Preclinical study of LMP1-RNAi-based anti-tumor therapy in EBV-positive nasopharyngeal carcinoma

RNA interference (RNAi) treatment has been proven to be an important therapeutic approach in cancer based on downregulation of target-oncogenes, but its clinical efficacy still needs further investigation. LMP1 is usually presented by Epstein-Barr virus (EBV)-positive tumor cells like EBV-associated nasopharyngeal carcinoma (NPC) and acts as an oncogene in tumorigenesis. However, the mechanism of LMP1 as a proto-oncogene in nasopharyngeal carcinoma is still unclear. Two sequence-specific shRNAs 1 and 2 were designed to target the different nucleotide loci of EBV latent antigen LMP1 gene and a series of in vivo and in vitro experiments were performed to investigate the therapeutic effect of sequence-specific shRNAs targeting LMP1 and its related molecular mechanisms in EBV-positive NPC. LMP1-shRNA2 generated a truncated LMP1 mRNA and protein, whereas LMP1-shRNA1 completely blocked LMP1 mRNA and protein expression. Both LMP1-shRNAs inhibited the proliferation and migration of NPC cells overexpressing LMP1 (NPC-LMP1) as well as the NPC-associated myeloid-derived suppressor cell (MDSC) expansion in vitro. However, LMP1-shRNA2 maintained the immunogenicity of NPC-LMP1 cells, which provoked MHC-class I-dependent T cell recognition. LMP1-shRNAs inhibited tumor growth in nude mice but did not reach statistical significance compared to control groups, while the LDH nanoparticle loaded LMP1-shRNAs and the antigen-specific T cells induced by NPC-LMP1 cells treated with LMP1-shRNA2 significantly reduced tumor growth in vivo. LMP1-RNAi-based anti-tumor therapy could be a new hope for the clinical efficacy of RNAi treatment of tumors like NPC.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

In vivo and in vitro study of co-expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma / Estudo in vivo e in vitro da coexpressão de LMP1 e Cripto-1 em carcinoma nasofaríngeo

Abstract Introduction: Nasopharyngeal carcinoma, an epithelial-derived malignant tumor which because of its anatomical location and atypical early symptoms, when diagnosed invasion and metastasis often have occurred. This requires a better understanding of the development mechanism, identifying diagnostic markers, and developing new treatment strategies. Objective: To study the relationship of LMP1 and Cripto-1 in nasopharyngeal carcinoma. Methods: The expression of LMP1 and Cripto-1 in specimens obtained from nasopharyngeal carcinoma patients (n = 42) and nasopharyngitis patients (n = 22) were examined. The expression of LMP1 and Cripto-1 in LMP1-negative and LMP1-positive (CNE1-LMP1) cells were also examined. Results: The expression of LMP1 and Cripto-1 was significantly higher in nasopharyngeal carcinoma than in nasopharyngitis (p < 0.05). Their expression in nasopharyngeal carcinoma with metastasis were significantly higher than that without metastasis (p < 0.05), which was correlated with TNM staging (p < 0.05). High Cripto-1 expression and high proliferation rate were seen in CNE1-LMP1 cells. Conclusions: The expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma is positively related. Their co-expression might contribute to the proliferation and metastasis of nasopharyngeal carcinoma.

Resumo Introdução: O carcinoma nasofaríngeo é um tumor maligno derivado do epitélio de localização anatômica recôndita e sintomas iniciais atípicos; quando diagnosticado, frequentemente invasão e metástases já ocorreram. Isso requer uma melhor compreensão do seu mecanismo de desenvolvimento, identificação dos marcadores diagnósticos e desenvolvimento de novas estratégias de tratamento. Objetivo: Estudar a relação de LMP1 e Cripto-1 no carcinoma nasofaríngeo. Método: A expressão de LMP1 e Cripto-1 em espécimes obtidos de pacientes com carcinoma de nasofaringe (n = 42) e pacientes com nasofaringite (n = 22) foi analisada. A expressão de LMP1 e Cripto-1 em células LMP1-negativas e LMP1-positivas (CNE1-LMP1) também foi analisada. Resultados: A expressão de LMP1 e Cripto-1 foi significantemente maior na presença de carcinoma nasofaríngeo do que na nasofaringite (p < 0,05). Sua expressão em carcinomas com metástase foi significantemente maior do que em casos sem metástase (p < 0,05), o que se correlacionou com o estadiamento TNM (p < 0,05). Uma alta expressão de Cripto-1 e alta taxa de proliferação foram observadas nas células CNE1-LMP1. Conclusões: A expressão de LMP1 e Cripto-1 é positivamente relacionada com carcinoma nasofaríngeo. Sua coexpressão pode ser atribuída à proliferação e metástase do tumor.

