Oncogene (2004) 23, 9–20
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ORIGINAL PAPERS
Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via
differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein
kinase signaling pathways
Jung-A Choi1,4, Moon-Taek Park1,4, Chang-Mo Kang1, Hong-Duck Um2, Sangwoo Bae1,
Kee-Ho Lee3, Tae-Hwan Kim1, Jae-Hong Kim4, Chul-Koo Cho1, Yun-Sil Lee1, Hee Yong Chung5
and Su-Jae Lee1,*
1
Laboratory of Radiation Effect, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea; 2Laboratory of
Experimental Pathology, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea; 3Laboratory of Molecular
Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea; 4Graduate School of Biotechnology, Korea
University, Seoul 136-701, Korea; and 5Department of Microbiology, College of Medicine, Hanyang University, Seoul 133-791,
Korea
It has been well known that Ras signaling is involved in
various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we
show the opposing roles of Ha-Ras and Ki-Ras genes in
the modulation of cell sensitivity to ionizing radiation.
Overexpression of active isoform of Ha-Ras (12V-HaRas) in Rat2 cells increases resistance to the ionizing
radiation. Constitutive activation of phosphoinositide-3kinase (PI3K) and Akt is detected specifically in 12V-HaRas-overexpressing cells. The specific PI3K inhibitor
LY294002 inhibits PI3K/Akt signaling and potentiates
the radiation-induced apoptosis, suggesting that activation
of the PI3K/Akt signaling pathway is involved in the
increased radio-resistance in cells overexpressing 12VHa-Ras. Overexpression of activated Ki-Ras (12V-KiRas), on the other hand, markedly increases radiation
sensitivity. The p38 mitogen-activated protein kinase
(MAPK) activity is selectively enhanced by ionizing
radiation in cells overexpressing 12V-Ki-Ras. The specific
p38 MAPK inhibitor, PD169316, or dominant-negative
p38 MAPK decreases radiation-induced cell death. We
further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras
involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing
irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38
MAPK. In addition, introduction of cells with RacN17, a
dominant-negative mutant of Rac, resulted in a marked
inhibition of radiation-induced Bax translocation and
apoptotic cell death as well as p38 MAPK activation.
Taken together, these findings explain the opposite effects
of Ha-Ras and Ki-Ras on modulation of radiosensitivity,
*Correspondence: S-J Lee, Laboratory of Radiation Effect, Korea
Institute of Radiological and Medical Sciences, Gongneung-Dong,
Nowon-Ku, Seoul 139-706, Korea; E-mail: [email protected]
This work was supported by the Nuclear R&D Program of the
Ministry of Science and Technology in Korea
Received 15 April 2003; revised 6 July 2003; accepted 7 July 2003
and suggest that differential activation of PI3K/Akt and
Rac/p38 MAPK signaling by Ha-Ras and Ki-Ras may
account for the opposing response to the ionizing
radiation. These data provide an explanation for the
diverse biological functions of Ras isozymes, and partly
accounts for the differential response of transformed cells
to anticancer treatments.
Oncogene (2004) 23, 9–20. doi:10.1038/sj.onc.1206982
Keywords: 12V-Ki-Ras; 12V-Ha-Ras; radiation-induced
apoptosis;
PI3K/Akt;
Rac/p38
MAPK;
Bax
translocation
Introduction
Signaling from the small GTPase, Ras, has been under
intense investigation in the past decade due to the
importance of the Ras as a switch mediating pathways
that control transcriptional activation of genes governing such critical and diverse cellular functions as
proliferation, development, differentiation, and apoptosis (Ayllon and Rebollo, 2000; Crespo and Leon, 2000;
Kolch, 2000; Brooks et al., 2001; Cheng and Meinkoth,
2001; Fedorov et al., 2001; Pervaiz et al., 2001; Radhika
and Dhanasekaran, 2001). It is not surprising, therefore,
that deregulation of Ras activity by mutation may have
such profound consequences for the cell. Mutations
conferring constitutive activation of Ras occur frequently in many types of human cancers (Bos, 1989;
Ahuja et al., 1990), and oncogenically mutated Ras can
also confer radiation resistance (FitzGerald et al., 1985;
Sklar, 1988; Ling and Endlich, 1989; McKenna et al.,
1990; Hermens and Bentvelzen, 1992; Miller et al., 1993;
Pirollo et al., 1993; Bernhard et al., 1996; Bernhard et al.,
2000). Evidence for Ras-mediated radioresistance has
been reported in both rodent and human tumor cell lines
including NIH3T3 fibroblasts, REF fibroblasts, DLD-1
colon carcinoma, and HT1080 fibrosarcoma. Recent
studies demonstrated that the phosphoinositide-3-kinase
(PI3K) pathway plays a critical role in mediating
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
10
enhanced radioresistance by the Ras oncogene (Rosenzweig et al., 1997; Hosoi et al., 1998; Gupta et al., 2001;
Grana et al., 2002). The studies showed that the PI3K
inhibitor wortmannin or LY294002 sensitized cells
bearing a constitutively active Ras mutant to ionizing
radiation. Inhibition of the PI3K downstream target
p70S6K by rapamycin, of the Raf–MEK–MAPK
pathway (MEK, mitogen-activated protein kinase
kinase; MAPK, mitogen-activated protein kinase) by
PD98059, or of the Ras–MEK kinase–p38 pathway
by SB203580 had no effect on radiation survival
in cells with oncogenic Ras (Gupta et al., 2001)
(http://mct.aacrjournals.org/cgi/content/full/2/4/-R16).
Furthermore, the expression of active PI3K in cells with
wild-type Ras resulted in increased radiation resistance
that could be inhibited by LY294002 (Gupta et al.,
2001). However, overexpression of Ras or the presence
of activated Ras does not increase radioresistance in all
contexts (Harris et al., 1990; Alapetite et al., 1991).
According to some reports, introduction of a
mutated Ras did not increase radioresistance in
human retinoblasts, fibroblasts, and keratinocytes
(Gupta et al., 2000). Moreover, in some circumstances,
oncogenic Ras has even been reported to increase
radiation sensitivity (Harris et al., 1990; Alapetite et al.,
1991).
Although a number of studies demonstrate that
mutated Ras can clearly modulate the survival and
radiation sensitivity, the signaling pathways by which
this is accomplished are not fully understood and are
likely to be cell type dependent. The fact that oncogenic
transformation and radiosensitivity modulation can be
uncoupled is shown by the inability of the transforming
oncogenes Myc and Abl to confer radioresistance
(Pirollo et al., 1993). Therefore, although several
signaling pathways downstream of Ras have been fairly
well defined, and their contribution to Ras-mediated
transformation has been well studied, it remains unclear
as to which of these pathways also contribute to Rasmediated radiosensitivity modulation.
