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Staurosporine modulates radiosensitivity and radiation-inducedapoptosis in U937 cells
HOW-RAN GUO1, CHIA-HSIN CHEN1, SHENG-YOW HO2,3, YUAN-SOON HO4,
RONG-JANE CHEN1 & YING-JAN WANG1
1Department of Environmental and Occupational Health, and 2Institute of Basic Medical Sciences, National Cheng Kung
University, Medical College, Tainan, 3Sinlau Christian Hospital, Tainan, Taiwan, and 4Institute of Biomedical Technology,
Taipei Medical University, Taipei, Taiwan
(Received 24 July 2005; revised 20 January 2006; accepted 23 January 2006)
AbstractPurpose: The present study aims at investigating the involvement of several genes in the cell cycle distribution and apoptosisin U937 cells, a cell line lacking functional p53 protein, after combined treatment with staurosporine and irradiation.Materials and methods: Using a DNA fragmentation assay, flow cytometry and western blot analysis, the molecular basis forthe effects of staurosporine in combination with the irradiation of leukemia cells was investigated.Results: Our results indicated that combined treatment led to an increased apoptotic cell death in U937 cells, which iscorrelated with the phosphorylation of the V-Jun sarcoma virus 17 oncogene homolog (c-JUN) NH2-terminal kinase protein(JNK), the activation of caspases, the increase in B cell leukemia/lymphoma 2 (Bcl-2) associated X protein (Bax), thedecrease in Bcl xL protein (Bcl-XL) levels, the loss of mitochondria membrane potential and the release of cytochrome c.Conclusions: Abrogation of the G2 checkpoint should be an effective strategy against p53-deficient leukemia cells toirradiation-induced cell killing.
Keywords: Apoptosis, cell cycle checkpoints, chemical modifiers, leukaemias, radiotherapy
Introduction
Ionizing radiation (IR) is one of the most effective
tools in the clinical treatment of cancer. However,
the success of radiotherapy is far from assured.
Increasing the sensitivity of tumor cells to the lethal
effects of radiation has the potential to improve the
efficacy of radiotherapy (Pawlik & Keyomarsi 2004).
The induction of DNA double-strand breaks was
considered the major mechanism of IR-induced cell
death. The cellular response to DNA damage
involves a cell-cycle arrest at both the G1/S and
G2/M transitions; these checkpoints maintain viabi-
lity by preventing the replication or segregation of
damaged DNA. The G1 arrest involves the p53-
mediated induction of p21 Wild-type p53-activated
Fragment 1/Cyclin-dependent kinase inhibitor
(p21WAF1/CIP1), whereas the G2 arrest involves
the inactivation of cell cycle p34 cdc2 kinase protein
(p34cdc2 kinase) (Maity et al. 1994). Following
DNA damage, p53-deficient cells fail to arrest at G1
and accumulate at the G2/M transition.
The status of p53 is pivotal for the response of
tumor cells to IR. Irradiation of cells with wild-type
p53 gene elevates the level of cellular p53 protein
and regulates the expression of a variety of down-
stream effector genes. Mutations in the p53 gene are
involved in acquired and intrinsic treatment resis-
tance in human tumors and render tumor cells
refractory to many anticancer therapies (Kinzler &
Vogelstein 1996, Giaccia & Kastan 1998). The
radioresistance of tumor cells lacking p53 may be a
consequence of a diminished ability to undergo
apoptosis in vitro and in vivo (Lowe et al. 1994).
Thus, the use of chemical modifiers as radio-
sensitizers in combination with low-dose irradiation
may increase the therapeutic effect by overcoming a
high apoptotic threshold.
Correspondence: Dr Ying-Jan Wang, Department of Environmental and Occupational Health, National Cheng Kung University Medical College, 138
Sheng-Li Road, Tainan, Taiwan 704. Tel: 886 6 235 3535 ext. 5804. Fax: 886 6 2752484. E-mail: [email protected]
Int. J. Radiat. Biol., Vol. 82, No. 2, February 2006, pp. 97 – 109
ISSN 0955-3002 print/ISSN 1362-3095 online � 2006 Taylor & Francis
DOI: 10.1080/09553000600589149
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Depending on the stimulus that initiates apoptosis,
different caspase cascades are activated (Rocha et al.
2000). Among the different caspases, caspase-3 plays
a major role in the effector phase of apoptosis
induced by a variety of stimuli (Abu-Qare & Abou-
Donia 2001). It has been demonstrated that the
activation of caspase-3 is regulated by at least two
mechanisms: One involves a direct pathway from
caspase-8 and the other is mediated by capase-9
following the release of cytochrome c from mito-
chondria (Shimizu et al. 1999). Upstream of the
caspase cascades pathway, mitogen-activated protein
kinase (MAPK) signal transduction pathways have
been shown to play a key role in the cellular response
to extracellular stimuli (Davis 1994, Cano &
Mahadevan 1995, Herskowitz 1995, Waskiewicz &
Cooper 1995). Three major mammalian MAPK
subgroups have been identified, including extra-
cellular signal-regulated kinase (ERK), c-JUN (V-Jun
sarcoma virus 17 oncogene homolog) NH2-terminal
kinase (JNK), and p38 MAPK (Ip & Davis 1998).
