Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat
cells is mediated by a p73-dependent pathway which targets the epigenetic
integrator UHRF1
Mahmoud Alhosin a,1
, Abdurazzag Abusnina a,1
, Mayada Achour a
, Tanveer Sharif a
, Christian Muller b
,
Jean Peluso b
, Thierry Chataigneau a
, Claire Lugnier a
, Vale´rie B. Schini-Kerth a
, Christian Bronner a,2
,
Guy Fuhrmann a,2,
*
a
CNRS UMR 7213 Laboratoire de Biophotonique et Pharmacologie, Faculte´ de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
b
CNRS UMR 7200 Laboratoire d’Innovation The´rapeutique, Universite´ de Strasbourg, Faculte´ de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
1. Introduction
The p73 gene produces a protein homolog to p53 with similar
functions [1]. p73 regulates the transcription of several p53 target
genes, including the apoptosis-regulating gene PUMA, a Bax
transactivator, and the cell cycle regulatory genes p21Waf1/Cip1
[2] and p16INK4A
[3]. Transcriptional activation of these genes leads
to the induction of cell-cycle arrest and/or apoptosis [2]. It is
therefore expected that many p53-responsive genes could also be
targets of p73, especially those genes which respond to DNA
damage and could initiate cell cycle arrest and/or apoptosis. This
could explain that cells lacking functional p53 have the ability to
undergo apoptosis through a p53-independent pathway when p73
is expressed [4]. Apart its p53-mimetic activity, p73 is also a major
component of specific signaling cascades, like the caspaseindependent
cell death (CICD) [5].
Under physiological conditions, the basal expression of the
p73 gene is kept extremely low and is only up-regulated in
response to cellular stress [1]. This explains that, in spite of
extensive searches, mutations of the p73 gene are rarely
detected in primary tumors [6], but aberrant hypermethylation
of the p73 promoter region and subsequent inactivation of
the p73 gene have been reported in acute lymphoblastic
leukemia (ALL) [7]. The consequences of this hypermethylation
and its maintenance are not clearly understood but they likely
target the expression of specific genes involved in the DNA
damage response. We hypothesized that UHRF1 (Ubiquitin-like,
containing PHD and RING Finger domains, 1) could be a major
Biochemical Pharmacology 79 (2010) 1251–1260
A R T I C L E I N F O
Article history:
Received 28 October 2009
Accepted 14 December 2009
Keywords:
Apoptosis
Caspase
Thymoquinone
Tumor suppressor protein p73
UHRF1
A B S T R A C T
The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully
characterized. We therefore attempted to identify the up- and down-stream events involved in the
activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and
epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed
the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute
lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation
of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment
triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the
mitochondrial membrane potential (DCm). TQ-induced apoptosis, confirmed by the presence of
hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent
changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic
down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all
involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression,
reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that
TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent
mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.
ß 2009 Elsevier Inc. All rights reserved.
* Corresponding author at: CNRS UMR 7213, Laboratoire de Biophotonique et
Pharmacologie, Faculte´ de Pharmacie, 74 route du Rhin, B.P. 60024, 67401 Illkirch,
France. Tel.: +33 3 68 85 41 33; fax: +33 3 68 85 43 13.
E-mail address: guy.fuhrmann@unistra.fr (G. Fuhrmann).
1
Co-equal first author.
2
Co-equal senior author.
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
0006-2952/$ – see front matter ß 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2009.12.015
effector of the p73 deregulation, since this nuclear protein is
known to be over-expressed in numerous cancer cell lines and
tissues [8–11].
Several studies have shown that UHRF1 participates in the
control of cell proliferation and cell cycle transition from G1 to S, by
regulating the expression of several genes, including RB1 and
p16INK4A
[12,13]. This suggests that pathological over-expression of
UHRF1, by repressing permanently the expression of specific
tumor suppressor genes, could induce disorders in the G1/S
progression and consequently promote tumor development [10].