The three major isoforms of this small GTPase
protein are Ha-Ras, Ki-Ras, and N-Ras. They share a
high degree of sequence homology. The only region that
exhibits significant sequence divergence is the final 24
residues of the protein, the hypervariable domain at the
COOH termini. Although they are almost identical,
distinct cellular functions for the Ras isoforms have
started to accumulate. For example, oncogenic Ki-Ras
but not oncogenic Ha-Ras blocks integrin b1-chain
maturation in colon epithelial cells (Yan et al., 1997).
More compelling observations on the functional distinctiveness of Ras isoforms came from experiments
with transgenic animals showing that only Ki-Ras is
essential for normal mouse development. Ha-Ras or
N-Ras knockouts showed no distinct phenotypes,
whereas a Ki-Ras knockout was embryonic lethal at
12–14 days of gestation (Joneson and Bar-Sagi, 1997;
Prior and Hancock, 2001).
Furthermore, opposing roles of Ki-Ras and Ha-Ras
genes in the regulation of redox signals has been
observed (Santillo et al., 2001; Cuda et al., 2002);
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Ha-Ras protein increases the number of cells undergoing apoptosis after oxidative stress, conversely the KiRas has a protective effect.
Distinct biological functions of Ras isoforms are
exerted through the selective activation of downstream
effectors (Joneson and Bar-Sagi, 1997; Campbell et al.,
1998), such as the serine/threonine kinases Raf, the p110
catalytic subunit of PI3K, and Ral-GDS, the exchange
factor for Ral GTPase. Raf proteins initiate a kinase
cascade that leads to MEK and extracellular-regulated
kinase (ERK) activation, which can influence various
cytosolic events and gene transcription in the nucleus
(Hagemann and Rapp, 1999). Raf/ERK signaling has
been shown to suppress apoptosis in many experimental
cell systems, although in some systems the Raf/ERK
activity can enhance apoptosis (Lander et al., 1996;
Pennisi, 1997; Cuda et al., 2002). PI3K that consistently
prevents apoptosis in many cell systems, generate PIP3,
which promotes a kinase cascade involving Akt, and the
substrates of Akt include proteins that modulate the
apoptotic signaling pathway (Datta et al., 1999). RalGDS can activate CDC42 and Rac, which activate
various MEK kinases that are involved in the regulation
of the stress-activated protein kinase/c-Jun N-terminal
kinase (SAPK/JNK) and p38 MAPK pathways (Johnson, 1999). SAPK/JNK and p38 MAPK are most
frequently associated with the induction of apoptosis
(Chuang et al., 2000; Stadheim et al., 2000; Kunz et al.,
2001). In addition, it has been reported that SAPK/JNK
and p38 MAPK act at an early step prior to dysfunction
of mitochondria and caspase activation in several cell
types. For example, the stimulation of p38 MAPK is an
early regulatory event for the cadmium-induced apoptosis in human promonocytic cells (Galan et al., 2000;
Cheng et al., 2001).
Ras proteins activate their effectors by direct interaction. Despite the fact that the amino-acid sequences are
almost identical, recent studies have demonstrated that
the Ras isoforms can differentially activate PI3K and
Raf. For example, H-Ras is a more potent activator of
PI3K than K-Ras (Yan et al., 1998), whereas K-Ras
recruits Raf-1 to the cell membrane more efficiently than
H-Ras (Yan et al., 1998; Voice et al., 1999). In addition,
K-Ras is a more potent activator of the Rac pathway
than H-Ras (Walsh and Bar-Sagi, 2001). These hierarchies appear to result, at least in part, from differences
in the mechanisms of membrane attachment of the Ras
isoforms (Yan et al., 1998; Walsh and Bar-Sagi, 2001).
We investigated distinct signaling pathways downstream of Ras isozymes in the modulation of cellular
response to the ionizing radiation. The Ha-Ras protein
increases resistance to the ionizing radiation by activation of the PI3K/Akt signaling pathway. Ki-Ras, on the
other hand, decreases the radiation resistance by
enhancing Bax translocation via activation of a p38
MAPK- dependent pathway. These findings link Ras
activity to the modulation of cell sensitivity to ionizing
radiation, providing an explanation for the divergent
biological activities of Ras isozymes, and may account
for the differential response of transformed cells to
anticancer agents.
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
11
Results
Differential modulation of radiosensitivity by activated
Ki-Ras and Ha-Ras
Rat2 cells were stably transfected with activated Ha-Ras
(12V-Ha-Ras) or Ki-Ras (12V-Ki-Ras) to determine
their role in the response to ionizing radiation. The
expression of mutant forms of Ki-Ras and Ha-Ras was
confirmed by Western blot analysis as shown in
Figure 1a. The cells were irradiated with the indicated
doses of ionizing radiation and clonogenic survival
assays were performed. 12V-Ha-Ras-overexpressing
cells showed increased clonogenic survival following
radiation compared to the parental cells. Surprisingly,
however, the clonogenic survival curves for cells overexpressing 12V-Ki-Ras indicated that they were much
more sensitive to radiation than the parental cells
(Figure 1b).
We next investigated whether the differential clonogenic survival by activated Ha-Ras and Ki-Ras was
associated with the differential regulation of apoptosis.
To examine radiation-induced apoptosis, cultures were
treated with 4 Gy radiation, and were stained with
Figure 2 Differential modulation of radiation-induced apoptosis
by overexpression of 12V-Ha-Ras and 12V-Ki-Ras. Parental Rat2,
and 12V-Ha-Ras-, or 12V-Ki-Ras-overexpressing Rat2 cells were
treated with 4 Gy of g-radiation. (a) Cells were stained with
Hoechst 33258 and apoptotic cells were scored at 24 and 48 h after
irradiation. The data represent averages of three independent
experiments with standard deviations. (b) Sandwich enzyme assay
for quantitative determination of cytoplasmic histone-associated
DNA fragments was performed using a Cell Death Detection
ELISA Kit to detect the degree of apoptosis 48 h after irradiation.
