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Etoposide increased the numbers of cells with sub-G1 DNA content in a concentration-dependent
manner

In our previous studies we have investigated the role of MCL-1 in DNA damage response
by using a low concentration of etoposide 17], 21]. To further our understanding of the differences in the cellular responses to treatment
with low compared to high concentrations of etoposide, mouse embryo fibroblasts (MEFs)
were treated with etoposide at different concentrations and analyzed for their apoptotic
response after 18 h. Treatment of MEFs with etoposide induced cell death, which was
verified by the appearance of sub-diploid G1 peaks in flow cytometry analysis of permeabilized
cells stained with propidium iodide. As shown in Fig. 1a, approximately 22% cells underwent apoptosis following treatment with 1.5 µM etoposide
for 18 h, compared to 60% with 15 µM and 65% with 150 µM etoposide. A representative
experiment showing the raw data from flow cytometry is shown in Additional file 1: Figure S1.

Fig. 1. Concentration-dependent etoposide-induced apoptosis. a Flow cytometry analysis of MEFs was performed to determine the percentage of cells
with DNA content below the threshold for cells in G1, following 18 h treatment with
1.5, 15 or 150 µM etoposide. Data shown are mean + SD from seven independent experiments
with three replicates each. b MEFs were treated with 1.5, 15 or 150 µM etoposide for 3, 6 or 18 h. Con indicates
extract from untreated cells. Total cell proteins were probed with anti-Caspase-3
antibody. Vinculin was used as the loading control. Figures are representative of
three independent experiments showing similar results.

We next investigated the activation of caspase-3 in response to different concentrations
of etoposide. Western blot analysis with caspase-3 antibody showed that 150 µM of
etoposide induces robust cleavage of caspase-3 within 6 h while 1.5 or 15 µM activate
caspase-3 only after 18 h (Fig. 1b). These results confirm that a low concentration of etoposide (1.5 µM) is indeed
able to induce apoptosis in MEFs following treatment for less than 24 h. Thus, these
conditions were subsequently used to compare effects of etoposide in inducing measurable
cell death, allowing us to more carefully query any differences in the molecular responses
to the various concentrations of drug.

Low or high concentrations of etoposide have different effects on transcriptional
regulation by p53

Tumor suppressor protein p53 plays an important role in DNA damage-induced apoptosis,
at least partly by acting as a transcription factor to direct the expression of apoptotic
mediators. We investigated the effect of etoposide on two representative transcriptional
targets of p53, the BH3-only pro-apoptotic protein PUMA and cell cycle inhibitor p21
^CIP1/WAF1
. Our results showed that treatment of cells with 15 µM of etoposide induced up-regulation
of PUMA protein expression (known to be directly correlated with its p53-dependent
transcription) within 1 h, which increased further at 3 and 6 h, before declining
after 18 h (Fig. 2a, upper panel). A similar increase was observed in p21
^CIP1/WAF1
mRNA levels, which showed an increase at 30 min and remained elevated even after 18 h
incubation (Fig. 2b, upper panel). When cells were treated similarly with 1.5 µM etoposide, which can
still induce substantial cell killing, no increase in the expression of either PUMA
protein or p21
^CIP1/WAF1
mRNA levels was observed over the same time periods (Fig. 2a, b, lower panels). These results suggested that the cell death observed in response
to treatment with 1.5 µM etoposide was independent of these two transcriptional events
mediated by p53.

Fig. 2. Etoposide effect on p53-mediated transcriptional events. a MEFs were treated with 15 µM of etoposide (upper panel) or 1.5 µM of etoposide (lower panel) for 1, 3, 6 or 18 h. Total cell proteins were probed with anti-PUMA antibody. Vinculin
or actin was used as the loading control. b MEFS were treated with 15 µM of etoposide (upper panel) or 1.5 µM of etoposide (lower panel) for 30 min, 1, 3, 6 or 18 h. Total RNA was isolated and reverse transcribed into
cDNA. The expression of p21
^CIP1/WAF1
was determined by RT-PCR using specific primers. ?-actin was used a loading control.
Figures are representative of three independent experiments.

