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Carcinogenesis, Vol. 21, No. 11, 2073-2077, November 2000
© 2000 Oxford University Press

Carcinogenesis

Role of the ß isoform of 14-3-3 proteins in cellular proliferation and oncogenic transformation

Yoshihiro Takihara2, Yoshiko Matsuda1 and Junichi Hara1

Department of Medical Genetics and Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University and
1 Department of Developmental Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 14-3-3 proteins are associated with proto-oncogene and oncogene products. Here, we generated NIH 3T3 cells overexpressing the ß isoform of the 14-3-3 proteins (14-3-3 ß) to examine the function of this isoform in cellular proliferation and oncogenic transformation. Overexpression of 14-3-3 ß in NIH 3T3 cells stimulated cell growth and supported anchorage-independent growth in soft agar medium and tumor formation in nude mice. To elucidate the molecular mechanisms of 14-3-3 ß-mediated NIH 3T3 transformation, we examined the activity of mitogen-activated protein kinase (MAPK) after serum stimulation. Overexpression of 14-3-3 ß augmented MAPK activity after serum stimulation, and MAPK activity correlated well with the amount of 14-3-3 ß expression. The colony-forming ability of NIH 3T3 cells overexpressing 14-3-3 ß in soft agar medium was efficiently abolished by exogenous expression of a dominant-negative mutant of MEK1 and 14-3-3 ß physically interacted with Raf-1 in these cells. These findings indicate that 14-3-3 ß has oncogenic potential, mainly through enhancement of Raf-1 activation and resultant augmentation of signaling in the MAPK cascade.

Abbreviations: MAPK, mitogen-activating kinase; PKC, protein kinase C; RA, retinoic acid.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The 14-3-3 proteins are highly conserved and are found in a broad range of organisms (1). The remarkable conservation of the 14-3-3 protein structures during evolution implies their fundamental importance in cellular physiology. These proteins were first implicated in the regulation of tyrosine and tryptophan hydroxylases (2) and protein kinase C (PKC) (3,4), and other findings suggested that they participate in mitogenic signaling pathways (5). The 14-3-3 proteins are specific phosphoserine-binding proteins (6) and interact with many proto-oncogene and oncogene products (5), e.g. Raf-1 (7–13), B-Raf (14,15), polyoma middle T antigen (16), Bcr (11,17), cdc25 phosphatases (18), phosphatidyl inositol 3-kinase (19), phosphorylated BAD (20), glucocorticoid receptor (21), insulin-like growth factor I receptor (22), insulin receptor substrate I (22) and cbl (23). There is, however, little direct evidence from in vivo studies concerning a role for 14-3-3 proteins in cellular proliferation and transformation.

In this study we established NIH 3T3 cells overexpressing the ß isoform of the 14-3-3 protein (14-3-3 ß) and showed that it has a role in the proliferation and oncogenic transformation of NIH 3T3 cells. We provide further evidence suggesting that augmentation of signaling in the mitogen-activated protein kinase (MAPK) cascade is required for 14-3-3 ß-mediated oncogenic transformation of NIH 3T3 cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Transfection
Cells were plated at a density of 5x105 per 90 mm dish 24 h before transfection. A plasmid, pSGß, was constructed by inserting a 1270 bp EcoRI–XbaI DNA fragment of a cDNA, Rae-1209, encoding 14-3-3 ß, into the mammalian expression vector pSG5 (24). Twenty micrograms of pSGß and 1 µg of pSV2neo (25) were precipitated by calcium phosphate and then added to the cells (26); after 48 h, 400 µg/ml of G418 (Gibco-BRL, Rockville, MD, USA) was added. After 14–21 days, G418-resistant colonies were isolated and propagated in G418-containing medium. Cells were also transfected with 20 µg of pMEK1DN (27) and 1 µg of pSV2hygro (28) and selected with 100 µg/ml of hygromycin B (Sigma Chemicals, St Louis, MO, USA). Exogenous expression of 14-3-3 ß and MEK1DN was detected by northern and immunoblot analyses.

Characterization of the growth properties
NIH 3T3 transfectants were seeded as described above and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS) and 200 µg/ml G418. The medium was changed every third day and cell numbers were counted daily. Doubling times were calculated as described above. When several successive cell counts showed no increase in number, the saturation density was recorded. All data shown are the means and standard deviations from two independent experiments. Statistical analysis was done using Student's t-test. Differences were considered significant when P values were <0.01.