In agreement with our hypothesis, it has been shown that the
activation of different cell cycle checkpoints during DNA damageinduced
apoptosis leads to a down-regulation of UHRF1 [9]; such
deregulation has been described to be dependent on the p53/
p21WAF1/CIP1
pathway [14]. It should be noted that reduction of
UHRF1 expression solely can suppress proliferation and induce
apoptosis of cancer cells whose p53 is inactivated [15]. This
suggests that UHRF1 functions as a component in the DNA damage
response pathways and that it plays a role in the maintenance of
genomic stability. In this point of view, accumulating evidences
have shown that UHRF1 acts as a multi-modular protein involved
in the maintenance of the chromatin status and its propagation
during cell division. Indeed, UHRF1 binds to methylated DNA and
recruits DNA methyltransferase 1 (DNMT1) and histone deacetylase
1 (HDAC1) through the SRA (SET and RING finger Associated)
domain [13,16–19]. These protein-protein interactions precede S
phase entry and could be required for cell cycle progression
[10,20]. UHRF1 therefore, ensures the crosstalk between DNA
methylation and histone modifications, promoting the maintenance
of the epigenetic code and its transmission from a mother
cell to the descent cells [21]. For these reasons it is suspected that a
down-regulation of UHRF1 in response to DNA damage has
dramatic consequences on the cell viability.
A number of studies have shown that thymoquinone (TQ) is a
potent cytotoxic and genotoxic drug over a broad range of human
cancer cells [22]. It has been suggested that TQ, as a DNA damaging
agent, is a potential reactive oxygen species (ROS) producer which
exerts its anti-cancer effects by inhibiting cell growth, arresting cell
cycle progression and inducing subsequently apoptosis [23–29].
p53-dependent [26] and p53-independent pathways [23,27] have
been evidenced to explain the cellular actions of TQ. In HCT-116
colorectal cancer cells, TQ-induced apoptosis involves an upregulation
of both p53 and p21WAF1/CIP1
expressions, concomitantly
with a down-regulation of the expression of the anti-apoptotic
protein Bcl-2 [26]. In p53-null myeloblastic leukemia HL-60 cells
[27] and in p53-null osteosarcoma MG63 cells [28], the anti-cancer
activities of TQ involve alterations of the Bax/Bcl2 ratio and caspase
activations, but the precise mechanisms remain unknown.
The aim of the present study is to determine in the p53deficient
Jurkat cell line [30] whether an activation of the p73 gene,
via a TQ-induced DNA damage, could target the anti-apoptotic
UHRF1 gene with subsequent cell cycle arrest and apoptosis. Our
results show that TQ produces intracellular ROS, promotes a DNA
damage-related cell cycle arrest and triggers apoptosis through the
activation of a p73-dependent mitochondrial and cell cycle
signaling pathway, followed by a down-regulation of UHRF1.
We hypothesize that this p73-dependent down-regulation of
UHRF1 prevents epigenetic code replication and thus hinders the
‘‘cancer signature’’ to be inherited by the daughter cancer cells.
2. Material and methods
2.1. Cell culture, treatment and transfection
The human leukemic T-cell line Jurkat (clone E6-1) was
cultured as previously described [13]. A 100 mM solution of TQ
(Sigma–Aldrich, St. Louis, MO, USA) was prepared in 100% DMSO
(DiMethylSulfOxide; Millipore S.A.S., Molsheim, France) and
appropriate working concentrations were prepared with the cell
culture medium; the final concentration of DMSO was of 0.1% in
both control and treated conditions. Transient transfections of p73
siRNA duplex (sc-36167; Santa Cruz Biotechnologies, Santa Cruz,
CA, USA), UHRF1 siRNA duplex (50
-GGUCAAUGAGUACGUC-
GAUdTdT-30
; [13]) or scramble siRNA duplex (50
-GGACUCUCG-
GAUUGUAAGAdTdT-30
; [13]) were performed with lipofectamine
2000 (Invitrogen, Eugene, OR, USA), following the manufacturer’s
recommendations. Transient transfections with pSG5 or pSG5UHRF1
plasmid were performed as previously described [31].
Experiments using a specific inhibitor of caspase-3 (Z-DEVD-FMK)
were carried out according to the manufacturer’s instructions
(Millipore S.A.S.).
