day 4; ‡: P< 0.05 vs day 6.
36
Table 1. PCR Primer Sequences, PCR product size and GenBankTM accession
number
PCR product size and
GenBankTMaccession
number
Primers
PCR Primer Sequences 5’-3’
forward and reverse
GAPDH
GAGGGGCCATCCACAGTCTT
TTCATTGACCTCAACTACAT
506 bp, XM221254
TRF1
ATCTGGTGTCTCCCTTACGG
GTGCAGTGATGTCCGACAAC
154 bp, RNU65656
TRF2
GGACAGCGAGACACTAAAGGC
CTGGATGACAATGTCTGCTTC
250 bp, NM031055
Bax
CCAAGAAGCTGAGCGAGTGTCTC
AGTTGCCGTCTGCAAACATGTCA
146 bp, NM017059
Bcl-2
AGCTGCACCTGACGCCCTT
CAGCCAGGAGAAATCAAACAGAGG
308 bp, NM016993
Figure 1
A
ct
1
0.1
50μ
μm
50μ
μm
50μ
μm
50μ
μm
50μ
μm
50μ
μm
24h
48h
B
50
C
A(+)/PI(-)
A(+)/PI(+)
40
ct
0.1
1
Bax
30
GAPDH
20
Bax/Bcl-2
% of cells
Bcl2
10
0
ct
0.1
24h
1
ct
0.1
48h
1
*
4
2
0
ct
0.1
1
Figure 2
% of Sa-b-gal positive
cells
A
*
40
*
20
*
50μ
μm
0
ct
10
1
0.1
0.05
50μ
μm
ct
0.01
0.1
B
10μ
μm
10μ
μm
10μ
μm
10μ
μm
ct
0.1
C
0.1
Viable cells (% control)
D
10μ
μm
0.1
^
§
‡
1250
1000
ct
750
0.1
1
10
300
1
^§
‡#
^
§
^
§
100
^
§
^
^
^ §
*§ ‡
# #
^
§ §
‡ ‡
200
0
E
0
1
2
3
4
day
50μ
μm
0.1
α tubulin
*
Area (%)
GAPDH
0
ct
400
50μ
μm
0.1
actin
50
50μ
μm
1
ct
*
100
0.1
1
*
200
*
0
ct
0.1
1
protein expression
(%)
ct
Fluorescence
intensity(pixel/area)
F
600
400
actin
αtubulin
*
200
*
0
Figure 3
A
1
Sub G
4.06
34.90
G0/G1
57.53
39.49
Chk2
S
14.27
17.06
18S
G2/M
22.02
8.74
>4N
3.71
2.38
ct
mRNA
expression(%)
ct
C
B
ct
0.1
Sub G
3.06
7.01
G0/G1
67.01
21.86
S
10.21
39.46
G2/M
18.18
20.87
>4N
2,75
14.87
1
120
*§
80
40
0
ct
0.1
0.1
0.1
ct
0.1
1
Figure 4
A
ct
0.1
B
1
TRF2
TRF1
TRF1
300
200
*
100
0
ct
C
TRF1
0.1
1
GAPDH
*
mRNA expression
(%)
protein expression
(%)
GAPDH
TRF2
ct
TRF2
0.1
1
ct
400
TRF2
TRF1
300
*
200
*
10 0
0
ct
0.1
1
0.1
50μ
μm
50μ
μm
50μ
μm
50μ
μm
50μ
μm
50μ
μm
1
Figure 5
A
B
siTRF2
10
noTrfx siCT
TRF2
50
100
noTrfx siCT siTRF1
TRF1
GAPDH
GAPDH
C
Sa-b-gal/ giemsa
A/PI
50μ
μm
10μ
μm
50μ
μm
50μ
μm
10μ
μm
50μ
μm
50μ
μm
10μ
μm
50μ
μm
50μ
μm
10μ
μm
50μ
μm
50μ
μm
10μ
μm
50μ
μm
100siTRF1
D
E
% of Sa-b-gal
positive cells
40
*
50
A(+)/PI(-)
A(+)/PI(+)
40
30
*
% of cells
10siTRF2
50siTRF2
100siTRF2
siCT
Sa-b-gal
20
10
0
30
20
10
0
siCT 100
50
siTRF2
10
100
siTRF1
siCT 100
50
siTRF2
10
100
siTRF1
Figure 6
A
B
0.1
1
10
protein expression (%)
ct
Ph-p38
Total-p38
Ph-ERK 1/2
Total-ERK 1/2
Ph-JNK 1/2
300
TRF1
TRF2
200
‡
*
§
Ph-JNK1/2
*
§
400
*
200
SB
SP PFT
Dox 1
#§
25
SP PFT
Dox 0.1
#§
§
ct
0.1
SB
1
10
SP
PFT
Dox 0.1
D
0.1
SB
0
*
0
ct
0.1
50
% of Sa-b-gal
positive cells
Ph-ERK1/2
1
C
100
% of cells 48h
MAPK activation
600
Ph-p38
§
0
ct
*§ ‡
§ §
‡
100
Total JNK 1/2
800
‡
A(+)/PI(-)
80
A(+)/PI(+)
60
40
20
0
ct
1
SB
SP PFT 0.1
Dox 1
SB
SP PFT
Dox 0.1
Figure 7
B
A
Dox 0.1
ct
0.1
SP
SB
p53
50μ
μm
protein expression (%)
GAPDH
50μ
μm
ct
*
240
§
160
Dox 0.1
*
80
0
ct
0.1
SP
SB
50μ
μm
50μ
μm
Dox 0.1
SP
SB
Dox 0.1
Figure 8
Doxorubicin 0.1
JNK
JNK and p53
inhibitors
Doxorubicin 1
p38
p38
TRF2
TRF2
p53
TRF1
SENESCENCE
APOPTOSIS
APOPTOSIS
Figure 9
A
day 0
day 15
day 6
58.52%
53.19%
24.40%
PI staining
8.72%
day 21
91.28%
41.48%
46.81%
75.60%
Cell size (FSC-H)
D
B
day
0
6
21
events
TRF2
TRF1
GAPDH
day 0
Median CV
351.95 37.34
day 6
539.61
36.83
day 15
day 21
509.33
415.99
40.28
34.86
protein expression
(%)
Cell size (FSC-H)
TRF2
160
TRF1
120
80
40
0
0
6
day
C
% of Sa-b-gal
positive cells
*
*
80
*§
ct
0,1
60
*
40
*§ ‡
20
0
0
4
6
day
21
21