PFT-? failed to rescue the cells from etoposide-induced apoptosis

To determine whether higher concentrations of etoposide may be inducing apoptosis
through the transcription-dependent actions of p53, we used the small molecule inhibitor
PFT-?. PFT-? has been shown to interfere with the expression of p53-inducible genes
12], 22], 23]. Although originally identified as a selective inhibitor of p53-induced transcription,
it is now known to have other p53 independent functions 24]. Pre-treatment of cells with 30 µM PFT-?, followed by 18 h treatment with either
1.5 or 15 or 150 µM of etoposide failed to rescue the cells from DNA damage-induced
apoptosis (Fig. 3a). In parallel experiments, cells were pre-treated with PFT-? and subsequently exposed
to UVC radiation to induce DNA damage mediated apoptosis. PFT-? similarly failed to
exert any effect on UV treatment–induced apoptosis.

Fig. 3. Effect of PFT-? on etoposide-induced apoptosis. a MEFs, pre-treated with 30 ?M PFT-? followed by 18 h treatment with 1.5, 15 or 150 µM
etoposide, were analyzed by flow cytometry to determine percentage of cells having
sub-G1 DNA content. Cells were treated in parallel with UVC and analyzed after 18 h.
Control indicates normally proliferating cells. Columns represent percentage of cells
having sub-G1 DNA content, as analyzed by flow cytometry. Data are mean + SD of three
independent experiments with three replicates each. b mRNA was extracted from untreated (0 h), 6 and 18 h treatment with 15 µM of etoposide
(black bars) or pre-treatment with PFT-? followed by etoposide (open bars). The relative expression of p21
^cip1/waf1
was determined by qRT-PCR (mean ± SD from three independent experiments). The values
of p21
^cip1/waf1
were normalized to ?-actin. c, d Parallel studies used 1.5 or 15 µM etoposide. Sub-G1 population of MEFs was measured
as in A for cells that were untreated (Control), pre-treated with 30 ?M PFT-? followed
by etoposide for 3 h (PFT-?-Etop 3 h), etoposide alone for 19 h (Etop 19 h), pre-treated
with PFT-? followed by etoposide for 19 h (PFT-?-Etop 19 h), washed after 3 h of co-treatment,
followed by further incubation for 18 h (PFT-?-Etop washed) and washed after 3 h,
and PFT-? was added back for 18 h (PFT-?-Etop + PFT-?). Columns represent percentage
of cells having sub-G1 DNA content; representative of three experiments with similar
results.

The inability of PFT-? to rescue cells treated with any concentration of etoposide
or exposure to UV radiation was intriguing. We therefore confirmed the efficacy of
PFT-? in inhibiting the transcriptional up-regulation of p21
^CIP1/WAF1
gene expression. MEFs were pre-treated with PFT-? followed by addition of 15 µM etoposide
for various time periods. PFT-? effectively inhibited the up-regulation in p21
^CIP1/WAF1
for up to 6 h (Fig. 3b), suggesting that the drug was indeed effective in suppressing p53-regulated transcription.
However, it failed to do so in cells that were treated for 18 h, a result that may
be explained based on studies showing that the half-life of PFT-? is approximately
5 h in physiological conditions 25]. We can conclude that blocking p53-dependent transcriptional events does not affect
etoposide-induced cell death.

In another experimental approach, cells were incubated with PFT-? and subsequently
treated with etoposide for 18 h, or treated with PFT-? and etoposide for 3 h only,
and the drug washed out prior to incubation in the presence or absence of PFT-? for
18 h. Cell death was assessed by measurement of sub-G1 DNA content. While no death
is detected after 3 h of etoposide treatment (not shown), 3 h treatment with either
1.5 or 15 ?M etoposide, followed by washing and further incubation, was sufficient
to induce cell death, almost as well as continual exposure to etoposide. However,
this was not affected by the presence of PFT-?, despite its ability to block p53-mediated
transcription (Fig. 3b–d). Together, these experiments support the conclusion that etoposide-induced cell
death is not due to an effect on p53-dependent transcription.