The data represent averages of three independent experiments with
standard deviations. (c) After 48 h, DNAs were extracted and
electrophoresed on 1.5% agarose gel and visualized by UV light
after ethidium bromide staining. The Rat2-Ki-Ras lane shows a
more extensive DNA ladder following irradiation, indicating more
DNA cleavage than the other lanes. A representative of three
independent experiments is shown
Figure 1 Differential modulation of clonogenic survival after
radiation by activated Ha-Ras (12V-Ha-Ras) and Ki-Ras (12V-KiRas). Exponentially growing Rat2 cells were stably transfected with
activated-Ha-Ras (12V-Ha-Ras) or -Ki-Ras (12V-Ki-Ras). (a)
Expression levels of 12V-Ha-Ras and 12V-Ki-Ras were determined
by Western blot analysis using antibody specific for Ha-Ras or KiRas, respectively. (b) The Rat2 cells expressing activated forms of
Ha-Ras or Ki-Ras were plated and treated with indicated doses of
g-radiation. After 10–15 days, the plates were stained with trypan
blue and scored for colony formation. The data represent average
values of triplicate experiments with standard deviations
Hoechst 33258 at 24 and 48 h and examined microscopically. Figure 2a shows that the number of
apoptotic cells induced by radiation in Ha-RasV12expressing cells was less than 15%, but a dramatic
increase in the number of apoptotic cells was observed in
12V-Ki-Ras-overexpressing cells (up to 40%), compared
to the parental cells (less than 25%). Using a sandwich
enzyme immunoassay, quantitative analysis of cytoplasmic histone-associated DNA fragments was also performed, and the results showed that a marked increase
in apoptosis was observed in cells expressing 12VKi-Ras (Figure 2b). To confirm these results further, the
DNAs from cells expressing 12V-Ki-Ras and 12V-HaRas were analysed for ladder formation after 48 h of
4 Gy irradiation. As shown in Figure 2c, the DNA from
12V-Ki-Ras-expressing cells was more extensively
cleaved than the parental or 12V-Ha-Ras-expressing
cells after irradiation. These results demonstrate that in
contrast to Ha-Ras, activated Ki-Ras augments radiation-induced apoptotic cell death in Rat2 fibroblasts.
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Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
12
To elucidate whether the high apoptotic index in Rat2
cells expressing 12V-Ki-Ras cells was due to overexpression of activated Ki-Ras or clonal variation, we
transiently transfected Rat2 and L929 fibroblasts with a
retroviral vector containing activated Ki-Ras
(MFGpuro-12V-Ki-Ras). The transient expression of
12V-Ki-Ras clearly repressed clonogenic survival after
radiation, and markedly enhanced radiation-induced
apoptotic cell death in both types of cells, compared to
cells transfected with the control retroviral vector (data
not shown), thereby reproducing the higher apoptotic
index found in 12V-Ki-Ras-expressing cells constitutively expressing activated Ki-Ras.
Role of PI3K/Akt pathway in the increased
radioresistance in cells overexpressing 12V-Ha-Ras
Since it has been reported that mutant Ras activates
PI3K by direct binding, which in turn activates Akt
(Joneson and Bar-Sagi, 1997; Campbell et al., 1998), we
determined the level of PI3K activity and phospho-Akt
in 12V-Ha-Ras- and 12V-Ki-Ras-overexpressing cells.
PI3K activity is high in Ha-Ras-expressing cells relative
to Ki-Ras-expressing or parental cells (Figure 3a). The
constitutive activation of Akt is also apparent only in
cells expressing 12V-Ha-Ras, but not in the parental and
12V-Ki-Ras-expressing cells (Figure 3a). The PI3K/Akt
signaling pathway has been known to enhance survival
after a variety of cellular stresses; however, the nature of
its response to ionizing radiation has not been established. To determine if the increased resistance to the
Figure 3 Activation of PI3K and Akt is involved in increased
radioresistance in cells overexpressing 12V-Ha-Ras. (a) Cell lysates in
exponentially growing parental Rat2, and 12V-Ha-Ras-, or 12V-KiRas-overexpressing Rat2 cells were prepared and immunoprecipitated
with PI3K antibody (anti-p85). Immune complexes were then
adsorbed using protein-A sepharose and subjected to a PI3K assay.
The products of the reactions were analysed by thin layer
chromatography, and visualized by autoradiogaphy. A representative
autoradiogram of PI3K assay is shown and the position of
phosphatidylinositol phosphate (PIP) is indicated (upper panel).
Rat2, and 12V-Ha-Ras- or 12V-Ki-Ras-overexpressing Rat2 cells
were prepared and phosphorylated/activated Akt were detected by
Western blot analysis with antibody specific for phosphorylated-Akt.
The data represent a typical experiment conducted three times with
similar results (bottom panel). (b) Rat2 and 12V-Ha-Ras-overexpressing Rat2 cells were treated with 4 Gy of g-radiation in the
presence or absence of LY294002 (10 mm). Cells were harvested 36 h
after irradiation and PI3K activity was analysed. The data represent a
typical experiment performed three times with similar results (upper
panel). Phosphorylated/activated Akt was detected by Western blot
analysis with an antibody specific for phosphorylated/activated Akt
(lower panel). (c) Rat2- and 12V-Ha-Ras-overexpressing Rat2 cells
were treated with 4 Gy of g-radiation in the presence or absence of
LY294002. Cells were harvested 48 h after irradiation and DNA
contents were analysed after PI staining (Cont; control, LY;
LY294002, IR; ionizing radiation). The data represent averages of
three independent experiments. Error bar, SD (upper panel). Rat2and 12V-Ha-Ras-overexpressing Rat2 cells were transiently transfected with dominant-negative Akt or control vector and were treated
with 4 Gy of g-radiation. Cells were harvested 48 h after irradiation
and cell death was analysed by PI staining. (pCMV5; control vector,
IR; ionizing radiation, DN-Akt; dominant-negative Akt). The data
represent averages of three independent experiments. Error bar, SD
(bottom panel)
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radiation in Ha-Ras transfected cells is directly correlated with PI3K signaling pathway, we examined the
effect of the PI3K inhibitor, LY294002, on Akt
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
13
phosphorylation and radiation-induced apoptosis. Cells
treated with LY294002 completely inhibited PI3K
activity and Akt phosphorylation, and decreased
resistance to the ionizing radiation in 12V-Ha-Rasexpressing cells (Figure 3b). Further evidence of the
involvement of the PI3K/Akt pathway was obtained by
expressing dominant-negative Akt in cells, resulting in
the suppression of Ha-Ras-mediated resistance to
ionizing radiation (Figure 3c). These results suggest
that the constitutive activation of the PI3K/Akt signaling pathway is involved in the increased radioresistance
in cells expressing 12V-Ha-Ras.
