| ObjectiveEpstein-Ban virus (EBV) is now the gamma subfamily of potentially oncogenic herpesviruses. In 1964, Tony Epstein succeeded in culturing the lymphoma cells which was observed by Denis Burkitt in regions of equatorial Africa. Epstein, Achong, and Barr identified the virus. EBV is a toroid-shaped double-stranded DNA virus. Its diameter is about 180nm. It has glycoprotein spikes on the outer envelope. The EBV genome is a liner double-stranded 184 kbp DNA. EBV is associated with many disease, primary infections in adolescence or early adult life manifest as IM (infectious Mononucleosis) and malignancies such as Burkitt's lymphoma, nasopharyngeal carcinoma, gastric carcinoma, posttransplantation lymphoma and lymphoma in immunodeficient patients.The association between EBV and gastric carcinoma was first reported by Burke et al in 1990. EBV DNA was demonstrated in more than 80% of gastric carcinomas of the lymphoepithelioma type by PCR and ISH. Subsequently, Shibata and Weiss demonstrated EBV infection in gastric adenocarcinomas of ordinary histology. 7 They reported that EBV is present in almost all carcinoma cells in EBV positive cases. Interest has increased with a report that 69 of 999 (6. 9%) cases of gastric carcinoma from Japan were EBV positive using ISH for the EBV encoded small RNAs (EBERs). Among them, the lymphoepithelioma type constituted only eight cases and the remaining cases were typical adenocarcinomas. In contrast to Burkitt s lymphoma and nasopharyngeal carcinoma, which are endemic in equatorial Africa and Southeast Asia, respectively, EBV positive gastric carcinoma is a non-endemic disease distributed throughout the world.However, there are some regional differences in the incidence of EBV positive gastric carcinoma as a proportion of all cases of gastric carcinoma, with the highest (16～18%) in the USA and Germany, and the lowest (4.3%) in China.There are 3 major types of EBV latent infection. Latencyâ…¢is characteristic of LCLs (lymphoblastoid cell lines) and lymphomas in AIDS or post-transplant patients. This involves of expression of all viral nuclear antigens EBNAs 1, 2, 3A, 3B, 3C and LP; 3 kides of latent membrane proteins LMP1, 2A, 2B; the small nonpolyadenylated RNAs and BARTs. Typeâ… andâ…¡latency has limited EBV gene expression. Since the EBNA3s are the most important targets of CTL, so latencyâ… andâ…¡are not easy to be cleared by CTL, the virus infection and host immune find a balance site, make EBV succeed to stay in the host. Gastric tumors express the Qp-initiated EBNA1 EBER and BARTs, but not LMP1. How does EBV infected cells escape from host immune surveillance? Maybe it is because of silencing of C promoter and no expression of the main targets (EBNA2 3s) of.CTL. The methlylation of C promoter binding factors bind region make it become silent. Despite our growing understanding of the role of EBV in the pathogenesis of disease, the optimal management of EBV-associated tumor remains unsatisfactory. Alternatively, EBV-positive tumor cell growth might be reversed by targeting EBNA1 or LMP1 transcription using ribozymes or antisense oligonucleotides. Many of these strategies remain at the " proof of principle" stage. Furthermore, except of immunosuppression patient the:EBV associated malignancies show highly stricted latent antigen expression, with loss of the immunodominant EBNA3A, 3B, 3C proteins, emphasizes the difficulties inherent in trying to target these tumors by immunologic means. Unless it is proves possible to active C promoter and express these immunodominant tatgets in tumor cells. For instance, by using demethylating agent such as 5'-azacytidine to resue Cp transcription in Burkitt's cell lead to activation of the C promoter and expression this family of proteins.This study tries to test if azacytidine can be helpful in gastric cancer treatment by using in vitro infected EBV positive cell lines. And furthermore we checked if Cp can be rescued by shRNA targeting DNMT.MATERIALS AND METHODSCell Lines and CultureGastric cancer cell line AGS was maintained in Ham F-12 medium (Sigma, St. Louis, MO) supplemented with 10% FBS (Gibco-BRL, Rockville, MD), penicillin (40 U/ml), and streptomycin (50 mg/ml). NUGC3 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 10% FBS and antibiotics. The neomycin-resistant EBVpositive GC cell lines were maintained in complete culture medium containing 500 g of G418 (Sigma-Aldrich Fine Chemicals, St. Louis, Mo.)