PES inhibits etoposide–induced apoptosis and cell cycle checkpoint response

Since we had shown that etoposide-induced cell death is independent of p53’s transcriptional
regulation, we tested the effect of pre-treatment with 10 µM PES. PES is a small molecule
that prevents the association of p53 to the mitochondria 13], 26]. Western blot analysis of mitochondrial extracts following etoposide treatment showed
a dramatic increase in mitochondrial p53 abundance, which was blocked in PES-treated
cells (data not shown). To directly assess the effect of PES on p53’s interaction
with a known binding partner at the mitochondria, p53 was immunoprecipitated and the
immunoprecipitate was probed for BCL-xL levels. As shown in Fig. 4a, PES disrupted the p53/BCL-xL interaction. It should be noted that PES had little
to no effect on total cellular p53 levels (see other data below). Furthermore, pre-treatment
of MEFs with PES dramatically inhibited etoposide-induced generation of sub-G1 DNA
content, particularly at earlier times (Fig. 4b). Moreover, PES was able to effectively block death of cells that were treated with
a 10–100 fold higher concentration of etoposide over a period of 18 h, as well as
cell death induced by UVC treatment (Fig. 4c). Together, this provides further evidence that the primary means by which etoposide
induces cell death is via the transcription-independent functions of p53.

Fig. 4. Effect of PES on etoposide-induced cell death. a MEFs were either untreated (Con) or treated with etoposide (Etop) for 6 h, or pre-treated
with 10 ?M of PES followed by 1.5 ?M of etoposide for 6 h. Mitochondrial extracts
were immunoprecipitated with anti-p53 antibody and probed for Bcl-xL. The first lane (IP Con IgG) represents mitochondrial extracts from untreated (Con) cells immunoprecipitated
with rabbit IgG alone Input p53 represents the starting material unbound to the Agarose
G beads, probed for the presence of p53. b MEFs were treated with either 1.5 µM etoposide alone or pre-treated with 10 ?M of
PES and followed by 1.5 µM etoposide for various times. Columns represent percentage
of cells having sub-G1 DNA content. A one-way ANOVA was carried out to compare the
treatment groups. Post hoc comparison using the Tukey HSD test indicated significant
inhibitory effect of PES treatment on the percentage of cells having sub-G1 DNA content.
Data shown are mean + SD from 3 independent experiments with three replicates each.
c Analysis of MEFs was done as in B; cells were pre-treated with 10 ?M PES followed
by 18 h treatment with 1.5, 15 or 150 µM etoposide. Cells were treated in parallel
with UVC and followed for 18 h. Statistical analysis was as in b. Data are mean + SD of three repeat analyses.

The transcription independent death pathway of p53 has been suggested to occur via
several possible pathways: p53 may act as a ‘super’ BH3-only protein and may interact
with the multi domain anti-apoptotic BCL-2 family members to liberate pro-apoptotic
members from inhibitory complexes 27]–29] or p53 can interact directly with pro-apoptotic BAK to release cytochrome C 29]. Yet another model proposed that stress-induced cytosolic p53 is sequestered by soluble
anti-apoptotic BCL-X
_L
and transcriptional activation of PUMA displaces p53, which then activates monomeric
cytosolic BAX to induce apoptosis 10]. Our results showing that activation of PUMA was not required are not consistent
with the latter possibility, and likely support a role for p53 as a BH3-only protein
at the mitochondria.

We next investigated the cell cycle status of the MEFs in the various treatment conditions.
Our results showed that in response to treatment with etoposide, MEFs undergo a DNA
damage-induced arrest at the G2/M phase of the cell cycle and apoptotic cells with
sub-G1 levels of DNA can be detected, as expected (Fig. 5a). Interestingly, while PFT-? had no effect, treatment of cells with PES overrides
the etoposide-induced DNA damage checkpoint at G2/M (Fig. 5a, b). Similar results were obtained when HeLa cells were pre-treated with PES and
followed by treatment with etoposide (data not shown). Etoposide is known to activate
checkpoint response, which delays the progression through the cell cycle. Since the
progression of cell cycle from G2 to mitosis is driven by cyclin dependent kinase
CDK1 30], it is a prime target of DNA damage response proteins for instigation of G2/M arrest.
The CDK inhibitor p21
^CIP1/WAF1
has been shown to be up-regulated at both G1/S and G2/M checkpoints and studies of
Ding et al. have reported an increase in its expression in response to etoposide treatment
in a p53-dependent manner 31]. We therefore determined whether the ability of PES to alter cell cycle effects of
etoposide is due to any effect on down-regulation of p21
^CIP1/WAF1
expression. As seen in Fig. 5c, PES treatment had no apparent effect on etoposide-induced p21
^CIP1/WAF1
transcription. Again, this finding supports the suggestion that etoposide’s effects
are mediated largely through p53’s functions at the mitochondria, unrelated to transcriptional
regulation.