Among these, the JNK pathway is involved in many
forms of stress-induced apoptosis and is the pathway
most strongly activated by stress stimuli, such as
ultraviolet (UV) and IR (Johnson et al. 1996).
Abrogation of the G2 checkpoint has been
associated with the sensitivity of tumor cells to
DNA-damaging agents (Powell et al. 1995, Bracey
et al. 1997). Staurosporine (STP) was originally
isolated from a Streptomyces species as an inhibitor
of protein kinase C (PKC) (Omura et al. 1977). STP
and its analogues have anti-tumor properties alone
and have also been shown to abrogate the G2
checkpoint and to sensitize tumor cells to DNA-
damaging agents (Gil et al. 2003). It has been
reported that the abrogation of the radiation-induced
G2 checkpoint by STP and its analogues is
associated with radiosensitivity in several tumor cell
lines, including colorectal tumor cells, fibrosarcoma
cells and ovarian carcinoma cells (Heerdt et al. 2000,
Wang et al. 2001, Zaugg et al. 2001, Playle et al.
2002). However, the molecular basis for the effects of
STP in combination with the irradiation of leukemia
cells has seldom been thoroughly studied. In this
study, we investigated the involvement of several
genes in the cell cycle distribution and apoptosis in
U937 cells, a cell line lacking functional p53 protein,
after combined treatment with STP and IR.
Materials and methods
Cell culture, drug treatment, and irradiation conditions
U937 cells, a human pre-monocytic leukemia cell
line, were obtained from the American Type Culture
Collection. U937 cells were cultured in RPMI
1640 medium (developed by Moore et al. at Roswell
Park Memorial Institute) (Life Technology, Grand
Island, NY, USA) supplemented with antibiotics
containing 100 U/ml penicillin, 100 mg/ml strepto-
mycin (Life Technology, Grand Island, NY, USA),
and 10% heat-inactivated fetal calf serum (FCS)
(HyClone, South Logan, UT, USA.), at 378C in a
5% carbon dioxide atmosphere. For exposure to
staurosporine (Sigma Chemical Co. St Louis, MO,
USA.), the reagent was added in concentrated form
to the culture medium and mixed gently. The
cultures were then incubated for the times indicated
in the figures. Irradiation was performed with 6 MV
X-rays using a linear accelerator (Digital M Meva-
tron Accelerator, Siemens Medical Systems, CA,
USA) at a dose rate of 5 Gy/min. An additional 2 cm
of tissue-equivalent bolus was placed on the top of
a plastic tissue culture flask to ensure electronic
equilibrium, and 10 cm tissue-equivalent material
was placed under the flask to obtain full back-scatter.
DNA fragmentation assay
The control and treated cells were grown in 75-T
culture flasks. After treatment, both groups of cells
were harvested, washed twice with ice-cold phosphate
buffered saline (PBS), suspended in TNE (10 mM
tris[Hydroxymethyl]aminomethane, HCl [Tris-HCl],
pH 7.6; 140 mM sodium chloride; and 1 mM
Disodium ethylenediaminetetraacetate [EDTA]),
and lysed at 378C in 4 ml of extraction buffer
(10 mM Tris-HCl, pH 8.0; 0.1 M EDTA, pH 8.0;
20 mg/ml pancreatic RNase; and 0.5% Sodium dode-
cyl sulphate [SDS]). (All chemicals are purchased
from Sigma Chemical Co. St Louis, MO, USA). After
2 h, proteinase K was added to a final concentration of
100 mg/ml, and the mixture was incubated for another
3 h at 508C. The DNA was extracted twice with equal
volumes of phenol, and once with chloroform-isoamyl
alcohol (24:1 V:V). The DNA was then precipitated
with 0.2 volumes of sodium acetate, pH 4.8, and
2.5 volumes of ethanol at 7208C overnight, then
pelleted at 13000 g for 1 h. The samples underwentelectrophoresis in a 1.5% agarose gel. The DNA wasmade visible by ethidium bromide (EtBr) staining.
