Carcinogenesis vol.24 no.9 pp.1549±1559, 2003
DOI: 10.1093/carcin/bgg113
Forced expression of antisense 14-3-3b RNA suppresses tumor cell growth in vitro
and in vivo
Akinori Sugiyama1, Yohei Miyagi2, Yuko Komiya1,
Nobuya Kurabe1, Chifumi Kitanaka3, Naoko Kato1,
Yoji Nagashima4, Yoshiyuki Kuchino3 and Fumio
Tashiro1,5
1
Department of Biological Science and Technology, Faculty of Industrial
Science and Technology, Tokyo University of Science, Yamazaki 2641,
Noda-shi, Chiba 278-8510, 2Division of Tumor Pathology, Kanagawa Cancer
Center Research Institute, Nakao 1-1-2, Asahi-ku, Yokohama
241-0815, 3Division of Biophysics, National Cancer Center Research
Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045 and 4Department of
Second Division of Pathology, Yokohama City University School of
Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan
5
To whom correspondence should be addressed
Email: [email protected]
The 14-3-3 family proteins are key regulators of various
signal transduction pathways including malignant transformation. Previously, we found that the expression of the
14-3-3b gene is deregulated as well as c-myc gene in aflatoxin B1 (AFB1 )-induced rat hepatoma K1 and K2 cells. To
elucidate the implication of 14-3-3b in tumor cell growth,
in this paper we analyzed the effect of forced expression of
antisense 14-3-3b RNA on the growth and tumorigenicity
of K2 cells. K2 cells transfected with antisense 14-3-3b
cDNA expression vector diminished their growth ability
in monolayer culture and in semi-solid medium. Expression level of vascular endothelial growth factor mRNA was
also reduced in these transfectants. Tumors that formed
by the transfectants in nude mice were much smaller and
histologically more benign tumors, because of their
decreased level of mitosis compared with those of the parental cells. Frequency of apoptosis detected by terminal
deoxynucleotidyl transferase-mediated dUTP nick end
labeling assay was increased in the transfectant-derived
tumors accompanying the inhibition of angiogenesis. In
addition, over-expression of 14-3-3b mRNA was observed
in various murine tumor cell lines. These results suggest
that 14-3-3b gene plays a pivotal role in abnormal growth
of tumor cells in vitro and in vivo.
Introduction
The 14-3-3 proteins are highly conserved and are found in
broad range of organisms (1). These proteins have attracted
interest because they participate in important cellular events
including cell division and apoptosis (2). The 14-3-3 proteins
are phosphoserine-specific binding proteins and interact with
many proto-oncogene and oncogene products such as Raf-1
Abbreviations: AFB1 , aflatoxin B1 ; DMEM, Dulbecco's modified Eagle's
medium; FCS, fetal calf serum; IGF-II, insulin-like-growth factor-II; INT,
1-p-iodophenyl-p-nitrophenyl-5-phenyltetrazolium chloride; ODNs, oligodeoxynucleotides; TUNEL, terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling; VEGF, vascular endothelial growth factor; vWF,
von Willebrand factor.
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Oxford University Press. All rights reserved
(3±6), Bcr/Bcr-Abl (7), polyoma middle T antigen (8), phosphatidylinositol 3-kinase (9), protein kinase C (10), ASK-1
(11), BAD (12), Cdc25 (13,14), FKHRL1 transcription factor
(15), keratin cytoskelton (16) and TERT telomerase subunit
(17). However, the functional analysis of the 14-3-3 proteins in
oncogenic transformation in vitro and in vivo is very limited.
Recently Takihara et al. (18) reported that the enforced
expression of 14-3-3b RNA in NIH3T3 cells confers on them
a tumorigenicity in nude mice through the stimulation of
mitogen-activated protein kinase (MAPK) cascade. Nonetheless, there are conflicting reports that 14-3-3s related proteins
are strongly down-regulated in SV40-transformed keratinocytes, and in SV40- and v-Ha-ras-transformed epithelial cells
(19,20). Moreover, the enforced expression of 14-3-3z in
combination with ras or raf-1 cannot transform normal
mouse fibroblasts (21). Thus, the real function of 14-3-3 proteins in oncogenic transformation is still vague.
In the course of the study of aflatoxin B1 (AFB1 ) hepatocarcinogenesis, we found that the expression of the 14-3-3b gene
was deregulated together with the c-myc gene, which is known
to cooperate with Raf-1 kinase for a cellular transformation, in
AFB1 -induced rat hepatocellular carcinoma K1 and K2 cells
(22,23). Mutation of 14-3-3b gene locus was also detected in
these cells (24). On the other hand, the mutation and/or deregulated expression of ras family oncogenes and suppressive
oncogene such as p53 and Rb were not detected in K1 and
K2 cells (24,25). Therefore, it is highly possible that 14-3-3b
gene cooperates with c-myc gene in cellular transformation.
