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Biochemistry and Biophysics |
Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka 565-0871 [J. H., M. O., Y. K., K. H., Y. N., K. A., K. T., T. K., Y. M.], and Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata 990-9585 [H. K.], Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various cellular stimuli that control cell growth and differentiation cause a rapid increase in the enzymatic activity of a family of serine/threonine kinases known as the MAP kinase family. The MAP kinase family has been classified into three subfamilies: ERKs, including ERK1 and ERK2; stress-activated protein kinases, also termed JNKs, including JNK1 (46 kDa) and JNK2 (55 kDa); and p38 kinase, a homologue of the yeast HOG1 (high-osmolarity glycerol response-1) kinase (17) . We recently reported that both ERK and JNK are activated by cisplatin-induced DNA damage and are required for cell survival after cisplatin treatment (18) . The involvement of MEK upstream of BAD phosphorylation (19) and the promotion of cell survival by the Ras-MAPK signaling pathway by phosphorylation of BAD at Ser-112 (20, 21, 22) were reported recently. However, the effect of certain DNA-damaging agents on the PI-3K-Akt-BAD cascade remains to be elucidated. Therefore, we sought to determine whether the PI-3K-Akt-BAD cascade plays a role in the cellular stress response to the chemotherapeutic agent cisplatin, which damages DNA through the formation of bifunctional platinum adducts. For our study, we used both Caov-3 human ovarian cancer cells, which are resistant to cisplatin, and A2780 human ovarian cancer cells, which are sensitive to cisplatin. Here we provide evidence that cisplatin but not transplatin, which does not readily damage DNA (23 , 24) , induced the activation of Akt and phosphorylation of BAD. Moreover, inhibition of Akt and BAD markedly decreased cell viability after treatment with cisplatin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß antibody, were obtained from New England
Biolabs (Beverly, MA). Rabbit polyclonal anti-HA antibody was obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Cell Titer
96-cell proliferation assay kit was obtained from Promega (Madison,
WI).
Cell Cultures.
Human ovarian papillary adenocarcinoma cell line Caov-3 was obtained
from American Type Culture Collection (Rockville, MD). Human ovarian
cancer cell line A2780, derived from a patient prior to treatment, was
kindly provided by Dr. T. Tsuruo (Institute of Molecular and Cellular
Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C.
Hamilton (Fox Chase Cancer Center, Philadelphia, PA; Refs. 25
, 26
). The cells were cultured at 37°C in DMEM with 10% FBS in
a water-saturated atmosphere of 95% O2 and 5%
CO2.
Constructs.
The vector encoding the various HA-tagged forms of Akt, either
wild-type or kinase-dead (K179M mutant), and the various forms of
HA-tagged BAD, either wild-type (pCDNA3-BAD) or mutants
(pCDNA3-BADS136A, pCDNA3-BADS112A, and pCDNA3-BAD S to A at 112 and
136) used in this study have been described previously
(12)
.
Clone Selection.
Caov-3 and A2780 cells were transfected for 12 h in 6-well tissue
culture plates with 2 µg of the empty vector (CMV-6); with CMV-6
containing the gene for HA-tagged wild-type Akt or HA-tagged Akt K179M
and the neomycin resistance gene; with the empty vector (pCDNA3), which
contains a neomycin resistance gene; or with pCDNA3-BAD,
pCDNA3-BADS136A, pCDNA3-BADS112A, or pCDNA3-BAD S to A at 112 and
136, using Lipofectamine plus (Life Technologies, Gaithersburg,
MD; Ref. 18
). Clonal selection was performed by adding
geneticin to the medium at a final concentration of 200 µg/ml 2 days
after the transfection. After 3 weeks, several clones were isolated
using cloning rings. Selected clones were then maintained in medium
supplemented with geneticin (100 µg/ml), and only low-passage cells
(passage < 10) were used for the experiments described
here. For analysis of the levels of ectopically expressed Akt and BAD
protein products, empty vector (CMV-6)-, wild-type Akt-, or
AktK179M-expressing Caov-3 cells or empty vector (pCDNA3)-, wild-type
BAD-, BADS112A-, BADS136A-, and BAD2SA-expressing Caov-3 cells cultured
in 100-mm dishes were lysed in ice-cold HNTG buffer [50 mM
HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton
X-100, 1.5 mM MgCl2, 1 mM
EDTA, 10 mM sodium PPi, 100
µM sodium orthovanadate, 100 mM NaF, 10
µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride (27)
]. The lysate samples
were immunoprecipitated with anti-HA antibody. Immune complexes were
precipitated with protein A-Sepharose, and the isolated proteins were
analyzed by electrophoresis on 8% SDS-polyacrylamide gels. Transfer to
nitrocellulose, Western blotting with anti-Akt or anti-BAD antibody,
and washing were performed as described elsewhere (27)
.
