Forced expression of antisense 14-3-3ß 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, Japan
2 Division of Tumor Pathology, Kanagawa Cancer Center Research Institute, Nakao 1-1-2, Asahi-ku, Yokohama 241-0815, Japan
3 Division of Biophysics, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
4 Department 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: ftashir{at}rs.noda.tus.ac.jp
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Abstract
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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 14-3-3ß 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 14-3-3ß in tumor cell growth, in this paper we analyzed the effect of forced expression of antisense 14-3-3ß RNA on the growth and tumorigenicity of K2 cells. K2 cells transfected with antisense 14-3-3ß 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 14-3-3ß mRNA was observed in various murine tumor cell lines. These results suggest that 14-3-3ß gene plays a pivotal role in abnormal growth of tumor cells in vitro and in vivo.
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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Introduction
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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 (36), 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 14-3-3ß 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 14-3-3
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 14-3-3
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 14-3-3ß 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 14-3-3ß 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 14-3-3ß gene cooperates with c-myc gene in cellular transformation.
In this paper, to investigate whether the over-expression of the 14-3-3ß gene implicates in neoplastic phenotype of K2 cells, we established K2 cells expressing reduced level of 14-3-3ß mRNA by the stable transfection with antisense 14-3-3ß 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 14-3-3ß and c-myc oligodeoxynucleotides (ODNs) the K2 cell growth was diminished in a synergistic manner. We also found that the 14-3-3ß gene was over-expressed in various tumor cell lines. Thus, it seems likely that the 14-3-3ß gene plays an important role in tumor cell growth perhaps through the cooperation with the c-myc gene.
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Materials and methods
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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 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, 3'-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).
Construction of expression vectors and isolation of stable transfectants
The EcoRIXbaI DNA fragment containing the complete coding region and partial 3'-untranslated region of rat 14-3-3ß cDNA (22) were inserted into the EcoRIKpnI 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 µg 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 14-3-3ß RNA based on the reduction of endogenous 14-3-3ß 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 14-3-3ß transfectant, because their expression levels of 14-3-3ß protein were much lower than those of the other antisense transfectants. The empty vector transfectant, V11, was selected as a control transfectant.
Northern blot analysis
Total RNAs were prepared from various cells including antisense 14-3-3ß cDNA-transfected cells and various adult Fisher 344 rat tissues by the acid guanidinium thiocyanatephenolchloroform extraction methods (26). Fisher 344 rats were obtained from Charles River Japan (Kanagawa, Japan). Aliquots of total RNA (20 µg) from each sample were electrophoresed on 1% agarose6% formaldehyde gel and transferred to Hybond N+ Nylon membrane (Amersham, Arlington Height, IL), and the filter was hybridized with 32P-labeled 1.3 kb EcoRIXbaI fragment of rat 14-3-3ß cDNA (22) at 42°C, 0.9 kb SmaIXbaI fragment of neor gene of pIRESneo vector (Clontech, Palo Alto, CA) at 42°C, 0.7 kb BamHIPstI fragment of PCR amplified rat 14-3-3
cDNA (27) at 42°C, 0.5 kb EcoRIHindIII 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 TrisHCl, 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 µg of the lysates were subjected to 12% SDSPAGE and transferred to Clear Blot Membrane (ATTO, Tokyo, Japan). 14-3-3ß protein was detected with 1/1000 diluted anti-rat 14-3-3ß polyclonal antibody in 20 mM TrisHCl 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 14-3-3ß 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 14-3-3ß, 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 14-3-3ß protein was densitometorically quantified with the NIH Image 1.62 ppc program using actin as an internal control.
Effect of antisense 14-3-3ß RNA expression on in vitro cell growth
Cells (2.5 x 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 x 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-5-phenyltetrazolium chloride (INT, Sigma) for 24 h and the number of colonies (>0.07 mm in diameter) was counted (31).