In this paper, to investigate whether the over-expression of
the 14-3-3b gene implicates in neoplastic phenotype of K2
cells, we established K2 cells expressing reduced level of
14-3-3b mRNA by the stable transfection with antisense
14-3-3b cDNA expression vector and analyzed their growth
ability in vitro and in vivo. As we would expect, the antisense
transfectants diminished their growth ability in the soft agar
medium as well as in the monolayer culture. Tumors that
formed by these transfectants in nude mice were much smaller
and histologically more benign tumors. In these tumors, high
frequency of apoptosis and inhibition of angiogenesis were
also observed. Moreover, by the simultaneous addition of
antisense 14-3-3b and c-myc oligodeoxynucleotides (ODNs)
the K2 cell growth was diminished in a synergistic manner. We
also found that the 14-3-3b gene was over-expressed in various
tumor cell lines. Thus, it seems likely that the 14-3-3b gene
plays an important role in tumor cell growth perhaps through
the cooperation with the c-myc gene.
Materials and methods
Cells
K1 and K2 cells established from AFB1 -induced rat hepatomas, were cultivated in collagen-coated culture dishes with Dulbecco's modified Eagle's
medium (DMEM) supplemented with 5% fetal calf serum (FCS) as described
previously (23). The cells were maintained at 37 C in a humidified atmosphere
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A.Sugiyama et al.
of 5% CO2 in air. Rat glioblastoma C6 cells and rat neuroblastoma B35, B50,
B65, B103 and B104 cells were a generous gift of Dr M.Rosenblum (University of California, San Francisco, CA) and Dr D.Shubert (The Salk Institute
for Biological Studies, La Jolla, CA). Rat peripheral nerve tumor RT4-AC,
RT4-D and RT4-E cells were obtained from Dr N.Sueoka (University of
Colorado, Boulder, CO). Rat hepatoma AH66tc, AH70Btc, 30 -mRLN-31,
dRLh84, dRLa74 and H4IIE, rat mammary tumor RMT-1 M2 cells, rat kidney
tumor ENUT-1 cells, rat adrenal pheochromocytoma PC12 cells and rat gliosarcoma 9L cells were supplied by the Japanese Cancer Research Resources
Bank (Tokyo, Japan).
Effect of antisense 14-3-3b RNA expression on in vitro cell growth
Cells (2.5 105 ) were plated in 35-mm dishes containing DMEM supplemented with 1% FCS and cultivated for various times. The viable cell number was
determined by exclusion of 0.3% trypan blue. At least 300 cells were counted
for each determination using TATAI hemacytometer. For soft agar assay, cells
(4 103 ) were suspended in 0.3% agar medium containing 2.5 or 10% FCS
and layered on 0.5% agar-coated 35-mm dish and cultivated for 3 weeks. The
colonies formed were stained with 0.25% 1-p-iodophenyl-p-nitrophenyl-5phenyltetrazolium chloride (INT, Sigma) for 24 h and the number of colonies
(40.07 mm in diameter) was counted (31).
Construction of expression vectors and isolation of stable transfectants
The EcoRI±XbaI DNA fragment containing the complete coding region and
partial 30 -untranslated region of rat 14-3-3b cDNA (22) were inserted into the
EcoRI±KpnI site of pcDNA3 expression vector (Invitrogen, Carlsbad, CA).
The antisense orientation of insert was confirmed by DNA sequence analysis.
The pcDNA3 empty vector was used as a control vector. K2 cells were
transfected with 5 mg of expression vector DNA using DOTAP transfection
reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instruction. After 14 days of selection by 1 mg/ml G418 (Life
Technologies, Gaithersburg, MD), resistant clones were expanded and analyzed for the expression level of antisense 14-3-3b RNA based on the reduction
of endogenous 14-3-3b mRNA and protein expression levels by northern and
western blots, respectively. Two transfected cell lines, A16 and A17, were
selected for further experiments as an antisense 14-3-3b transfectant, because
their expression levels of 14-3-3b protein were much lower than those of the
other antisense transfectants. The empty vector transfectant, V11, was selected
as a control transfectant.
Tumorigenicity in nude mice
K2 and its derived cell lines including V11, A16 and A17 cells (1 107 /200 ml
phosphate-buffered saline/flank) were subcutaneously inoculated into 5-weekold athymic nude mice (BALB/c Jcl nu/nu, Clea Japan, Tokyo, Japan). Tumor
volume was calculated as (short axis2 long axis)/2. The tumor tissues were
fixed in 10% formalin and embedded in paraffin. Sections were subjected to
staining with hematoxylin and eosin, terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay (32) and immunohistochemistry. All mice were kept under a 12:12 h light:dark cycle at 22±24 C at
the experimental animal facility, Tokyo University of Science. Standard
laboratory feed (MR standard, Nousan LTD, Kanagawa, Japan) and tap
water were given ad libitum. Mouse care and handling conformed to the NIH
guidelines for animal research. The experimental protocols were approved by
the Institutional Animal Care and Use Committee.