Fig. 5. Effect of PES on cell cycle events. a Representative flow cytometry profiles of MEFs, with control (no treatment), treatment
with 1.5 µM etoposide for 18 h, pre-treatment with 30 ?M of PFT-? followed by 18 h
treatment with 1.5 µM etoposide or pre-treatment with 10 ?M of PES followed by 18 h
treatment with 1.5 µM etoposide. Percentage of cells having sub-G1 DNA content is
indicated. Data shown are representative of six experiments with similar results.
bColumns represent distribution of cells in G1, S and G2/M phases of cell cycle, as analyzed
by flow cytometry. Data are mean + SD of three independent experiments with three
replicates each. c MEFs were treated for various times with 15 µM of etoposide either alone or pre-treated
with 10 ?M of PES. Total RNA was isolated and reverse transcribed into cDNA. The expression
of p21
^cip1/waf1
was determined using specific primers in RT-PCR. ?-actin was used as a loading control.
Representative of three independent experiments. d MEFs were untreated (Con) or treated with 1.5 µM of etoposide for 3 or 6 h alone
or pre-treated with 10 ?M PES. Total cell proteins were probed with anti-Ser345 CHK1,
anti-CHK1, anti-phospho-Tyr 15 CDK1, anti-CDK1, Cyclin B1 antibodies, and anti-vinculin
as the loading control.

We next investigated the effect of PES treatment on CDK1/Cyclin B1 activity. Phosphorylation
of CDK1 at the inhibitory site, Tyr15, is a key event controlling the G2/M switch.
MEFs were treated with either etoposide alone or in combination with PES for 3 or
6 h and expression of phospho-Tyr15-CDK1 was investigated by immunoblotting. As shown
in Fig. 5d, an increase in phospho-Tyr15-CDK1 was observed at 3 and 6 h post treatment with
etoposide, as expected in cells blocked at G2/M. Pre-treatment with PES decreased
the etoposide-induced CDK1 phosphorylation at both 3 and 6 h. We next examined the
effect of PES on Cyclin B1 expression, which is an absolute requirement for CDK1 activity.
Similar to phospho-Tyr15-CDK1, an increase in Cyclin B1 expression was observed at
3 and 6 h post-etoposide treatment. Interestingly, the Cyclin B1 level was much lower
in the presence of PES. A decrease in phospho-Tyr15-CDK1/Cyclin B1 expression is consistent
with cells escaping the G2 checkpoint arrest that normally occurs in response to etoposide
treatment. Activation of Checkpoint kinase 1 (CHK1) blocks the entry into mitosis
by phosphorylating members of CDC25 family of phosphatases, which activate cyclin
B1-CDK1 through dephosphorylation of Thr14 and Tyr15 32], 33]. Hence, we next examined whether the inhibitory effect of PES on Tyr15-CDK1 was the
consequence of CHK1 inhibition. We probed the same membrane with anti-phospho-Ser345
CHK1 antibody. Our results showed that, as expected, CHK1 was activated in response
to etoposide treatment at 3 and 6 h. Pre-treatment with PES resulted in a marked reduction
in CHK1 activation which would eventually impede the cell cycle arrest by allowing
the cells to enter mitosis. These results highlight several possible mechanisms by
which PES treatment is overriding the etoposide-induced cell cycle arrest.

Treatment with etoposide causes G2 arrest by p53-dependent and independent pathways
and either pathway can adequately cause G2 arrest 16]. The p53-dependent pathway can exert its inhibitory effects on cell cycle progression
through either direct binding of p21
^cip1/waf1
to CDK1 34], down-regulation of CDK1/Cyclin B1 protein levels 16] or p21
^cip1/waf1
mediated prevention of inhibitory phosphorylation of P130 and P107 by CDKs, which
in turn represses the transcription of several genes required for progression through
G2/M 35]. The p53-independent pathway on the other hand is regulated by DNA damage response
kinases ATM and ATR 36]. Our results showed that in response to treatment with 1.5 µM of etoposide, the cells
arrest at G2 through the p53-independent pathway and that PES is able to bypass it
by inhibiting the phosphorylation of CHK1 on Ser 345 (Fig. 5d). These data contradict the findings of Balaburski et al. who showed that PES treatment
leads to G2/M arrest by inhibiting the activity of Anaphase promoting complex/cyclosome
and thus prevents the degradation of Cyclin B1 37]. The reason for this apparent discrepancy is not clear as Balaburski et al. used
HeLa cells in their studies and we have also confirmed that our results, first observed
in MEFs, are also observed in HeLa cells.

