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Journal of Virology, September 2007, p. 9299-9306, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00537-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Epstein-Barr Virus Latent Membrane Protein 2A Mediates Transformation through Constitutive Activation of the Ras/PI3-K/Akt Pathway{triangledown}

Makoto Fukuda{dagger} and Richard Longnecker*

Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611

Received 14 March 2007/ Accepted 11 June 2007


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ABSTRACT
 
Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.


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INTRODUCTION
 
Epstein-Barr virus (EBV) ubiquitously infects the majority of humans and is an important human tumor virus that is causally associated with various lymphoid and epithelioid malignancies (20, 53). The underlying mechanism of how EBV persists in humans and how the virus contributes to cancer is still poorly understood. Primary human B lymphocytes infected in vitro with EBV become immortalized establishing lymphoblastoid cell lines (LCLs). This process constitutes an in vitro model for the contribution of EBV to B lymphoid disease. EBV gene expression in LCLs is restricted to six nuclear antigens (EBNA1, -2, -3A, -3B, -3C, and -LP), three integral membrane proteins (latent membrane protein 1 [LMP-1], -2A, and -2B), two nonpolyadenylated RNAs (EBER-1 and -2), and the BamHI A rightward transcripts (BARTs) (20, 24, 53). Among the EBV genes expressed in LCLs, along with EBNA1, LMP2A is routinely detected in most EBV-related malignancies (20, 24, 48, 53). Due to this persistent expression, LMP2A may be an important risk factor in EBV-associated tumorigenesis.

LMP2A consists of a long N-terminal tail, 12 membrane-spanning domains, and a short C-terminal tail and forms aggregates in patches on the surfaces of latently infected cells (17, 23). The N-terminal tail of LMP2A contains eight constitutively phosphorylated tyrosine residues and several proline-rich regions that are critical for the ability of LMP2A to interact with cellular proteins (17, 23). The LMP2A N-terminal intracellular region contains multiple functional domains, including an immunoreceptor tyrosine-based activation motif (ITAM) homologous to that found in the immunoglobulin {alpha} and immunoglobulin ß signaling subunits of the B-cell receptor (BCR) (13). LMP2A associates with Src family protein tyrosine kinases (PTKs) and Syk PTK that normally form part of the BCR signaling complex (6, 13, 14). LMP2A alters normal BCR signaling and as a consequence prevents BCR-induced lytic replication in LCLs grown in tissue culture (30). In addition, we have shown that LMP2A regulates BCR-induced EBV reactivation and apoptosis through tyrosine phosphorylation (15). Studies using transgenic mice have shown that LMP2A provides developmental and survival signals to BCR-negative B cells through constitutive activation of the Ras/phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in LMP2A transgenic mice (7, 8, 39). Unlike the situation in B cells, targeting of LMP2A to the epidermis of transgenic mice is not associated with any alteration in normal epithelial differentiation and growth (22).

Previous studies have shown that LMP2A has transforming capabilities, alters epithelial cell motility, and inhibits epithelial cell differentiation (9, 37, 41). Many of these observed effects of LMP2A on normal epithelial biology may be related to the activation of the PI3-K/Akt pathway by LMP2A (41, 46) and the promotion of cell survival by LMP2A through the activation of the PI3-K/Akt pathway (16, 39). Furthermore, LMP2A expression is important in epithelial cell clone outgrowth following infection of epithelial cells (31, 32). Although there is some similarity in the function of LMP2A, such as the activation of the Syk PTK in epithelial cells (28), other studies suggest that differences exist such as the phosphorylation of LMP2A in epithelial cells by the Csk PTK (42).

In this study, to determine the effect of LMP2A on cellular transformation in nonhematopoietic cells, LMP2A was stably expressed in the human keratinocyte cell line HaCaT and the gastric carcinoma cell line HSC-39 and colony formation in soft agar was monitored. LMP2A-mediated colony formation in soft agar was not observed in HaCaT cells, while LMP2A-expressing HSC-39 cells formed colonies in soft agar. In addition, LMP2A did not increase levels of activated Ras in HaCaT cells but did in HSC-39 cells. The LMP2A-mediated transformation activity in HSC-39 cells was inhibited in the presence of specific inhibitors of Ras or PI3-K, indicating that LMP2A activates the Ras/PI3-K/Akt pathway to mediate cell transformation in HSC-39 cells. These findings provide new insight regarding specific cellular pathways utilized by LMP2A to induce not only cell survival but also cell transformation in nonhematopoietic cells.