Northern blot analysis
Total RNAs were prepared from various cells including antisense 14-3-3b
cDNA-transfected cells and various adult Fisher 344 rat tissues by the acid
guanidinium thiocyanate±phenol±chloroform extraction methods (26). Fisher
344 rats were obtained from Charles River Japan (Kanagawa, Japan). Aliquots
of total RNA (20 mg) from each sample were electrophoresed on 1% agarose±
6% formaldehyde gel and transferred to Hybond N‡ Nylon membrane
(Amersham, Arlington Height, IL), and the filter was hybridized with 32 Plabeled 1.3 kb EcoRI±XbaI fragment of rat 14-3-3b cDNA (22) at 42 C, 0.9 kb
SmaI±XbaI fragment of neor gene of pIRESneo vector (Clontech, Palo Alto,
CA) at 42 C, 0.7 kb BamHI±PstI fragment of PCR amplified rat 14-3-3s
cDNA (27) at 42 C, 0.5 kb EcoRI±HindIII fragment of PCR amplified
human vascular endothelial growth factor (VEGF) cDNA (28) at 37 C and
0.7 kb BamHI fragment of PCR amplified rat insulin-like growth factor-II
(IGF-II) cDNA (29) at 42 C in solution containing 50% formamide as
described previously (30). Developed X-ray films were scanned in a Macintosh
Performa 6410 computer and the expression levels of these mRNAs were
densitometrically quantified with the NIH Image 1.62 ppc program (National
Institute of Health, Bethesda, MD) using 28S ribosomal RNA as a internal
control.
Western blot analysis
Cells were lysed in SDS sample buffer (62.5 mM Tris±HCl, pH 6.8, 2% SDS,
10% glycerol) without bromophenol blue and 2-mercaptoethanol, and sonicated for 4 s. The lysates were heated at 95 C for 5 min and then cleared by
centrifugation. Protein concentration was determined by BCA-kit (Pierce,
Rockford, IL), and then 1/20 vol of 2-mercaptoethanol was added to the
lysates. Aliquots of 30 mg of the lysates were subjected to 12% SDS±PAGE
and transferred to Clear Blot Membrane (ATTO, Tokyo, Japan). 14-3-3b
protein was detected with 1/1000 diluted anti-rat 14-3-3b polyclonal antibody
in 20 mM Tris±HCl pH 7.4-buffered saline containing 0.02% Tween 20
(TBST) and 2.5% non-fat dried milk for 12 h at 4 C. The antibody was
prepared using glutathione-S transferase-rat 14-3-3b fusion protein as an antigen in our laboratory. Actin, Raf and c-Myc were detected with 1/400 diluted
anti-actin antibody (Sigma, St Louis, MO), 1/1000 diluted anti-Raf-1 antibody
(Calbiochem, San Diego, CA) and 1/100 diluted anti-c-Myc monoclonal
antibody (Oncogene Research Products, Cambridge, MA), respectively, in
TBST containing 2.5% non-fat dried milk for 12 h at 4 C. After washing
with the same buffer, the filters were incubated with 1/5000 diluted horseradish
peroxidase-conjugated anti-rabbit IgG (Biosource, Camarillo, CA) for 14-3-3b,
actin and Raf-1 or horseradish peroxidase-conjugated anti-mouse IgG
(Vector, Burlingame, CA) for c-Myc and the Enhanced Chemiluminescence
Reagent (Amersham). Developed X-ray films were scanned in a Macintosh
Performa 6410 computer and the expression level of 14-3-3b protein was
densitometorically quantified with the NIH Image 1.62 ppc program using
actin as an internal control.
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TUNEL assay
Sections of paraffin-embedded tumors were used. Apoptosis was evaluated
using TUNEL assay kit (ApopTag; Intergen, Purchase, NY). To quantify the
relative number of apoptotic cells in tumors, the number of TUNEL-positive
cells per field (40) was counted in the 20±40 randomly chosen fields of three
independent tumors formed by each transfectant. The data were expressed as
the mean number of apoptotic cells/field SE.
Immunohistochemistry
The formalin-fixed and paraffin-embedded specimens were sectioned and
immunostained using the diaminobenzidine-based detection methods in conjunction with standard avidin±biotin enhancement system as described previously (33). The specimens were boiled in 10 mM citrate buffer (pH 6.8) to
renature antigen. Anti-human von Willebrand factor (vWF) mouse monoclonal
antibody (DAKO, Glostrup, Denmark) was used at a 1:50 dilution. Nuclei were
counterstained with hematoxylin.
Treatment with antisense ODNs
The sequence of antisense ODNs corresponding to the flanking region of
translational initiation site of rat 14-3-3b, raf-1 and c-myc were 50 -TACTGGTACCTGTTT-30 (22), 50 -CTGTCTGTGCTCCAT-30 (34) and 50 -CACGTTGAGGGGCAT-30 (35), respectively. The nonsense ODN consisting of random
base sequence used for the control experiment was 50 -CTACGGAAGTAGACT-30 . Lipofectamin, a cationic lipid (Life Technologies) was used to
increase the uptake of ODNs into cells (36). K2 cells were seeded in DMEM
containing 1% FCS at 2.5 104 cells/well of 48-well dish, pre-incubated with
4 mg/ml Lipofectamine in serum-free DMEM for 20 min, and then treated with
various concentrations of ODNs. After 4 h, the medium containing ODNs and
Lipofectamin was replaced with DMEM containing 1% FCS and ODNs.
ODNs were added every 12 h, and after 48 h the number of viable cells were
determined.
Statistical analysis
All data were expressed as mean SE of the indicated number of experiments.
Comparisons of data were carried out using ANOVA followed by Tukey
post-hoc test for multi-group comparisons or Student's t-test. Differences
were considered statistically significant at P 5 0.05. The software package
KaleidaGraph 3.6 (Synergy Software, Reading, PA) was used for the statistical
analysis.