/ml.Establishment of EBV-infected GC CellsNU-GC-3 cells were infected with rEBV (containing the Neor gene de scribed above) using a"cell-to-cell" infection procedure as described previously. Cells were maintained in culture medium containing G418 (500 g/ml).Infection proceduresOne or 2days before infection, epithelial cells to be used as virus recipients were detached by treating them with 2mM EDTA-PBS and were seeded into a 12-well culture plate at 5 10 cells in 2ml of the appropriate medium per well. On the day of infection, all culture medium was replaced with the same volume of fresh medium. For cocultivation, after sIgG cross-linking, 1ml of rEBV-infected Akata (Ak+) cell suspension (6 105ml) was added to the cultures. Both cultures were then incubated for 3 days at 37 C in 5% CO2, with replacement of half of the medium with fresh medium on day 2. The FCS concentration of the culture medium was reduced to 5% during the infection period to prevent cell overgrowth. After completion of the infection step (day 3), the cocultivation cultures were gently but thoroughly washed four times with PBS to remove residual viable virus donor cells, and 2 ml of fresh medium containing 10% FCS was added again. On day 4 or 5, the cells were reseeded into 96 at 102 to 104/ml per well in culture medium containing an appropriate concentration of G418 for selection (500g/ml). Immunofluorescence.The infected cells were smeared on a slide glass and fixed with a 1:1 mixture of acetone and methanol. The expression of EBNA proteins was visualized by the anticomplement immunofluorescence method. A human serum reactive to EBNA proteins was used as a primary antibody, and fluorescein isothiocyanateconjugated anti-human C3C antibody (DaKo) was used as a secondary antibody.ImmunoblottingCell lysates and immunoprecipitates were resolved by SDS PAGE on 8% gels, and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Membranes were blocked with 5% nonfat dry milk in Trisbuffered saline (TBS-M [pH 7.6]), and probed with first antibody, After the first antibody reaction for 2 hr at room temperature or 4℃over night, membranes were washed in TBS-Tween 20 (0.1%) solution followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG. Membranes were visualized with anECLWestem blotting kit (Amersham Biosciences).RT-PCR AnalysisTotal RNA was extracted from cells with Trizol reagent (Gibco-BRL), and then treated with DNase 1 (Gibco-BRL). One microgram of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Gibco-BRL) using 100 pmol of random hexamer (Takara, Otsu, Japan) for cDNA synthesis. cDNA aliquots were then subjected to PCR analyses using primer pairs specific for Cp Wp Qp or EBER, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each PCR cycle consisted of denaturation at 94℃for 3 min, annealing at 55℃for 45 sec and extension at 72℃for 1 min for 30 cycles (Cp Wp Qp) or 25 cycles (EBER, GAPDH). Sequences of prim ers are listed in Tableâ… . The PCR products (7.5 1 each) were electrophoresed in 2% agarose gel, transferred to Hybond N+(Amersham), and specifically amplified DNA was detected by the Gene Images 3'-oligolabeling module (Amersham) and ECL detection reagents (Amersham). The quality of RNA was checked by PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.Plasmids, Transfection, and IsolationAnneal the siRNA template oligonucleotides and ligate annealed siRNA template insert into pSilencer (Ambion, Inc Products) then transform E. coli with the ligation products. Pick clones, isolate plasmid DNA, and digest with BamHâ… and Hindâ…¢to confirm the presence of the siRNA template insert. we sequence the insert to confirm that there are no unwanted mutations. OriP is useful in primate cells since the vector showed high efficiency of stable transfection for EBV positivwe cells. So we transfer U6 promoter and shRNA template fragment into Orip vector. ShRNA sequence see table 1.Transfection: Seed a six-well tissue culture plate with 1-2×105 cells in 2ml of DMEM supplemented with 10% fetal calf serum. Incubate