2.2. Cell proliferation, viability and apoptosis assays
Cells were seeded on 6-multiwell plates at a density of 2 Â 106
cells/well, grown for 24 h and exposed to TQ at different
concentrations for different times. Cell proliferation rate was then
assessed by colorimetric assay using the CellTiter 961
AQueous
One Solution Cell Proliferation Assay (MTS), following the
manufacturer’s recommendations (Promega, Charbonnie`res-lesBains,
France). Cell viability rate was determined by cell counting
using the trypan blue exclusion method (Invitrogen). The viability
rate was obtained by dividing the number of trypan blue-negative
cells by the total number of cells. Cell apoptosis rate was assessed
by flow cytometer (BD FACSCalibur system, BD Biosciences, San
Diego, CA, USA) using the Annexin V-FITC/propidium iodide (PI)
apoptosis assay (BD Biosciences), following the manufacturer’s
recommendations. CellQuest software (BD Biosciences) was used
for the analysis of the data. At least 10,000 events were recorded,
iteratively increased if possible, and represented as dot plots.
2.3. Cell cycle phase distribution analysis and quantitation of
hypodiploid sub-G0/G1 cell population
Cells were plated in 80 cm2
culture flasks at a density of
1.5 Â 105
cells/ml, grown for 24 h and exposed to TQ at different
concentrations for different times. Cells were then prepared as
previously described [32]. Cellular DNA content was assessed by
flow cytometry in either a Guava EasyCyte Plus HP system (Guava
Technologies, Hayward, CA, USA) or a BD FACSCalibur system (BD
Biosciences).
2.4. Assessment of DNA fragmentation pattern
Genomic DNA was prepared according to the manufacturer’s
instructions (Qiagen, Courtaboeuf, France), separated by electrophoresis
on a 1% agarose gel and visualized under UV light with
ethidium bromide.
2.5. Analysis of the production of ROS metabolites
Treated and untreated cells, seeded at an initial density of
2 Â 106
cells/well in 6-well plates, were stained for 30 min at room
temperature with a 1 mmol/l dihydroethidium (DHE; Sigma
Aldrich) solution and then subjected to flow cytometric analyses
(BD FACSCalibur, BD Biosciences). 10,000 events were recorded per
experiment.
2.6. Mitochondrial membrane potential measurement
Cells grown as described above, were incubated for 15 min at
37 8C in PBS supplemented with 40 nM of [3,30
-DihexyloxacarboM.
Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–12601252
cyanine iodide] – DiOC6; Sigma Aldrich – and 1 mg/ml PI, followed
by FACS analysis (BD FACSCalibur, Becton Dickinson). At least,
5000 cells were analyzed for each sample.
2.7. Western blot analysis
Proteins from cell lysates were extracted, separated on 10–15%
SDS-polyacrylamide gels and transferred to membranes as
previously described [13,33]. Immunoblotting was performed by
using either a mouse monoclonal anti-p73 antibody (BD Biosciences
Pharmingen), a rabbit polyclonal anti-p16 antibody
(Proteogenix, Oberhausbergen, France), a rabbit polyclonal anticleaved
caspase-3 antibody (Cell Signaling Technology, Danvers,
MA, USA), a mouse monoclonal anti-UHRF1 antibody [31], a mouse
monoclonal anti-DNMT1 antibody (Stressgen Biotechnologies,
Victoria, BC Canada), a rabbit polyclonal anti-HDAC1 antibody
(USBiological, Swampscott, MA, USA), or a mouse monoclonal antibeta
actin antibody (Abcam, Paris, France), according to the
manufacturer’s instructions. Membranes were then incubated
with the appropriate horseradish peroxidase-conjugated secondary
antibody. Signals were visualized as previously described [13]
and subjected to optical densitometric (OD) quantification by
using NIH’s Image J software.
2.8. Statistical analysis
Data were presented in a bar graph form, expressed as
means Æ S.E.M. from at least three independent experiments and
statistically subjected to the one-way ANOVA test. Significance levels
were defined in accordance with the standard notation.
3. Results
3.1. TQ inhibits cell growth and induces cell cycle arrest of Jurkat cells
The Jurkat cell line was used to identify and characterize the
molecular mechanisms induced by TQ. We first analyzed the
effects of TQ on the growth parameters of this cell line. Cell
proliferation (Fig. 1A) and cell viability (Fig. 1B) following TQ
treatment, were decreased in a concentration-dependent manner.
In our experimental conditions, calculated concentrations of TQinduced
half-maximal effects on cell proliferation and viability
were respectively of 24.2 Æ 0.3 mM and 24.3 Æ 0.2 mM for 24 h of
treatment. When treated with TQ for 48 h, these values were
23.3 Æ 0.2 mM and 23.1 Æ 0.4 mM respectively (data not illustrated).
These results indicate that Jurkat cells respond to TQ within 24 h and
at the previously published concentrations [24–28].