Results
Effect of enforced expression of antisense 14-3-3b RNA on
cell growth in vitro
In order to analyze the function of 14-3-3b protein in tumor
cell growth, we introduced antisense 14-3-3b cDNA expression vector into K2 cells, which are over-expressing 14-3-3b
Tumorigenesis by 14-3-3b protein
protein (22). Northern blot analysis revealed that although in
A16 and A17 transfectants exogenous antisense 2.4 kb RNA
was not detected perhaps due to the instability of doublestranded mRNA, the 2.9 kb endogenous 14-3-3b mRNA
expression was diminished to 55 and 36% of the parental
control cells, respectively (Figure 1A). The additional band
at 1.9 kb may be 14-3-3h mRNA (37). Expression of the neor
gene inserted in the expression vector as a selection marker
was clearly detected in all of the isolated cell lines including
the empty vector-transfected V11 cells. These results suggest
that in A16 and A17 cells the endogenous 14-3-3b mRNA
expression could be suppressed by the enforced expression of
antisense 14-3-3b RNA. To confirm the assumption, the
expression level of 14-3-3b protein in these cells was analyzed
by western blotting with anti-14-3-3b antibody. As expected,
the expression levels of 27 kDa 14-3-3b protein in both A16
and A17 cells were reduced to 65 and 59% compared with that
of the parental K2 cells, respectively, without any effects on
the actin expression (Figure 1B). While, in the vacant vectorintroduced V11 cells the expression level of 14-3-3b protein as
well as actin was similar to that of the parental K2 cells. These
results indicate that forced expression of antisense 14-3-3b
RNA specifically suppresses the endogenous expression of
14-3-3b protein. We could not isolate the transfectants whose
expression levels of 14-3-3b protein were lower than those of
A16 and A17 cells, showing the possibility that more severe
down-regulation of 14-3-3b protein does not support the
Fig. 1. Suppression of 14-3-3b expression by transfection with antisense
cDNA. (A) Expression of endogenous and exogenous 14-3-3b mRNA in
the antisense 14-3-3b cDNA expression vector-transfected K2 cells. Aliquots
of 20 mg total RNAs from the parental K2 cells, empty vector-transfected
V11 cells and antisense cDNA-transfected A16 and A17 cells were
electrophoresed on 1% agarose±6% formaldehyde gel and transferred to
a nylon filter. (Upper panel) The filter was hybridized with 32 P-labeled
14-3-3b cDNA and exposed to X-ray film. (Middle panel) The same filter
was deprobed and rehybridized with 32 P-labeled neor cDNA as a probe.
(Lower panel) Picture of ethidium bromide stained 28S ribosomal RNAs is
indicated to allow comparison of total amount of RNA employed. (B)
Expression level of 14-3-3b protein in the transfectants. Aliquots of 30 mg
cell lysates from the transfectants were electrophoresed on 12% SDS±PAGE
and transferred to Clear Blot Membrane (ATTO). Western blot analysis
was performed using a polyclonal antibody against rat 14-3-3b protein
(upper) and actin (lower), respectively.
survival or growth of the transfectants. Therefore, we further
analyzed the role of 14-3-3b in K2 cell growth in vitro and
in vivo using A16 and A17 transfectants. To determine
whether the enforced expression of antisense 14-3-3b RNA
causes a diminution of cell proliferation, the cell growth rates
of the transfectants were assessed over 3 days. As shown in
Figure 2A, the growth rates of both A16 and A17 cells in the
medium supplemented with 1% FCS were diminished to 51.8
and 48.3%, respectively, as compared with that of the parental
K2 cells after 3 days. The growth rate of vacant vectorintroduced V11 cells was similar to that of K2 cells. We
also examined the effect of antisense 14-3-3b RNA on the
anchorage-independent growth of K2 cells. The transfectants
were cultured in the soft agar for 3 weeks and after staining
with INT the number of colonies (>0.07 mm in diameter) was
counted. The colony-forming abilities of A16 and A17 transfectants in the presence of 10% FCS were diminished to 32.6
and 62.9% of the control K2 cells, respectively (Figure 2B). In
the presence of 2.5% FCS the colony-forming abilities of A16
and A17 cells were extremely reduced and estimated to be 21.4
and 25.8% of the control K2 cells, respectively (Figure 2B
and C); whereas, the colony-forming ability of V11 cells was
comparable with that of K2 cells in both 2.5 and 10% FCS.
These results suggest that 14-3-3b protein participates in the
growth control of K2 cells in soft agar medium as well as in
monolayer culture.
Reduction of tumorigenicity by suppression of endogenous
14-3-3b expression
We next investigated the effect of suppression of 14-3-3b
expression on the tumorigenicity of K2 cells. Empty vectortransfected V11 cells formed progressively growing solid
tumors in all mice as well as the parental K2 cells, when
1 107 cells were subcutaneously inoculated into the left
flanks of nude mice (Figure 3A). At 41 days, the average
volume of tumors developed by K2 and V11 were estimated
to be 4.00 and 3.91 cm3 , respectively (Figure 3B). Even though
solid tumors were developed in all mice by the inoculation of
1 107 A16 and A17 cells into the right flanks, both cells
produced much smaller tumors as compared with those of K2
and V11 cells (Figure 3A and B). After 41 days of the injection, the resulting tumors were fixed with 10% formalin and
stained with eosin and hematoxylin for histological examination. As shown in Figure 3C and D, histological examination
indicated that malignant features such as mitoses and/or
nuclear atypia in tumors formed by A16 and A17 cells
were markedly diminished as compared with those of K2and V11-derived tumors. The number of mitotic cells in
A16- and A17-derived tumors was 46.7 and 42.8% of those
in K2-derived tumors, respectively.