for 18-24 hours at 37℃in a CO2 humidified incubator or until the cells are 40-60% confluent. Dilute 1-2 micrograms of DNA into 100 microliters serum free DMEM in a sterile tube. To this tube, add 6 microliters of PLUS Reagent found in the LIPOFECTAMINETM PLUS Reagent kitInvitrogen 18324). Allow mixture to stand for 15 minutes at room temperature. In a second tube dilute 4 microliters of LIPOFECTAMINETM Reagent into 100 microliters serum free DMEM.. Combine the two solutions, mixing gently and incubate at room temperature for 10-15 minutes. Wash cells once with 2 ml serum free DMEM. For each transfection, add 0.8 ml serum-free medium to each tube containing the LIPOFECTAMINETM PLUS Reagent-DNA complexes. Mix gently and overlay the complexes onto cells.. Incubate the cells for 3 hours at 37℃in a CO2 humidi fied incubator. Remove the DNA-containing medium and replace with 2 ml of DMEM supplemented with 10% fetal calf serum. Incubate cells at 37℃in a CO2 humidified incubator for an additional 48-72 hours. Change the medium to complete mediem with 500ug/ml hygromycin. And incubate for another 21 days.RESULT1 In vitro cultured gastric cancer EBV positive cell lines are latencyâ… , express only EBNA1 but not EBNA2, 3s and LMP1 (fig 2). The results of the im- munofluorescence of EBNA are showed in fig 1. And EBER expression was checked by RT-PCR (fig 3).2 After treatment of 5-azacytidine, Cp can be activated in AGS/EBV and NUGC3/EBV cell lines. In AGS/EBV and NUGC3/EBV cell lines the only active promoter is Qp but not Cp or Wp (fig 4). After treatment of 5'-azacytidine as the shown concentration for 5 days we can see the activation of Cp (fig 5, 6). And no change in Wp and Qp (fig 7)3 After treatment of 5-azacytidine, AGS/EBV and NUGC3/EBV cell lines express LMP1 (fig 8).4 Furthermore repression of DNMT1 can be seen after 5-azacytidine treatment and is dose dependent (fig 9).5 The expression of DNA methylation enzymes in Burkitt' lympnoma cell lines by RT-PCR. In typeâ… latency they have higher expressions of DNMT1 and DNMT3b but lower expression of DNMT3a. (fig 10)6 Suppression the expression of DNMT1 by using siRNA cannot activate Cp. The expression of DNMT1 after transfection of siRNA is shown in fig 11. we can see the expression of DNMT1 was suppressed. But there was no activation of C promoter (fig 12).7 Suppression the expression of DNMT1 and DNMT3b by using siRNA cannot activate Cp sufficiently. The expression of DNMT1 after transfection of siRNA is shown in fig 13. we can see that the expression of DNMT1 was suppressed but the expression of 3b was higher. And when 3 b was suppressed DNMT1 became higher. Both suppressing DNMT1 and 3 b we can see the activation of Cp but is not sufficient. (Fig 14)DiscussionThere are 3 major types of latent infection of EBV. Typeâ…¢latency includes in vitro transformed B cell-lymphoblastoid cell lines (LCLs), some of the Burkitt's lymphoma cell lines and Acquired Immunodeficiency Syndrom (AIDS) lymphoma, posttransplantation lymphoma. This involves of expression of 6 viral nuclear antigens EBNA1, 2, 3A, 3B, 3C and-LP; 3 latent mem- brane proteins LMP1, LMP2A and LMP2B; the small nonpolyadenylated RNAs (EBERs) and BamHI A RNAs (BARs). In latencyâ… orâ…¡, the expressed genes are very limited. EBNA3s are the most important targets of the CTL, so latencyâ… andâ…¡cells are easy to escape from the immune responses.The association between EBV and gastric carcinoma was first reported by Burke et al in 1990. EBV DNA was demonstrated in more than 80% of gastric carcinomas of the lymphoepithelioma type by PCR and ISH. Subsequently, Shi bata and Weiss demonstrated EBV infection in gastric adenocarcinomas of ordi nary histology. They reported that EBV is present in almost all carcinoma cells in EBV positive cases. Interest has increased with a report that 69 of 999 (6. 