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MATERIALS AND METHODS
 
Cell lines and media. HaCaT cells (3) were maintained in Dulbecco's modified Eagle's medium (DMEM), and HSC-39 cells (51) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1,000 U/ml penicillin, and 1,000 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. HaCaT cells are spontaneously immortalized human keratinocytes. HSC-39 cells, a human signet ring cell gastric carcinoma cell line, were kindly provided by K. Yanagihara. LMP2A-expressing HSC-39 cells and vector control cells were previously described (16).

Antibodies and reagents. Rat monoclonal antibody against LMP2A (14B7-1-1) was described previously (12). Anti-phospho-Akt (serine 473) antibody and Akt antibody were purchased from New England Biolabs. Anti-rat and anti-rabbit antibodies conjugated to horseradish peroxidase were purchased from Amersham and New England Biolabs, respectively. Genistein, a specific tyrosine kinase inhibitor, and manumycin A, a potent inhibitor of farnesyltransferase, were obtained from Calbiochem. LY294002, a PI3-K-specific inhibitor, was obtained from Cell Signaling Technologies.

Production of recombinant retroviruses. GP293 cells were transiently transfected with 2 µg of the vector pBAMHYGRO (52) or pMP2LMP2A (25) and 2 µg of VSV-G (vesicular stomatitis virus glycoprotein G envelope). At 48 h transfection, culture supernatant was harvested and filtered through a 0.45-µm filter. HaCaT cells were transduced by incubation with retrovirus-containing GP293 supernatant and Polybrene at 4 µg/ml overnight. The medium was removed, and the cells were supplied with fresh complete medium and cultured for 48 h before the addition of selection medium containing hygromycin at 0.4 mg/ml. Seven single clones were isolated after 2 to 3 weeks.

Transfection. Cells (5 x 106) were suspended in 400 µl of complete DMEM or RPMI 1640 medium with 25 µg of pcDSR{alpha}-human H-RasV12 or pcDSR{alpha}-human H-RasN17 plasmids (gift from H. Kitayama, Kyoto University) or the vector pBJ5 plasmid (47). Cells were then transfected with the Gene Pulser (Bio-Rad, Hercules, Calif.) at 300 V and 960 µF of capacitance in a 0.4-cm electrode gap cuvette (Bio-Rad). HaCaT or HSC-39 cells were supplied with fresh complete medium and cultured for 48 h before the addition of selection medium containing G418 at 0.8 mg/ml. Single clones were isolated after 2 to 3 weeks. For propagation, cells were maintained in medium containing G418 at 0.4 mg/ml. LMP2A-expressing HSC-39 cells were supplied with fresh complete medium and cultured for 48 h before the addition of selection medium containing 0.8 mg/ml G418 and 0.4 mg/ml hygromycin. Single clones were isolated after 2 to 3 weeks. For propagation, cells were maintained in medium containing 0.4 mg/ml G418 and 0.4 mg/ml hygromycin.

Ras activation assay. The activation of Ras was evaluated using a Ras activation assay kit (Upstate, Charlottesville, VA) according to the manufacturer's protocol. Briefly, cells (1.0 x 107 cells) were serum starved for 2 h, washed twice with cold phosphate-buffered saline and lysed with 1 ml of Mg2+ lysis/wash buffer (MLB). Aliquots of lysates were set aside to allow quantitation of total Ras by immunoblotting. The remainder of the lysates was precleared by glutathione-agarose, 10 µg of Raf-1 Ras-binding domain (RBD)-conjugated agarose was added, and the mixture was incubated at 4°C for 1 h. Raf-1 RBD-conjugated agarose is a glutathione S-transferase (GST) fusion protein, corresponding to the human RBD (residues 1 to 149) of Raf-1; provided that it is bound to glutathione-agarose, it specifically binds to and precipitates Ras-GTP from cell lysates. After the beads were washed three times with MLB, they were suspended in 2x Laemmli sample buffer, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using 1 µg/ml anti-Ras antibody (RAS10) as a primary antibody, and visualized using the ECL Western blotting detection system (Amersham Pharmacia Biotech) and by densitometry of corresponding bands using a computing densitometer with ImageJ software (National Institute of Mental Health, Bethesda, MD).