activity, respectively. Transfection of 12V-Ki-Ras-expressing cells with a dominant-negative mutant of p38 MAPK
markedly inhibited radiation-induced apoptosis, whereas
dominant-negative ERK had no effect on the cell death
caused by ionizing radiation. In addition, transfection of
12V-Ki-Ras-expressing cells with RacN17, a dominantnegative form of Rac1, significantly repressed p38 MAPK
activity and radiation-induced apoptosis (Figure 5d),
suggesting that Rac is positioned upstream of p38 MAPK
in the radiation-induced apoptotic signaling pathway in
Activation of p38 MAPK by ionizing radiation in cells
overexpressing 12V-Ki-Ras
The best-known Ras signaling pathways are the MAPK
cascades, which include the extracellularly regulated
ERK pathway, the stress-activated SAPK/JNK pathway, and the p38 MAPK pathway. To determine the
involvement of MAPK in the modulation of radiationinduced apoptosis by Ras, we first investigated the
activation of the MAPK signal transduction pathway in
cells expressing different isoforms of Ras. As shown in
Figure 4a, the expression of both Ras led to the
constitutive activation of MEK/ERK and JNK. Interestingly, however, p38 MAPK activity was much higher
in the 12V-Ki-Ras-expressing cells than the parental or
cells expressing 12V-Ha-Ras. Moreover, p38 MAPK
activity was markedly increased following ionizing
irradiation in 12V-Ki-Ras-overexpressing cells, but not
in Rat2 cells or 12V-Ha-Ras-overxpressing cells
(Figure 4b). In addition, radiation caused increased
phosphorylation of ERK in Ki-Ras-expressing cells, but
not JNK, suggesting selective activation of p38 MAPK
and ERK by radiation in cells overexpressing Ki-Ras.
Activation of p38 MAPK is involved in the increased
radiation-induced apoptosis in 12V-Ki-Rasoverexpressing cells
As MAPKs are regulated differentially by Ha-Ras and KiRas, we tested whether differential activation of MAPKs
influences the radiation-induced apoptosis. We pretreated
the cells with PD98059, a specific inhibitor of MEK/ERK,
or PD169316, a specific inhibitor of p38 MAPK, and
performed Hoechst 33258 staining. Pretreatment of 12VKi-Ras-expressing cells with PD169316 inhibited radiation-induced apoptotic cell death in a dose-dependent
manner; apoptosis was almost completely blocked by
30 mm PD169316 (Figure 5a). However, the MEK/ERKspecific inhibitor PD98059 did not affect radiation-induced
apoptosis of the 12V-Ki-Ras-expressing cells (Figure 5b).
To confirm further the role of p38 MAPK in enhanced
apoptosis in cells expressing 12V-Ki-Ras following radiation, we transfected cells with plasmids expressing
dominant-negative mutants of ERK or p38 MAPK and
examined the radiation-induced apoptotic cell death. As
shown in Figure 5c, transient transfection of dominantnegative ERK or p38 MAPK markedly reduced the
cellular level of phosphorylated ERK or the p38 MAPK
Figure 4 Activation of p38 MAPK by ionizing irradiation in cells
overexpressing 12V-Ki-Ras. (a) Exponentially growing parental
Rat2, and 12V-Ha-Ras- or 12V-Ki-Ras-overexpressing Rat2 cells
were harvested, and phosphorylated/activated MEK, ERK, JNK,
and p38 MAPK were assessed by Western blot analysis with
antibodies specific for phospho-MEK, -ERK, -JNK, and -p38
MAPK. The p38 MAPK activity was determined with an immune
complex kinase assay using GST-ATF-2 as a substrate. (b) Parental
Rat2, and 12V-Ha-Ras- or 12V-Ki-Ras-overexpressing Rat2 cells
were treated with 4 Gy of g-radiation. After 36 h, cells were harvested
and phosphorylated/activated ERK and JNK were assessed by
Western blot analysis with antibodies specific for phospho-ERK and
-JNK. The p38 MAPK activity was determined with an immune
complex kinase assay using GST-ATF-2 as a substrate
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Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
14
cells overexpressing 12V-Ki-Ras. Taken together, these
results suggest that the selective activation of p38 MAPK
signaling pathway by ionizing radiation is involved in the
increased radiation-induced apoptosis in the 12V-Ki-Rasexpressing cells.
Overexpression of 12V-Ki-Ras enhances ionizing
radiation-radiation-induced apoptosis through increased
intracellular redistribution of Bax, cytochrome c release,
and caspase 3 activation
To evaluate the contribution of the mitochondrial
pathway to the enhanced apoptosis seen with activated
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Ki-Ras following radiation, we examined cytochrome c
release into the cytosol of irradiated 12V-Ki-Ras- or
12V-Ha-Ras-transformed cells. Figure 6a shows that a
much higher level of cytosolic cytochrome c was
detected in Ki-Ras-expressing cells than in Ha-Rastransfected cells at 36 h after radiation. In addition, the
activation of caspase 3 and poly ADP-ribose polymerase
(PARP) cleavage was detected in 12V-Ki-Ras-expressing cells along with the cytochrome c release into the
cytosol (Figure 6b), in agreement with previous studies
suggesting that caspase 3 activation follows cytochrome
c release (Bossy-Wetzel and Green, 1999; Nagase et al.,
2001; Pan et al., 2001). These results indicate that
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
15
mitochondrial cytochrome c release and caspase 3
activation are essential mediators in the process of
enhanced apoptosis following radiation in 12V-Ki-Rasexpressing cells.
Since it has been shown that translocation of Bax
from the cytosol to the mitochondria causes a decline of
mitochondrial membrane potential subsequent to cytochrome c release and caspase activation (Yoshino et al.,
2001), we investigated whether the expression of
activated Ki-Ras enhances radiation-induced mitochondrial translocation of Bax. Figure 6c shows that ionizing
radiation dramatically increased the translocation of
Bax from cytosol to the mitochondria in a timedependent manner, without changing the protein levels
of Bcl-2, Bcl-xL, and Bax (data not shown). Indirect
immunofluorescence staining of Bax also clearly indicated its translocation to the mitochondria after
radiation treatment in cells expressing 12V-Ki-Ras
(Figure 6d). These results suggest that enhanced
radiation-induced cell death in 12V-Ki-Ras-expressing
cells involves alterations in the intracellular redistribution of Bax protein.