Cytotoxicity.
Cell viability (18)
was assessed by the addition for
1 h of cisplatin or transplatin 1 day after seeding 1.0 x 102
test cells into 96-well plates,
followed by exchanging the medium with fresh medium. Because a
significant level of DNA repair after cisplatin-induced DNA damage was
detected 5 days later but not after 24–72 h (28)
, the
number of surviving cells was determined 5 days later by determination
of the absorbance at 590 nm (A590 nm)
of the dissolved formazan product after addition of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
inner salt for 1 h as described by the manufacturer (Promega). By
the fifth day, the cells without treatment were growing exponentially
and had achieved a density of 6.0–6.5 x 103 cells/well. All experiments were carried out
in quadruplicate, and viability is expressed as the ratio of the number
of viable cells with cisplatin or transplatin treatment to the number
without treatment.
Assay of Akt Kinase Activity.
Cells were incubated in the absence of serum for 16 h and then
treated with various materials. Cells were then washed twice with PBS
and lysed in ice-cold lysis buffer [20 mM Tris (pH 7.4),
150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1
mM EGTA, 2.5 mM sodium
PPi, 1 mM ß-glycerophosphate, 1
mM sodium orthovanadate, 1 µg/ml leupeptin, and 1
mM phenylmethylsulfonyl fluoride]. The extracts were
centrifuged to remove cellular debris, and the protein content of the
supernatants was determined using the Bio-Rad protein assay reagent.
Two hundred fifty µg of protein from the lysate samples were
incubated with gentle rocking at 4°C overnight with immobilized Akt
antibody cross-linked to agarose hydrazide beads. After Akt was
selectively immunoprecipitated from the cell lysates, the
immunoprecipitated products were washed twice in lysis buffer and twice
in kinase assay buffer [25 mM Tris (pH 7.5), 10
mM MgCl2, 5 mM
ß-glycerophosphate, 0.1 mM sodium orthovanadate and 2
mM DTT], and the samples were resuspended in 40 µl of
kinase assay buffer containing 200 µM ATP and 1 µg
GSK-3 fusion protein. The kinase reaction was allowed to proceed at
30°C for 30 min and stopped by the addition of Laemmli SDS sample
buffer (29)
. Reaction products were resolved by 15%
SDS-PAGE followed by Western blotting with a phospho-GSK-3 antibody.
For analysis of the total amount of Akt, 250 µg of protein from the
lysate samples were resolved by 8% SDS-PAGE, followed by Western
blotting with anti-Akt antibody. For analysis of the effect of
ectopically expressed Akt on Akt activity, empty vector (CMV-6)-,
wild-type Akt-, or AktK179M-expressing Caov-3 cells grown in 100-mm
dishes were treated with 1 mM cisplatin for 3 h. The
lysate samples were immunoprecipitated with anti-HA antibody. Immune
complexes were precipitated with protein A-Sepharose, and the kinase
reaction was carried out in the presence of cold ATP and GSK-3
fusion protein, as described above.
Phosphorylation of BAD.
Cells cultured in 100-mm dishes were transfected with 4 µg of
pCDNA3-BAD, using Lipofectamine plus. At 72 h after transfection,
serum-deprived cells were treated with various materials. They were
then washed twice with PBS and lysed in ice-cold HNTG buffer
(27)
. The lysate samples were immunoprecipitated with
phospho-BAD (Ser-112) or phospho-BAD (Ser-136) antibody. Immune
complexes were precipitated with protein A-Sepharose, and the isolated
proteins were analyzed by electrophoresis on 8% SDS-PAGE. Transfer to
nitrocellulose, Western blotting with phospho-BAD (Ser-112) or
phospho-BAD (Ser-136) antibody, and washing were performed as described
elsewhere (27)
.