Since suppression of apoptosis is essential for tumor progression (38), using TUNEL assay we further examined the
extent of apoptosis in tumors developed by the transfectants.
As shown in Figure 4A, the apoptotic cells were substantially
observed in tumors developed by both A16 and A17 cells. The
apoptotic index of tumors developed by A16 and A17 cells
were estimated to be 3.1- and 2.9-fold as compared with those
of K2 cells, respectively (Figure 4B).
Several lines of evidence show that angiogenesis is required
for the growth, expansion and metastasis of solid tumors
(39,40). We therefore observed the angiogenesis of tumors
derived from the transfectants using anti-vWF monoclonal
antibody. As shown in Figure 5A, the tumors derived from
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A.Sugiyama et al.
Fig. 2. Inhibition of K2 cell growth by enforced expression of antisense 14-3-3b RNA. (A) Cells were seeded in 1% FCS-containing medium at 2.5 105 cells per
35-mm dish and cultivated for various times. Each value is the average SE of triplicate culture dishes. P 5 0.001 compared with parent K2 cells.
(B) Cells were seeded in 0.3% agar medium containing 10% FCS or 2.5% FCS at 4 103 cells per 35-mm dish, and cultivated for 3 weeks. Then colonies
formed (>0.07 mm in diameter) were counted after staining with INT. Each value is average SE of triplicate culture dishes. P 5 0.01, P 5 0.01 compared
with parent K2 cells. (C) After 3 weeks, photographs of colonies formed were taken. Bars represent 5 mm.
A16 and A17 cells had a lower microvessel density as compared with those from K2 and V11 cells. The number of vWFpositive cells by A16 and A17 were estimated to be 34.8 and
20.8% of the control K2 cells, respectively (Figure 5B).
Angiogenesis in hepatocellular carcinoma has been reported
to be largely influenced by VEGF and IGF-II (41±43). Therefore, we analyzed the expression level of VEGF mRNA in the
transfectants by northern blotting. In A16 and A17 cells, the
expression levels of VEGF mRNA were markedly downregulated and estimated to be 38 and 27% of the parent K2
cells, respectively (Figure 5C). In V11 cells, the expression
level of VEGF transcript was comparable with that of K2 cells.
On the other hand, the expression of IGF-II mRNA was hardly
detected in any cells including the parental K2 cells, although
the significant expression was observed in P1 rat brain used as
a control. These results suggest that 14-3-3b implicates in the
regulation of VEGF gene expression.
Cooperation among 14-3-3b, Raf-1 and c-Myc in K2 cell
growth
It has been reported that the maintenance of active conformation of Raf-1 kinase by the association with 14-3-3 protein is
required for the Ras/MAPK signal transduction pathway (3±6).
Moreover, the expression of c-myc gene, which synergizes
with ras, raf-1 and bcl-2 oncogenes in cell transformation
(44±49), is deregulated in K2 cells (50). We examined, therefore, the effect of antisense 14-3-3b, raf-1 and c-myc ODNs on
the K2 cell growth to clarify the mechanism of abnormal cell
growth and tumorigenicity of K2 cells. As shown in Figure 6,
as compared with nonsense ODN, the proliferation of K2 cells
was efficiently inhibited by the antisense 14-3-3b, raf-1 or
c-myc ODNs dependent on the increase in the amount of
ODNs added, and their inhibition levels were similar to each
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other. The inhibition observed in the presence of nonsense
ODN is perhaps due to the non-specific inhibition of protein
synthesis as observed previously (35). To confirm that these
antisense ODNs specifically suppress each corresponding
protein, we analyzed the expression level of 14-3-3b, Raf-1
and c-Myc in the respective antisense ODN-treated K2 cells by
western blot using actin as an internal control. In the treatment
with 10 mM each of the antisense ODN, the levels of 14-3-3b,
Raf-1 and c-Myc proteins were decreased to 54, 29 and 73% of
the nonsense ODN-treated control, respectively (Figure 6B).
On the other hand, the expression levels of actin and the other
two proteins except for the protein treated by the corresponding antisense ODNs were not changed, showing that the antisense ODNs specifically down-regulated the expression level
of corresponding proteins.
To examine the cooperation among 14-3-3b, Raf-1 and
c-Myc in the K2 cell growth, K2 cells were treated with the
various combinations of the corresponding antisense ODNs.
As shown in Figure 6C, the combinatorial treatment with the
antisense ODNs of 14-3-3b and raf-1 inhibited the K2 cell
growth in an additive manner. While, synergistic inhibition
was observed by the treatment of antisense c-myc ODN with
antisense 14-3-3b or raf-1 ODNs. Simultaneous treatment
with the three ODNs severely inhibited K2 cell growth in a
synergistic manner. These results support the assumption
that 14-3-3b maintains the active form of Raf-1 kinase and
synergizes the c-Myc function in cell proliferation.