Flow cytometry
1.56106 human pre-monocytic leukemia U937 cells
were suspended with ice-cold PBS and fixed in 70%
ethanol at 7208C for at least 1 h. After fixation, the
cells were washed twice, incubated in 0.5 ml of 0.5%
Triton X-100/PBS at 378C for 30 min with 1 mg/ml
of RNase A, and stained with 0.5 ml of 50 mg/ml
propidium iodide for 10 min. Fluorescence emitted
from the propidium iodide-DNA complex was
analysed at 488 nm/600 nm (excitation/emission
wavelength) by fluorescence-activated cell sorter
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(FACScan flow cytometry). The population of nuclei
in each phase of the cell cycle was determined using
established CellFIT DNA analysis software (Becton
Dickenson, San Jose, CA, USA).
Western blot analysis
Treated and untreated cells were rinsed three times
with ice-cold PBS pelleted at 8006g for 5 min, and
lysed in 500 ml of freshly prepared extraction buffer
(10 mM Tris-HCl pH 7, 140 mM sodium chloride,
3 mM magnesium chloride, 0.5% [v/v] Nonidet P-
40 [NP-40], 2 mM phenylmethylsulfonyl fluoride,
1% [w/v] aprotinin, and 5 mM dithiothreitol
[DTT]), for 20 min on ice. The extracts were
centrifuged for 30 min at 10,0006g. Proteins were
loaded at 50 mg/lane on 12% [w/v] SDS-polyacryla-
mide gel (SDS-PAGE), blotted, and probed using
specific antibodies, including p19, p21/Cip1, p27,
proliferating cell nuclear antigen (PCNA), Retino-
blastoma-binding protein (RBBP), B-cell leukemia/
lymphoma 2 (bcl-2), bax, bcl-xl, cyclin A, cyclin B,
cyclin E, cdc2, cyclin dependent kinase 2 (cdk2),
retinoblastoma (RB2), cyclin D3, cdk4, caspase 3,
caspase 8, caspase 9, poly(ADP-ribose) polymerase
(PARP), cytochrom C, JNK, phospho-JNK (Trans-
duction Laboratories, Lexington, KY), and
phospho-cdc2 (specific for phospho-Tyr 15, Cell
Signaling, Beverly, MA, USA). Caspase 3, caspase 9,
PARP, phospho- cdc2, JNK and phospho-JNK were
detected using a chemiluminescence (ECL) detec-
tion system (Amersham Life Science, Arlington
Heights, IL, USA). Others immunoreactive bands
were visualized through incubation with the colori-
genic substrates, nitro blue tetrazolium, and 5-
bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP)
(Sigma Chemical Co.) The expression of Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH)
was used as the control for equal protein loading.
Analysis of mitochondrial transmembrane potential
The change in mitochondrial transmembrane potential
was monitored by flow cytometry. Briefly, U937 cells
were collected and suspended in 1 ml PBS. Mito-
chondrial transmembrane potential was measured
directly using 40 nM 3,30- dihexyloxacarbocyanine
(DiOC6(3); Molecular Probes, Eugene, Ore., USA).
Fluorescence was measured after staining the cells for
15 min at 378C.
Statistical analysis
Data are expressed as mean+SD. Statistical
significance was determined by using the Student’s
t-test for comparison between the means. Difference
was considered significant when p5 0.05.
Results
STP enhances apoptotic cell death and abrogates the G2
arrest induced by 5 Gy IR
As shown in Figure 1A, treatment with 5 Gy IR
alone for 24 h did not induce DNA fragmentation in
U937 cells. In contrast, pretreatment with a low dose
Figure 1. Characterization of apoptosis in U937 cells treated with
low-dose of STP combined with IR. (A) Time course of DNA
fragmentation after treatment with STP 10 nM and STP 50 nM
for 24 h as the positive control was analysed by 1.8% agarose gel
electrophoresis. (B) Cells were pre-treated with 10 nM STP for 1
or 4 h before 5 Gy IR for various time periods. IR alone was
treated for 24 h. (C) Quantification of apoptotic cells treated with
10 nM STP and 5 Gy IR alone or combination for 18 h. Apoptotic
fraction was recognized as sub-G1 population of cell cycle
measured by flow cytometry. (C: control; IR: 5Gy irradiation);
*p5 0.05 vs. STP.
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of STP (10nM), 1 or 4 h before 5 Gy IR induced
DNA fragmentation at 18 and 15 h, respectively.
DNA fragmentation was observed at 24 h in the
positive control group (treated with STP 50 nM),
whereas STP 10 nM alone did not induce DNA
fragmentation in a time-dependent manner (Figure
1B). Quantitative analyses of DNA fragmentation
showed that combined treatment with STP and 5 Gy
IR induced an approximate 20% apoptotic cell death
rate, whereas, STP or 5 Gy IR alone induced less
than a 10% of apoptosis rate (Figure 1C).
Figure 2 shows the cell cycle progression in U937
cells treated with STP and 5 Gy IR. When cells were
treated with 5 Gy IR alone, 62% of the cells were in
G2/M phase. When treated with 10 nM STP and 5
Gy IR, 20% of the cells were in G2/M phase, and
21% of the cells were in sub-G1 phase, respectively.
