© 2000 Nature America, Inc. 0929-1903/00/$15.00/⫹0
www.nature.com/cgt
Cre-loxP-mediated bax gene activation reduces growth rate
and increases sensitivity to chemotherapeutic agents in
human gastric cancer cells
Koga Komatsu,1 Susumu Suzuki,1 Tooru Shimosegawa,1 Jun-ichi Miyazaki,2 and
Takayoshi Toyota1
1
Department of Internal Medicine (III), Tohoku University School of Medicine, Sendai, Japan; and
Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka, Japan.
2
Dysregulation of apoptosis may be closely related to the development of cancer and its chemoresistance. Overexpression of Bax,
an inducer of apoptosis, has led to increased cell death in a variety of cancer cell lines. In this study, we investigated the effect of
Bax overexpression in two gastric cancer cell lines, MKN-28 and MKN-45, using a Cre-loxP-mediated inducible expression system.
After induction of bax, both cell lines showed decreased proliferation, partially due to increased cell death. Furthermore,
Bax-expressing MKN-28 cells were more sensitive to cisplatin. These results indicate that up-regulation of the bax gene may provide
a novel strategy for the treatment of gastric cancer. Cancer Gene Therapy (2000) 7, 885– 892
Key words: Bax; Cre-loxP system; chemotherapy; Bcl-2; apoptosis.
A
n increased understanding of the genes involved in
the regulation of apoptosis (or programmed cell
death) has led to the hypothesis that dysregulation of
apoptosis underlies tumor development, progression, and
its resistance to therapies.1 Thus, therapies directed at
altering the levels of expression of apoptosis-regulating
genes to increase the cell death response may be a useful
strategy for treating cancer. Therefore, it is important to
know the effects of modulating these genes in cancer cells.
Members of the bcl-2 gene family, including bcl-2,
bcl-X, bax, bad, and others, play a crucial role in the
regulation of apoptosis.2 The members of this family act
as either inducers or inhibitors of apoptosis by controlling each other’s homo- or heterodimerization. In particular, the Bax protein has been postulated to accelerate apoptosis and to counter the death repressor
function of Bcl-2, and a model has been proposed in
which the ratio of Bcl-2 to Bax determines whether or
not the apoptotic pathway progresses.2,3 Genetic alterations in these genes are often observed in gastric
cancer.4 – 8 Therefore, bcl-2 and bax may be closely
related to the ability of gastric cancer cells to survive.
There have been several studies on the effect of
enhancing Bax expression in cancer cells. It has been
shown that Bax has a tumor suppressor function in a
transgenic mouse brain tumor model9 and in breast
cancer cells transplanted in severe combined immunodeficient mice,10 and gene transfer of bax results in
strong antitumoral activity in non-small cell lung cancer
in vivo.11 Several studies have shown that overexpressed
Bax enhanced apoptosis after treatment with chemotherapeutic agents in human breast cancer cells,12,13 ovarian
cancer cells,14 erythroleukemia cells,15 and rat glioma
cells,16 and after treatment with radiation in human
breast cancer cells.17 However, the effect of enhanced
Bax expression in gastric cancer has not been reported.
In the present study, we examined the effects of
induced Bax overexpression in gastric cancer cells,
rather than the effects of stable Bax overexpression. We
used the Cre-loxP-mediated site-specific recombination
system to regulate bax gene expression.18 This system
can be used to turn on the expression of a transgene that
has been integrated into the host chromosomes in a
silent form, by infecting cells with a Cre-expressing
adenovirus (Ad) vector.19 We applied this system to two
gastric cancer cell lines: MKN-28 cells, which overexpress Bcl-2 protein and have a mutation in the p53 gene,
and MKN-45 cells, which lack a detectable level of Bcl-2
protein and have the wild-type p53 gene.5,20 Our study
shows that overexpressed Bax reduces the growth rate of
these cells and sensitizes MKN-28 cells to cisplatin
(CDDP).
Received July 23, 1999; accepted November 28, 1999.
Address correspondence and reprint requests to Dr. Koga Komatsu,
Department of Internal Medicine (I), Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. E-mail address:
[email protected]
MATERIALS AND METHODS
Cancer Gene Therapy, Vol 7, No 6, 2000: pp 885– 892
Cells and culture conditions
Two human gastric cancer cell lines, MKN-28 and MKN-45,
were purchased from the Japanese Cancer Research Re-
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
sources Bank (Tokyo, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, Mo), 10%
heat-inactivated fetal bovine sera (Life Technologies,
Karlsruhe, Germany), 2 mM L-glutamine, and 1% penicillinstreptomycin.