Because cell growth is a result of the progression of the cells
through the different phases of the cell cycle, we next determined
the effects of TQ on the cell cycle distribution (Fig. 1C). Slight
modifications were already detectable at 10 mM of TQ and a
significant accumulation of the cell population in G0/G1 phase was
observed for higher concentrations. It appears therefore that TQ is
able to inhibit the growth of Jurkat cells by promoting cell cycle
arrest at the G0/G1 phase.
3.2. TQ induces apoptosis in Jurkat cells
We next investigated whether TQ could induce apoptosis in
Jurkat cells. As shown in Fig. 2A, increasing concentrations of TQ
are associated with increasing number of apoptotic cells. TQ
began to trigger apoptosis at 10 mM while at 30 mM, a major
proportion of cells was concerned. As expected, the calculated
half-maximal effect of TQ on apoptosis was 24.7 Æ 0.3 mM (24 h
treatment). For 48 h of treatment with TQ, this value was
23.2 Æ 0.3 mM (results not illustrated). Hence, these data were
consistent with those obtained from cell proliferation assays. As a
next step, cell cycle phase distribution analysis was focused on the
detection of specific G0/G1 apoptotic cells; as shown in Fig. 2B,
increasing concentrations of TQ led to increasing number of
hypodiploid sub-G0/G1 cells. In agreement with these data, the
intensity of the genomic DNA smears of the TQ-treated Jurkat cells
increased in a concentration-dependent manner (Fig. 2C). Finally
all these results demonstrate the occurrence of a p53-independent
DNA damage-related apoptosis in the p53-mutated Jurkat cells,
when exposed to TQ.
Fig. 1. Concentration-dependent effects of TQ on proliferation, cell viability and cell
cycle of Jurkat cells. Cells were exposed to TQ at the indicated concentrations and
incubated for 24 h. (A) Cell proliferation rate was assessed by colorimetry using the
MTS assay. (B) Cell viability rate was assessed by cell counting using trypan blue dye
exclusion assay. The absolute value obtained for each TQ-treated sample is
expressed in a second step as percent relative to the corresponding absolute value
obtained for the untreated sample and set at 100. (C) Cell cycle distribution was
assessed by a capillary cytometry detection assay. Cell number in G0/G1, S or G2/M
phase was determined and expressed as percent relative to the total cell number.
Values are means Æ S.E.M. of three experiments (n = 3); statistically significant: **,
p < 0.01; ***, p < 0.001 (versus the corresponding untreated group).
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–1260 1253
3.3. TQ induces the generation of ROS and the breakdown of DCm in
Jurkat cells
We suspected that TQ, like many quinone compounds (e.g.
denbinobin), can trigger apoptosis by generating ROS [34], that in
turn should induce mitochondrial membrane disruption. We
therefore determined by flow cytometry the levels of the DNA
intercalating fluorescent marker ethidium which is produced after
intracellular oxidation of DHE. As shown in Fig. 3A, cells exposed to
increasing concentrations of TQ exhibited enhanced accumulation
of intracellular ROS. When compared to untreated cells, a 77%
increase of the fluorescence emitted by the ROS-induced oxidation
of DHE was detected in cells treated with 100 mM of TQ (Fig. 3A
and B). In parallel, the effects of TQ on the mitochondrial
membrane potential status were investigated by determining
the uptake rate of DiOC6, a mitochondrial specific and voltagedependent
fluorescent dye. Fig. 4 shows that the number of cells,
emitting high fluorescence levels, decreases when 20 mM or higher
concentrations of TQ were used, indicating a dramatic drop of
DCm. These results suggest that the DNA-damaging agent TQ
induces apoptosis by producing ROS metabolites and triggering
mitochondrial membrane potential loss in the p53-mutated Jurkat
cells.
3.4. p73 is up-regulated and UHRF1 is down-regulated in TQ-treated
Jurkat cells
In order to define the nature of the cell cycle checkpoint
signaling pathway which could be activated in response to the TQinduced
DNA damage, we first examined the expression status of
p73. In the absence of TQ, the expression of p73 in Jurkat cells was
faintly detectable; however when concentrations of TQ reached
10 mM, a sharp increase in the expression of p73 was observed
after either 24 h or 48 h of treatment (Fig. 5A and B). This upregulation,
observed in a concentration-dependent manner,
concerned both a and b isoforms of p73 and was correlated with
increasing expression levels of the tumor suppressor protein
p16INK4A
(Fig. 5A). Interestingly, p73 re-expression was accompanied
by a transient over-expression of caspase-3 cleaved subunits,
only observed in cells exposed to 10 mM of TQ. At higher
concentrations of TQ however, caspase-3, became undetectable
in their active conformation (Fig. 5A).