Western blotting. Cells were washed with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 for the detection of phosphorylated proteins and other proteins, lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 10 µg/ml leupeptin, and protein levels were quantitated by Dc protein assay (Bio-Rad, Hercules, CA). Equivalent amounts of protein were subjected to heat denaturation at 70°C for 10 min. Proteins were resolved by SDS-PAGE, transferred to Immobilon-P (Millipore, Bedford, MA), and blocked with 4% skim milk at room temperature for 1 h. Membranes were incubated in Tris-buffered saline (TBST) (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) with primary antibodies for 2 h and then in appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h. Following incubation, the membranes were visualized using the ECL detection kit. Quantitative data were obtained using ImageJ software.

Colony formation assay. The colony formation assay technique was used as an in vitro assay for anchorage-independent cell growth, a parameter indicative of transformation (11). Wells in a six-well plate (Falcon) were covered with 0.5% SeaPlaque agarose (FMC, Rockland, ME) or Bacto agar (Difco) containing DMEM and 10% FBS. HaCaT cells (1 x 104 cells or 2 x 104 cells) from each HaCaT clone in 0.3 or 0.33% SeaPlaque agarose or Bacto agar containing DMEM and 10% FBS were then added to the base. Cells were fed with fresh top agar (0.3% or 0.33% agar in DMEM with 10% FBS) every week. HSC-39 cells (1 x 104 cells) were suspended in 0.4% SeaPlaque agarose or Bacto agar in RPMI 1640 medium (or DMEM) containing 3% FBS. Cells from each HSC-39 clone were plated in six-well plates precoated with 0.5% SeaPlaque agarose or Bacto agar containing 3% FBS in RPMI 1640 medium (or DMEM). After 3 weeks of incubation, colonies were counted and measured. Cells were fed with fresh top agar (0.4% agar in RPMI 1640 medium or DMEM with 10% or 3% FBS) every week. In some experiments, the Ras inhibitor (manumycin A) or PI3-K inhibitor (LY294002) was added to the agar medium to a concentration of 10 µM or 25 µM and replenished in DMEM or RPMI 1640 medium upon feeding. All assays were performed in triplicate.


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RESULTS
 
Anchorage-independent cell growth of HaCaT and HSC-39 cells expressing LMP2A. LMP2A activates the PI3-K/Akt pathway in B cells and epithelial cells (16, 41, 46). In addition, LMP2A transforms the HaCaT human keratinocyte cell line in part through the activation of the PI3-K/Akt pathway (41). To further investigate the importance of this activation in LMP2A-mediated cell transformation, HaCaT cells were retrovirally transduced with a LMP2A expression vector and the vector control. Following transduction, hygromycin-resistant, stable transfected clones were selected and verified for LMP2A expression and Akt phosphorylation by Western blot analysis (Fig. 1A). To confirm LMP2A-mediated cell transformation in HaCaT cells, we tested colony formation in soft agar, a test for transformation activity. In our experiments, inefficient colony formation was observed in parental, vector control and LMP2A-expressing HaCaT cells (diameter under 200 µm) (Fig. 1B). To assess the effect of LMP2A on transformation activity in another cell line, previously constructed LMP2A-expressing HSC-39 cells (a human gastric carcinoma cell line) and vector control cells (16) were also tested using colony formation in soft agar. LMP2A-expressing HSC-39 cells formed colonies that were significantly larger (diameter over 200 µm) than the size of colonies in parental and vector control HSC-39 cells (Fig. 1C).