Gastric cancer cell lines MKN-45 and MKN-28 stably
transfected with inducible bax
The plasmid pCAG-CAT-bax-neo was constructed as follows:
Human Bax-␣ full-length cDNA was produced by reverse
transcriptase-polymerase chain reaction (RT-PCR), and inserted into the EcoRI site of pCY4B, a derivative of the
pCAGGS expression vector.21 The sequence of the Bax-␣
cDNA was confirmed. The loxP-CAT-loxP unit (loxP-flanked
chloramphenicol acetyl-transferase (CAT) gene) was cut out at
the XbaI-BamHI site of the pCAG-CAT-Z plasmid22 (kindly
provided by Dr. Araki, Kumamoto University School of Medicine, Kumamoto, Japan), and inserted into the XbaI-HindIII
site between the promoter and the Bax cDNA of pCY4B-Bax.
The neomycin-resistance gene, under control of the thymidine
kinase gene promoter, was inserted into the SalI site of the
plasmid, resulting in pCAG-CAT-bax-neo. MKN-45 and
MKN-28 cells were seeded into 10-cm dishes and transfected
20 hours later with 20 ␮g/dish of this plasmid DNA by
calcium-phosphate coprecipitation.23 Cells were washed after
24 hours and fed with 10 mL of fresh medium. After another
24 hours, the medium was changed to the selective medium
containing 400 ␮g/mL active G418. The resulting G418-resistant clones that were derived from MKN-45 and MKN-28 were
isolated. The amount of CAT in the extracts from these cells
was determined using the CAT enzyme-linked immunosorbent
assay kit (Boehringer Mannheim, Mannheim, Germany). The
protein concentration of the cell extracts was determined using
a protein assay kit (Bio-Rad, Richmond, Calif). Two stable
transfectants, 45CB1 and 28CB2, derived from MKN-45 and
MKN-28, respectively, were shown to produce Bax mRNA
upon infection with the Cre-expressing Ad vector, AdCAGCre,19 and were used in the following study.
Ad vector infection
As a control for the Ad-mediated gene transfer,
Adex1CAlacZ,24 a ␤-galactosidase (␤-gal)-expressing Ad vector under the transcriptional control of the ␤-actin promoter
and cytomegalovirus enhancer,21 was used. During the exponential growth phase, 45CB1 and 28CB2 cells were plated in
6-well culture plates at a density of 5 ⫻ 105 cells/well 24 hours
before Adex1CAlacZ infection. Immediately before infection,
the culture medium was removed, and suspensions of Ad were
added over the monolayers at various multiplicities of infection
(MOIs) (from 0 to 100). After a 24-hour incubation with
complete culture medium, ␤-gal expression was evaluated
using 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (XGal) as the substrate, as described previously.19
Detection of Cre-loxP-based DNA recombination and
recombinant bax expression
Cells were lysed with 0.5% sodium dodecyl sulfate and 100
␮g/mL proteinase K. DNA was extracted from the lysates with
phenol/chloroform (1:1 vol/vol), precipitated with ethanol, and
dissolved in 10 mM tris(hydroxymethyl)aminomethane (Tris)HCl (pH 7.5) and 1 mM ethylenediaminetetraacetic acid. A
total of 1 ␮g of DNA was subjected to 35 cycles of PCR
amplification, with each cycle consisting of 1 minute at 94°C, 1
minute at 64°C, and 2 minutes at 72°C using a DNA Thermal
cycler 480 (Perkin Elmer Cetus, Branchburg, NJ). The primers
used for amplification of the sequences within the bax gene
were BA1 (5⬘-AGCTGCAGAGGATGATTGCC-3⬘) and BA2
(5⬘-ACAAAGATGGTCACGGTCTG-3⬘); the sequences between the CAG promoter and the Bax cDNA were CA1
(5⬘-CTGCTAACCATGTTCATGCC-3⬘) and BA3 (5⬘-CTCCATGTTACTGTCCAGTTC-3⬘).
For RT-PCR analysis, mRNA was isolated from cells using
the QuickPrep mRNA purification kit (Pharmacia, Piscataway,
NJ). First-strand cDNA was generated from 120 ng of mRNA
with the First-Strand cDNA Synthesis Kit (Pharmacia). A total
of 35 cycles of amplification were carried out, each of which
consisted of denaturation (1 minute at 94°C), annealing (1
minute at 62°C), and primer extension (1 minute at 72°C). The
primers used to amplify the sequences between the area
upstream of the splice donor in the CAG promoter and the
Bax cDNA were CA2 (5⬘-CTCTGACTGACCGCGTTACT3⬘) and BA3. The primers used to amplify the sequences of
␤-actin mRNA were AC1 (5⬘-GAGCTGCGTGTGGCTCCC3⬘) and AC2 (5⬘-TGGCATGGGGGAGGGCATA-3⬘); the
primers used to amplify the sequences specific to endogenous
Bax mRNA were BA3 and BA4 (5⬘-AGGCGGCGGCGGGAGCGG-3⬘).
For Northern blot analysis, total RNA was extracted using
Isogen (Nippon Gene, Toyama, Japan). A total of 15 ␮g of
total RNA was separated on a 1% agarose gel containing 0.66
M formaldehyde, transferred onto a nylon membrane filter
(Amersham, Arlington Heights, Ill), and probed with 32Plabeled human bax-␣ full-length cDNA and human ␤-actin
cDNA (Wako, Osaka, Japan).