In addition, 46% of the cells were in G1 phase of
combined treatment, significantly more than those
treated with 5 Gy IR alone (24%). These results
suggest that combined treatment can override
IR-induced G2/M arrest and increase the number
of apoptotic cells through increasing G1 phase
progression.
Expression of cell cycle regulators in U937 cells treated
with STP and IR
To examine the molecular mechanisms of the
abrogation of G2/M arrest and the increase in the
G1 phase in U937 cells treated with STP and IR, we
studied the expression of cell cycle G1/S and G2/M
regulatory proteins. Figure 3 illustrates that 5 Gy IR
alone resulted in a remarkable increase in the
accumulation of cyclin A and cyclin B as well as a
slight accumulation of cdc2. The observations
suggested that 5 Gy IR may lead to phosphorylation
of cdc2 on Thr-14 and/or Tyr-15 and thus result in a
G2/M arrest. Figure 3 shows that phosphorylation of
cdc2 on Tyr-15 could be detected in cells treated
with 5 Gy IR, whereas STP in combination with IR
inhibited the phosphorylation of cdc2 stimulated by
Figure 2. Cell cycle progression in U937 cells treated with STP
and IR. Proportion (A) and quantification (B) of cell cycle phase
was analyzed by flow cytomety. U937 cells were treated with STP,
IR or combination for 18h. #p5 0.05 vs. 5 Gy alone.
Figure 3. Effects of combined treatment with STP and IR on the
expression of cell cycle regulators. Cells were treated with 10 nM
STP, 5 Gy IR alone or combined treatment for 18 h. (A) The
expression of G2/M-relating proteins was shown. (B) The
expression of G1/S-relating proteins was shown. The level of total
GAPDH protein was used as controls for equal loading of protein
in different lanes.
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IR alone. These results indicated that STP may
abrogate the G2/M arrest induced by 5 Gy IR
through decreasing phosphorylation of cdc2 on Tyr-
15. Figure 3 also showed that combined treatment
with STP and 5 Gy IR increased G1/S regulatory
proteins such as p21, cyclin E and Rb2, as opposed
to 5 Gy IR alone. The expression of CDK4 and p27
decreased slightly when compared to 5 Gy IR alone.
These results indicated that combined treatment
could override G2/M arrest through decreasing the
expression of G2/M-related proteins such as cyclin
A, cyclin B, cdc2 and reducing phosphorylation of
cdc2 on Tyr-15, whereas an increased G1 phase
occurred through regulatory proteins such as cyclin
E, p21, and Rb2.
Cytochrome c-mediated caspases activation in U937 cells
treated with STP and IR
Figure 4A shows the western blot analysis of the pro-
caspase enzymes and their activated cleavage forms.
All the caspase-3, caspase-9, and caspase-8 cleavage
forms could be detected in U937 cells with
combined STP and IR treatment. Poly (ADP-ribose)
polymerase, PARP, has been identified as a substrate
for caspase-3. The cleavage of PARP by the activated
caspase-3 results in the formation of an 85 kDa C-
terminal fragment. Our results also showed that the
specific cleavage of PARP could be found in cells
with combined treatment (Figure 4B). To further
confirm the contribution of caspase activation in the
induction of apoptosis in cells, a selective peptide
inhibitor for activated caspase-3 was used. We found
that the pre-incubation of cells with inhibitor
Z-VAD-FMK (a broad inhibitor of caspase-3, -6, -7)
could attenuate the apoptotic cell death of combined
treatment with STP and IR (Figure 4C).
The fluorescent probe 3,30- dihexyloxacarbocy-
nine was used to measure the mitochondrial
transmembrane potential (DCm), and the release of
mitochondrial cytochrome c was detected by Wes-
tern blotting. Figure 5 shows that minor changes in
DCm and a slight release of cytochrome c could be
observed in cells treated with either STP or IR alone.
However, combined treatment caused an obvious
change in the mitochondrial potential (DCm) and in
the release of cytochrome c, when compared to the
control and STP or IR alone (Figure 5A). Mean-
while, the expression levels of the Bax proteins were
elevated in the cells with combined treatment when
compared to treatment with STP or IR alone,
whereas the expression levels of the Bcl-xL proteins
declined with combined treatment (Figure 5B).
These results suggested that cytochrome c-mediated
the caspase-9 and caspase-3 activation involved in
the induction of apoptosis in cells with combined
treatment.