Activation of p38 MAPK is required for the radiationinduced Bax translocation in 12V-Ki-Ras-overexpressing
cells
To assess the role of activation of p38 MAPK in the
process of Bax translocation to the mitochondria and
cytochrome c release, we pretreated 12V-Ki-Ras-expressing cells with PD169316, an inhibitor of p38 MAPK
(Figure 7a), or transiently expressed dominant-negative
p38 MAPK (Figure 7b), and examined the intracellular
distribution of the Bax protein and cytochrome c by
immunoblot analysis with mitochondrial and cytosolic
fractions. We found that the pretreatment almost
completely prevented Bax translocation to the mitochondria, release of cytochrome c into the cytosol,
caspase 3 activation, and PARP cleavage (Figure 7a and
b). Indirect immunofluorescence staining of Bax clearly
showed that its translocation to the mitochondria is
dependent on the activation of p38 MAPK signaling
pathway (Figure 7c). In addition, transfection of 12VKi-Ras-expressing cells with RacN17, a dominant-
negative Rac1, significantly repressed radiation-induced
Bax translocation and cytochrome c release as well as
p38 MAPK activation in 12V-Ki-Ras-overexpressing
cells (Figure 7d). Conversely, the introduction of the
constitutive active form of Rac1 (RacV12) clearly
enhanced radiation-induced p38 MAPK activation,
Bax translocation, and apoptotic cell death (data not
shown). These results indicate that p38 MAPK functions as an important effector of the radiation-induced
apoptosis in 12V-Ki-Ras-expressing cells by facilitating
Bax translocation to the mitochondria and cytochrome
c release into the cytosol.
Discussion
Recently, the evidences for distinct cellular functions for
the Ras isoforms have been accumulated in various
studies. The differential biological functions of Ras
proteins are exerted through the selective activation of
downstream effectors. In this study, we demonstrate
that Ha-Ras and Ki-Ras protein significantly differ in
their abilities to modulate response to the ionizing
radiation and to activate downstream effectors. The HaRas protein increases resistance to ionizing radiation by
activation of the PI3K/Akt signaling pathway; Ki-Ras,
on the other hand, decreases the radiation resistance by
enhancing Bax translocation via activating an Rac/p38
MAPK-dependent pathway.
A clear difference was observed in the abilities of
Ha-Ras and Ki-Ras to enhance radiation-induced
apoptosis. The overexpression of active isoform of HaRas (12V-Ha-Ras) in Rat2 cells increases resistance to
the ionizing radiation. The constitutive activation of
PI3K and Akt is detected specifically in 12V-Ha-Rasexpressing cells. The PI3K inhibitor LY294002 or
dominant-negative Akt attenuates Ha-Ras-induced
radioresistance, suggesting that the activation of PI3K/
Akt has a critical role in the induction of radioresistance
in cells overexpressing 12V-Ha-Ras. These results were
consistent with recent reports demonstrating that PI3K
is an important mediator of Ras-induced radiation
resistance (Gupta et al., 2001; Grana et al., 2002),
and that PI3K mediates survival-associated signaling
Figure 5 Effects of p38 MAPK inhibition on radiation-induced apoptosis in 12V-Ki-Ras-overexpressing cells. (a) Rat2 cells overexpressing 12VKi-Ras were treated with 4 Gy of g-radiation in the presence or absence of the indicated doses of PD169316. The p38 MAPK activity was
determined with an immune complex kinase assay using GST-ATF-2 as a substrate (upper panel). Apoptosis was determined by Hoechst 33258
staining and scored for apoptotic changes in nuclear morphology 36 h after irradiation. The data represent averages of three independent
experiments. Error bars, SD (lower panel). (b) Rat2 cells overexpressing 12V-Ki-Ras were treated with 4 Gy of g-radiation in the presence or
absence of PD98059 (10 mm). ERK activity was detected by an immune complex kinase assay using myelin basic protein (MBP) as a substrate
(upper panel). Apoptosis was determined by Hoechst 33258 staining and scoring for apoptotic changes in nuclear morphology 36 h after
irradiation. The data represent averages of three independent experiments. Error bar, SD (lower panel). (c) Rat2 cells overexpressing 12V-Ki-Ras
were transiently transfected with dominant-negative p38 MAPK (DN-p38), -ERK (DN-ERK) or control vector and were treated with 4 Gy of gradiation. Cells were harvested at 36 h and p38 MAPK activity was determined with an immune complex kinase assay using GST-ATF-2 as a
substrate. Phosphorylated/activated ERK1/2 was detected by Western blot analysis with antibodies specific for phospho-ERK1/2. The data
represent a typical experiment conducted three times with similar results (left panel). Apoptosis was determined by Hoechst 33258 staining and
apoptotic changes in nuclear morphology were scored at 36 h after irradiation. The data represent averages of three independent experiments. Error
bars, SD (right panel). (d) Rat2 cells overexpressing 12V-Ki-Ras were transiently transfected with dominant-negative Rac (RacN17) or control
vector and were treated with 4 Gy of g-radiation. Cells were harvested at 36 h and phosphorylated/activated p38 MAPK and ERK1/2 were detected
by Western blot analysis with antibodies specific for phospho-p38 MAPK or -ERK1/2. The data represent a typical experiment conducted three
times with similar results (left panel). Apoptosis was determined by Hoechst 33258 staining and apoptotic changes in nuclear morphology were
scored at 36 h after irradiation. The data represent averages of three independent experiments. Error bars, SD (right panel)
Oncogene
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
16
apoptotic cell death in response to radiation. The
activation of caspase 3 and PARP cleavage was detected
in 12V-Ki-Ras-expressing cells along with the cyto-
Figure 6 Ionizing radiation enhances Bax translocation, cytochrome c release, and caspase 3 activation in cells overexpressing
12V-Ki-Ras. Rat2 cells overexpressing 12V-Ki-Ras or 12V-Ha-Ras
were treated with 4 Gy of g-radiation. (a) After 36 h, cytosolic
fractions were prepared and cytochrome c was detected by Western
blot analysis. The data represent a typical experiment conducted
four times with similar results. (b) After 24 and 48 h, whole cell
lysates were prepared and assessed by Western blot analysis for
caspase 3 and PARP. The data represent a typical experiment
conducted three times with similar results. (c) After 24 and 48 h,
mitochondrial fraction was prepared and Bax was detected by
Western blot analysis using anti-Bax antibody. The data represent
a typical experiment conducted four times with similar results. (d)
Cells overexpressing 12V-Ki-Ras were treated with 4 Gy of gradiation. After 36 h, cells were incubated with Mitotracker and
stained with FITC-conjugated anti-Bax antibody. The slides were
analysed by a confocal laser scanning microscope. Mitochondrial
localization of Bax was defined by yellow spots indicating overlap
of FITC and Mitotracker Red
pathway that allows cells to tolerate a variety of cellular
insults better, including serum deprivation, extracellular
matrix detachment, and DNA damage (Yao and
Cooper, 1995; Khwaja et al., 1997; Kulik et al., 1997).