Over-expression of 14-3-3b mRNA in various tumors
The data described above strongly supported the idea that
14-3-3b gene could be classified as a proto-oncogene. We
examined, therefore, the expression level of 14-3-3b mRNA
in various murine tumor cell lines including hepatomas
Tumorigenesis by 14-3-3b protein
Fig. 3. Effect of antisense 14-3-3b RNA expression on tumorigenicity of K2 cells. K2 cells and the transfectants were injected into the flanks of nude mice
at the concentration of 1 107 cells per flank alone or together with V11 and A16 or A17. (A) After 41 days, photographs of tumors formed were taken.
(B) After 41 days, the average volumes of tumors formed in the right and left flanks of three to six mice were estimated and represented with the mean value SE.
P 5 0.01 compared with the parental K2 cells. (C) Histological analysis of tumors formed by the transfectants. After 41 days of the injections of transfectants,
the resulting tumors were fixed in 10% formalin and stained with hematoxylin and eosin for histological analysis. Arrowheads show mitosis and/or nuclear
atypia. Bars represent 20 mm. (D) The number of mitotic cells was counted at a lower magnification (40). Each value is the average SE of 20±40 fields of
triplicate tumor sections. P 5 0.01 compared with the parent K2 cells.
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A.Sugiyama et al.
Fig. 4. High frequency of apoptosis in tumors generated by antisense 14-3-3b cDNA transfectants. The tumor sections as described in Figure 3 were also analyzed
by TUNEL assay. (A) Photographs show typical TUNEL-positive cells in tumors formed by the transfectants. The arrowheads indicate TUNEL-positive cells.
Bars present 50 mm. (B) The number of TUNEL-positive cells was counted at a lower magnification (40). Each value is the average SE of
20±40 fields of triplicate tumor sections. P 5 0.01 compared with the parent K2 cells.
(K1, K2, AH66tc, AH70Btc, 30 -mRLN-31, dRLh84, dRLa74
and H4IIE), mammary tumor RMT-1 M2 cells, kidney tumor
ENUT-1 cells, adrenal pheochromocytoma PC12 cells, which
are well known to differentiate into sympathetic neuron-like
cells in the presence of nerve growth factor (51), peripheral
nerve tumors (RT4-AC, RT4-D and RT4-E), glioblastoma C6
cells, gliosarcoma 9L cells and neuroblastoma cells (B35, B50,
B65, B103 and B104). Northern blot analysis was carried out
using 32 P-labeled 1.3 kb EcoRI±XbaI fragment of rat 14-3-3b
cDNA. As shown in Figure 7A and B, high levels of 14-3-3b
mRNA expression were detected in all tumors examined as
compared with those of corresponding adult rat tissues except
for the mammary tissue. Nonetheless, the expression levels of
14-3-3b mRNA in hepatomas such as AH66tc, dRLa74 and
H4IIE, mammary tumor RMT-2 M2 and kidney tumor ENUT-1
were lower than that of K2 cells. To confirm the possibility
that the over-expression of the 14-3-3b is implicated in the
proliferation of various tumors like K2 cells, glioblastoma C6
cells were also treated with 14-3-3b antisense ODNs. As
shown in Figure 7C, the growth of C6 cells was efficiently
inhibited in a dose-dependent manner, while the nonsense
ODNs showed only a slight effect. These results suggest that
deregulated expression of 14-3-3b gene participates in malignant transformation of various types of tumors as well as
AFB1 -induced rat hepatoma K2 cells.
Lack of 14-3-3s mRNA expression in normal rat liver and K2
cells
14-3-3s is specifically expressed in epithelial cells (19,20) and
a p53-regulated inhibitor of G2 /M progression (27). Recently
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Vercoutter-Edouart et al. (52) reported that the expression
of 14-3-3s protein is strongly down-regulated in breast
cancer cell lines and primary breast carcinomas. To examine
whether the down-regulation of 14-3-3s is implicated in
hepatocarcinogenesis, we analyzed the expression level of
14-3-3s mRNA in K2 and its derived cell lines including
V11, A16 and A17 cells and various rat tissues by northern
blot. As shown in Figure 8, the expression of 14-3-3s mRNA
was hardly detected in K2 and its derived cell lines and in the
various tissues including the liver. While, only in the testis,
placenta and newborn rat skin the significant expressions were
observed. These results show that the 14-3-3s gene expression
is extremely tissue-specific and is completely deficient in the
normal liver. Thus, the silencing of 14-3-3s gene expression
observed in K2 and its derived cell lines might not implicate in
their abnormal growth and tumorigenicity.