Activation of JNK signaling pathway in U937 cells
treated with STP and IR
To investigate whether the JNK signaling pathway
was involved in U937 cells with combined treatment,
phosphorylated (active form) JNK protein was
detected by western blotting. Our results showed
that the activation of JNK increased slightly at
15 – 30 min when treated with 5 Gy IR and 10 nM
STP alone, whereas, a prolonged activation of JNK
could be observed from 30 – 120 min after combined
treatment (Figure 6A, 6B). We used the specific
inhibitor of JNK (SP600125) to further confirm the
involvement of the JNK signaling pathway in the
apoptosis and cell cycle regulation altered by STP
combined with 5 Gy IR. Pretreatment of the U937
cells with SP600125 resulted in an alteration of cell
cycle progression and an inhibition of apoptotic cells.
As shown in Figure 6C, when U937 cells treated
with SP600125 alone or in combination with 5 Gy
IR, the proportion of cells in G2/M phase increased
to 65.74% and 59.44%, respectively. These results
supported the theory that basal JNK plays an
important role in G2/M transition. In addition, a
diminution of apoptotic cells was observed in cells
pretreated with SP600129 in comparison with
combined treatment (4.8% vs. 20.1%). The propor-
tion of cells at G2/M phase arrest was 36.7% when
pretreated with SP600125 (Figure 6C), and was
20.75% without the pretreatment (Figure 6D).
Prolonged activation of the JNK pathway could
trigger an increased apoptosis in U937 cells treated
with STP combined with 5 Gy IR.
Discussion
G2/M arrest and apoptosis are commonly observed
in cells treated with DNA-damaging agents, includ-
ing irradiation (Miyata et al. 2001b). It has been
shown that early apoptosis could be induced in U937
cells by a high dose of irradiation, whereas G2/M
arrest was induced by a low dose of radiation.
Shinomiya et al. (2000) found that U937 cells
treated with low dose (5 Gy) IR could induce G2/
M arrest at 12 – 36 h, then cells were released from
the blockade and entered G1 phase at 36 – 48 h,
when sub-G1 fraction, namely apoptosis, become
obvious. However, after high dose (20 Gy) IR
treatment, the S and G2/M phase fractions rapidly
disappeared, and an increase of sub-G1 fraction was
detectable in 6 h. They suggested that an execution
of apoptosis after high dose (20 Gy) IR is an early
and premitotic event, whereas apoptosis following
low dose (5 Gy) IR occurs after the release of G2/M
blockage (postmitotic event) and may be executed at
G1 phase. Moreover, many studies indicated that
cancer cells with mutant or deleted p53 such as
Staurosporine modulates radiosensitivity in U937 cells 101
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U937 cells, exploit the absence of G1/S checkpoint
but accumulate in G2/M phase in response to
irradiation (Koniaras et al. 2001, Matsui et al.
2001). The G2/M arrest means the existence of
DNA damage repair prior to potential activation
of apoptosis and may be a crucial determinant of
radioresistance. Some studies implicated that
chemical compounds capable of abrogating G2/M
arrest and stimulating apoptosis are clinical avail-
able to override radioresistance (Yao et al. 1996,
Miyata et al. 2001a). According to these studies, we
combined the treatment of low-does STP with low
dose irradiations in the current study to overcome
the radioresistance and G2/M arrest induced by low
Figure 4. Caspases related mechanisms of apoptosis in U937 cells. (A) Western blot analyses of caspase- 3, -8, -9 cleavage forms in U937
cells treated with STP(10nM), IR (5 Gy) alone, or combined treatment for 18 h. Cleavage forms of caspases proteins represent its activation.
(B) The changes of PARP protein levels were detected by Western blotting. The level of total GAPDH protein was used as controls for equal
loading of protein in different lanes. (C) Apoptosis of U937 cells was assessed 18 h after combined treatment with STP (10 nM) and IR
(5 Gy), with or without preincubation with caspases inhibitors Z-VAD-FMK (40 mM), ZB4 (25 mM), Z-IETD-FMK (25 mM);
*p50.05 vs. combined treatment; #p50.05 vs. control.
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does of irradiation in p53 deleted cancer cells. A
study, that irradiated U937 cells with 5 Gy IR and
observed a prominent G2/M arrest in 12 – 36 h when
apoptosis fraction was not obvious (Shinomiya et al.
2000). Our results indicated that the combined
treatment with 10 nM STP and 5 Gy IR for 18 h
could induce remarkable apoptosis when compared
to STP or IR treatment alone (Figure 1). Meanwhile,
G2/M arrest was overridden, and the G1 phase
proportion was increased (Figure 2). In addition,
pretreatment with STP followed by 5 Gy IR for 18 h
induced more apoptotic cells compared to STP or 5
Gy IR alone (Figure 1). We believe that STP can
override the G2/M arrest induced by low dose (5 Gy)
IR and thus lead to earlier apoptosis. Our data
implicated that the balance between the extent of
DNA damage and the duration of G2/M arrest might
determine whether irradiated cells would survive or
undergo apoptosis.