For analysis of the total amount of BAD, 250 µg of protein from the lysate samples were resolved by 8% SDS-PAGE, followed by Western blotting with anti-BAD antibody. For analysis of the effect of ectopically expressed BAD on BAD phosphorylation, empty vector (pCDNA3)-, wild-type BAD-, BADS112A-, BADS136A-, and BAD2SA-expressing Caov-3 cells grown in 100-mm dishes were treated with 1 mM cisplatin for 3 h. The lysate samples were immunoprecipitated with anti-HA antibody. Immune complexes were precipitated with protein A-Sepharose, and the isolated proteins were resolved by 8% SDS-PAGE, followed by Western blotting with phospho-BAD (Ser-112) or phospho-BAD (Ser-136) antibody, as described above.
Statistics.
Statistical analysis was performed using Student’s t test,
and P < 0.01 was considered significant.
Data are expressed as the mean ± SE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and at the indicated concentrations for 3 h (Fig. 1B)
. Cell lysates were immunoprecipitated with immobilized
anti-Akt antibody, followed by addition of GSK-3 fusion protein and
Western blotting with anti-phospho-GSK-3/ß antibody. Activation of
Akt by cisplatin in Caov-3 cells was detected at 1 h, reached a
plateau from 3 h through 6 h, and declined thereafter (Fig. 1A
, top row). We confirmed that the total amount
of Akt in each lane was the same (Fig. 1A
, middle
row). Activation of Akt by cisplatin in A2780 cells was
also detected at 1 h, reached a plateau at 3 h, and declined
thereafter (Fig. 1A
, bottom row). Cisplatin
induced the activation of Akt in a dose-dependent manner in Caov-3
(Fig. 1B)
and A2780 cells (data not shown). It is known that
cisplatin but not transplatin forms covalent cross-links between the N7
position of adjacent guanine or adenine-guanine residues (23
, 24)
. Treatment with transplatin had no apparent effect on Akt
activation, whereas cisplatin clearly induced Akt activation in Caov-3
(Fig. 1C
, top row) and A2780 (Fig. 1C
,
bottom row) cells. Because Akt is an effector of survival
signaling downstream from PI-3K, we next determined whether stimulation
of both types of cells with cisplatin could increase the activity of
Akt through a PI-3K-dependent mechanism. Both types of cells were
stimulated with cisplatin in the presence or absence of wortmannin, a
PI-3K inhibitor, and the kinase activity of Akt was assayed. The
induction of Akt activity by cisplatin was inhibited by wortmannin
(Fig. 1C
, Lane 3). These results indicate that
only the DNA-damaging cisplatin isomer activates Akt activity in both
types of cells through a PI-3K-dependent mechanism.
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, top rows) or A2780 cells (Fig. 2
, bottom rows)
were transfected with pCDNA3-BAD and exposed to 1
mM cisplatin for 3 h. Cell lysates were
immunoprecipitated with either anti-phospho-BAD (Ser-112; Fig. 2A
) or anti-phospho-BAD (Ser-136; Fig. 2B
)
antibody, followed by Western blotting with the same antibody. Whereas
transplatin had no effect on BAD phosphorylation (Fig. 2
, Lane
4), cisplatin induced the phosphorylation of BAD at Ser-112 and
Ser-136 in Caov-3 and A2780 cells (Fig. 2
, Lane 2).
Moreover, we confirmed that the total amount of BAD in each lane from a
given cell line was the same (Fig. 2C)
. Because cisplatin
induced BAD phosphorylation, the relative amount of nonphosphorylated
BAD was reduced by cisplatin, suggesting that cisplatin-induced BAD
phosphorylation indicated the inhibition of the proapoptotic
effect of BAD. In accordance with reports showing that Akt
phosphorylates BAD specifically at Ser-136 (12
, 13)
,
cisplatin-induced phosphorylation of BAD at Ser-136 was inhibited
by wortmannin (Fig. 2B
, Lane 3). On the other
hand, cisplatin-induced phosphorylation of BAD at Ser-112 was not
inhibited by wortmannin (Fig. 2A
, Lane 3). The
presence of two phosphorylation sites on BAD suggests that the
simultaneous activation of different survival pathways may result in
the concomitant phosphorylation of BAD Ser-112 and Ser-136 by different
kinase cascades (12)
. In addition, the possibility that
MEK is involved upstream of BAD phosphorylation has been reported
(19)
. Therefore, we examined the effect of PD98059, an MEK
inhibitor, on BAD phosphorylation. Although pretreatment with PD98059
had no effect on the phosphorylation of BAD at Ser-136 (Fig. 2B
, Lane 5), it attenuated the phosphorylation of
BAD at Ser-112 (Fig. 2A
, Lane 5).