There are several lines of evidence that UHRF1 deregulation
may impair the control of G1/S transition during cell cycle
checkpoint activation [8,9,14]. Since such activation is known to
occur during the DNA damage-related apoptosis of TQ [23–26], we
analyzed the expression levels of UHRF1 and its partners DNMT1
and HDAC1 in TQ-treated Jurkat cells. As shown in Fig. 5A,
treatment of Jurkat cells with TQ (up to 10 mM) resulted in a faint
decrease in the expression levels of UHRF1 and DNMT1. In
contrast, the expression levels of HDAC1 appeared to increase.
However at higher concentrations of TQ, all the three proteins
became undetectable. Taken together, these results also observed
after 48 h of TQ treatment (Fig. 5B), indicate that TQ-induced
apoptosis in Jurkat cells is associated with an activation of the cell
cycle checkpoint regulator p73 and a down-regulation of the
UHRF1/DNMT1/HDAC1 complex.
3.5. TQ rapidly induces apoptosis, cell cycle arrest and deregulation of
p73 and UHRF1 expressions in Jurkat cells
In order to determine more precisely the chronology of the
cellular and molecular events induced by TQ, we analyzed the
time-course effects of TQ on Jurkat cells, at a concentration
corresponding to its half-maximal activity (25 mM). As shown in
Fig. 6A and B, a notable number of cells in apoptosis appeared
within 3 h of treatment, suggesting that the cell cycle progression
is rapidly slowing down after TQ exposition. Accordingly,
significant accumulation of cells in G0/G1 phase could be
evidenced after 6 h of treatment (Fig. 6C). Interestingly, increased
levels of p73 could be detected within 3 h of TQ exposition
(Fig. 6D); this up-regulation is accompanied with a progressive
down-regulation of UHRF1, in association with the appearance of a
lower molecular weight form (Fig. 6D). Moreover the upregulation
of p73 was also correlated with a marked overexpression
of caspase-3 cleaved subunits. Thus, for the concentrations
of TQ above 10 mM that showed an absence of cleaved
caspase-3 after 24 h or 48 h (Fig. 5A and B), there was an increased
expression of the activated caspase-3 in the early hours of
treatment. These results suggest that TQ-induced apoptosis is
linked to a rapid deregulation of p73 and UHRF1 expressions in
Jurkat cells.
Fig. 2. Concentration-dependent apoptosis induced by TQ in Jurkat cells. Cells were
exposed to TQ at the indicated concentrations and incubated for 24 h. (A) Cell
apoptosis rate was assessed by capillary cytometry using the Annexin V-FITC
staining assay. The number of apoptotic cells is expressed as percent relative to the
total cell number. Values are means Æ S.E.M. of three experiments (n = 3); statistically
significant: ***, p < 0.001 (versus untreated group). (B) Hypodiploid sub-G0/G1 cell
rate was assessed by cytometry detection assay. Cell number was determined and
expressed as percent relative to the total cell number. Values are means Æ S.E.M. of
three experiments (n = 3); statistically significant: *, p < 0.05; ***, p < 0.001 (versus
untreated group). (C) Genomic DNA fragmentation was analyzed on an agarose gel, as
described in ‘‘Section 2’’. The observed patterns are representative of three different
experiments.
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–12601254
3.6. Knockdown of p73 counteracts TQ-induced UHRF1 downregulation,
cell cycle arrest and apoptosis in Jurkat cells
To determine whether p73 could act in Jurkat cells as a main
regulator of the apoptotic signaling pathway activated by TQ, we
attempted an acute depletion of p73 by transfecting TQ-treated
cells with specific siRNA against p73. As shown in Fig. 7A, p73
knockdown restored UHRF1 expression at levels similar to those
observed for untreated cells; accordingly a normalization of the
expression level ratio of the two proteins could be evidenced
(Fig. 7B). The knockdown of p73 also allowed reactivation of cell
cycle progression since the percentage of the cell populations in
G0/G1 and S phase is significantly inversed when compared to
that of TQ-treated cells transfected with scramble siRNA (Fig. 7C).