Figure 1
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  		FIG. 1. Effect of LMP2A expression on anchorage-independent cell growth in HaCaT and HSC-39 cells. (A) HaCaT cells were stably retrovirally transduced with LMP2A or vector control expression constructs. After 2 h of serum starvation, whole-cell extracts were separated by SDS-PAGE, and the expression levels of LMP2A (54 kDa), phospho-specific Akt (P-Akt) (on serine 473) (60 kDa) were determined by immunoblotting in parental (P), vector control (Vector), and LMP2A-expressing HaCaT cells. The lower panel shows equal loading of total Akt (T-Akt). (B) The ability of LMP2A to induce anchorage-independent cell growth in HaCaT cells. Parental, vector control (Vector), or LMP2A-expressing HaCaT cells (1 x 104 cells) were cultured in soft agar for 3 weeks. Colonies were defined as cell clusters greater than 200 µm in size and scored. Representative images from one of three independent experiments. (C) The ability of LMP2A to induce anchorage-independent cell growth in HSC-39 cells. Parental, vector control (Vector), or LMP2A-expressing HSC-39 cells (1 x 104 cells) were cultured in soft agar for 3 weeks. Colonies were defined as cell clusters greater than 200 µm in size and scored. Representative images from one of three independent experiments are shown. The average numbers of foci of parental (P), vector control (V), or LMP2A-expressing (2A) HSC-39 cells formed in a well from three independent experiments are shown in the graph; error bars indicate standard deviations. Data were analyzed by Student's t test. Values that were statistically significantly different from each other (P < 0.001) are indicated (**).

Expression of activated Ras in LMP2A-expressing HaCaT and HSC-39 cells. Previous studies from our laboratory demonstrated that LMP2A mediates survival of murine transgenic LMP2A-expressing B cells through the constitutive activation of the Ras/PI3-K/Akt pathway (39). Ras proteins are members of the superfamily of small GTPases that are important in a variety of signaling pathways leading to cell proliferation, differentiation, and transformation (43). To further investigate the importance of constitutive activation of the Ras/PI3-K/Akt pathway in anchorage-independent cell growth, activated Ras was isolated by immunoprecipitation with a GST fusion protein containing the RBD of Raf-1. As shown in Fig. 2, LMP2A-expressing HSC-39 cells expressed higher levels of activated Ras than parental and vector control cells did. In contrast, the level of activated Ras in LMP2A-expressing HaCaT cells was similar to those of parental and vector control cells (Fig. 2).


Figure 2
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  		FIG. 2. Expression of activated Ras in LMP2A-expressing HaCaT and HSC-39 cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) HaCaT and HSC-39 cells were cultured for 2 h without serum. Ras activation was evaluated by pulling down active GTP-loaded Ras with a GST fusion protein containing the RBD of Raf-1 (GST-Raf1-RBD) and blotting with anti-Ras antibody. Equal quantities of Ras in the extracts were confirmed by immunoblotting a fraction of the total cell lysates taken before pulling down GST-Raf1-RBD. (B) The extent of expression of activated Ras and total Ras protein from panel A was quantified using ImageJ software. The relative level was calculated as the ratio of activated Ras to total Ras protein levels. The ratios are normalized to the level of Ras activity of the parental cells, which was set at 1. Results are expressed as the means ± standard deviations (error bars) for three experiments.

Inhibition of the Ras/PI3-K/Akt pathway suppresses anchorage-independent cell growth in HSC-39 cells. Ras family members stimulate multiple effector-mediated signaling pathways, including the Raf/MEK/ERK (extracellular signal-regulated kinase), PI3-K/Akt, and RalGDS pathways (43). To explore the role of the Ras/PI3-K/Akt pathway on LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth, we examined the effect of manumycin A, a specific Ras inhibitor, or LY294002, a specific PI3-K inhibitor, on Akt phosphorylation in LMP2A-expressing HaCaT and HSC-39 cells. The specificity of Akt phosphorylation downstream of Ras activation or PI3-K activation was confirmed by the inhibition of Akt serine 473 phosphorylation after treatment with manumycin A at 10 µM or LY294002 at 25 µM for 1 h in both LMP2A-expressing HaCaT and HSC-39 cells. LY294002 inhibited Akt phosphorylation in both LMP2A-expressing HaCaT and HSC-39 cells; however, manumycin A did not inhibit Akt phosphorylation in LMP2A-expressing HaCaT cells but did in LMP2A-expressing HSC-39 cells (Fig. 3A). We next tested the effect of manumycin A or LY294002 on anchorage-independent cell growth in LMP2A-expressing HSC-39 cells. In regards to both number and size, colony formation was significantly reduced by manumycin A and LY294002 in LMP2A-expressing HSC-39 cells (Fig. 3B).