Discussion
In this report, we demonstrated that the reduction of overexpressed 14-3-3b protein in AFB1 -induced rat hepatoma K2
cells by the transfection with antisense 14-3-3b cDNA expression vector resulted in the significant suppression of their
growth rate in monolayer culture and colony formation ability
in semi-solid medium (Figure 2). Tumors formed in nude mice
by these antisense transfectants, whose expression levels of
VEGF mRNA were markedly diminished, were much smaller
and histologically more benign tumors compared with those of
the empty vector-introduced V11 cells and the parental K2
Tumorigenesis by 14-3-3b protein
Fig. 5. Inhibition of angiogenesis and VEGF mRNA expression by enforced expression of antisense 14-3-3b RNA. (A) Analysis of angiogenesis in tumors
generated by K2, V11, A16 and A17 transfectants. After 41 days of the injection of the transfectants, the sections of tumors were immunostained with anti-vWF
monoclonal antibody using the diaminobenzidine-based detection method in conjunction with standard avidin±biotin enhancement system. Arrowheads indicate
neovascularization (brown stain). Bars present 50 mm. (B) The number of vWF-positive cells was counted at a lower magnification (40). Each value is the
average SE of 20±40 fields of triplicated tumor sections. P 5 0.01 compared with that of parental K2 cells. (C) Suppression of VEGF mRNA expression in
antisense 14-3-3b cDNA transfectants. Aliquots of 20 mg total RNAs from the parental K2, V11, A16 and A17 cells and P1 rat brain as a positive control were
electrophoresed on 1% agarose±6% formaldehyde gel and transferred to a nylon filter. (Upper panel) The filter was hybridized with 32 P-labeled human VEGF
cDNA and exposed to an X-ray film. (Middle panel) The same filter was deprobed and rehybridized with 32 P-labeled rat IGF-II cDNA as a probe. (Lower panel)
Picture of ethidium bromide stained 28S ribosomal RNAs is indicated to allow comparison of total amount of RNA employed.
cells (Figures 3±5). In addition to these results, we found that
over-expression of 14-3-3b mRNA was detected in various
types of tumor cell lines and the growth of C6 cells was also
inhibited by antisense 14-3-3b ODNs (Figure 7). It has been
reported that NIH3T3 cells acquire tumorigenicity by the
enforced expression of sense 14-3-3b cDNA (18). Therefore,
the 14-3-3b gene could be classified as a proto-oncogene and
its deregulated expression may implicate in malignant transformation of various types of tumors, although there is a
conflicting report that binding of 14-3-3b to the C-terminus
of Wee1 increases Wee1 stability and kinase activity, and
arrests cell cycle at G2 / M phase (53).
14-3-3s (also called HME or stratifin) is an epithelial cell
marker and down-regulated in the epithelial cells transformed
by SV40 or c-Ha-ras oncogene (19,20). It has been also
reported that the expression of 14-3-3s protein is markedly
down-regulated in breast cancer cell lines MCF-7 and MDAMB-231 and in primary breast carcinomas (52,54). These
reports indicate that 14-3-3s is a tumor suppressor and its
down-regulation largely participates in the neoplastic transformation of breast epithelial cells. Nonetheless, our results
showed that the expression of 14-3-3s mRNA was limited in
the testis, placenta and newborn rat skin, while the expression
in K2 and its derived cell lines was hardly detected as well as
the normal rat liver (Figure 8). Thus, it seems likely that downregulation of 14-3-3s may not deeply implicate in hepatocarcinogenesis.
Verification of various cooperative genetic lesions in multiple steps of carcinogenesis is a major issue in cancer research.
Cooperating oncogenes are categorized as those, which
complement each other in signal transduction pathways of
many growth factors (55). This means that the growth advantage acquired by the synergic action of multiple oncogenes is
the result of a balanced mitogenic signal, which constitutively
stimulates both cell cycle progression and cell survival.
Although only over-expression of c-myc gene without serum
causes apoptosis, c-myc seems to be very important in
oncogene synergy (56,57). It has been reported that c-myc
1555
A.Sugiyama et al.
Fig. 6. Effect of antisense 14-3-3b, raf-1 and c-myc ODNs on K2 cell proliferation. (A) Dose-dependent effects of antisense 14-3-3b, raf-1 and c-myc ODNs
on the K2 cell growth. K2 cells (2.5 104 ) were treated with various concentrations of nonsense and antisense rat 14-3-3b, raf-1 and c-myc ODNs in the presence
of lipofectamine. Antisense and nonsense ODNs were added every 12 h. After 48 h from the beginning of treatment, the number of viable cells was counted by
exclusion of 0.3% trypan blue. Each value is average SE of triplicate culture dishes. P 5 0.01 compared with the nonsense ODN treatment at the same
concentration. (B) Expression level of 14-3-3b, Raf-1 and c-Myc proteins in the antisense ODNs-treated cells. Aliquots of 20 mg cell lysates from the cells
treated with 10 mM each of ODNs were electrophoresed on 12% SDS±PAGE and transferred to Clear Blot Membrane (ATTO). Western blot was performed with
specific antibodies against 14-3-3b, Raf-1, c-Myc and actin as indicated. N and A represent nonsense and antisense ODNs, respectively. Star represents the
expression of protein corresponding to the antisense ODN treatment. (C) Cooperation among 14-3-3b, Raf-1 and c-Myc in the K2 cell growth. K2 cells were
treated with various combinations of antisense 14-3-3b, raf-1 and c-myc ODNs in the same conditions described in (A) and after 48 h the number of viable cells
was counted. Each value is average SE. P 5 0.01 and P 5 0.01 compared with the nonsense ODN treatment at the same concentration and the single
treatment of the corresponding ODN, respectively.
1556
Tumorigenesis by 14-3-3b protein
Fig. 7. Over-expression of 14-3-3b mRNA in various murine tumor cell lines. (A) Northern blot analysis of 14-3-3b mRNA expression in various tumor cell lines.