STP is a PKC inhibitor and also a strong inducer
of apoptosis at doses 25 – 100 times higher than the
current study, when applied as a single agent (Bossy-
Wetzel et al. 1998). It has been hypothesized that a
strategy could be developed that would permit the
exploitation of the G2 checkpoint to obtain a
therapeutic index in the treatment of cancers lacking
a G1 checkpoint which requires functional p53. The
lack of a G1 checkpoint is common in more than
50% of cancers containing p53 mutation (Levine
1997), which is a critical component for the
induction of apoptosis in response to DNA damage
(Lowe et al. 1993a, 1993b). Using this strategy,
normal cells would arrest in the G1 after DNA
damage from irradiation or chemotherapy, whereas
Figure 5. (A) Induction of mitochondrial dysfunction in U937 cells. Cells were treated with STP (10 nM), IR (5 Gy) alone or combined
treatment for 18 h. Then, cells were incubated with 3,30- dihexyloxacarbocynine and analyzed by flow cytometry. (B) Induction of
cytochrome C release in U937 cells treated with STP, IR alone and combined treatment for 18 h. Expressions of Bax, and Bcl-xL protein
levels were detected by Western blot using specific antibodies.
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cancer cells with a defective G1 checkpoint would
progress through the S-phase and into the G2 phase.
Therefore, abrogation of the G2 checkpoint would
be more detrimental to the cancer than the normal
cells. STP and its analogs have been reported to
increase the radiosensitivity of p53-deficient cancer
cells by the abrogation of the G2 checkpoint (Yao
et al. 1996, Busby et al. 2000, Kihara et al. 2000,
Wang et al. 2001). However, the mechanisms by
which the cell cycle or other key factors override G2/
M arrest in U937 cells with combined STP and IR
treatment have seldom been completely elucidated.
It has been reported that STP inhibits cell growth
at both G1 (low concentration) and G2/M (high
concentration) phases, and/or induces apoptosis
in human cancer cells (Bossy-Wetzel et al. 1998).
Figure 6. The activation of JNK in U937 cells. (A and B) Cells were treated with 5 Gy IR, 10 nM STP alone or combined treatment for
various time period. Expression of JNK phosphorylation was detected by Western blot using specific phospho- JNK antibody. The level of
total GAPDH protein was used as controls for equal loading of protein in different lanes. (C) Cell cycle progression and apoptosis after
treated with 5 Gy IR, 10 nM STP, 25 mM JNK inhibitor SP600125 alone or combined treatment with or without pretreatment of 25 mM
SP600125. Proportion of cell cycle phase and apoptosis were measured by flow cytometry for 18 h. The proportion of cell cycle phase and
apoptosis after different treatment was indicated in (D); #p5 0.05 vs. combined treatment.
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A study on murine embryonic fibroblasts (MEF)
found that p53 and p16 functions were not essential
for STP induced G1 arrest, whereas Rb played an
important role in determining the degree of G1 arrest
observed in the first cell cycle following the exposure
to STP. In addition, MEF from mice lacking Rb
genes showed approximately a 70% reduction in the
capacity to arrest in the G1 phase following STP
treatment (Orr et al. 1998). Schnier et al. showed
that STP might promote the hypophosphorylation of
Rb leading to G1 arrest in bladder carcinoma cells
and found that levels of CDK inhibitor proteins p21
and p27 levels were increased. They concluded that
G1 arrest at the Rb dependent checkpoint may
prevent the activation of cyclin E/CDK2 through
stabilizing its interaction with inhibitor proteins p21
and p27 (Schnier et al. 1996). These studies
suggested that the G1 arrest induced by low dose
STP (less than 10 nM) may result from the
accumulation of Rb and p21, which is consistent
with our observation as shown in Figure 3B. We
suggested that Rb accumulation (Figure 3B) may also
play an important role in STP-triggered overriding of
G2/M arrest induced by 5 Gy IR. A more recent
report indicated that exposure of U937 cells ectopi-
cally-expressing Bcl-2 to the combination of 7-
hydroxystaurosporine (UCN-01) plus 2 Gy IR leads
to a reduction in cell proliferation, and that this
phenomenon appears to involve a non-apoptotic
mechanism (Cartee et al. 2002). Despite failing to
enhance apoptosis, UCN-01 treatment abrogated IR-
induced G2/M arrest, enhanced activation of CDK1,
promotion of G0/G1 arrest, and dephosphorylation
of Rb which is similar to our current study. The
expression of Bcl-2 has been shown to prevent the
induction of apoptosis by a variety of stimuli,
suggesting that Bcl-2 protein seems likely to function
as an antagonist of a central mechanism operative in
cell death (Hockenbery et al. 1993, Zhong et al. 1993,
Lin et al. 2004). These might contribute to the non-
apoptotic mechanism in Bcl-2 over-expressed U937
cells exposed to UCN-01 plus IR.