Figure 3
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  		FIG. 3. Effect of manumycin A or LY294002 on Akt phosphorylation and anchorage-independent cell growth in LMP2A-expressing cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) HaCaT and HSC-39 cells were cultured for 1 h without serum, and then the cells were incubated for 1 h without manumycin A (control [C]) or with manumycin A (M) (10 µM) or LY294002 (L) (25 µM). Equal amounts of protein from the respective cells were separated by SDS-PAGE. The levels of phospho-specific Akt (P-Akt) (on serine 473) (60 kDa) were determined by immunoblotting. The bottom blots show equal loading of protein and expression of Akt (T-Akt) (60 kDa). The extent of expression of P-Akt and T-Akt proteins was quantified using ImageJ software. The relative level was calculated as the ratio of P-Akt to T-Akt protein levels. Results are expressed as the mean ± standard deviations (error bars) for three experiments. (B) LMP2A-expressing HSC-39 cells (1 x 104 cells) were cultured in soft agar without manumycin A (Control) or with manumycin A (10 µM) or LY294002 (25 µM) for 3 weeks. Colonies were defined as cell clusters greater than 400 µm in size and scored. Representative images from one of three independent experiments are shown. The average number of foci formed from LMP2A-expressing HSC-39 cells cultured without manumycin A (control [C]) or with manumycin A (M) or LY294002 (Ly) in a well from three independent experiments is shown; error bars indicate standard deviations. Data were analyzed by Student's t test. Values that were statistically significantly different (P < 0.05) from each other are indicated (*).

Effects of Ras mutants on Akt phosphorylation and anchorage-independent cell growth in HSC-39 and HaCaT cells. In order to confirm that the absence of Ras signaling impairs the ability of LMP2A to drive anchorage-independent cell growth, LMP2A-expessing HSC-39 cells were transfected with vector control or the dominant-negative RasN17 mutant (Ras17N), and individual clones were selected. As expected, dominant-negative Ras17N almost completely abolished LMP2A-mediated Akt phosphorylation and significantly inhibited both the number and size of colonies in anchorage-independent growth assays in LMP2A-expressing HSC-39 cells (Fig. 4A and B); however, the vector control did not inhibit LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in LMP2A-expressing HSC-39 cells (data not shown).


Figure 4
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  		FIG. 4. Dominant-negative Ras mutant (RasN17) inhibits Akt phosphorylation and anchorage-independent cell growth in LMP2A-expressing HSC-39 cells. (A) LMP2A-induced Akt phosphorylation was inhibited in HSC-39 cells expressing both LMP2A and RasN17. Parental (P), vector control (V), LMP2A-expressing (2A), and LMP2A-RasN17-coexpressing (2A + N17) HSC-39 cells were cultured for 1 h without serum. Equal amounts of protein from the respective cells was separated by SDS-PAGE. The levels of phospho-Akt (P-Akt) (serine 473) (60 kDa) were determined by immunoblotting. The bottom blots show equal loading of protein and expression of Akt (T-Akt) (60 kDa). The extent of expression of P-Akt and T-Akt proteins was quantified using a densitometer with ImageJ software. The relative level was calculated as the ratio of P-Akt to T-Akt protein levels. Results are expressed as the means ± standard deviations (error bars) for three experiments. (B) Soft agar colony formation of LMP2A-RasN17-coexpressing HSC-39 cells. LMP2A-expressing and LMP2A-RasN17-coexpressing (LMP2A+N17) HSC-39 cells (1 x 104 cells) were cultured in soft agar for 3 weeks. Colonies were defined as cell clusters greater than 400 µm in size and scored. Representative images from one of three independent experiments are shown. The average number of foci formed in a well from three independent experiments are shown; error bars indicate standard deviations. Data were analyzed by Student's t test. Values that were statistically significantly different (P < 0.05) from each other are indicated (*).

