Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A) on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC). This study establishes a foundation for further study on EBVaGC and its gene therapy.


Introduction
Epstein-Barr virus (EBV), a gamma herpes virus, has been associated with a variety of human malignancies such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), and gastric cancer (GC) [1]. EBV is detected in the tissue of about 10% of gastric carcinoma cases throughout the world [2,3]; furthermore, EBV is associated with 80%-100% of the rare lymphoepithelioma-like gastric carcinomas [4] and is also present in 35% of gastric stump carcinomas [5]. Latent membrane protein 2A (LMP2A) is an EBVencoded protein that has been implicated in regulating viral latency and pathogenesis within infected cells [6]. Recent studies revealed that about 50% EBV-associated gastric carcinoma (EBVaGC) expressed LMP2A. Gastric cancer is one of the most common malignant tumors in the world. It is very difficult to remove and eliminate tumor cells completely by surgical, radio therapeutic, and chemotherapeutic methods. Therefore, developing an immunotherapeutic method to treat gastric cancer is of critical importance. LMP2A contains many potential T-cell activating epitopes which can trigger cytotoxic T lymphocytes (CTL), thus it is an ideal target gene of immunotherapy. However, the pathogenic role of LMP2A in tumor progression is not well understood. So making an intensive study of LMP2A is of important meaning to reveal the disease mechanism of EBV. In the present study, LMP2A gene was cloned into adenoviral vector to generate recombinant adenovirus, which could introduce LMP2A gene into gastric cancer cells, in order to evaluate the possible effects of LMP2A expression on human gastric cancer cell line. Our study provides more complete understanding of the role of LMP2A in EBV latency and tumorigenesis and establishes a foundation for further study on gastric cancer and its gene therapy.

Cell Culture and
Medium. Type 1 EBV strain positive B95-8 cells were used for amplifying LMP2A gene, and human embryonic kidney cell line HEK293 were used for packing recombinant adenovirus. The human GC cell line SGC-7901 was purchased from ATCC. All cell lines were maintained in high glucose DMEM medium supplemented with 100 mL/L fetal bovine serum (FBS). The culture medium contained 100 U/mL of penicillin and 100 μg/mL of streptomycin (GIBCO BRL, Gathersburg, Md) at 37 • C in 5% CO 2 .

Target Gene and Primers.
The RT-PCR product of LMP2A was obtained from B95-8 cell line genome RNA. The specific primers were as follows: 5 -GGCAGATCTATG-GGGTCCCTAGAAATGGTG-3 (sense strand) and 5 -GCGATATCTTATACAGTGTTGCGATATGG-3 (antisense strand), and Bgl II and EcoR V restriction sites and protective bases were introduced into sense and antisense primers, respectively, as underlined. The predicted length of PCR product is about 1500 bp.

Recombinant Retroviral Vector
Construction. PCR amplified target gene LMP2A and shuttle vector pAdTrack-CMV were digested with Bgl II and EcoR V. The digested products were purified and ligated with DNA ligation I to form transfer plasmid of pAdTrackCMV-2A. Then it was linearized by PmeI and cotransformed into BJ5183 bacterial cells with adenovirus genomic DNA plasmid of pAdeasy-1. The homologous recombinant adenoviral plasmid named pAd-2A was confirmed by PCR, restriction endonuclease analyses and sequencing. The control retroviral vector pAd-LacZ was constructed in the same manner.

Production and Propagation of Recombinant Adenoviruses in HEK293.
Four micrograms of recombinant adenoviral vector DNA, after Pac I digestion and ethanol precipitation, were transfected into HEK293 cells in each 25 cm 2 flask at 50%-70% confluence. Transfection was accomplished using the LipofectAMINE reagent from Life Technologies (Gaithersberg, Md). Transfected cells were monitored for GFP expression and collected 7-10 days after transfection, when about 1/3 cells appeared cytopathic effect (CPE), by scraping cells off flasks and pelleting them along with any floating cells in the culture. After centrifugation, the cell pellet was resuspended in 2.0 mL sterile phosphate-buffered saline (PBS) and lysed for four consecutive freeze/thaw cycles, and the virus was collected from the supernatant. One third of viral lysate was used to infect HEK293 cells in a 25 cm 2 flask. The infection efficiency could be conveniently followed with green fluorescent protein (GFP). Three to four days later, viruses were harvested as described above. This process was repeated 1-3 times. At this point, viral titers were often high enough to use for gene transfer experiments in cultured cells. Viral titer was determined by virus titer determination kit following the manufacturers recommendations (Ben Yuan Zheng Yang Company, Beijing, China), which was up to 3.16 × 10 9 pfu/mL.

Infection of SGC Cells with Recombinant Adenoviruses.
When cultures of SGC cells were 70% confluence, the cells were incubated with recombinant adenoviruses at a proper MOI at 37 • C overnight. The cells then were washed once with PBS, and fresh medium was added. At 4 days after infection, the infection efficiency was estimated by using fluorescence microscopy to detect GFP-expression. The infected SGC cells were harvested, and transcriptional expression of LMP2A in SGC cells were examined by RT-PCR followed by Southern blot analysis.