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and A2780 cells (Fig. 3C)
expressing a
kinase-deficient Akt (AktK179M) was compared with that of an empty
vector (CMV-6)-expressing control line. AktK179M cells express an Akt
derivative rendered kinase-inactive by a point mutation within the
catalytic domain (5
, 30
, 31)
. We first confirmed the
overexpression of ectopically expressed Akt protein products (Fig. 3A
, bottom row) and the negative effects of the
expression of HA-AktK179M on Akt activity (Fig. 3A
,
top row). The viability of Caov-3 cells was not
affected by increasing concentrations of cisplatin of >100
µM. Further titrations revealed
IC50 values of 380 and 422
µM for parental and empty vector-expressing
Caov-3 cells, respectively (Table 1)
. In contrast, the AktK179M-expressing Caov-3 cells exhibited an
IC50 as low as 84 µM,
indicating a >5.2-fold greater sensitivity to cisplatin than the empty
vector-expressing Caov-3 cells (Fig. 3B
and Table 1
). On the
other hand, the IC50 value of A2780 cells was 84
µM (Table 1)
. The AktK179M-expressing A2780
cells exhibited IC50 values of 57
µM, indicating a >1.5-fold greater sensitivity
to cisplatin than the empty vector-expressing A2780 cells (Fig. 3C
and Table 1
).
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. In further titrations, no
significant effect was observed with transplatin even at 250
µM, indicating that the requirement for
sensitization by kinase-deficient Akt depends on the stereospecific
DNA-binding properties of cisplatin, consistent with the results for
the activation of Akt (Fig. 1C)
. Expression of wild-type Akt
did not affect the sensitivity to cisplatin compared with the control
line (Fig. 3, B and C)
. Thus, the sensitization
to cisplatin observed in the kinase-deficient Akt-expressing cells
appeared to be attributable to interference with activated Akt. We
confirmed these results with other clonal derivatives of Akt (data not
shown).
Interference of Phosphorylated BAD at Ser-112 or Ser-136 Sensitizes
Caov-3 and A2780 Cells to Cisplatin but not Transplatin.
We next examined whether the phosphorylation of BAD is also required
for cell viability after cisplatin treatment of Caov-3 (Fig. 4B)
and A2780 cells (Fig. 4C)
. To test the
importance of BAD Ser-112 and Ser-136 for cell viability, we
transfected Caov-3 and A2780 cells with mutant BAD constructs in which
Ser-112 (BADS112A), Ser-136 (BADS136A), or both (BAD2SA) were converted
to alanine so that BAD could no longer be phosphorylated at these sites
(12)
. We first confirmed the overexpression of ectopically
expressed BAD protein products (Fig. 4A
, bottom
row) and the negative effects of the expression of BADS112A,
BADS136A, or BAD2SA on BAD phosphorylation (Fig. 4A
,
top row). BADS112A-, BADS136A-, and BAD2SA-expressing Caov-3
cells exhibited IC50s of 77, 78, and 56
µM, respectively, indicating more than 6.0-,
5.9-, and 8.4-fold greater sensitivity, respectively, to cisplatin than
the empty vector (pCDNA3)-expressing Caov-3 cells (Fig. 4B
and Table 2
). On the other hand, BADS112A-, BADS136A-, and BAD2SA-expressing A2780
cells exhibited IC50 values of 52, 53, and 36
µM, indicating more than 1.5-, 1.5-, and
2.3-fold greater sensitivity, respectively, to cisplatin than the empty
vector (pCDNA3)-expressing A2780 cells (Fig. 4C
and Table 2
).
|
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. Interestingly, exogenous expression
of either BADS112A or BADS136A in Caov-3 and A2780 cells decreased the
viability after treatment with cisplatin to a level intermediate
between that of cells expressing exogenous wild-type BAD and cells
expressing BAD2SA. Transplatin had no discernible effect on the
BADS112A-, BADS136A-, or BAD2SA-expressing Caov-3 cells at
concentrations at which the viability after treatment with cisplatin
was <40% (Fig. 4D)
. Thus, the sensitization to cisplatin
observed in the mutant BAD-expressing cells appeared to depend on the
stereospecific DNA-binding properties of cisplatin, which is consistent
with the results for the phosphorylation of BAD (Fig. 2)
, and the
sensitization appeared to be attributable to interference with BAD
phosphorylated at Ser-112 and Ser-136. We confirmed this result with
other clonal derivatives of BAD (data not shown).