PES enhances deacetylation of Lys373/382 of p53

The stability and activity of p53 is regulated by post-translational modifications
38]. In particular, acetylation of lysine residues in the C-terminal regulatory domain
of p53 has been shown to correlate well with the stability and activity of p53 39]. We examined the effect of PES on the acetylation of Lys 373/382 of p53. Since MEFs
used in the study were transformed using SV40 large T antigen, the p53 is constitutively
acetylated at Lys 373/382. Interestingly when cells were treated with either PFT-?
or PES alone, a decrease in Lys 373/382 acetylation of p53 was observed (data not
shown). However, treatment of MEFs with PFT-? was unable to exert any effect on p53
acetylation when added with etoposide, while PES added in the presence of etoposide
resulted in reduced acetylation on residues 373 and 382 (Fig. 6a).

Fig. 6. Effect of drugs on p53 acetylation and complex formation. a MEFs were either left untreated (Con) or treated with etoposide (Etop) alone or in
combination with either PFT-? or PES for 3 h. Total cell extracts were prepared. The
blot was probed with anti-Lys373/382 p53 antibody or anti-p53 as a loading control.
b MEFs were either untreated (Con) or treated with etoposide (Etop) for 3 h, or pre-treated
with either 30 µM of PFT-? or 10 ?M of PES followed by 1.5 ?M of etoposide for 3 h.
Total cell extracts were immunoprecipitated with anti-p53 antibody and probed for
HDAC1 and Mdm2. The first lane (IP Con IgG) represents PES and etoposide-treated extracts
immunoprecipitated with rabbit IgG alone to indicate position of the IgG heavy chain
(IgGH). Input Mdm2 represents the starting material unbound to the Agarose G beads,
probed for the presence of Mdm2.

PES mediated deacetylation of p53 is not regulated by Mdm2-HDAC1

Mdm2 has been shown to negatively regulate acetylation of p53 38], 40]. The effect of Mdm2 on p53 acetylation was reported to result following recruitment
of a complex containing Histone deacetylase 1 (HDAC1) 41]. We therefore sought to determine whether p53 in normally proliferating MEFs was
bound to Mdm2 and whether treatment with PES would enhance recruitment of Mdm2-HDAC1
complex. We used co-immunoprecipitation studies to determine these interactions. Our
results showed that in untreated MEF cells, p53 interacts with both endogenous Mdm2
as well as HDAC1. However, pre-treatment of cells with either PFT-? or PES resulted
in an increase in Mdm2-independent p53/HDAC1 association. We did not observe an effect
on the HDAC1 association upon treatment with etoposide alone. It is noteworthy that
despite recruitment of HDAC1 to p53 in PFT-? treated MEFs, no deacetylation of p53
on Lys373/382 is observed in cells co-treated with etoposide (Fig. 6a, b) suggesting that presence of HDAC1 alone may not be sufficient for deacetylation.

p53 is transiently activated and stabilized in response to various stimuli by post-translational
modifications. These modifications include phosphorylation 42], which has been shown to interfere with the ability of Mdm2 to negatively regulate
p53 43] and acetylation, which has been shown to promote p53 stability 41]. It is well established that Mdm2 ubiquitinates p53 on lysine residues 373/382 and
hence acetylation of these residues prevents the ubiquitin mediated turnover of p53
38], 42]. Therefore, our finding that p53 is associated with Mdm2 and HDAC1 in MEFs that normally
express p53 acetylated on Lysine 373/382 residues are intriguing. One explanation
for these observations could be that the presence of another protein partner is required
in the complex for efficient deacetylation. The pre-treatment with either PFT-? or
PES, which affect p53 very differently, both caused increased recruitment of HDAC1
to p53. In this context it should be mentioned that recruitment of HDAC1 is Mdm2 independent
and while PES causes deacetylation in the presence or absence of etoposide treatment,
PFT-? deacetylates only when added alone. Etoposide and small molecule inhibitors
PFT-? and PES have distinct activities related to their effects on p53 and perhaps
the composition of the complex formed is a reflection of that. These observations
are indeed intriguing and necessitate further investigation.