To verify whether activation of Ras is sufficient for Akt phosphorylation and anchorage-independent cell growth in HaCaT and HSC-39 cells, we analyzed the effect of the constitutively active V12 Ras mutant (RasV12) on Akt phosphorylation and anchorage-independent growth. As shown in Fig. 5A and B, expression of activated Ras readily induced Akt phosphorylation in HaCaT (clone 1 [Cl-1], Cl-2, Cl-3, and Cl-4) and HSC-39 (Cl-1, Cl-2, and Cl-3) cells. Interestingly, despite Akt activation by activated Ras, the HaCaT cell clone (Cl-1) did not exhibit anchorage-independent cell growth similar to the LMP2A-expressing cell clones. In HaCaT clone Cl-2, only two colonies were observed (Fig. 5A). In addition, as expected, activated Ras induced anchorage-independent growth in HSC-39 cells (Cl-1, Cl-2, and Cl-3 [Fig. 5B]).


Figure 5
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  		FIG. 5. Effect of active Ras mutant (RasV12) on the induction of Akt phosphorylation and anchorage-independent cell growth in HaCaT and HSC-39 cells. (A and B) Vector (V), LMP2A-expressing (2A), and RasV12-expressing HaCaT cells (A) and HSC-39 cells (B) were cultured for 1 h without serum. Equal amounts of protein from the respective cells were separated by SDS-PAGE. The levels of phospho-Akt (P-Akt) (serine 473) (60 kDa) were determined by immunoblotting. The bottom blots show equal loading of protein and expression of Akt (T-Akt) (60 kDa). The extent of expression of P-Akt and T-Akt proteins was quantified using ImageJ software. (Bottom left) The relative level was calculated as the ratio of P-Akt to T-Akt protein levels. Results are expressed as the means ± standard deviations (error bars) for three experiments. (Right) Soft agar colony formation of RasV12-expressing HaCaT (Cl-1 and Cl-2) (A) and HSC-39 (Cl-1, Cl-2, and Cl-3) cells (B). Cells (1 x 104 cells) were cultured in soft agar for 3 weeks. Colonies were defined as cell clusters greater than 200 µm in size and scored. Representative images from one of three independent experiments are shown. (Bottom right) The average number of foci formed in a well from three independent experiments is shown; error bars indicate standard deviations.

Effect of manumycin A or LY294002 on EGF-induced Akt phosphorylation in HaCaT cells. To further explore the Ras/PI3-K/Akt pathway in HaCaT cells, the phosphorylation of Akt following epidermal growth factor (EGF) treatment of HaCaT cells was analyzed. Akt is activated by several growth factors (21), including EGF, which activates the PI3-K/Akt pathway through the activation of Ras (21, 36), which can be partially blocked by the dominant-negative Ras17N mutation (21, 36).

To further explore the Ras/PI3-K/Akt cascade in HaCaT cells, we tested the effect of manumycin A or LY294002 on EGF-induced Akt phosphorylation in HaCaT cells. As shown in Fig. 6, EGF induced Akt phosphorylation in HaCaT cells. An EGF receptor inhibitor (a tyrosine kinase inhibitor), genistein, inhibited the EGF-induced Akt phosphorylation in HaCaT cells. In addition, LY294002 also inhibited EGF-induced Akt phosphorylation; however, manumycin A did not inhibit EGF-induced Akt phosphorylation (Fig. 6). These results suggest that EGF may activate the PI3-K/Akt pathway without Ras activation in HaCaT cells similar to what is observed with LMP2A expression.