Measurement of cell viability by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay
45CB1 and 28CB2 cells were seeded into 96-well plates at a
density of 1 ⫻ 104 cells/well at 24 hours before AdCAG-Cre19
infection, Adex1CAlacZ24 infection, or a mock infection.
Immediately before infection, the culture medium was removed from the wells, and suspensions of AdCAG-Cre or
Adex1CAlacZ at a MOI of 20 were placed onto the cell
monolayers for 1 hour. The cells were then cultured in
complete medium. After incubating for 24, 48, and 72 hours,
cell viability was determined by the MTT assay (Cell Proliferation Kit I, Boehringer Mannheim), which measures viable cell
dehydrogenase activity.25 Absorbance of the fluid was read on
a model 450 microplate reader (Bio-Rad). Cell proliferation
was proportional to the absorbance at the test wavelength (550
nm) minus the absorbance at the reference wavelength (695
nm). The cell viability on each day was expressed as the
absorbance relative to that at 0 hours.
Analysis of DNA fragmentation
DNA fragmentation was analyzed by agarose gel electrophoresis. Cells (2 ⫻ 106) were plated in 6-cm dishes and incubated
the following day with Ad vectors (at a MOI of 20) for 48
hours. Both adherent and floating cells were collected, pelleted
by centrifugation, and incubated in 0.1 mL of 10 mM Tris-HCl
(pH 7.4) containing 10 mM ethylenediaminetetraacetic acid
and 0.5% Triton X-100 at 4°C for 10 minutes. Samples were
subsequently microfuged at 4°C for 10 minutes at 13,000 ⫻ g,
and the supernatants were incubated for 1 hour at 37°C with
ribonuclease A (final concentration 0.4 mg/mL). Next, 2 ␮L of
20 mg/mL proteinase K was added, and incubation was continued for 1 hour. The DNA was then purified and analyzed by
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
electrophoresis in 2.0% agarose gels. The DNA in the gels was
visualized under ultraviolet light after staining with ethidium
bromide.
Transient transfection into MKN-45 cells and analysis
by X-Gal staining
The expression plasmid vectors pCAGBax and pCAG␤GN (a
generous gift of Dr. K. Mitani, University of California Los
Angeles School of Medicine, Los Angeles, Calif), which harbor
the human bax-␣ or lacZ (with a nuclear localization signal)
genes under the transcriptional control of the ␤-actin promoter
and cytomegalovirus enhancer,21 were used for transient transfection into MKN-45 cells. MKN-45 cells (5 ⫻ 105) were plated
on 35-mm dishes and transfected with 2.5 ␮g of pCAGBax or
empty pCXN2 plasmid and 0.25 ␮g of pCAG␤GN plasmid
using the calcium-phosphate method.23 After 48 hours, cell
monolayers were washed twice with phosphate-buffered saline
(PBS), and the adherent cells were fixed with 0.2% glutaraldehyde in PBS. ␤-gal activity was detected using X-Gal as a
substrate, as described previously.19 The number of cells with
a blue-stained nucleus was determined by microscopic examination. Total RNA was extracted from cells using Isogen
(Nippon Gene), and RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer Cetus). The primers CA2
and BA3 were used to detect exogenous Bax mRNA; the
primers AC1 and AC2 were used to detect ␤-actin mRNA.
Drug sensitivity of the cells after the induction of bax
To evaluate the death-promoting activity of Bax in the presence of antitumor drugs, the Bax-induced 28CB2 clone (B28CB2) and the parental 28CB2 clone (P-28CB2) were compared. B-28CB2 and P-28CB2 cells were seeded into 96-well
plates at a density of 1 ⫻ 104 cells in 100 ␮L of medium per
well and incubated for 24 hours to induce all of the tumor cells
to enter the exponential growth phase. A total of 100 ␮L of
medium containing various concentrations of drugs was then
added to each well. Four agents were used: CDDP, mitomycin
C (MMC), etoposide (VP-16), and camptotecin (CPT) (Sigma). The plates were incubated for 24 hours, and cells were
washed once with PBS and incubated for an additional 48
hours in drug-free medium. The antitumoral activity of the
drugs was evaluated by the MTT assay as described above. The
concentration of each drug yielding 50% inhibition of cell
growth (IC50) was calculated using a curve-fitting algorithm.
Data analysis
The Mann-Whitney U test and the two-tailed Student’s t test
were applied when appropriate.
RESULTS
Efficiency of Ad-mediated gene transfer in MKN cells
To test the efficiency of the Ad-mediated gene transfer, 45CB1 and 28CB2 cells were infected with
Adex1CAlacZ at various MOIs. At a MOI of 20, lacZ
gene expression was detected in ⬎90% of the cells of
both lines at 24 hours postinfection (data not shown).