The CDK were recognized as key regulators of cell
cycle progression through their association with
regulatory subunits called cyclins (Pietenpol &
Stewart 2002, Maggiorella et al. 2003). Deregulation
of CDK activation or overexpression of cyclin D and
cyclin E has been frequently found in human cancers
(Pietenpol & Stewart 2002). p21 is a down stream
effector of p53 that mediates both G1 and G2/M
phase arrest (Harada & Ogden 2000, Ando et al.
2001). Nevertheless, there is sufficient evidence
showed that the up-regulation of p21 can be
independent of functional p53 protein (Ding et al.
2001, Sato et al. 2002). In the present study, we
found an obvious induction of p21 expression,
however, no significant change was found in the
expression of PCNA protein (Figure 3B). One study
has shown that the ovarian carcinoma cell line
SKOV-3, with a mutation of p53, is radioresistant
because of a loss of radiation-induced p21up-
regulation (Fan et al. 1998). CDKIs p27 and p21
regulate the G1/S transition of the cell cycle by
inhibiting cyclins D, E, and A. It also has been
reported that the human glioblastoma multiform
lines which were sensitive to ionizing radiation
showed a transient increase in the CDKIs p27 and
p21 within 24 h after exposure to radiation (Yao
et al. 2003). In our experiment, we observed a
marked induction of p21 and an inhibition of cyclin
A in cells with combined treatment. Meanwhile, the
G1 phase was increased. Cyclin E is essential for
progression through the G1 phase of the cell cycle
and initiates DNA replication by interacting with and
activating its catalytic partner, CDK2. The reduced
expression of p27Kip1 has been reported to correlate
with tumor progression and poor survival (Catzavelos
et al. 1997, Mori et al. 1997). Our observations imp-
licated that, when combined with 5 Gy IR, the induc-
tion of p21 by STP may result in the reduction in
radioresistance of U937 cells through overriding G2/
M arrest and enhancing early apoptosis (Figure 3B).
In addition, the kinase activity associated with Cyclin
B-cdc2 is critical for the G2/M transition, and the
activity of Cyclin B-cdc2 complex is regulated by the
positive regulator cdc25c and the two negative
regulators Wee1 and Myt1. The protein kinase
Wee1 phosphorylates cdc2 on Tyr-15, and the kinase
Myt1 phosphorylates cdc2 on Thr-14 and Tyr-15.
Both kinases are therefore capable of inactivating the
Cyclin B-cdc2 complex, leading to G2/M arrest
(O’Connell et al. 2000). Therefore, an increase in
Tyr-15 phosphorylated cdc2 represented one of the
definite markers indicating G2/M arrest. A significant
increase in the phosphorylation of cdc2 on Tyr-
15 was observed in cells treated with 5 Gy IR, which
resulted in decreased kinase activity and, therefore, a
G2/M arrest (Figure 3A). However, the phosphoryla-
tion of cdc2 on Tyr-15 was reduced through STP
combined with IR, providing the evidence of the
involvement of p21 in G1 phase arrest induced by
STP. Taken together, we suggest that STP combined
with IR enhances apoptosis and alters the cell cycle
progression through the induction of p21, the most
obvious change found in our study. Since the U937
cells used in the current study had a mutated, inactive
p53 gene, the increase in G1 by the induction of
p21 might be independent of the effect of p53 (Zaugg
et al. 2001).
Caspases are synthesized as pro-enzymes, which
are activated by the cleavage of their subunit.
Caspases-2, -8, -9, and caspase-10, termed apical
caspases, are earlier stimulated in the apoptotic
process and then activate their effector caspases
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(namely caspase-3, -6, and -7) (Hengartner 2000).
Among the apical caspases, caspase-9 is activated in
response to internal insults such as DNA damage,
whereas caspase-8 and caspase-10 are effectors of the
death receptor-mediated apoptotic signaling path-
way, which is initiated by Fas (Hengartner 2000). In
addition, interactions among the Bcl-2 family pro-
teins (Bax, Bak, Bcl-2, Bcl-xl, ect.) stimulate the
release of cytochrome c to induce apoptosis (Gross
et al. 1999, Huang & Strasser 2000). Overexpression
of Bcl-2 or Bcl-xL blocks the release of cytochrome c
from mitochondria and inhibits apoptosis (Kluck
et al. 1997, Verheij & Bartelink 2000). In U937 cells,
expression of caspase-3 precursor product appeared
to be higher than caspase-8 and caspase-9 precursor
products. Our results suggest that combined treat-
ment manipulates multiple cellular targets that
trigger different apoptotic cascades: One leads to
the release of mitochondrial cytochrome c and
activates caspase-9, while the other causes activation
of the membrane death receptors thus resulting in
the activation of caspase-8 (Figure 4). Similar results
have been observed in Jurket cells and normal T
lymphocytes, in which the activation of caspase-8 by
PUVA led to a consequent activation of caspase-9
and caspase-3 (Martelli et al. 2004).We suggest
combined treatment can result in the activation of
two apoptotic pathways, caspase-8 and caspase-9.