Tumorigenicity in nude mice
K2 and its derived cell lines including V11, A16 and A17 cells (1 x 107/200 µl phosphate-buffered saline/flank) were subcutaneously inoculated into 5-week-old athymic nude mice (BALB/c Jcl nu/nu, Clea Japan, Tokyo, Japan). Tumor volume was calculated as (short axis2 x 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 transferase-mediated dUTP nick end labeling (TUNEL) assay (32) and immunohistochemistry. All mice were kept under a 12:12 h light:dark cycle at 2224°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.
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 (x40) was counted in the 2040 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 avidinbiotin 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 14-3-3ß, raf-1 and c-myc were 5'-TACTGGTACCTGTTT-3' (22), 5'-CTGTCTGTGCTCCAT-3' (34) and 5'-CACGTTGAGGGGCAT-3' (35), respectively. The nonsense ODN consisting of random base sequence used for the control experiment was 5'-CTACGGAAGTAGACT-3'. 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 x 104 cells/well of 48-well dish, pre-incubated with 4 µg/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 < 0.05. The software package KaleidaGraph 3.6 (Synergy Software, Reading, PA) was used for the statistical analysis.
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Results
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Effect of enforced expression of antisense 14-3-3ß RNA on cell growth in vitro
In order to analyze the function of 14-3-3ß protein in tumor cell growth, we introduced antisense 14-3-3ß cDNA expression vector into K2 cells, which are over-expressing 14-3-3ß 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 double-stranded mRNA, the 2.9 kb endogenous 14-3-3ß 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 14-3-3
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 14-3-3ß mRNA expression could be suppressed by the enforced expression of antisense 14-3-3ß RNA. To confirm the assumption, the expression level of 14-3-3ß protein in these cells was analyzed by western blotting with anti-14-3-3ß antibody. As expected, the expression levels of 27 kDa 14-3-3ß 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 vector-introduced V11 cells the expression level of 14-3-3ß protein as well as actin was similar to that of the parental K2 cells. These results indicate that forced expression of antisense 14-3-3ß RNA specifically suppresses the endogenous expression of 14-3-3ß protein. We could not isolate the transfectants whose expression levels of 14-3-3ß protein were lower than those of A16 and A17 cells, showing the possibility that more severe down-regulation of 14-3-3ß protein does not support the survival or growth of the transfectants. Therefore, we further analyzed the role of 14-3-3ß in K2 cell growth in vitro and in vivo using A16 and A17 transfectants. To determine whether the enforced expression of antisense 14-3-3ß 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 vector-introduced V11 cells was similar to that of K2 cells. We also examined the effect of antisense 14-3-3ß 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 14-3-3ß protein participates in the growth control of K2 cells in soft agar medium as well as in monolayer culture.

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Fig. 1. Suppression of 14-3-3ß expression by transfection with antisense cDNA. (A) Expression of endogenous and exogenous 14-3-3ß mRNA in the antisense 14-3-3ß cDNA expression vector-transfected K2 cells. Aliquots of 20 µg total RNAs from the parental K2 cells, empty vector-transfected V11 cells and antisense cDNA-transfected A16 and A17 cells were electrophoresed on 1% agarose6% formaldehyde gel and transferred to a nylon filter. (Upper panel) The filter was hybridized with 32P-labeled 14-3-3ß cDNA and exposed to X-ray film. (Middle panel) The same filter was deprobed and rehybridized with 32P-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 14-3-3ß protein in the transfectants. Aliquots of 30 µg cell lysates from the transfectants were electrophoresed on 12% SDSPAGE and transferred to Clear Blot Membrane (ATTO). Western blot analysis was performed using a polyclonal antibody against rat 14-3-3ß protein (upper) and actin (lower), respectively.
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Fig. 2. Inhibition of K2 cell growth by enforced expression of antisense 14-3-3ß RNA. (A) Cells were seeded in 1% FCS-containing medium at 2.5 x 105 cells per 35-mm dish and cultivated for various times. Each value is the average ± SE of triplicate culture dishes. *P < 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 x 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 < 0.01, **P < 0.01 compared with parent K2 cells. (C) After 3 weeks, photographs of colonies formed were taken. Bars represent 5 mm.