Cancer Gene Therapy, Vol 7, No 6, 2000
887
Establishment of inducible bax transfectant clones
derived from MKN-45 and MKN-28 gastric cancer
cells
To examine the effects of Bax overexpression in gastric
cancer cells, we first produced MKN-45 and MKN-28
transfectants bearing an expression plasmid, pCAGCAT-bax-neo, which directs expression of the bax gene
upon Cre-mediated excision of the loxP-flanked CAT
gene located between the CAG promoter and the bax
gene. Several transfectant clones were tested for their
expression levels of the CAT gene. Clone 45CB1, which
was derived from MKN-45, and clone 28CB2, which was
derived from MKN-28, were found to express high levels
of CAT and were used in the subsequent study. These
clones did not express Bax mRNA until they were
induced to express it upon infection with the Creexpressing Ad vector AdCAG-Cre.19
Detection of Cre-loxP-based DNA recombination and
induction of bax gene expression
To confirm that the Cre-loxP-based DNA recombination
occurred in the 45CB1 and 28CB2 cells after AdCAGCre infection, PCR and RT-PCR analyses were performed. DNA was extracted from each kind of tumor
cell 24 hours after infection with AdCAG-Cre or
Adex1CAlacZ at a MOI of 20. The 5⬘ primer CA1 and
the 3⬘ primer BA3 were designed to hybridize with the
junctional sequence between the CAG promoter and the
bax gene (Fig 1A). Amplification of DNA from AdCAGCre-infected cells using these primers produced a DNA
fragment of 400 bp, whereas this DNA fragment was not
amplified from the DNA of Adex1CAlacZ-infected
cells, although both DNAs produced the same 300-bp
fragment by PCR, using primers BA1 and BA2 (Fig 2A).
Next, mRNA was extracted from tumor cells at 8 and
24 hours after infection with AdCAG-Cre or
Adex1CAlacZ. To amplify the junctional sequences, the
5⬘ primer CA2 and the 3⬘ primer BA3 were used (Fig
1B). A 400-bp fragment was amplified from the cells
infected with AdCAG-Cre at 8 and 24 hours. This 5⬘
primer was designed to hybridize with the upstream
sequence of the splicing donor site. Therefore, this
400-bp fragment was specific for the mRNA from the
recombined transgene. It was not amplified from the
mRNA of Adex1CAlacZ-infected cells, although both
mRNAs produced the same 250-bp fragment specific for
␤-actin mRNA (Fig 2B). These results indicate that the
AdCAG-Cre infection efficiently induced Bax expression
through the Cre-mediated excision of the CAT gene
sequence from the CAG-CAT-bax transgene.
Northern blot analysis revealed that the expression
levels of Bax mRNA, compared with ␤-actin mRNA,
increased ⬃2-fold in 45CB1 cells and ⬃3-fold in 28CB2
cells at 24 hours after AdCAG-Cre infection (Fig 3A).
To distinguish endogenous and exogenous Bax mRNAs,
both of which were the same size (1.0 kb), RT-PCR was
performed using the 5⬘ primer CA2 or BA4 and the 3⬘
primer BA3. Only the mRNA derived from the exog-
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
Figure 1. Structures of the CAG-CAT-Bax gene before and after
Cre-mediated excision of the CAT gene in relation to genomic PCR
analysis (A) and RT-PCR analysis (B). The small arrows indicate the
positions and directions of the primers used for PCR. The expected
sizes of PCR products are indicated.
enously transfected bax gene (CAGBax) increased after
AdCAG-Cre infection (Fig 3B).
Effect of Cre-mediated bax gene activation on MKN
cells
To analyze the effect of bax gene activation on cell
viability, 45CB1 and 28CB2 cells were either infected
with AdCAG-Cre or Adex1CAlacZ at a MOI of 20 or
mock-infected and cultured for 3 days. The MTT assay
was performed each day thereafter to determine cell
viability over time. In Bax-expressing 45CB1 cells, decreased proliferation was seen up to day 3, compared
with 45CB1 cells that were infected with Adex1CAlacZ
or mock-infected (Fig 4A). In contrast, Bax-expressing
28CB2 cells showed decreased proliferation only on day
1 (compared with cells infected with Adex1CAlacZ),
and up to day 2 (compared with mock-infected cells)
(Fig 4B). There was no significant difference in viability
Figure 2. Cre-mediated DNA recombination and induction of bax
expression. A: PCR analysis of DNA from 45CB1 cells (lanes 2 and
3) and 28CB2 cells (lanes 4 and 5) at 24 hours after infection with
Adex1CAlacZ (lanes 2 and 4) or AdCAG-Cre (lanes 3 and 5),
pCAG-CAT-neo DNA (negative control for PCR, lane 6) and pCAGCAT-bax-neo DNA (positive control for PCR, lane 7). Lane 1
contains size markers. B: RT-PCR analysis of poly(A⫹)-RNA from
45CB1 cells (lanes 1, 2, 5, and 6) and 28CB2 cells (lanes 2, 3, 7, and
8) at 8 hours (lanes 1– 4) and 24 hours (lanes 5– 8) after infection with
Adex1CAlacZ (odd lanes) or AdCAG-Cre (even lanes). Lane 9, size
markers. Cre-mediated bax gene expression was seen only in the
cells infected with AdCAG-Cre. The primers used in these PCR
procedures are indicated in Figure 1.