Anchorage independence assay
Cells were suspended at a density of 1x104/ml in 0.4% low melting point agarose (Gibco-BRL) in DMEM supplemented with 5% FCS, and were poured over 0.53% agarose in the same medium. Two weeks after plating, a total of 200 single cells and colonies was randomly counted under a phase-contrast Nikon microscope (type Diaphot-TMD). The proportion of the cells that had formed colonies (>0.07 mm in diameter) was defined as the colony-forming ability (29,30). Each experiment was performed twice.

Tumorigenicity assay
Six-week-old nude mice (BALB/c nu/nu; Clea Japan, Tokyo, Japan) were injected subcutaneously on the flank with 1x106 cells suspended in 200 µl of phosphate-buffered saline (31). The latency period was defined as the time required to develop visible tumors. Mice inoculated with control cells were inspected regularly for 24 weeks.

Immunoblot analysis
Cells were lysed in buffer containing 100 mM NaCl, 10 mM Tris–HCl pH 7.6, 1 mM EDTA and 100 µg/ml phenylmethylsulphonyl fluoride. An equal volume of 2x SDS sample buffer was added and samples were clarified by boiling, sonication and centrifugation. The clarified extracts were separated by electrophoresis on a 10% SDS–polyacrylamide gel. Proteins were transferred to nylon membranes, immunoblotted with primary antibody raised against bacterially synthesized 14-3-3 ß protein, rabbit anti-Mek1 polyclonal antibody, mouse anti-phospho-Erk monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rat anti-c-Myc monoclonal antibody (9E10) (TAGO, Burlingame, CA, USA), and visualized with horseradish peroxidase-conjugated anti-rabbit, rat and mouse IgG antibodies (Cappel, Durham, NC, USA) and enhanced chemiluminescence detection reagents (DuPont, Wilmington, DE, USA) (32).

Immunoprecipitations
A confluent dish of NIH 3T3 cells was rinsed with ice-cold phosphate-buffered saline (PBS) and were lysed in TBST (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, supplemented with 1 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, 10 mM ß-glycerophosphate, 5 mM NaF, 10 mM sodium pyrophosphate and 1 mM orthovanadate). The lysate was precipitated with an anti-14-3-3 ß antibody and protein G–Sepharose (Pharmacia, Uppsala, Sweden) for 1 h at 4°C. Immunoprecipitates were washed with TBST and subjected to immunoblot analysis with an anti-Raf-1 antibody (Santa Cruz Biotechnology).

MAPK assay
Cells were lysed by directly adding lysis buffer containing 20 mM Tris–HCl (pH 8.0), 20 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 2 mM EGTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and 0.1% Triton X-100 in a total volume of 200 µl. Extracts (15 µl) were then assayed by adding 10 µl of substrate buffer containing 6 mM substrate peptide, HEPES, 300 µM sodium orthovanadate and 0.05% sodium azide (pH 7.4), and 5 µl of ATP buffer containing 0.3 mM [{gamma}-32P]ATP (300 µCi/ml) and 90 mM MgCl2. After a 30 min incubation at 37°C, 10 µl of 300 mM orthophosphoric acid was added to terminate the reaction. Thirty microliters of each sample was spotted on to phosphocellulose disks, washed three times for 30 min in 0.5% phosphoric acid, and washed once for 5 min in distilled water. The radioactivity on each disk was then determined by scintillation counting (33).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Overexpression of 14-3-3 ß induces tumorigenic transformation of NIH 3T3 cells
We isolated cDNAs for five isoforms of the mouse 14-3-3 proteins, which correspond to early responsive genes to retinoic acid (RA) in F9 cells (34). One of the cDNAs, Rae-1209, encodes mouse 14-3-3 ß. The Rae-1209 cDNA was subcloned into a mammalian expression vector, pSG5 (24), and co-transfected into NIH 3T3 cells with a pSV2neo vector as a selection marker (25). We then established six independent clones overexpressing 14-3-3 ß and examined two representative clones, Nß-1 and Nß-2, in detail. The levels of 14-3-3 ß mRNAs in the Nß-1 and Nß-2 cells were 12- and seven-fold higher than those in the control NIH 3T3 cells, respectively (Figure 1Go and Table IGo). The overexpression of 14-3-3 ß in these cells was confirmed by immunoblot analysis (data not shown).