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Reduction of tumorigenicity by suppression of endogenous 14-3-3ß expression
We next investigated the effect of suppression of 14-3-3ß expression on the tumorigenicity of K2 cells. Empty vector-transfected V11 cells formed progressively growing solid tumors in all mice as well as the parental K2 cells, when 1 x 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 x 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 K2- and 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.

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Fig. 3. Effect of antisense 14-3-3ß 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 x 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 < 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 µm. (D) The number of mitotic cells was counted at a lower magnification (x40). Each value is the average ± SE of 2040 fields of triplicate tumor sections. *P < 0.01 compared with the parent K2 cells.
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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).

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Fig. 4. High frequency of apoptosis in tumors generated by antisense 14-3-3ß 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 µm. (B) The number of TUNEL-positive cells was counted at a lower magnification (x40). Each value is the average ± SE of 2040 fields of triplicate tumor sections. *P < 0.01 compared with the parent K2 cells.
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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 A16 and A17 cells had a lower microvessel density as compared with those from K2 and V11 cells. The number of vWF-positive cells by A16 and A17 were estimated to be 34.8 and 20.8% of the control K2 cells, respectively (Figure 5B).

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Fig. 5. Inhibition of angiogenesis and VEGF mRNA expression by enforced expression of antisense 14-3-3ß 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 avidinbiotin enhancement system. Arrowheads indicate neovascularization (brown stain). Bars present 50 µm. (B) The number of vWF-positive cells was counted at a lower magnification (x40). Each value is the average ± SE of 2040 fields of triplicated tumor sections. *P < 0.01 compared with that of parental K2 cells. (C) Suppression of VEGF mRNA expression in antisense 14-3-3ß cDNA transfectants. Aliquots of 20 µg total RNAs from the parental K2, V11, A16 and A17 cells and P1 rat brain as a positive control were electrophoresed on 1% agarose6% formaldehyde gel and transferred to a nylon filter. (Upper panel) The filter was hybridized with 32P-labeled human VEGF cDNA and exposed to an X-ray film. (Middle panel) The same filter was deprobed and rehybridized with 32P-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.
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Angiogenesis in hepatocellular carcinoma has been reported to be largely influenced by VEGF and IGF-II (4143). 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 down-regulated 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 14-3-3ß implicates in the regulation of VEGF gene expression.
Cooperation among 14-3-3ß, 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 (36). Moreover, the expression of c-myc gene, which synergizes with ras, raf-1 and bcl-2 oncogenes in cell transformation (4449), is deregulated in K2 cells (50). We examined, therefore, the effect of antisense 14-3-3ß, 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 14-3-3ß, raf-1 or c-myc ODNs dependent on the increase in the amount of ODNs added, and their inhibition levels were similar to each 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 14-3-3ß, 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 µM each of the antisense ODN, the levels of 14-3-3ß, 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.

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Fig. 6. Effect of antisense 14-3-3ß, raf-1 and c-myc ODNs on K2 cell proliferation. (A) Dose-dependent effects of antisense 14-3-3ß, raf-1 and c-myc ODNs on the K2 cell growth. K2 cells (2.5 x 104) were treated with various concentrations of nonsense and antisense rat 14-3-3ß, 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 < 0.01 compared with the nonsense ODN treatment at the same concentration. (B) Expression level of 14-3-3ß, Raf-1 and c-Myc proteins in the antisense ODNs-treated cells. Aliquots of 20 µg cell lysates from the cells treated with 10 µM each of ODNs were electrophoresed on 12% SDSPAGE and transferred to Clear Blot Membrane (ATTO). Western blot was performed with specific antibodies against 14-3-3ß, 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 14-3-3ß, Raf-1 and c-Myc in the K2 cell growth. K2 cells were treated with various combinations of antisense 14-3-3ß, 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 < 0.01 and **P < 0.01 compared with the nonsense ODN treatment at the same concentration and the single treatment of the corresponding ODN, respectively.
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To examine the cooperation among 14-3-3ß, 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 14-3-3ß 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 14-3-3ß 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 14-3-3ß maintains the active form of Raf-1 kinase and synergizes the c-Myc function in cell proliferation.