Effect of Cisplatin on Phosphorylation of BAD in
Kinase-deficient Akt-expressing Cells.
To examine whether Akt acts upstream of BAD, we examined whether
cisplatin induces the phosphorylation of BAD in kinase-deficient Akt
(AktK179M)-expressing Caov-3 cells. Although cisplatin induced the
phosphorylation of BAD at Ser-136 in empty vector (CMV6)-expressing
cells, cisplatin did not induce the phosphorylation of BAD at Ser-136
in the kinase-deficient Akt (AktK179M)-expressing cells (Fig. 5B)
. Interestingly, the phosphorylation of BAD at Ser-112
induced by cisplatin was detected in both empty vector
(CMV6)-expressing cells and kinase-deficient Akt (AktK179M)-expressing
cells (Fig. 5A)
. On the other hand, cisplatin-induced Akt
activity was not changed in cells transfected with BAD mutants (data
not shown). These data confirm that Akt functions upstream of the
phosphorylation of BAD at Ser-136.
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DISCUSSION |
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PI-3K signaling is very important in mitogenesis, protein synthesis, membrane ruffling, and cell-cycle progression (33 , 34) . Sequence homology studies have revealed that the PI-3K domain is present in a variety of proteins active in DNA repair (35) . In addition, it has been shown that wortmannin induces marked radiosensitivity in murine fibroblasts and human tumor cells and blocks the induction of p53 after DNA damage (36) . Thus, PI-3K signaling is suspected to be involved in DNA repair. The PI-3K-Akt-BAD cascade is reported to suppress the apoptotic death induced by a variety of stimuli, including growth factor withdrawal, cell-cycle discordance, and loss of cell adhesion in a number of cell types (5, 6, 7, 8, 9, 10, 11) . However, until recently there had not been any studies addressing the role of the PI-3K-Akt-BAD cascade in the DNA repair in cells treated with chemotherapeutic DNA-damaging drugs. This is the first report showing that cisplatin induces the PI-3K-Akt-BAD cascade and that this cascade might be necessary for DNA repair in human ovarian cancer cells treated with cisplatin.
Resistance to cisplatin is a multifactorial phenomenon, the elements of which may be placed in three general categories: (a) reduced accumulation of cisplatin, (b) elevated levels of glutathione and metallothionein, and (c) increased DNA damage tolerance or repair (37, 38, 39, 40) . Because cisplatin acts by forming DNA-DNA cross-links (both intrastrand and interstrand) and DNA-protein cross-links, resulting in DNA damage, the repair of the affected DNA clearly is an important mechanism of resistance to cisplatin (41) . However, the mechanism of this DNA repair is not completely clear. In general, drug-induced apoptosis is dependent on the balance between cell cycle checkpoints and DNA repair mechanisms (42) . Thus, the intracellular signaling that modulates apoptosis may be involved in DNA repair and may be an appropriate target for strategies to overcome the resistance to chemotherapeutic DNA-damaging drugs. This study showed that cisplatin treatment led to activation of Akt and phosphorylation of BAD, and that overexpression of kinase-deficient Akt or BAD caused sensitization to the cytotoxic effects of cisplatin in both sensitive and resistant cells. Thus, the activation of these cascades by cisplatin and the related function of these cascades appeared to be not very different between sensitive and resistant cells, as is also the case in the activation of the ERK and JNK cascades by cisplatin (18) . The reason that Caov-3 is cisplatin-resistant and A2780 is cisplatin-sensitive is unclear. However, both Akt-dependent phosphorylation of BAD Ser-136 and ERK-dependent phosphorylation of BAD Ser-112 seemed to be necessary for maintaining cell viability following the genotoxic stress of cisplatin, leading to resistance to cisplatin in the case of sensitive cells and greater resistance to cisplatin in the case of resistant cells; therefore, blockage of these cascades may be the candidate for strategies to reverse the resistance to cisplatin.