between the cells infected with Adex1CAlacZ and the
mock-infected cells. The morphological changes seen in
the Bax-expressing cells were cell shrinkage and fragmentation, which were usually associated with apoptotic
cell death (Fig 5A). In addition, agarose gel electrophoresis revealed that DNA fragmentation occurred in
45CB1 cells after infection with AdCAG-Cre, but not
after infection with Adex1CAlacZ (Fig 5B). These results indicated that the decreased growth rate after the
induction of bax expression was caused at least in part by
the increasing rate of spontaneous apoptosis in these
cells.
Transient transfection with bax into MKN-45 cells
In the previous experiment, decreased proliferation was
seen in 45CB1 cells after the induction of bax expression,
and we speculated that this decreased proliferation was
caused by an increase in spontaneous apoptosis. Therefore, overexpressed Bax might trigger cell death by itself
in 45CB1 cells. To examine this possibility, we cotrans-
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
Figure 3. Cre-mediated bax gene activation in 45CB1 and 28CB2
cells. A: Northern blot analysis revealed that the bax-␣ mRNA,
relative to the expression of ␤-actin mRNA, increased ⬃2-fold in
45CB1 cells and ⬃3-fold in 28CB2 cells at 24 hours after AdCAGCre infection. B: Competitive RT-PCR analysis of exogenous and
endogenous bax expression. PCR primers were designed to detect
the exogenous (CAGBax) and endogenous Bax mRNA, which
generated PCR bands of 400 bp and 250 bp, respectively.
889
Figure 4. Effect on the cell viability of 45CB1 cells (A) and 28CB2
cells (B) after Cre-mediated bax gene activation. Cells were cultured
3 days after infection with AdCAG-Cre (f) or Adex1CAlacZ (F) at a
MOI of 20 or after mock infection (䡺). The number of viable cells
was measured by the MTT assay. The points show absorbance
relative to that obtained at 0 hours. Results are represented as
means ⫾ SD from two independent trials, each consisting of
triplicate experiments. In 45CB1 cells, a distinct difference in growth
rates was observed between cells with AdCAG-Cre infection and
those with Adex1CAlacZ infection or mock infection, up to day 3. In
28CB2 cells, a distinct difference in growth rates was observed
between cells with AdCAG-Cre infection and those with
Adex1CAlacZ infection or mock infection on day 1. Statistical data
in (A) and (B): ⴱ, Not significant; ⴱⴱ, P ⬍ .05; ⴱⴱⴱ, P ⬍ .005; ⴱⴱⴱⴱ, P ⬍
.001; ⴱⴱⴱⴱⴱ, P ⬍ .0001 (two-tailed Student’s t test).
parent 28CB2 cells (P-28CB2). However, B-28CB2 cells
did not show any change in sensitivity to VP-16.
DISCUSSION
fected the bax expression vector (pCAGBax) or empty
vector (pCXN2, as a control) with the lacZ expression
vector (pCAG␤GN) into parental MKN-45 cells and
determined the number of lacZ-positive cells by X-Gal
staining. The lacZ-positive MKN-45 cells were markedly
reduced at 48 hours posttransfection when cotransfected
with the Bax expression plasmid compared with control
plasmid (0.25 ⫾ 0.3% vs. 12.2 ⫾ 2.1%, P ⬍ .01,
Mann-Whitney U test). RT-PCR was used to detect the
expression of exogenously transfected bax in the cells
transfected with the pCAGBax plasmid (Fig 6). We
conclude, therefore, that Bax expression in MKN-45
cells leads to increased cell death.
Previous studies have shown that stable expression of
Bax is permissive for apoptosis but does not necessarily
induce death in mammalian cells.3,13,14,17 However, it
Induction of bax expression sensitizes 28CB2 cells to
some chemotherapeutic drugs
As shown in Figure 4B, Bax-expressing 28CB2 cells
showed no significant difference in cell proliferation
compared with uninduced 28CB2 cells after day 3 and
were considered to stably express Bax. We examined the
sensitivity of these cells to various chemotherapeutic
agents. Bax-induced 28CB2 cells (B-28CB2) that had
been infected with AdCAG-Cre showed increased sensitivity to some chemotherapeutic agents (Fig 7), with a
significant decrease in the IC50 of CDDP (P ⬍ .001),
MMC (P ⬍ .005), and CPT (P ⬍ .001) compared with
Cancer Gene Therapy, Vol 7, No 6, 2000
Figure 5. A: Effect of bax gene activation on the cell morphology of
45CB1 and 28CB2 cells. Cells were cultured for 48 hours after
Adex1CAlacZ or AdCAG-Cre infection and photographed by phasecontrast microscopy at ⫻100 magnification. After bax induction,
many small dead cells were observed. B: Agarose gel electrophoresis of DNA from 45CB1 cells treated with Adex1CAlacZ or AdCAGCre. Lane 1, size markers. DNA samples were fractionated by
electrophoresis in 2% agarose gels and stained with ethidium
bromide.