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  		Fig. 1. Northern blot analysis of 14-3-3 ß mRNAs. Total RNAs were prepared from NC-1, NC-2 and NC-3 (three control NIH 3T3 cell lines established after transfecting pSV2neo) and from Nß-1 and Nß-2 (two independently established NIH 3T3 cell lines overexpressing 14-3-3 ß). The signals of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown in the lower part of the figure as a control for the amount of RNA loaded in each lane. The positions and sizes of the mRNAs detected by the 14-3-3 ß probe are indicated on the right. The 1.7 kb band corresponds to exogenous 14-3-3 ß mRNA, and the 2.8 and 1.2 kb bands to endogenous 14-3-3 ß mRNA.

 

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  		Table I. Growth properties of NIH 3T3 cells overexpressing 14-3-3ß
 
In DMEM supplemented with 5% FCS, Nß-1 and Nß-2 cells showed significantly faster doubling times and significantly higher saturation density than the control NIH 3T3 cells (P<0.01) (Table 1Go). However, in medium supplemented with 0.5 % FCS, there was no significant difference in the doubling times between these two transfectants and the control NIH 3T3 cells (data not shown). Interestingly, the Nß-1 and Nß-2 cells formed colonies in soft agar medium within 2 weeks with an efficiency ranging from 2 to 5.5%, whereas the control NIH 3T3 cells remained as single cells (Table IGo and Figure 2Go). Similar growth properties were observed in four other independently established NIH 3T3 clones overexpressing 14-3-3 ß (data not shown). The higher the levels of 14-3-3 ß in the transfectants, the stronger were the stimulatory effects on their growth properties, such as growth rates, saturation density and colony-forming ability in soft agar medium (Table IGo). The colonies derived from Nß-1 and Nß-2 cell lines were smaller than those derived from the NIH 3T3 cell line transformed by the activated H-ras gene (Figure 2Go).



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  		Fig. 2. Anchorage-independent growth of NIH 3T3 cells overexpressing 14-3-3 ß. The NIH 3T3 cell lines were seeded in soft agar medium and representative colonies were observed by microscopy (100x magnification). NC-1, an NIH 3T3 cell line trasfected with pSVneo; ras, an NIH 3T3 cell line transformed by the activated H-ras gene; Nß-1 and -2, NIH 3T3 cell lines overexpressing 14-3-3 ß. The colonies derived from Nß-2 cells tended to be smaller than those derived from Nß-1 cells.

 
Moreover, in all the nude mice injected with Nß-1 or Nß-2 cells, tumors developed within 4–12 weeks, whereas in no mouse injected with control NIH 3T3 cells did a tumor develop in the 24 week observation period (Table IGo and Figure 3Go). The time required to produce tumors of at least 3 mm diameter was considered the latency period. The latency period was longer in Nß-1 and Nß-2 cell lines than in activated ras-transformed NIH 3T3 cell line. After re-establishing several G418-resistant cell lines from the tumors in nude mice, we confirmed that they had properties essentially similar to those of their ancestral Nß-1 or Nß-2 cells (data not shown). These results indicate that additional mutations had not been induced in these cells during passage in nude mice, and that overexpression of 14-3-3 ß leads to tumorigenesis.



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  		Fig. 3. Tumorigenicity of NIH 3T3 cell lines overexpressing 14-3-3 ß. Cells (1x106) suspended in 200 µl PBS were injected subcutaneously in nude mice. The tumours derived from Nß-1 and Nß-2 cell lines were observed 2 and 8 weeks after injection, respectively. No tumors were observed in mice injected with control NIH 3T3 cells in the 24 week observation period (Table IGo).