Total RNAs were extracted from hepatoma cells (K1, K2, AH66tc, AH70Btc, 30 -mRLN-31, dRLh84, dRLa74 and H4IIE), mammary tumor RMT-1 M2 cells,
kidney tumor ENUT-1 cells, adrenal pheochromocytoma PC12 cells, peripheral nerve tumor cells (RT4-AC, RT4-D and RT4-E), glioblastoma C6 cells,
gliosarcoma 9L cells and neuroblastoma cells (B35, B50, B65, B103 and B104). Aliquots of 20 mg total RNAs were electrophoresed on 1% agarose±6%
formaldehyde gel and hybridized with 32 P-labeled 1.3 kb EcoRI±XbaI fragment of rat 14-3-3b cDNA. (B) Northern blot analysis of 14-3-3b mRNA expression
in adult rat tissues. Total RNAs were extracted from the adult rat liver, brain and kidney. The expression level of 14-3-3b was analyzed by northern blot.
(C) Effects of antisense 14-3-3b ODNs on the C6 cell growth. C6 cells (2.5 104 ) were treated with various concentrations of nonsense and antisense rat 14-3-3b
ODNs in the presence of lipofectamine. Antisense and nonsense ODNs were added every 12 h. After 48 h from the beginning of treatment, the number of viable
cells was counted by exclusion of 0.3% trypan blue. Each value is average SE of triplicate culture dishes. P 5 0.01 compared with the nonsense ODN
treatment at the same concentration.
Fig. 8. Expression level of 14-3-3s mRNA in various rat tissues. Total RNAs were extracted from the parental K2 cells, the antisense 14-3-3b cDNA
transfectants, various adult Fisher 344 rat tissues and newborn rat skin. Aliquots of 20 mg total RNAs were electrophoresed and analyzed by northern blot using
32
P-labeled 0.7 kb BamHI±PstI fragment of PCR amplified rat 14-3-3s cDNA as a probe.
1557
A.Sugiyama et al.
synergizes with ras, raf-1 and bcl-2 oncogenes in cell transformation (44±49). Moreover, by the interaction with Bcl-2,
Raf-1 kinase has been reported to prevent myeloid cell apoptosis (58). In this experiment, we observed that the treatment
with antisense 14-3-3b and raf-1 ODNs inhibited K2 cell
growth in an additive manner. On the other hand, synergistic
inhibition was observed by the addition of antisense c-myc
ODNs in combination with antisense 14-3-3b and/or raf-1
ODN (Figure 6C). Therefore, it is quite possible that 14-3-3b
synergizes with c-Myc in cell transformation through the
activation of Raf-1 kinase.
Previously, we reported that 14-3-3b gene is over-expressed
in K2 cells, in which the c-myc gene expression is also deregulated (23). Furthermore, K1 cells, another cell line established
from AFB1 -induced rat hepatoma, also over-expresses both
c-myc and 14-3-3b genes (23,24). In both cells, mutations in
suppressive oncogenes such as p53 and Rb and oncogene such
as c-Ki-ras, c-Ha-ras and N-ras were not detected (24,25,59),
although point mutation has been detected by others in the ras
family genes in AFB1 -induced rat hepatomas (60,61). In this
study we found that the reduction of over-expressed 14-3-3b
protein by the enforced expression of antisense 14-3-3b
RNA caused the severe suppression of tumorigenicity of
K2 cells in nude mice (Figure 3). Thus, the activation of
c-myc and 14-3-3b genes might be critical steps in AFB1
hepatocarcinogenesis.
It is well known that angiogenesis is essential for the development and progression of solid tumors. Without the ability to
recruit new blood vessels, it is likely that most tumors could
not grow beyond a limited size (40). Thus, it seems that tumors
continuously produce angiogenic factors, permitting tumor
cell growth and expansion. On the other hand, the inhibition
of angiogenesis by endostatin, a 20 kDa C-terminal fragment
of collagen XVIII, causes substantial apoptosis in tumors (62).
Furthermore, it has been reported that angiogenic factors such
as VEGF, IGF-II and basic fibroblast growth factor implicate
in angiogenesis during hepatocarcinogenesis (41±43,63). In
this study we found that in tumors formed by the antisense
A16 and A17 transfectants, a high level of apoptosis was
induced (Figure 4). Moreover, the expression level of VEGF
mRNA in A16 and A17 cells was significantly diminished and
angiogenesis in tumors formed by these transfectants was also
decreased (Figure 5). Therefore, it is highly possible that the
deregulated expression of 14-3-3b gene largely participates in
tumorigenic angiogenesis through the constitutive stimulation
of VEGF mRNA expression, and the severe inhibition of
tumorigenicity together with apoptosis observed in tumors
formed by the antisense transfectants is mediated, at least in
part, by the inhibition of angiogenesis. However, at present we
cannot rule out the possibility that anti-apoptotic function of
14-3-3b implicates in tumorigenicity through the suppression
of proapoptotic factors such as BAD (12), forkhead family
transcription factor FKHRL1 (15) and ASK-1 kinase (11).
In summary, our results support the idea that 14-3-3b protein, a regulator of various signal transduction pathways, is a
real proto-oncogene and its deregulated expression largely
implicates in various neoplastic transformations including
AFB1 hepatocarcinogenesis.
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Received April 20, 2002; revised June 17, 2003; accepted June 24, 2003
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