Over-expression of 14-3-3ß mRNA in various tumors
The data described above strongly supported the idea that 14-3-3ß gene could be classified as a proto-oncogene. We examined, therefore, the expression level of 14-3-3ß mRNA in various murine tumor cell lines including hepatomas (K1, K2, AH66tc, AH70Btc, 3'-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 32P-labeled 1.3 kb EcoRIXbaI fragment of rat 14-3-3ß cDNA. As shown in Figure 7A and B, high levels of 14-3-3ß 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 14-3-3ß 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 14-3-3ß is implicated in the proliferation of various tumors like K2 cells, glioblastoma C6 cells were also treated with 14-3-3ß 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 14-3-3ß gene participates in malignant transformation of various types of tumors as well as AFB1-induced rat hepatoma K2 cells.

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Fig. 7. Over-expression of 14-3-3ß mRNA in various murine tumor cell lines. (A) Northern blot analysis of 14-3-3ß mRNA expression in various tumor cell lines. Total RNAs were extracted from hepatoma cells (K1, K2, AH66tc, AH70Btc, 3'-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 µg total RNAs were electrophoresed on 1% agarose6% formaldehyde gel and hybridized with 32P-labeled 1.3 kb EcoRIXbaI fragment of rat 14-3-3ß cDNA. (B) Northern blot analysis of 14-3-3ß mRNA expression in adult rat tissues. Total RNAs were extracted from the adult rat liver, brain and kidney. The expression level of 14-3-3ß was analyzed by northern blot. (C) Effects of antisense 14-3-3ß ODNs on the C6 cell growth. C6 cells (2.5 x 104) were treated with various concentrations of nonsense and antisense rat 14-3-3ß 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 < 0.01 compared with the nonsense ODN treatment at the same concentration.
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Lack of 14-3-3
mRNA expression in normal rat liver and K2 cells
14-3-3
is specifically expressed in epithelial cells (19,20) and a p53-regulated inhibitor of G2/M progression (27). Recently Vercoutter-Edouart et al. (52) reported that the expression of 14-3-3
protein is strongly down-regulated in breast cancer cell lines and primary breast carcinomas. To examine whether the down-regulation of 14-3-3
is implicated in hepatocarcinogenesis, we analyzed the expression level of 14-3-3
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 14-3-3
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 14-3-3
gene expression is extremely tissue-specific and is completely deficient in the normal liver. Thus, the silencing of 14-3-3
gene expression observed in K2 and its derived cell lines might not implicate in their abnormal growth and tumorigenicity.
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Discussion
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In this report, we demonstrated that the reduction of over-expressed 14-3-3ß protein in AFB1-induced rat hepatoma K2 cells by the transfection with antisense 14-3-3ß 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 cells (Figures 3
5). In addition to these results, we found that over-expression of 14-3-3ß mRNA was detected in various types of tumor cell lines and the growth of C6 cells was also inhibited by antisense 14-3-3ß ODNs (Figure 7). It has been reported that NIH3T3 cells acquire tumorigenicity by the enforced expression of sense 14-3-3ß cDNA (18). Therefore, the 14-3-3ß 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 14-3-3ß to the C-terminus of Wee1 increases Wee1 stability and kinase activity, and arrests cell cycle at G2/M phase (53).
14-3-3
(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 14-3-3
protein is markedly down-regulated in breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas (52,54). These reports indicate that 14-3-3
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 14-3-3
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 down-regulation of 14-3-3
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 synergizes with ras, raf-1 and bcl-2 oncogenes in cell transformation (4449). 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 14-3-3ß 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 14-3-3ß and/or raf-1 ODN (Figure 6C). Therefore, it is quite possible that 14-3-3ß synergizes with c-Myc in cell transformation through the activation of Raf-1 kinase.
Previously, we reported that 14-3-3ß 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 14-3-3ß 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 14-3-3ß protein by the enforced expression of antisense 14-3-3ß RNA caused the severe suppression of tumorigenicity of K2 cells in nude mice (Figure 3). Thus, the activation of c-myc and 14-3-3ß 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 (4143,63). 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 14-3-3ß 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 14-3-3ß 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 14-3-3ß 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.