However, we failed to detect the activation of PI3K/Akt
pathway in cells overexpressing activated Ki-Ras,
suggesting differential activation of downstream effector
molecules by different Ras isozymes.
In contrast to Ha-Ras, stable or transient transfection
of cells with activated Ki-Ras markedly increased
Oncogene
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
17
chrome c release into the cytosol, in agreement with
previous studies suggesting that caspase 3 activation
follows cytochrome c release (Bossy-Wetzel and Green,
1999; Nagase et al., 2001; Pan et al., 2001). These results
indicate that mitochondrial cytochrome c release and
caspase 3 activation are essential mediators of enhanced
apoptosis following radiation observed in Ki-Ras-overexpressing cells. We also found that activated Ki-Ras
enhances radiation-induced cell death through cytochrome c release following intracellular redistribution of
cell death activator Bax. Analysis of various Bax
mutations indicated that cell death was only observed
when the Bax protein was capable of translocating to the
mitochondria (Nechushtan et al., 1999). Bax translocation to the mitochondria has been shown to reduce
mitochondrial membrane potential, enhance cytochrome c release from mitochondria, and activate
caspases (Desagher et al., 1999; Finucane et al., 1999).
In this study, we observed the activation of ERK and
p38 MAPK by ionizing radiation in cells overexpressing
12V-Ki-Ras. Inhibition of the MEK/ERK pathway by
using the MEK inhibitor, PD98059 or by expressing
dominant-negative ERK did not show any changes in
apoptotic cell death. These results were consistent with
others who previously showed that the inhibition of
MEK activity does not influence radioresistance (Belka
et al., 2000; Gupta et al., 2000, 2001; Grana et al., 2002).
However, inhibition of the p38 MAPK activity by p38
Figure 7 Inhibition of p38 MAPK attenuates radiation-induced Bax
translocation, cytochrome c release, and caspase 3 activation in cells
overexpressing 12V-Ki-Ras. (a) Rat2 cells overexpressing 12V-Ki-Ras
were treated with 4 Gy radiation in the presence or absence of 30 mm
PD169316. After 36 h, mitochondrial fraction was prepared and
mitochondria-localized Bax and HSP60 were detected by Western
blot analysis using anti-Bax or -HSP60 antibody. Cytosolic fractions
were prepared and released cytochrome c from mitochondria was
detected by Western blot analysis using anti-cytochrome c antibody.
Whole cell lysates were prepared and assessed by Western blot analysis
for caspase 3 and PARP. The data represent a typical experiment
conducted at least three times with similar results (b) Rat2 cells
overexpressing 12V-Ki-Ras were transiently transfected with dominant-negative p38 MAPK (DN-p38) or control vector and were treated
with 4 Gy of g-radiation. After 36 h, mitochondrial fraction was
prepared and mitochondria-localized Bax and HSP60 were detected by
Western blot analysis using anti-Bax or -HSP60 antibody. Cytosolic
fractions were prepared and released cytochrome c from mitochondria
was detected by Western blot analysis using anti-cytochrome c
antibody. Whole cell lysates were prepared and assessed by Western
blot analysis for caspase 3 and PARP. The data represent a typical
experiment conducted three times with similar results. (c) Cells
overexpressing 12V-Ki-Ras were treated with 4 Gy of g-radiation in
the presence or absence of 30 mm PD169316. After 36 h, cells were
incubated with Mitotracker and stained with FITC-conjugated antiBax antibody. The slides were analysed by a confocal laser scanning
microscope. Mitochondrial localization of Bax was defined by yellow
spots indicating overlap of FITC and Mitotracker Red. (d) Cells
overexpressing 12V-Ki-Ras were transiently transfected with dominant-negative Rac (RacN17) or control vector and were treated with
4 Gy of g-radiation. After 36 h, the mitochondrial fraction was
prepared and mitochondria-localized Bax and HSP60 were detected
by Western blot analysis using anti-Bax or -HSP60 antibody. Cytosolic
fractions were prepared and released cytochrome c from mitochondria
was detected by Western blot analysis using anti-cytochrome c
antibody. Whole cell lysates were prepared and assessed by Western
blot analysis for PARP. The data represent a typical experiment
conducted three times with similar results
MAPK inhibitor, PD169316, or by expressing dominantnegative p38 MAPK markedly attenuated radiationinduced apoptosis, suggesting that the activation of p38
MAPK is involved in Ki-Ras-induced radiosensitivity.
We also found that the inhibition of p38 MAPK almost
completely prevented Bax translocation to the mitochondria, release of cytochrome c into the cytosol,
caspase 3 activation, and PARP cleavage. These results
are consistent with the fact that p38 MAPK are
frequently associated with the induction of apoptosis
(Chuang et al., 2000; Stadheim et al., 2000; Kunz et al.,
2001), and that p38 MAPK acts at an early step prior to
dysfunction of mitochondria and caspase activation in
several cell types (Galan et al., 2000; Cheng et al., 2001).
The significant reduction of caspase 3 activation and
PARP cleavage after concomitant treatment with
radiation and p38 MAPK inhibitor in cells overexpressing Ki-Ras is consistent with our demonstration that
Bax translocation is regulated by p38 MAPK. Thus we
propose that in 12V-Ki-Ras-expressing cells, ionizing
radiation activates p38 MAPK, which promotes Bax
translocation to the mitochondria followed by cytochrome c release and caspase activation. The results of
the present study suggest that Bax translocation to the
mitochondria is subject to regulation by p38 MAPK in
response to concomitant introduction of radiation and
activated Ki-Ras. This finding is consistent with recent
studies demonstrating that NH2 (Goping et al., 1998)
and COOH termini (Nechushtan et al., 1999) of Bax are
rich in glycine and hydroxyl amino acids such as serine
and threonine that are potential targets of the p38
MAPK (Ghatan et al., 2000), and critical for regulating
the subcellular distribution of Bax. Phosphorylation of
either terminus may cause conformational change,
facilitating mitochondrial membrane insertion of Bax.
However, in contrast to Bcl-2 and Bad, evidence for Bax
phosphorylation is rare. Alternatively, it is conceivable
that while Bax is not directly phosphorylated, the
phosphorylation of a Bax-binding protein may facilitate
membrane targeting and insertion.