Consistently, a significant decrease in the number of apoptotic
cells could be observed (Fig. 7D). These data clearly demonstrated
that UHRF1 is down-stream of p73 in the TQ-induced apoptotic
pathway. In order to address unambiguously whether UHRF1 is
targeted by p73 and is involved in the apoptotic process, we
further determined the direct relationships between a p73
deregulation, modulations of UHRF1 expression and apoptosis
in Jurkat cells, in the absence of TQ. As shown in Fig. 8A, siRNAinduced
decrease of the basal expression levels of p73 upregulated
UHRF1 expression. Moreover UHRF1 siRNA which
induced the decrease of the basal expression levels of UHRF1
(Fig. 8B) significantly increased the number of apoptotic cells
when compared to the controls (Fig. 8C), showing that UHRF1
exhibits anti-apoptotic properties. Altogether these results
indicate that TQ triggers apoptosis in Jurkat cells through a
DNA damage response involving the activation of the tumor
suppressor protein p73, which is able to induce a down-regulation
of the expression of the anti-apoptotic protein UHRF1.
Fig. 3. Concentration-dependent ROS production induced by TQ in Jurkat cells. Cells were exposed to TQ at the indicated concentrations and incubated for 24 h. ROS
accumulation was assessed by flow cytometry after DHE incubation. (A) Shows in each histogram the levels of fluorescence. (B) Shows the overlapping fluorescence curves
obtained for untreated and TQ-treated (100 mM) cells. The data are representative of three independent experiments.
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–1260 1255
3.7. UHRF1 down-regulation in TQ-treated Jurkat cells leads to
apoptosis via a caspase-dependent mechanism
We suspected that TQ, at a concentration corresponding to its
half-maximal effects, could activate a p73-dependent caspasedependent
pathway which could then trigger the down-regulation
of UHRF1 and subsequently apoptosis. We therefore examined the
effects of a caspase-3 specific inhibitor on TQ activity in Jurkat cells.
Indeed, Z-DEVD-FMK was able to counteract the decrease of
UHRF1 expression (Fig. 9A) and the apoptotic process (Fig. 9B)
induced by 25 mM of TQ. Moreover, an acute overexpression of
UHRF1 after TQ treatment can partially rescue the cells from
apoptosis, thus mimicking the effects of a caspase-3 inhibitor
(Fig. 9C). The results suggest that the TQ-activation of the p73dependent
mitochondrial caspase-dependent pathway induces
UHRF1 down-regulation which explains, at least in part, the
observed apoptosis.
4. Discussion
It has been reported that the pro-apoptotic activity of TQ on
cancer cells occurs in p53 wild-type cells through an up-regulation
of both the tumor suppressor protein p53 and the cyclin dependent
kinase inhibitor p21Waf1/Cip1
which in turn induces G1 cell cycle
arrest and apoptosis [26]. In p53-null cells however, the molecular
mechanisms leading to TQ-induced mitochondrial reactivity are
poorly documented.
The present study demonstrates that TQ triggers apoptosis in
the p53-deficient Jurkat cell line through the production of ROS
and the activation of the cell cycle checkpoint regulator p73.
Accordingly it has been shown that ROS production can be an
efficient activator of p73 expression [35]. On the other hand,
different point mutations in the p53 gene have been evidenced in
Jurkat cells, mostly at the C-terminal basic domain [30], which is
required for the activation of the p21Waf1/Cip1
gene, for cell cycle
arrest and for apoptosis [36]. Thus, the p53-dependent pathway is
not involved in the effects of the ROS producer TQ on Jurkat cells.
Similarly to p53, the structural and functional homolog p73 can
promote cell cycle arrest and apoptosis when over-expressed [4].
Moreover, it has been reported that cellular stress signals can
induce endogenous expression of p73 in p53 null or mutant cells,
engaging a p53-independent apoptotic pathway [37,38]. Accordingly
p73 seems to act as a cellular gatekeeper by preventing the
proliferation of TQ-exposed Jurkat cells; obviously the sharp reexpression
of p73 that we observed in response to TQ, triggers G0/
Fig. 4. Concentration-dependent DCm disruption induced by TQ in Jurkat cells. Cells were exposed to TQ at the indicated concentrations and incubated for 24 h. DCm
alteration was assessed by flow cytometry using the DiOC6 staining assay. The percentage of damaged cells with depolarized mitochondrial membranes is indicated in each
histogram. The results are representative of three independent experiments.