 
Correlation of 14-3-3 expression and augmented MAPK activity after serum stimulation
We examined the kinetics of MAPK activity during serum stimulation in NIH 3T3 cells overexpressing 14-3-3 ß, and we assessed the relationship between expression of 14-3-3 ß and MAPK activity. Quiescent cultures of cells were incubated with 20% FCS for the indicated times. Cell lysates were prepared from cells and were examined for MAPK activity by a phosphocellulose filter binding assay, using a synthetic peptide substrate that is highly selective for p42/p44 MAPK (33). Basal MAPK activity showed little difference between control cells and cells overexpressing 14-3-3 ß, indicating that 14-3-3 ß had little stimulatory effect on MAPK activity in the absence of serum (Figure 4AGo). After serum stimulation, MAPK activity was stimulated 20-fold and 10-fold in Nß-1 and Nß-2 cells, respectively, while it was stimulated six-fold in control NIH 3T3 cells (Figure 4AGo). By immunoblot analysis with an anti-phospho-ERK monoclonal antibody, we confirmed that serum activation of MAPK was augmented in Nß-1 and Nß-2 cells (Figure 4BGo). The augmented MAPK activity after serum stimulation appeared to depend on the amount of 14-3-3 ß in the cells (Table IGo and Figure 4Go). We also showed that Raf-1 protein was detected in 14-3-3 ß immunoprecipitates from Nß-1 and Nß-2 cells (Figure 4CGo). Thus interaction of 14-3-3 ß and Raf-1 was presumed to result in augmented signaling in the MAPK cascade in these cells.





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  		Fig. 4. MAP kinase activity in NIH 3T3 cells overexpressing 14-3-3 ß. (A) Kinetics of MAP kinase activity observed after serum stimulation in NIH 3T3 cells overexpressing 14-3-3 ß. NC-1, control NIH 3T3 cells transfected with pSV2neo; Nß-1 and Nß-2, NIH 3T3 cells overexpressing 14-3-3 ß. The value relative to MAPK activity in quiescent cells is shown in the figure. The kinetics of MAP kinase activity in NC-1 were consistent with those in parental cells (data not shown). Each experiment was performed three times and the mean and standard deviation are shown. (B) Immunoblot analysis with an anti-phospho-Erk monoclonal antibody. The augmented MAPK activity 5 min after serum stimulation was confirmed by immunoblot analysis. Note that the activity was higher in Nß-1 cells than in Nß-2 cells. The protein bands were stained with amido black on the filter. Bands with a molecular mass similar to those of Erk1 and Erk2 are shown in the lower panel as a control for the amount of protein on the blots. (C) Co-immunoprecipitaion of 14-3-3 ß and Raf-1 from Nß-1 and Nß-2 cells. Lysates were prepared from these cells, immunoprecipitated with an anti-14-3-3 ß antibody and subjected to immunoblot analysis with an anti-Raf-1 antibody. In the control lane, a similar experiment was done using pre-immune rabbit serum to precipitate the complex.

 
Exogenous expression of MEK1DN caused reversion of the transformation phenotype in NIH 3T3 cells overexpressing 14-3-3 ß
We attempted to determine whether enhanced signaling in the MAPK cascade is responsible for the tumorigenic transformation of NIH 3T3 cells by overexpression of 14-3-3 ß. We co-transfected a MEK1DN expression vector (27) and a pSV2hygro vector as a selection marker (28), and established six independent hygromycin-resistant cell lines which were derived from either Nß-1 or Nß-2 cells. The exogenous epitope-tagged MEK1DN and endogenous MEK1 were independently detected by immunoblot analysis with a polyclonal antibody against mouse MEK1 (Figure 5Go), because the molecular mass of exogenous MEK1DN is significantly larger than that of endogenous MEK1. The amount of exogenous MEK1DN was much greater than the amount of endogenous MEK1. Exogenous MEK1DN expression was confirmed by detecting the same bands with a monoclonal antibody (9E10) against the c-Myc epitope (Figure 5Go). We examined the colony-forming ability of the transfectants in soft agar medium. Control cells derived from the Nß-1 and Nß-2 clones showed 3–4.5% colony-forming ability, which was essentially the same as that in the parental cells, whereas each of the six transfectants derived from Nß-1 or Nß-2 cells showed significantly reduced colony-forming ability (Figure 5Go). Morphologically, these cells appeared flatter than parental cells (data not shown). Thus, reversion of the transformation phenotype was caused by the exogenous expression of MEK1DN.