Ral-GDS is another Ras effector that can activate
CDC42 and Rac, which in turn activate various MEK
kinases that are involved in the regulation of the SAPK/
JNK and p38 MAPK pathways (Johnson, 1999). It is
well established that the Rac/p38 MAPK pathway is
activated by various types of membrane-associated
cellular signals induced by many apoptosis-inducing
agents. In this study, we also provided evidence of the
fact that Rac is involved in the Ki-Ras-mediated
enhancement of radiation-induced apoptosis. Inhibition
of Rac1 activity with RacN17, a dominant-negative
mutant of Rac1, in 12V-Ki-Ras-expressing Rat2 cells
significantly reduced radiation-induced apoptosis as well
as p38 MAPK activation, Bax translocation, and
cytochrome c release. Furthermore, introduction of
activated Rac1 (RacV12) into Rat2 cells resulted in an
increase in the activation of p38 MAPK and in the
sensitivity to ionizing radiation (data not shown),
demonstrating that Rac acts as an upstream regulator
of p38 MAPK during ionizing radiation-induced apoptosis. This is in good agreement with the recent study
Oncogene
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
18
showing that the Rac/p38 MAPK/Bax translocation
pathway has an essential role in the apoptosis induced
by UV irradiation (Eom et al., 2001). On the other hand,
it has been recently reported that the activation of PI3K
also leads to the activation of Rac GTPase (Rebollo and
Martinez, 1999), which acts as a downstream effector
molecule of Ras signaling pathways that control actin
polymerization, transcriptional activation, and cell
proliferation. However, we failed to detect PI3K
activation in cells overexpressing Ki-Ras; moreover,
pretreatment of LY294002, a specific inhibitor of PI3K,
did not affect radiation-induced p38 MAPK activation,
suggesting that the activation of Rac/p38 MAPK
signaling pathway by radiation in Ki-Ras-overexpressing cells is independent of PI3K signaling.
Recently, opposing roles of Ki-Ras and Ha-Ras genes
in the regulation of redox signals and apoptosis have
been observed (Santillo et al., 2001; Cuda et al., 2002).
The study shows that the Ha-Ras protein increases the
intracellular level of reactive oxygen species and the
number of cells undergoing apoptosis after oxidative
stress, conversely the Ki-Ras has a protective effect.
On the contrary, however, here we show that cells
overexpressing Ki-Ras are more sensitive than Ha-Rasexpressing cells to ionizing radiation. Moreover,
radiation increases the level of intracellular reactive
oxygen species more potently in Ki-Ras-overexpressing
cells than Ha-Ras-expressing cells (unpublished data).
We also find that the increased level of reactive oxygen
species in activated Ki-Ras-expressing cells after ionizing irradiation is dependent on the Ras/Rac/p38 MAPK
signaling pathway (unpublished data). Differences in the
regulation of reactive oxygen species level and apoptosis
by Ras isozymes in previous reports and our results are
likely to be stress type and cell type dependent.
In conclusion, in this study, we showed that the
sensitivity to ionizing radiation is differentially modulated by activated Ki-Ras and Ha-Ras. Our findings
explain the opposite effects of Ha-Ras and Ki-Ras on
radiation-induced apoptosis and suggest that differential
activation of Akt and p38 MAPK signaling may account
for the opposing response to ionizing radiation by HaRas or Ki-Ras. The Ha-Ras protein increases resistance
to ionizing radiation by activation of the PI3K/Akt
signaling pathway; Ki-Ras, on the other hand, decreases
the radiation resistance by enhancing Bax translocation
via activation of an Rac/p38 MAPK-dependent pathway.
These findings link Ras activity to the modulation of cell
sensitivity to ionizing radiation, provide an explanation
for the divergent biological activities of Ras isozymes,
and partly account for the differential response of
transformed cells to anticancer agents.
Materials and methods
Chemicals and plasmids
Monoclonal antibodies to H-Ras and K-Ras were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and
monoclonal antibodies to phosphorylated ERK, p38 MAPK
and JNK were obtained from Cell Signaling (Beverly, MA,
USA). Polyclonal antibodies to caspase 3 and PARP were
Oncogene
purchased from Upstate Biotechnology, and monoclonal
antibody to Bax, Bcl-2, and Bcl-XL was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). PD98059,
SB203580, and PD169316 were obtained from Calbiochem
(San Diego, CA, USA). pSPORT-Ki-Ras (V12) and pSPORTHa-Ras (V12) were gifts from Dr P Kirshmeier (ScheringPlough Research Institute). pCMV5-DN-p38, an expression
vector encoding dominant-negative mutant of p38 MAPK,
and pLCEP4-DN-ERK, a dominant-negative mutant of ERK,
were gifts from Dr Roger Davis (University of Massachusetts
Medical School, Worcester, MA, USA). pEXV-RacN17, an
expression vector encoding a dominant-negative mutant of
Rac1, and pEXV-RacV12, an expression vector encoding a
constitutively active form of Rac1, were gifts from Dr Alan
Hall (University College London, London, UK). pCMV5DN-Akt, an expression vector encoding a dominant-negative
mutant of Akt was a gift from Dr Richard A Roth (Stanford
University School of Medicine, Stanford, CA, USA).
Cell culture
Rat2 and L929 murine fibroblast cells were obtained from the
American Type Culture Collection (Rockville, MD, USA),
and were grown in RPMI 1640 medium supplemented with
10% fetal bovine serum (FBS, GIBCO) and antibiotics at 371C
in a humidified incubator with 5% CO2.
Irradiation
Cells were irradiated with 4 Gy using a g-cell irradiator with
137
Cs source (Atomic Energy of Canada, Ltd, Canada).
Stable transfection of the activated Ki-Ras (12V-Ki-Ras) and
activated Ha-Ras (12V-Ha-Ras) constructs into Rat2 cells
Using the Lipofectamin method (GIBCO/BRL), exponentially
growing Rat2 cells were transfected with expression plasmids
encoding 12V-Ki-Ras, 12V-Ha-Ras, or a control vector for 6 h
at 371C, and the cells were incubated with a standard medium
for 24 h. At that time G418 selection was begun at a
concentration just high enough to kill 100% of the parental
cells (400 mg/ml). After 10–15 days, colonies were picked by the
cloning cylinder technique and were expanded into cell lines.
Clonogenic survival assay
The clonogenic survival assay was performed as described
previously (Bache et al., 2001). Cells were seeded into 60-mm
dishes at densities sufficient to produce approximately 200
colonies per dish in the controls, and were incubated for 10–14
days. Colonies were fixed with 75% ethanol and 25% acetic
acid, and stained with trypan blue. The number of colonies
consisting of more than 50 cells was counted.