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
Figure 6. Gene expression of exogenous bax (pCAGBax) and
␤-actin in MKN-45 cells at 12 hours posttransfection. Each lane B
represents the pCAGBax-transfected cells, each lane C represents
the pCXN2-transfected (control) cells, and each lane M represents
size markers. Only pCAGBax-transfected cells showed exogenous
bax expression.
has been shown that Bax expression was sufficient to
induce apoptosis in Jurkat cells, using a reverse tetracycline-inducible system,26 and in some mammalian cells
that were transiently transfected.11,15,27 These different
Figure 7. Concentrations of each drug yielding 50% inhibition of cell
viability (IC50). The post-Bax-induced 28CB2 clone (u) and parental
28CB2 clone (䡺) were compared. Cells were seeded at a density of
1 ⫻ 104 cells/well in 96-multiwell plates and exposed to various
concentrations (0 –100 ␮g/mL) of each drug for 24 hours. At 48
hours postincubation, the number of viable cells was measured by
MTT assay. The IC50 was calculated using a curve-fitting algorithm,
and the data indicate the mean ⫾ SD for triplicate determinations.
The Bax-induced clone showed a decrease in the IC50 of CDDP (P ⬍
.001), MMC (P ⬍ .005), and CPT (P ⬍ .001), but not in the IC50 of
VP-16 (not significant). P values were determined by the two-tailed
Student’s t test.
results are partly attributable to differences in the experimental systems. In stable transfection, highly expressing
clones may be lost when the product of the transfected
gene is toxic to the cell, or some other protein may be
overproduced to counteract the effects of this product on
the selection process. Because of these potential problems, rather than investigating the effects of a stable
overexpression of Bax, we studied Bax expression in
gastric cancer cells using a Cre-loxP-based DNA recombination system.19 The results obtained using this system
indicated that enhanced Bax expression reduced cell
proliferation in gastric cancer cells, and especially in
MKN-45 cells. We did not perform cell cycle analysis,
but assumed that the decreased proliferation was partially caused by a higher rate of cell death, because more
dead cells and DNA ladders were seen in Bax-induced
cells than were seen in Adex1CAlacZ-infected cells. In a
previous report using stable Bax overexpression in a rat
glioma cell line, a decreased growth rate and increased
rate of spontaneous apoptosis were seen in the baxtransfected cells.16 Moreover, the transient transfection
assay in this study also supported the idea that overexpressed Bax induced cell death in MKN-45 cells. However, the Bax-mediated reduction of lacZ-positive cells
in the transient transfection assay seemed greater than
the reduction with Cre-loxP-mediated Bax-expressing
cells. We speculate that this difference is due to different
amounts of Bax expression. We could not determine the
Bax expression level in the transient transfection assay,
but many copies of plasmid DNA were likely taken up by
the cells with the calcium-phosphate coprecipitation
procedure. Conversely, the bax induction level in the
45CB1 clone was not very high (2-fold, as assessed by
Northern blotting). Therefore, only high levels of Bax
expression may accelerate cell death in MKN-45 cells.
Bax may not be a potent promoter of apoptosis as
reported previously.15,16 In addition, characteristics of
the target cells may greatly affect the outcome of Bax
overexpression. In previous studies using a reverse tetracycline-inducible system, Bax expression was able to
induce apoptosis in Jurkat cells,26 but it was not sufficient to induce apoptosis in R30C and MCF-7 breast
cancer cells.12 In our study, the reduced cell proliferation after bax induction was clearly observed in MKN-45
(45CB1) cells but was only transiently in MKN-28
(28CB2) cells. These differences may be due in part to
the expression levels of Bcl-2 in each cell line: MKN-45
has no detectable Bcl-2, whereas MKN-28 has it at high
levels.5 The levels of Bax in MKN-28 cells are probably
similar or rather low compared with MKN-45 cells (see
Bax mRNA levels at 0 hours in Fig 3A). Therefore, the
ratio of Bcl-2 to Bax is probably higher in MKN-28 cells
compared with MKN-45 cells. A previous report showed
that overexpression of Bcl-2 increased the half-life of
Bax in a leukemia cell line, and suggested the existence
of a feedback mechanism that may help to maintain the
ratio of Bcl-2 to Bax protein in a physiologically appropriate range.28 Therefore, it is possible that Bax turnover
was accelerated by Bax overexpression in MKN-28 cells,
although this may not fully explain the difference in the
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KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
effects of Bax overexpression on cell viability between
MKN-28 and MKN-45 cells. The exact roles played by
the Bax protein are rather difficult to define, in part
because Bax also forms complexes with other cellular
proteins and because different cells may tolerate different steady-state levels of Bax protein.