Figure 6
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  		FIG. 6. Effect of manumycin A or LY294002 on EGF-induced Akt phosphorylation in HaCaT cells. Cells were cultured for 1 h with (+) or without (–) serum (FBS) and then preincubated for 10 min with genistein (G) (30 µg/ml) or for 30 min with manumycin A (M) (10 µM) or LY294002 (L) (25 µM), and then cells were treated with EGF (10 ng/ml) for 10 min. Control cells (C) were not treated. Equal amounts of protein from the respective cells were separated by SDS-PAGE. The levels of phospho-Akt (P-Akt) (serine 473) (60 kDa) were determined by immunoblotting. The bottom blot shows equal loading of protein and expression of Akt (T-Akt) (60 kDa). The extent of expression of P-Akt and T-Akt proteins was quantified by using ImageJ software. The relative level was calculated as the ratio of P-Akt to T-Akt protein levels. Results are expressed as the means ± standard deviations (error bars) for three experiments.


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DISCUSSION
 
In this study, we have demonstrated that LMP2A induces activation of the Ras/PI3-K/Akt pathway and anchorage-independent cell growth in the human gastric carcinoma cell line HSC-39. In contrast, in the human keratinocyte cell line HaCaT, LMP2A also activates the PI3-K/Akt pathway, but there is an absence of anchorage-independent cell growth. In the presence of Ras and PI3-K inhibitors, LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth were inhibited in HSC39 cells, suggesting that the Ras/PI3-K/Akt pathway plays a key role in LMP2A-mediated cell transformation of HSC-39 cells. Our studies with HaCaT cells contrast with earlier studies by Scholle et al. in which they observed that LMP2A efficiently induced anchorage-independent cell growth in HaCaT cells (41). We see a similar activation of phosphorylated Akt in HaCaT cells as in the earlier studies upon expression of LMP2A, but this phosphorylation does not require Ras activation, since inhibition of Ras with manumycin A in our studies does not block Akt phosphorylation. As in the earlier studies, the phosphorylation of Akt was dependent on PI3-K, since the PI3-K inhibitor LY294002 blocked Akt phosphorylation in both HaCaT and HSC-39 cells. To further investigate this difference, we expressed the constitutively active RasV12 mutant and although the Ras mutant effectively induced Akt phosphorylation, it did not clearly induce anchorage-independent growth in HaCaT cells in contrast to HSC-39 cells in which colonies were observed, similarly to when LMP2A is expressed. Thus, the difference observed between our studies and the previous studies is likely related to earlier observations that indicate that benign and malignant clones of Ha-Ras-expressing HaCaT cells can be isolated (4, 5).

The activation of Ras by LMP2A is significant, since Ras is frequently mutated in human cancers and plays a key role in the conversion of a normal cell into a cancer cell. Ras proteins act as GTP/GDP-regulated molecular switches that modulate signal transduction pathways controlling cell proliferation, differentiation, and survival (43). Oncogenic mutations in Ras have been linked to many types of human cancer (19, 43). These mutations lock Ras proteins into a permanently activated state leading to constitutive stimulation of downstream signaling pathways. Ras stimulates multiple effecter-mediated signaling pathways, including the Ras/MEK/ERK, the PI3-K, and the RalGDS pathways (43). In turn, these pathways modulate the activities of transcription factors that regulate numerous genes responsible for cell transformation and tumorigenesis (1, 49, 50). It has been reported that transcription factor NF-{kappa}B contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras (10, 18). Our recent studies indicate that NF-{kappa}B is constitutively activated in B cells expressing LMP2A in transgenic mice (45), suggesting that NF-{kappa}B activation may play an important role in LMP2A-mediated transformation.