Preparation of membrane fractions
To gain the cytosol and membrane fractions, harvested cells
were suspended in fractionation buffer (10 mm Tris-HCl, pH
7.5, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 20 mm
leupeptin) and lysed by sonication. The lysate was centrifuged at
100 000 g for 1 h at 41C to obtain cytosol fraction (supernatant).
The pellet representing particulate proteins was resuspended in
the above buffer containing 0.1% Triton X-100 and lysed by
sonication. The lysate was centrifuged at 100 000 g for 1 h at 41C
to obtain membrane fraction (supernatant).
Preparation of cytosolic and mitochondrial fractions
Cells were collected and washed twice in ice-cold PBS,
resuspended in S-100 buffer (20 mm HEPES, pH 7.5, 10 mm
Differential modulation of radiosensitivity by Ras isozymes
J-A Choi et al
19
KCl, 1.9 mm MgCl2, 1 mm EGTA, 1 mm EDTA, mixture of
protease inhibitors), and incubated on ice for 20 min. After
20 min of incubation on ice, the cells were homogenized with a
Dounce glass homogenizer and a loose pestle (Wheaton,
Millville, NJ, USA) for 70 strokes. Cell homogenates were
spun at 1000 g to remove unbroken cells, nuclei, and heavy
membranes. The supernatant was respun at 14 000 g for 30 min
to collect the mitochondria-rich (the pellet) and the cytosolic
(the supernatant) fractions. The mitochondria-rich fraction
was washed once with the extraction buffer, followed by a final
resuspension in lysis buffer (150 mm NaCl, 50 mm Tris-HCl,
pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mm EGTA)
containing protease inhibitors for Western blot analysis.
Hoechst 33258 staining
Hoechst 33258 staining was performed as described previously
(Choi et al., 2001). Briefly, cells were fixed with 4%
paraformaldehyde for 30 min at room temperature, and then
washed once with PBS. Hoechst 33258 (50 ng/ml) was added to
the fixed cells, incubated for 30 min at room temperature, and
washed with PBS. Cells were mounted and examined by
fluorescence microscopy. Apoptotic cells were identified by the
condensation and fragmentation of their nuclei. The percentage of apoptotic cells was calculated as the ratio of apoptotic
cells to total cells counted 100. A minimum of 500 cells were
counted for each treatment.
Sandwich enzyme immunoassay
Both adherent and floating cells were collected 48 h after
g-radiation treatment and washed with ice-cold PBS. Quantitative determinations of cytoplasmic histone-associated DNA
fragments were performed using the Cell Death Detection
ELISA Kit (Boehringer Mannheim, Indianapolis, IN, USA).
Flow cytometric analysis of apoptosis
Apoptosis was identified and quantified by flow cytometry with
propidium iodide (PI) staining. Both adherent and floating cells
were collected after g-radiation treatment, washed with ice-cold
PBS, and fixed with 70% ice-cold ethanol overnight at 41C. Fixed
cells were washed twice with PBS and treated with 1 mg/ml
RNase for 30 min at 371C. Cellular DNA was stained with 50 mg/
ml PI in PBS containing 0.05% Nonidet P-40. Cells were then
analysed by FACScan flow cytometer (Becton Dickinson,
Franklin Lakes, NJ, USA). From the analysis of DNA
histograms, the percentages of cells in different cell cycle phases
were evaluated. Cells with DNA content less than the G1 phase
(sub-G1) were taken as apoptotic cells.
Extraction of DNA and agarose-gel electrophoresis
Parental Rat2, and 12V-Ha-Ras- and 12V-Ki-Ras-expressing
Rat2 cells were treated with 4 Gy of g-radiation and were lysed
in a lysis buffer (20 mm Tris/HCl, pH 8.0, 0.1 mm EDTA, 1%
SDS, and 0.5 mg/ml proteinase K). DNA samples were
extracted and separated by agarose-gel electrophoresis.
Western blot analysis
Western blot analysis was performed as described (Lee et al.,
1998). Briefly, cell lysates were prepared by extracting proteins
with lysis buffer (40 mm Tris-HCl (pH 8.0), 120 mm NaCl,
0.1% Nonidet-P40) supplemented with protease inhibitors.
Proteins were separated by SDS–PAGE and transferred onto a
nitrocellulose membrane. The membrane was blocked with 5%
nonfat dry milk in Tris-buffered saline and then incubated
with primary antibodies for 1 h at room temperature. Blots
were developed with a peroxidase-conjugated secondary
antibody, and proteins were visualized by enhanced chemiluminescence (ECL) procedures (Amersham, Arlington
Heights, IL, USA) according to the manufacturer’s recommendations.
Immune complex kinase assay
The immune complex kinase assay was performed as
previously described (Riabowol et al., 1989). Briefly, cell
lysates were incubated with an anti-p38 MAPK antibody (Cell
Signaling, Beverly, MA, USA), and immunocomplexes were
collected on protein A-Sepharose beads (Sigma, St Louis, MI,
USA) and resuspended in a kinase assay mixture containing
[g-32P]ATP (ICN) and GST-ATF-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a substrate. The proteins were
separated on SDS–PAGE, and the bands were detected by
autoradiography.
Immunofluorescence analysis
For immunofluorescence analysis, cells were fixed with 2%
paraformaldheyde, permeabilized with 0.1% Triton X-100 in
PBS, incubated with 25 nm Mitotracker Red CMXRos
(Molecular Probes, Inc., Eugene, OR, USA) for 30 min at
room temperature, and then washed three times with PBS.
Cells were then incubated with rabbit anti-human Bax diluted
1 : 200 in 5% FBS/PBS for 1 h at room temperature in a
humidified chamber. Excess antibody was removed by washing
the coverslips three times with PBS. Cells were then incubated
with biotin-labeled goat anti-rabbit IgG (Zymed Laboratories,
Inc., South San Francisco, CA, USA) diluted 1 : 200 in 5%
FBS/PBS, and protected from light at room temperature for
1 h. After washing three times with PBS, cells were incubated
with FITC-streptavidin diluted 1 : 200 in 5% FBS/PBS for 4 h.
After washing three times with PBS, coverslips were mounted
onto microscope slides using ProLong antifade mounting
reagent (Molecular Probes, Inc., Eugene, OR, USA). The
slides were analysed by a confocal laser-scanning microscope
(Leica Microsystems, Heidelberg, Germany).
Abbreviations
MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK1/2, extracellularregulated kinase 1 and 2; SAPK, stress-activated protein
kinase; JNK, c-Jun N-terminal kinase; PARP, poly
ADP-ribose polymerase; PI3K, phosphoinositide-3-kinase.
Acknowledgements
This work was supported by a Nuclear R&D Program from
the Ministry of Science and Technology in Korea.
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