9%) cases of gastric carcinoma from Japan were EBV positive using ISH for the EBV encoded small RNAs (EBERs). In contrast to Burkitts lymphoma and nasopharyngeal carcinoma, which are endemic in equatorial Africa and Southeast Asia, respectively, EBV positive gastric carcinoma is a non-endemic disease distributed throughout the world about 4-10%.The sheer range of EBV-associated diseases emphasizes the importance of developing either prophylactic vaccine to reduce disease burden or therapeutic strategies that specifically target virus-positive lesion. A number of approaches are currently being pursued towards these goals. They identified the major viral envelope glycoprotein gp350 as the dominant target of the neutralizing antibody response.Was the initial spur to development of a prophylactic EBV vaccine? However, it is now evident that neutralizing antibodies alone, or the combination of antibody and cell-mediaed responses to a single envelope component, are unlikely to offer long-lasting sterile immunity against an orally transmitted agent such as EBV. Nevertheless, a gp350-based vaccine could reduce the dose of infectious virus acquired orally and, particularly if combined with other vaccine antigens eliciting appropriate CD8+T cell response, could limit virus replication at the initial site of infection.Alternatively, EBV-positive tumor cell growth might be reversed by targeting EBNA1 or LMP1 transcription using ribozymes or antisense oligonucleotides. Many of these strategies remain at the "proof of principle" stage. Further- more, except of immunosuppression patient the EBV associated malignancies show highly stricted latent antigen expression, with loss of the immunodominant EBNA3A, 3B, 3C proteins, emphasizes the difficulties inherent in trying to target these tumors by immunologic means. Unless it is proves possible to active C promoter and express these immunodominant tatgets in tumor cells. For instance, by using demethylating agent such as 5-azacytidine to rescue Cp transcription in Burkitt's cell lead to activation of the C promoter and expression this family of proteins. In this study we try to find a new way for EBV positive gastric carcinoma therapy.We succeeded in getting EBV positive gastric cancer cell lines by using cell to cell infection. In vitro cultured EBV positive cell lines is typeâ… latency, only expressed EBNA1 and EBER but had no expression of EBNA2 3 s or LMP1. The Qp is the only active promoter in these cell lines (see result 1). Many researches have shown that methylation of the C promoter is critical in inactive of it. C promoter is silent in Burkitt's lymphoma, primary nasopharyngel carcinoma or gastric carcinoma.Because EBV is present in almost all carcinoma cells in EBV positive cases, the therapy for EBV induced disease focus on EBV. 5-azacytidine to rescue Cp transcription in Burkitt's cell lead to activation of the C promoter, but there is no report about Gastric carcinoma. In this research, we firstly found that the same rescue function can be seen in GC cell lines. After treatment of 5'-azacytidine, Cp can be activated in AGS/EBV and NUGC3/EBV cell lines and express LMP1 (fig 5, 8). Furthermore repression of DNMT1 can be seen after treatment and is dose dependent (fig 9). So we think 5'-azacytidine may has the function to repress the expression of DNMT. But in this study we cannot active Cp sufficiently by using siRNA to suppress the expression of DNMT (fig 14, 15). It may because that DNMT may not be sufficiently suppressed by siRNA or when one of the DNMT has been suppressed another will be activated (fig 13).Demethylating agents have been in clinical trials since 1967. 5-azacytidine has been used in combination with other agents to treat acute leukemia, and as a single agent to treat myelodysplastic syndrom. However, its most interesting use has been as a treatment for hemoglobinopathy including sickle cell anemia. For gastric carcinoma it is till at the in vitro research step. But there are many evidences show that 5-azacytidine may help to induce the transcription of tumor suppress genes. It is may be helpful for EBV positive gastric carcinoma therapy.Conclusion1 EBV positive gastric carcinoma cell lines can be constituted in vitro by using cell to cell infection, and they are typeâ… latency.2 After treatment of 5-azacytidine, Cp can be activated in AGS/EBV and NUGC3/EBV cell lines and express LMP1.3 Suppression of DNMT1 can be seen after 5-azacytidine treatment and is dose dependent.4 Cp cannot be sufficiently activated by using siRNA to suppress the expression of DNMT.
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