Our studies have also highlighted an important feature of LMP2A in regard to LMP2A function in different cell types. From our studies and other studies exploring the function of LMP2A in epithelial cells and other cell types (9, 37, 41), there appears to be three factors that are important in considering the effects of LMP2A on cell phenotypes. These include cell origin or type, sensitivity of the particular cell to transformation, and LMP2A-induced signaling within a particular cell. In B cells, LMP2A has dramatic effects on normal BCR signal transduction and can drive B-cell development and survival in the absence of normal BCR signals (8, 15, 29, 30, 40). However, in B cells, there has been no indication that LMP2A when expressed in the absence of other EBV proteins results in alteration of normal cell proliferation. In fact, LMP2A is not required for EBV transformation of human B cells (26, 27, 44). Although LMP2A is not required for EBV transformation of B cells, the analysis of EBV gene expression in EBV-associated cancers in comparison to LMP2A transgenic mice indicates that LMP2A may play a key role in tumorigenesis (38, 39). LMP2A or LMP2A transcripts are routinely detected in Hodgkin's lymphoma (20, 48), nasopharyngeal carcinoma (20, 48), and most recently, Burkitt's lymphoma (2), suggesting that LMP2A is a key factor important for the ability of EBV to contribute to human cancers. The initial studies suggesting that LMP2A may be involved in cell proliferation came from studies by Raab-Traub's and Tsai's research groups, demonstrating that LMP2A has dramatic effects on epithelial cells, such as the ability to confer anchorage-independent growth, tumors in nude mice, inhibition of epithelial cell differentiation, and activation of cell motility (9, 41). In addition to the methods described in this report, we have also tested the effect of LMP2A expression on the anchorage-independent cell growth in HaCaT cells in the same assay as previously done (41). LMP2A-expressing HaCaT cells formed large numbers of colonies (diameter under 200 µm); however, the size and number of the colonies were similar to the colonies of parental and vector control HaCaT cells (data not shown). Interestingly, studies with primary epithelial cell lines have shown that other factors are important in the transformation of epithelial cells, since expression of LMP2A alone does not induce growth transformation (35). Our current studies and the previous studies with HaCaT cells support this notion. An important distinction is that LMP2A activates PI3-K/Akt pathway in both HaCaT and HSC-39 cells, but whereas this activation is dependent on Ras in HSC-39 cells, Ras appears to be nonessential for this activation in at least some HaCaT cells. Finally, the signaling pathways induced by LMP2A may also have important consequences for the effects of LMP2A expression on the cell phenotype. In support of this idea, studies have shown that glycogen synthase kinase 3ß is phosphorylated and inactivated with concomitant nuclear beta-catenin accumulation in the majority of nasopharyngeal carcinoma specimens, but this is not observed in Hodgkin's lymphoma (33, 34, 35).

In conclusion, our current studies indicate that LMP2A activation of the Ras/PI3-K/Akt pathway is essential for the growth transformation of HSC-39 cells. The ability of LMP2A to constitutively activate the Ras/PI3-K/Akt pathway, a common event during cell survival and tumorigenesis, suggests that the viral protein plays a key role not only in EBV latency and persistence but also in the development in EBV-associated malignancies. We propose that the importance of LMP2A in tumorigenesis arises from a combination of relativistic effects of host cell type and origin, host cellular transformation activity, and LMP2A-induced signaling pathways. The present work, which has shown differences in LMP2A-mediated transformation activity between HaCaT and HSC-39 cells, indicates that intrinsic biological differences between various cell types must be taken into account for the detection of transformation activity. Hence, one can hypothesize that LMP2A may play not only a key role in ensuring EBV latency in healthy individuals but also may work as an enhancing tumorigenic factor in EBV-associated diseases.


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ACKNOWLEDGMENTS
 
R.L. is supported by Public Health Service grants CA62234, CA73507, and CA93444 from the National Cancer Institute and is a Stohlman Scholar of the Leukemia and Lymphoma Society of America. The research described in this article was supported in part by Philip Morris USA and by Philip Morris International (M.F.).

We thank K. Yanagihara for HSC-39 cells and H. Kitayama for human H-RasV12 and human H-RasN17 expression plasmids. We thank members of the Longnecker laboratory for help with these studies.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0467. Fax: (312) 503-1339. E-mail: r-longnecker{at}northwestern.edu Back

{triangledown} Published ahead of print on 20 June 2007. Back

{dagger} Present address: Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. Back


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Journal of Virology, September 2007, p. 9299-9306, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00537-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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