Initial reports of experiments with Bax overexpression
in breast and ovarian carcinomas demonstrated dramatic increases in sensitivity to chemotherapeutic
agents.12–14 Our study showed that overexpression of
Bax also sensitized MKN-28 gastric cancer cells to
treatment with CDDP, and with MMC and CPT, although weakly. In particular, the sensitivity to treatment
with CDDP was nearly doubled. Previous reports
showed that Bax could sensitize breast cancer cells13 and
lymphoid cells29 to CDDP-induced cell death. CDDP is
thought to act analogously to alkylating agents. Overexpressed Bax may sensitize the cells suffering from DNA
damage caused by DNA alkylation. Overexpression of
Bcl-2 and/or mutation of the p53 gene was implicated in
the CDDP resistance in ovarian cancer cells.30,31 Because MKN-28 cells overexpress Bcl-2 protein and have
a mutated p53 gene, overexpressed Bax might have
bypassed the need for the upstream signal molecules
such as p53. However, Bax overexpression failed to
sensitize human ovarian cancer cells lacking functional
p53 to another platinum analog (carboplatin).14
However, Bax overexpression did not sensitize
MKN-28 cells to etoposide/VP-16 in our study. Previously, two studies reported that Bax overexpression
failed to sensitize human ovarian tumor cells14 or erythroleukemia cells15 to etoposide/VP-16-induced apoptosis despite sensitizing these cells to some other agents. In
contrast, other investigators have reported that Bax
could sensitize these cells to etoposide/VP-16.13,29 Thus,
it remains to be elucidated whether Bax overexpression
has differential effects on CDDP- and etoposide/VP-16induced apoptosis among different cells.
The Cre-loxP-based recombination system has been
used extensively for tissue-specific gene activation or
deletion in transgenic mice32 and for excision, integration, and translocation of cellular genomes in eukaryotic
cells.33,34 Sakai et al19 reported efficient regulation of
gene expression using the Cre-expressing Ad vector in
mammalian cells stably transfected with the lacZ reporter gene. In the present study, we also showed that
the AdCAG-Cre vector could be effectively used to
activate the bax gene in human gastric cancer cell lines.
Therefore, the combination of the loxP-based on/off
switching unit and the AdCAG-Cre vector is a very
useful tool for manipulating the expression of a gene
whose product may be toxic to cells. In addition, this
system is also useful for comparing the pre-gene-expressing state with the post-gene-expressing state in the
same cell.
Many new antitumoral therapies have focused on the
mechanisms of inducing apoptosis.1 Therefore, it is
important to investigate the outcomes of exogenously
modulated apoptosis-regulating genes in cancer cells. In
this study, we showed that Bax overexpression in gastric
Cancer Gene Therapy, Vol 7, No 6, 2000
891
cancer cells resulted in decreased proliferation and
increased sensitivity to CDDP. These pro-apoptotic effects of Bax may prove beneficial in reducing tumors.
Thus, up-regulation of the bax gene could be expected to
be a useful strategy for the treatment of gastric cancer.
ACKNOWLEDGMENTS
We thank Drs. K. Araki and K. Mitani for the generous gift of
plasmids and Drs. I. Saito and K. Sakai for the generous gifts
of the Ad vectors Adex1CAlacZ and AdCAG-Cre, respectively.
REFERENCES
1. Martin SJ, Green DR. Apoptosis and cancer: the failure of
controls on cell death and cell survival. Crit Rev Oncol
Hematol. 1995;18:137–153.
2. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse
on the BCL2 family and cell death. Blood. 1996;88:386 –
401.
3. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that
accelerates programmed cell death. Cell. 1993;74:609 – 619.
4. Tahara E. Molecular mechanism of stomach carcinogenesis. J Cancer Res Clin Oncol. 1993;119:265–272.
5. Ayhan A, Yasui W, Yokozaki H, Seto M, Ueda R, Tahara
E. Loss of heterozygosity at the bcl-2 gene locus and
expression of bcl-2 in human gastric and colorectal carcinomas. Jpn J Cancer Res. 1994;85:584 –591.
6. Krajewska M, Fenoglio-Preiser CM, Krajewski S, et al.
Immunohistochemical analysis of Bcl-2 family proteins in
adenocarcinomas of the stomach. Am J Pathol. 1996;149:
1449 –1457.
7. Yamamoto H, Sawai H, Prucho M. Frameshift somatic
mutations in gastrointestinal cancer of the microsatellite
mutator phenotype. Cancer Res. 1997;57:4420 – 4426.
8. Ouyang H, Furukawa T, Abe T, Kato Y, Horii A. The bax
gene, the promoter of apoptosis, is mutated in genetically
unstable cancers of the colorectum, stomach, and endometrium. Clin Cancer Res. 1998;4:1071–1074.
9. Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T. Bax
suppresses tumorigenesis and stimulates apoptosis in vivo.
Nature. 1997;385:637– 640.
10. Bargou RC, Wagener C, Bommert K, et al. Overexpression of death-promoting gene bax-␣ which is downregulated in breast cancer restores sensitivity to different
apoptotic stimuli and reduces tumor growth in SCID mice.