Fig. 5. Effects of TQ on UHRF1, DNMT1, HDAC1, p73, p16 and cleaved caspase-3
expressions in Jurkat cells. Cells were exposed to TQ at the indicated concentrations
and incubated for 24 h (A) or 48 h (B). Immunoblotting analyses were performed as
described in ‘‘Section 2’’ with the corresponding antibodies. Specific bands were
detected with their expected apparent molecular weight. The data are
representative of at least three independent experiments.
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–12601256
G1 cell cycle arrest and apoptosis. This cell reactivity is likely a
down-stream effect of p73 since the knockdown of p73 in TQtreated
cells restored cell cycle progression and proliferation. It has
been observed that early increased expression of p73 in response
to cell stress is a consequence of an accumulation at the protein
level [39]. It could therefore be hypothesized that the proteasomemediated
proteolytic degradation of p73 is rapidly blocked after
TQ-treatment, leading to a stabilization of the protein.
To determine whether the cell cycle checkpoint regulator p73
possesses an operative activity, we analyzed the expression of
some of its down-stream effectors in TQ-treated Jurkat cells.
Interestingly, a transient TQ concentration-dependent up-regulation
of caspase 3 cleaved subunits has been shown. First of all, this
suggests that TQ exerts its apoptotic activity through caspasedependent
and caspase-independent pathways. p73, like p53, is
known to promote the activation of caspases [2]. However, only
p73 is involved in a caspase-independent cell death (CICD), which
protects cells from aneuploidy by inducing their death when
chromosome missegregation occurs [5]. Interestingly it has been
observed that the switch from a caspase-dependent to a caspaseindependent
pathway allows the progression from a specific cell
death state to another [40]. Jurkat cells treated with high
concentrations of TQ likely switch to a particular cell death
program which involves the p73-dependent CICD pathway.
We have previously shown in HCT116 cells, a colon cancer cell
line, that the activation of the p53-dependent p21Waf1/Cip1
pathway is able to down-regulate UHRF1 [14]. Here we show in
Jurkat cells, the most abundant known source of UHRF1 ([31];
personal laboratory observations), that a p73-dependent pathway
also regulates UHRF1. Indeed, TQ induces the activation of the cell
cycle checkpoint regulator p73, which in turn represses UHRF1
expression in the p53 mutant Jurkat cells. The knockdown of p73 in
either untreated or TQ-treated cells modulates UHRF1 expression,
indicating that UHRF1 is a down-stream effector of p73 in this cell
type. Our data also show that TQ down-regulates DNMT1, which is
not surprising considering that DNMT1 is a privileged partner of
UHRF1 [13]. Otherwise the variations of HDAC1 expression levels
after TQ treatment, at least during the first 24 h, remain to be
Fig. 6. Time-course of the effects of TQ on the number of apoptotic cells, cell cycle distribution and p73, UHRF1 and cleaved caspase-3 expressions in Jurkat cells. Cells were
exposed to 25 mM of TQ for the indicated times. (A) Shows the results of a representative apoptosis assay, as explained in the legend of Fig. 2A. Cells of the lower left quadrant
are viable; cells of the lower right quadrant are in apoptosis. The number of cells in apoptosis, expressed as percentage relative to the total cell number, is indicated. (B)
Recapitulates the percentage of cells in apoptosis. Values are means Æ S.E.M. of three experiments (n = 3); statistically significant: **, p < 0.01; ***, p < 0.001 (versus untreated
group). (C) Shows the cell cycle distribution, as explained in the legend of Fig. 1C. Values are means Æ S.E.M. of three experiments (n = 3); statistically significant: ***, p < 0.001
(versus the corresponding untreated group). (D) Shows representative immunoblotting results, as explained in the legend of Fig. 5.
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–1260 1257
elucidated. Interfering effects between direct and indirect actions
of TQ could however be invoked.