BAD function is modulated by phosphorylation at two sites, Ser-112 and
Ser-136 (14)
. The presence of two phosphorylation sites in
BAD also suggests that the simultaneous activation of different
survival pathways may result in the concomitant phosphorylation of BAD
Ser-112 and Ser-136 by different kinase cascades (12)
. Akt
primarily triggers BAD phosphorylation at Ser-136, and phosphorylation
at this site is sufficient to promote survival in cerebellar granule
cells (12)
. In other cell types, such as
interleukin-3-dependent hematopoietic cells, prevention of cell death
may involve both BAD Ser-112 and BAD Ser-136 (14)
. Thus,
the role of BAD Ser-112 is different, depending on the type of cell or
extracellular stimulus. Moreover, conflicting evidence regarding the
potential role of MEK-dependent BAD phosphorylation has emerged in
recent years. MEK inhibition had no effect on platelet-derived growth
factor-induced BAD phosphorylation (12)
. In contrast, MEK
inhibition was found to inhibit both interleukin-3 and granulocyte
macrophage colony-stimulating factor-dependent BAD
phosphorylation but to have no effect on cell survival
(19)
. It has been reported very recently that the
MAPK-activated pp90-ribosomal S6 kinase family catalyzed the
phosphorylation of BAD Ser-112 (20)
, and that the Ras-MAPK
pathway was involved in the phosphorylation of BAD Ser-112 (21
, 22)
and its function related to dissociation of BAD from
Bcl-XL (21)
. Cisplatin induced the
phosphorylation of BAD at both Ser-112 and Ser-136 (Fig. 2)
. Whereas
cisplatin-induced phosphorylation of BAD Ser-112 was MEK-dependent,
cisplatin-induced phosphorylation of BAD Ser-136 was
PI-3K-Akt-dependent (Figs. 2
and 5)
. In addition, cisplatin-induced
phosphorylation of both BAD Ser-112 and Ser-136 was involved in
maintaining cell viability after cisplatin treatment (Fig. 4)
. Thus,
our results suggest that the ERK and PI-3K-Akt signaling cascades
converge at BAD to suppress the apoptotic effect of BAD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Osaka University Medical School, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan. Phone: 81-6-6879-3354; Fax: 81-6-6879-3359;
E-mail: masa{at}gyne.med.osaka-u.ac.jp 
2 The abbreviations used are: MAP,
mitogen-activated protein; PI-3K, phosphatidylinositol 3-kinase;
PKB, protein kinase B; ERK, extracellular signal-regulated (protein)
kinase; JNK, c-Jun N-terminal protein kinase; MEK, MAP/ERK kinase;
transplatin, trans-diaminodichloroplatinum; MAPK, MAP
kinase; CMV, cytomegalovirus; HA, hemagglutinin.
Received 3/ 9/00. Accepted 8/23/00.
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 D. J Hausenloy, M. M Mocanu, and D. M Yellon Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning Cardiovasc Res, August 1, 2004; 63(2): 305 - 312. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, Y. Nishio, T. Hayasaka, A. Kimura, T. Ohta, M. Saito, J. Kawagoe, K. Takahashi, N. Yada-Hashimoto, et al. Inhibition of NF{kappa}B Increases the Efficacy of Cisplatin in in Vitro and in Vivo Ovarian Cancer Models J. Biol. Chem., May 28, 2004; 279(22): 23477 - 23485. [Abstract] [Full Text] [PDF]
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Z. Jin, F. Gao, T. Flagg, and X. Deng Nicotine Induces Multi-site Phosphorylation of Bad in Association with Suppression of Apoptosis J. Biol. Chem., May 28, 2004; 279(22): 23837 - 23844. [Abstract] [Full Text] [PDF]
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E. Arimoto-Ishida, M. Ohmichi, S. Mabuchi, T. Takahashi, C. Ohshima, J. Hayakawa, A. Kimura, K. Takahashi, Y. Nishio, M. Sakata, et al. Inhibition of Phosphorylation of a Forkhead Transcription Factor Sensitizes Human Ovarian Cancer Cells to Cisplatin Endocrinology, April 1, 2004; 145(4): 2014 - 2022. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, A. Kimura, Y. Ikebuchi, K. Hisamoto, E. Arimoto-Ishida, Y. Nishio, K. Takahashi, K. Tasaka, and Y. Murata Tamoxifen Inhibits Cell Proliferation via Mitogen-Activated Protein Kinase Cascades in Human Ovarian Cancer Cell Lines in a Manner Not Dependent on the Expression of Estrogen Receptor or the Sensitivity to Cisplatin Endocrinology, March 1, 2004; 145(3): 1302 - 1313. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, A. Kimura, Y. Nishio, E. Arimoto-Ishida, N. Yada-Hashimoto, K. Tasaka, and Y. Murata Estrogen Inhibits Paclitaxel-Induced Apoptosis via the Phosphorylation of Apoptosis Signal-Regulating Kinase 1 in Human Ovarian Cancer Cell Lines Endocrinology, January 1, 2004; 145(1): 49 - 58. [Abstract] [Full Text] [PDF]