J Clin Invest. 1996;97:2651–2659.
11. Coll JL, Negoescu A, Louis N, et al. Antitumor activity of
bax and p53 naked gene transfer in lung cancer: in vitro and
in vivo analysis. Hum Gene Ther. 1998;9:2063–2074.
12. Wagener C, Bargou RC, Daniel PT, et al. Induction of the
death-promoting gene bax-␣ sensitizes cultured breastcancer cells to drug-induced apoptosis. Int J Cancer. 1996;
67:138 –141.
13. Sakakura C, Sweeney EA, Shirahama T, et al. Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin
and etoposide. Surg Today. 1997;27:676 – 679.
14. Strobel T, Swanson L, Korsmeyer S, Cannistra SA. BAX
enhances paclitaxel-induced apoptosis through a p53-independent pathway. Proc Natl Acad Sci USA. 1996;93:14094 –
14099.
892
KOMATSU, SUZUKI, SHIMOSEGAWA, ET AL: ENHANCED BAX REDUCES CELL GROWTH IN GASTRIC CANCER
15. Kobayashi T, Ruan S, Clodi K, et al. Overexpression of bax
gene sensitizes K562 erythroleukemia cells to apoptosis
induced by selective chemotherapeutic agents. Oncogene.
1998;16:1587–1591.
16. Vogelbaum MA, Tong JX, Higashikubo R, Gutmann DH,
Rich KM. Transfection of C6 glioma cells with the bax
gene and increased sensitivity to treatment with cytosine
arabinoside. J Neurosurg. 1998;88:99 –105.
17. Sakakura C, Sweeney EA, Shirahama T, et al. Overexpression of bax sensitizes human breast cancer MCF-7 cells to
radiation-induced apoptosis. Int J Cancer. 1996;67:101–
105.
18. Abremski K, Hosse R, Sternberg N. Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell.
1983;32:1301–1311.
19. Sakai K, Mitani K, Miyazaki J. Efficient regulation of gene
expression by adenovirus vector-mediated delivery of the
Cre recombinase. Biochem Biophys Res Commun. 1995;
217:393– 401.
20. Matozaki T, Sakamoto C, Matsuda K, et al. Missense
mutations and a deletion of the p53 gene in human gastric
cancer. Biochem Biophys Res Commun. 1992;182:215–223.
21. Niwa H, Yamamura K, Miyazaki J. Efficient selection for
high-expression transfectants with a novel eukaryotic vector. Gene. 1991;108:193–200.
22. Araki K, Araki M, Miyazaki J, Vassalli P. Site-specific
recombination of a transgene in fertilized eggs by transient
expression of Cre recombinase. Proc Natl Acad Sci USA.
1995;92:160 –164.
23. Graham FL, Van Der Eb AJ. A new technique for the
assay of infectivity of human adenovirus 5 DNA. Virology.
1973;52:456 – 467.
24. Heike Y, Takahashi M, Kanegae Y, Sato Y, Saito I, Saijo
N. Interleukin-2 gene transduction into freshly isolated
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
lung adenocarcinoma cells with adenoviral vectors. Hum
Gene Ther. 1997;8:1–14.
Mosmann T. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. J Immunol Methods. 1983;65:55– 63.
Xiang J, Chao DT, Korsmeyer SJ. Bax-induced cell death
may not require interleukin 1␤-converting enzyme-like
proteases. Proc Natl Acad Sci USA. 1996;93:14559 –14563.
Hunter JJ, Parslow TG. A peptide sequence from Bax that
converts Bcl-2 into an activator of apoptosis. J Biol Chem.
1996;271:8521– 8524.
Miyashita T, Kitada S, Krajewski S, Horne WA, Delia D,
Reed JC. Overexpression of the Bcl-2 protein increases
the half-life of p21Bax. J Biol Chem. 1995;270:26049 –26052.
Simonian PL, Grillot DAM, Merino R, Nunez G. Bax can
antagonize Bcl-XL during etoposide- and cisplatin-induced
cell death independently of its heterodimerization with
Bcl-XL. J Biol Chem. 1996;271:22764 –22772.
Elipoulos AG, Kerr DJ, Herod J, et al. The control of
apoptosis and drug resistance in ovarian cancer: influence
of p53 and Bcl-2. Oncogene. 1995;11:1217–1228.
Perego P, Giarola M, Righetti SC, et al. Association
between cisplatin resistance and mutation of p53 gene and
reduced Bax expression in ovarian carcinoma cell systems.
Cancer Res. 1996;56:556 –562.
Orban PC, Chui D, Marth JD. Tissue- and site-specific
DNA recombination in transgenic mice. Proc Natl Acad Sci
USA. 1992;89:6861– 6865.
Sauer B, Henderson N. Cre-stimulated recombination at
loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res. 1989;17:147–161.
Gu H, Zou YR, Rajewsky K. Independent control of
immunoglobulin switch recombination at individual switch
regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993;73:1155–1164.
Cancer Gene Therapy, Vol 7, No 6, 2000