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  		Fig. 5. Effects of MEK1DN on transformation of NIH 3T3 cells by overexpression of 14-3-3 ß. Nß-1 and Nß-2 cells overexpressing 14-3-3 ß were co-transfected with a MEK1DN expression vector and a selection plasmid, pSV2hygro. Three representative transfectants of Nß-1 (Nß-1-DN1, -DN2 and -DN3) and three of Nß-2 (Nß-2-DN1, -DN2 and -DN3) were subjected to further analysis. Immunoblots with anti-MEK1 and anti-c-Myc epitope (9E10) antibodies are shown in the upper and lower panels, respectively. The upper panel shows the levels of exogenous c-Myc-tagged MEK1DN and endogenous MEK1. Exogenous c-Myc-tagged MEK1DN is indicated by an arrowhead. The colony-forming abilities of the transfectants in soft agar medium (%) are shown at the bottom of the lanes. `Nß-1-C' and `Nß-2-C' indicate control Nß-1 and Nß-2 cells transfected with pSV2hygro, respectively. The colony-forming abilities of these cells were essentially the same as those of each parental cell line. *The Nß-2-DN1 cells retained colony-forming ability at a frequency of <0.05%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we generated NIH 3T3 cells overexpressing 14-3-3 ß and examined the effect of 14-3-3 ß on cell proliferation and oncogenic transformation. The overexpression of 14-3-3 ß in NIH 3T3 cells showed significant growth-promoting activity and induced both anchorage-independent growth in soft agar medium and tumor formation in nude mice (Table IGo and Figures 2 and 3GoGo). The growth-promoting activity of 14-3-3 ß correlated well with the amount of 14-3-3 ß expression in NIH 3T3 cells. We next examined the activity of MAPK in NIH 3T3 cells overexpressing 14-3-3 ß after serum induction. The augmented activity of MAPK correlated well with the amount of 14-3-3 ß in the NIH 3T3 cells (Table IGo; Figures 1 and 4A and BGoGo). The growth promotion and the enhancement of MAPK activity in Nß-1 and Nß-2 cells were not overt in the absence of serum, but were obvious in the presence of serum (Table IGo and Figure 4AGo). It appears, therefore, that overexpression of 14-3-3 ß in NIH 3T3 cells augments the growth signal but does not itself activate the signal. We also expressed exogenous MEK1DN in NIH 3T3 cells overexpressing 14-3-3 ß and examined the effect of exogenous MEK1DN on anchorage-independent growth in soft agar medium. MEK1DN efficiently reduces signaling in the MAPK cascade and suppresses transformation of NIH 3T3 cells by Mos, activated Raf-1 and Ras (27). The colony-forming ability of these cells was efficiently reduced by exogenous expression of MEK1DN (Figure 5Go). We also showed that 14-3-3 ß physically interacts with Raf-1 in NIH 3T3 cells overexpressing 14-3-3 ß (Figure 4CGo) and that 14-3-3 ß can augment Raf-1 activity in budding yeast that had been genetically engineered to monitor mammalian Raf-1 activity (34). Although we did not show directly that overexpressed 14-3-3 ß augmented Raf-1 activity in these cells, these results suggest the following molecular mechanisms underlying 14-3-3 ß-mediated oncogenic transformation of NIH 3T3 cells: (i) enhanced signaling in the MAPK cascade has an important role in the oncogenic transformation of NIH 3T3 cells; (ii) 14-3-3 ß enhances signaling in the MAPK cascade by interacting with Raf-1. However, the possibility still remains that the transformation phenotype is accomplished through multiple activities of 14-3-3 ß, because there is evidence suggesting that 14-3-3 proteins interact with other molecules involved in cell proliferation and survival signals and cell cycle control such as PKC (3,4), phosphatidyl-3-kinase (19), phosphorylated BAD (20) and cdc25 phosphatases (18).

Recently we showed that overexpression of the five isoforms of the 14-3-3 proteins enhances Raf-1 activity in the genetically engineered budding yeast (34). All of these results strongly indicate that expression of 14-3-3 proteins has oncogenic potential in humans (35). Although it has been reported that expression of 14-3-3 proteins is significantly reduced in mammary carcinoma and squamous-cell carcinoma cells (36), further examination of the 14-3-3 proteins may shed new light on their role in carcinogenesis in humans.


   Notes
 
2 To whom correspondence should be addressed Email: takihara{at}biken.osaka-u.ac.jp Back


   Acknowledgments
 
We thank Drs K.Matsumoto, T.Akiyama, M.Nozaki and K.Inoue for their comments on the study and Dr K.Okazaki for the MEK1DN mutant plasmid. This work was supported in part by a Grant-in-Aid for Cancer Research, a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Osaka Cancer Foundation and the Osaka Cancer Research Foundation.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 29, 2000; revised July 28, 2000; accepted July 31, 2000.


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