The levels of caspases (caspase -1, -2, -3, -6, -7, -8,
-9, and -10) expression in cancer cell lines and
neoplastic tissues were lower than those in the
control specimens. It has been demonstrated that
impaired expression of caspases is due to gene
deletion, mutation, and hypermethylation (Philchen-
kov et al. 2004). Mutation or hypermethylation of
caspase-8 gene has been reported to be associated
with no or low protein expression in some cancer cell
lines such as head and neck cancer cells and
neuroblastomas (Mandruzzato et al. 1997, Teitz
et al. 2001, van Noesel et al. 2003). A study by Teitz
et al. (2002) also implicated that gene deletion,
mutation, and methylation result in caspase-9
inactivation. They found caspase-9 was either de-
leted or translocated to another chromosome in
neuroblastoma cell lines with N-Myc gene amplifica-
tion. Although there is no sufficient evidence
showing deletion, mutation, or hypermethylation of
caspase-8 and -9 genes in U 937 cells, we found the
basal levels of procaspase-8 and -9 pretty low. STP
alone or in combination with IR could increase the
expressions and activities of procaspase-8 and -9.
STP enhanced procaspase-9 expression and activity
has been reported in neuroblastoma cell lines with
remaining caspase-9 allel(s) (Teitz et al. 2002),
which is similar to our results (Figure 4A). Further-
more, Watson et al. also indicated that STP was able
to decrease DNA methylation status in H4IIE Rat
hepatoma cells through unknown mechanism (Wat-
son et al. 2004). However, further investigation
leading to a comprehensive understanding of the
caspase activation induced by the combined treat-
ment is needed.
Mitochondria play a central role in both extrinsic
and intrinsic apoptotic pathways (Gil et al. 2003).
They play an important role in apoptosis by releasing
of cytochrome c from themselves into the cytoplasm,
leading to the activation of the caspase-cascade
system (Liu et al. 1996, Zhivotovsky et al. 1998).
Figure 5 shows that minor changes in DCm and a
slight release of cytochrome c could be observed in
cells with STP and IR alone, compared to the control.
However, combined treatment caused an obvious
change in the mitochondrial potential (DCm) and the
release of cytochrome c, when compared to the
control and STP or IR alone. The Bcl-2 family
includes both death antagonists such as Bcl-2 and
Bcl-xL, and death agonists such as Bax, Bak, and Bad
(Cory & Adams 2002). The expression levels of Bax
proteins were elevated in cells with combined
treatment when compared to STP or IR alone,
whereas the expression levels of Bcl-xL proteins
declined with combined treatment (Figure 5B).
These results suggest that combined treatment with
STP and IR caused a significant change in trans-
membrane potential and subsequently increased the
release of mitochondrial cytochrome c in U937 cells.
The mitogen-activated protein kinase (MAPK)
signal transduction pathways are conserved in
eukaryotic cells (Cano & Mahadevan 1995). The
JNK group is strongly activated by pro-inflammatory
cytokines (Ip & Davis 1998) or by extracellular stress
such as irradiation and heat shock (Enomoto et al.
2000, 2001). Previous studies have shown that
phosphorylation and activation of JNK was related
to the activation of caspase in X-ray and heat-
induced apoptosis (Enomoto et al. 2000, 2001). Our
results indicated that the prolonged activation of
JNK found in cells with combined treatment might
be involved in the changes in G2/M phase arrest
induced by 5Gy IR alone (Figure 6). Some studies
demonstrated that prolonged activation of MAPK
increased the expression of p21 protein. For exam-
ple, low-dose irradiation led to prolonged activation
of the MAPK cascade in lung cancer cells, and the
ability of radiation to increase p21 was dependent on
the MAPK cascade (Carter et al. 1998). It has been
suggested that the basal level of JNK plays an
important role during G2/M transit, and that JNK-
mediated apoptosis is cell cycle-dependent but p53-
independent (Du et al. 2004, Mingo-Sion et al.
2004). Thus, p21 may be an important target for
JNK cell cycle effects in mutant p53 cells. These
studies correlated well with our current results that
combined treatment of STP and IR could override
106 H.-R. Guo et al.
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G2/M arrest via increasing the expression of p21
protein. However, the precise roles of p21 in
modulating this complex mechanism still need to
be further investigated.
Acknowledgements
This study was supported by the National Science
Council (NSC 93-2320-B-006-031 and NSC 93-2320-
B-006-049).
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Staurosporine modulates radiosensitivity in U937 cells 109
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