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J. Hayakawa, C. Depatie, M. Ohmichi, and D. Mercola The Activation of c-Jun NH2-terminal Kinase (JNK) by DNA-damaging Agents Serves to Promote Drug Resistance via Activating Transcription Factor 2 (ATF2)-dependent Enhanced DNA Repair J. Biol. Chem., May 30, 2003; 278(23): 20582 - 20592. [Abstract] [Full Text] [PDF]
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 C. Steenbergen, C. A. Afshari, J. G. Petranka, J. Collins, K. Martin, L. Bennett, A. Haugen, P. Bushel, and E. Murphy Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H268 - H276. [Abstract] [Full Text] [PDF]
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G. W. Krystal, G. Sulanke, and J. Litz Inhibition of Phosphatidylinositol 3-Kinase-Akt Signaling Blocks Growth, Promotes Apoptosis, and Enhances Sensitivity of Small Cell Lung Cancer Cells to Chemotherapy Mol. Cancer Ther., September 1, 2002; 1(11): 913 - 922. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, A. Kimura, K. Hisamoto, J. Hayakawa, Y. Nishio, K. Adachi, K. Takahashi, E. Arimoto-Ishida, Y. Nakatsuji, et al. Inhibition of Phosphorylation of BAD and Raf-1 by Akt Sensitizes Human Ovarian Cancer Cells to Paclitaxel J. Biol. Chem., August 30, 2002; 277(36): 33490 - 33500. [Abstract] [Full Text] [PDF]
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 D. G. Stupack and D. A. Cheresh Get a ligand, get a life: integrins, signaling and cell survival J. Cell Sci., January 10, 2002; 115(19): 3729 - 3738. [Abstract] [Full Text] [PDF]
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 B. Zhang, D. F. Spandau, and A. Roman E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis J. Virol., January 1, 2002; 76(1): 220 - 231. [Abstract] [Full Text] [PDF]
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J. Hayakawa, M. Ohmichi, K. Tasaka, Y. Kanda, K. Adachi, Y. Nishio, K. Hisamoto, S. Mabuchi, S. Hinuma, and Y. Murata Regulation of the PRL Promoter by Akt through cAMP Response Element Binding Protein Endocrinology, January 1, 2002; 143(1): 13 - 22. [Abstract] [Full Text] [PDF]
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K. Hisamoto, M. Ohmichi, Y. Kanda, K. Adachi, Y. Nishio, J. Hayakawa, S. Mabuchi, K. Takahashi, K. Tasaka, Y. Miyamoto, et al. Induction of Endothelial Nitric-oxide Synthase Phosphorylation by the Raloxifene Analog LY117018 Is Differentially Mediated by Akt and Extracellular Signal-regulated Protein Kinase in Vascular Endothelial Cells J. Biol. Chem., December 7, 2001; 276(50): 47642 - 47649. [Abstract] [Full Text] [PDF]
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 A. von Gise, P. Lorenz, C. Wellbrock, B. Hemmings, F. Berberich-Siebelt, U. R. Rapp, and J. Troppmair Apoptosis Suppression by Raf-1 and MEK1 Requires MEK- and Phosphatidylinositol 3-Kinase-Dependent Signals Mol. Cell. Biol., April 1, 2001; 21(7): 2324 - 2336. [Abstract] [Full Text]
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K. Hisamoto, M. Ohmichi, H. Kurachi, J. Hayakawa, Y. Kanda, Y. Nishio, K. Adachi, K. Tasaka, E. Miyoshi, N. Fujiwara, et al. Estrogen Induces the Akt-dependent Activation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells J. Biol. Chem., January 26, 2001; 276(5): 3459 - 3467. [Abstract] [Full Text] [PDF]
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),
wild-type Akt- (
), and AktK179M-expressing (•) Caov-3
(B and D) and A2780 (C)
cells after treatment with the indicated concentrations of cisplatin
(B and C) and transplatin
(D) as described in "Materials and Methods."
), and
BAD2SA-expressing (
) Caov-3 (B and D)
and A2780 (C) cells after treatment with the indicated
concentrations of cisplatin (B and C) and
transplatin (D) as described in "Materials and
Methods."