RT-PCR and Southern Blot Analysis.
Total RNA was extracted using Trizol RNA isolation reagent (Molecular Research Center, Cincinnati, Ohio). 1 μg of total RNA was transcribed using the Reverse Transcription System (Promega, USA). Total RNA was treated with DNase I (Roche, Basel, Switzerland) to remove contaminating genomic and adenoviral DNA before cDNA was prepared. Three microliters of cDNA for each sample were used to perform the PCR. PCR on total RNA (before reverse transcription) was used to control for DNA contamination in samples. The sequences of primers were as follows: 5 -ATGACTCATCTCAACACATA-3 (nt. 166874-166893), 5 -CATGTTAGGCAAATTGCAA-3 (nt. 166380-166361). Product size was 280 bp. As probe, the oligonucleotide: ATCCAGTATGCCTGCCTGTA (nt. 166862-166881) was used. Southern blot hybridization was performed after RT-PCR for confirmation. The amplified products were electrophoresed in 2% agarose gel, transferred onto a Hybond N + nylon membrane (Amersham Pharmacia Biotec, Ireland), and subjected to hybridization with 3 -end-DIGlabeled oligonucleotide probes. The hybridized signals were detected by alkaline phosphatase (AP) conjugated anti-DIG antibodies. The substrate of AP was CSPD (Roche Diagnostics, Germany). cDNAs from EBV immortalized lymphoblastoid cell lines (LCL) were used as positive controls, and those from EBV-negative SGC cells as negative controls.

Cell Cycle Analysis by Flow Cytometry.
For flow cytometry analysis of the DNA content, SGC cells infected with

Statistics Analysis.
All data are expressed as means ± SD. Statistical comparisons were made using the analysis of variance. P < 0.05 was considered significant.

Analysis of Recombinant Plasmid pAd-2A.
The fragments of PCR amplification and Pac I digestion of plasmid pAd-2A digested were 1500 bp, 37.58 kb, and 3.0 kb, respectively ( Figure 1). All the products were detected by 1% agarose gel electrophoresis as predicted. The expression of the LMP2A gene was verified by gene sequencing.

Preparation of Recombinant Adenovirus.
Expression was visible 24 h after transfection in 20%-30% of the cells, representing the fraction of the population that was transfected.  Comet-like foci, visualized with GFP fluorescence, began to appear at 4-5 days after transfection, indicating adenovirus was produced successfully (Figure 2). The adenovirusmediated gene LMP2A expression was verified in HEK 293 by RT-PCR. The expected 1500-bp fragment was amplified from pAd-2A transfected SGC cells, but not from pAd-LacZ transfected cells (Figure 3).

Discussion
Gastric cancer, with clinical prognosis, is the second leading cause of cancer-related mortality worldwide [7]. Recently, great attention has been paid to the association of EBV infection and gastric carcinoma. EBV is detected in the tissue of about 10% of gastric carcinoma cases throughout the world. In each case, 100% of carcinoma cells are infected with EBV. Analysis of EBV in carcinoma biopsies indicates that carcinoma is formed by the proliferation of a single EBV-infected cell [2]. These findings suggest that EBV plays an important role in the development of EBVpositive gastric carcinomas. LMP2A gene is expressed in vivo in humans with latent infections and most EBV-related malignancies [8][9][10][11]. Recent studies revealed that about 50% EBV-associated gastric carcinoma (EBVaGC) expressed LMP2A. Due to this persistent expression, LMP2A is thought to play an important role in EBV latency and may be an important risk factor in EBV-associated diseases.
It has been reported that LMP2A is involved in blocking B-cell specific signaling pathways and calcium mobilization, which might be advantageous for maintaining latent patterns of EBV infection and inhibiting EBV reactivation [12]. LMP2A is also reported to transform epithelial cells, greatly affect cell growth and differentiation pathways in a human keratinocyte cell line (HaCaT), and inhibit transforming growth factor-β1-induced apoptosis in HSC-39, through activation of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway [13,14]. EBV latent membrane protein 2A activates beta-catenin signaling in epithelial cells [15]. The emerging importance role of LMP2A in both B-cell and epithelial cell growth led us to evaluate the effect of LMP2A on cell cycle regulation in a GC cell line. TGF-β is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems [16]. TGF-β can induce immunoglobulin A class switching and play an important role in differentiation, growth, matrix formation, and the regulation of immune and inflammatory responses [17,18]. It is an inducer of apoptosis in BL and some gastric carcinoma cell lines [19][20][21][22] Abnormalities in cell cycle regulation lead to uncontrolled cell proliferation and the transformation into cancer cells [23,24]. Cell cycle analysis indicated that vAd-2Ainfected SGC cells had significantly more cells in S phase in comparison with parental and vector control SGC cells. Cyclins, CDKs, and CDKIs play important roles in controlling major checkpoints in the mammalian cell cycle [25]. Cyclin E overexpression occurred in 50%-60% of GC and adenocarcinomas [26] and is essential for progression from early to late G1 [27]. The cyclin E expressions of SGC detected by confocal microscopic analysis showed a positive linear correlation with S phase. Liang Bin et al. analyzed the relationship between cyclin E/CDK2 expression and clinicopathological characteristics of the tumors, indicating that overexpression of cyclin E protein was correlated with poorer differentiation advanced tumor stage and lymph node involvement [28], which was consistent with the report of Sakagnchi et a1. [29].
In summary, these results suggest that upregulated expression of cyclin E and S phases caused by LMP2A may be associated with the malignant potential in gastric cancer as well as potentiate metastases. LMP2A could increase GC cell proliferation via upregulating the expression of cyclin E and CDK2 which induced S phase arrest and inhibit apoptosis of SGC cells. These studies have provided a more complete understanding of the role of LMP2A in tumorigenesis of GC and established a foundation for further study on gastric cancer and its gene therapy.