We have previously observed that UHRF1 reduction after
activation of the p53-dependent pathway is consecutive of both
a transcriptional suppression and a protein degradation enhancement
[14]; this suggests that the p73-dependent downregulation
of UHRF1 we evidenced, likely results from the same
processes. Accordingly the lower molecular weight form we
observed within few hours after TQ-treatment could be related
to increased UHRF1 degradation after caspase activation. Indeed
a specific inhibitor of caspase-3 can recover UHRF1 expression
and rescue cells from apoptosis induced by TQ. Interestingly it
has been shown that repression of UHRF1 expression after
treatment with different inducers of cell cycle checkpoints
regulators is associated with cell cycle arrest in G0/G1 and cell
proliferation inhibition [9]. Although accumulating data have
highlighted a correlation between the reduction of UHRF1
expression and cell cycle arrest or apoptosis, the molecular
mechanisms which clearly explains this correlation remains
unknown [10]. In view of the important role that the epigenetic
integrator UHRF1 plays in the maintenance of the chromatin
status, its down-regulation should necessarily lead to the loss of
genome integrity and cell death. Accordingly, we observed that
UHRF1 knockdown in untreated cells, as well as an acute
overexpression of UHRF1 in TQ-treated cells, have direct
incidences of the cell apoptosis rate. UHRF1 appears therefore
to have in our experimental settings anti-apoptotic properties,
in agreement with previous observations [10,15,21]. One
interesting idea therefore is that preventing the epigenetic code
to be replicated after UHRF1 deregulation leads to the activation
of an apoptotic pathway. But this hypothesis needs further
investigations.
In conclusion, this is the first report which shows that a natural
compound induces apoptosis by acting on the epigenetic integrator
UHRF1. By using a p53 mutant cell line, we have shown that TQ
Fig. 7. Effects of p73 knockdown on TQ activity in Jurkat cells. Untransfected cells
(2; TQ), scramble siRNA (80 pmol) transfected cells (3; TQ + scr. siRNA) or p73
siRNA (80 pmol) transfected cells (4; TQ + siRNA p73), seeded at a density of
2 Â 105
cells/ml were grown 18 h before exposure to 10 mM TQ and further
cultured for 54 h. As a control, untransfected cells were cultivated without TQ for
72 h (1; Control). (A) Shows representative immunoblotting results, as explained in
the legend of Fig. 5. (B) Shows UHRF1/p73 expression level ratio, determined as
described in ‘‘Section 2’’. (C) Shows the cell cycle distribution, as explained in the
legend of Fig. 1C. (D) Shows the percentage of cells in apoptosis, as explained in
Fig. 2A. Values are means Æ S.E.M. of three experiments (n = 3); statistically
significant: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus the corresponding cell
condition 1);88, p < 0.01 (versus the corresponding cell condition 3).
Fig. 8. Direct relationships between p73 knockdown, UHRF1 deregulation and
apoptosis in Jurkat cells. Cells were seeded at a density of 2 Â 105
cells/ml and
grown after different transfection treatments for 48 h. (A) and (B) show
representative immunoblotting results, as explained in the legend of Fig. 5, and
obtained with protein extracts of untransfected cells (control) or cells transfected
with 80 pmol of either a scramble siRNA, a siRNA against p73 or a siRNA against
UHRF1. (C) Shows the percentage of cells in apoptosis after the different treatments,
as explained in legend of Fig. 2A. Values are means Æ S.E.M. of three experiments
(n = 3); statistically significant: ***, p < 0.001 (versus control);888, p < 0.001 (versus
scramble siRNA).
M. Alhosin et al. / Biochemical Pharmacology 79 (2010) 1251–12601258
produces ROS metabolites and acts through a p73-dependent
mitochondrial pathway which targets UHRF1 and likely DNMT1.
This pathway could involve either a caspase-dependent or
caspase-independent activation (see graphical abstract). However
the role of CICD on UHRF1 expression and activity remains unclear
and its study is currently under investigation. Our data also
highlight a new property of TQ which could be used to prevent the
epigenetic code to be transmitted from a mother cell to the
daughter cells.
Acknowledgments
This study was supported by grants of the Ligue contre le
Cancer, Comite´ du Haut-Rhin, France. Mahmoud Alhosin and
Mayada Achour are supported by fellowships from the Syrian
Higher Education Ministry. Abdurazzag Abusnina is supported by a
fellowship from the Libyan Higher Education Ministry. Tanveer
Sharif is supported by a fellowship from the Higher Education
Commission of Pakistan. The authors would like to thank Claudine
Ebel (IGBMC, Illkirch, France) for her scientific expertise and
Emmanuelle Georgi for skilled technical assistance.
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