Presencia del marcador lmp-1 del virus epstein-barr en linfomas de caninos / Presence of lmp-1 protein of epstein-barr virus in lymphomas of canine origin

RESUMEN El virus Epstein-Barr (EBV) es un virus de alta prevalencia en humanos que se asocia con tumores de la línea linfoide B. En caninos se dispone de pocos reportes sobre la presencia del EBV y su rol en esta especie. El objetivo del presente estudio fue determinar la presencia de la proteína latente de membrana del EBV (LMP-1) en tejidos obtenidos de 20 linfomas de caninos cuyo diagnóstico se había realizado durante un periodo de 10 años, entre 2004 y 2014. Los linfomas se reclasificaron mediante las nuevas clasificaciones histopatológicas para linfomas y se sometieron a inmunohistoquímica (IHQ) con los anticuerpos anti-CD79a, anti-CD3, anticuerpos específicos para linfocitos B y T, además de un anti-LMP-1 como marcador de la presencia del EBV. Se encontró que el linfoma más común fue el linfoma nodal de zona T con un 75% de los casos. Al realizar la inmunomarcación se encontraron 18 casos positivos a CD3, 2 casos positivos a CD79a y 6 casos positivos a LMP-1, lo que representa el 30% de positividad del EBV en linfomas. El análisis Ji cuadrado demostró significancia estadística entre la presencia del virus y la presencia del linfoma lo que sugiere, no solamente que el virus está circulando en la población canina, sino que además puede tener relación con la ocurrencia de esta neoplasia. Con relación a las variables demográficas, sólo en la raza Golden Retriever se demostró relación con la presencia del linfoma, pero no con la presencia del virus.

ABSTRACT Epstein Barr virus (EBV) is a human high prevalent virus associated with lymphoid B cells tumors development. In canines, few reports have been published regarding the presence of the virus in dogs but its role in this species remains unclear. The aim of this study was to determine the presence of LMP-1 protein of EBV in 20 canine lymphomas tissues which were previously diagnosed in a period of time between 2004 -2014. Lymphomas were reclassified in accordance with the new histopathological classifications for lymphomas and were stained by IHQ with anti-CD79a, anti-CD3 and anti-LMP-1; in addition, specific antibodies for B lymphocytes, T lymphocytes and EBV biomarker, respectively. It was found that the most common lymphoma was T-zone lymphoma in 75% of the cases of the study. The distribution of the cases regarding the immunostaining was: 18 positive cases with anti-CD3, 2 positive cases with anti-CD79a and 6 positive cases with anti- LMP-1. Positive cases of LMP-1 as a biomarker of the presence of EBV corresponded to the 30% of the cases of the study. Chi-square test showed statistical significance between the presence of the virus and the presence of lymphomas, which suggests not only that the virus is circulating in the canine population but also that could have implications in the development of the disease. Regarding demographic parameters, only the Golden Retriever breed showed a relationship with the presence of lymphoma, but not with the presence of the virus.

Advance on subtypes/variant of Epstein-Barr virus and relationship between subtypes/variants andEpstein-Barr virus related diseases / 国际儿科学杂志

Epstein-Barr virus( EBV )is a ubiquitous human gamma-1 herpesvirus,which is associated with human malignancies,such as nasopharyngeal carcinoma,Burkitt's lymphoma and Hodgkin lymphoma.Based on the polymorphism of amino acids in the polymorphic region of EBV genome,it can be classified into different subtypes/variants.By now,whether the subtypes/variants of EBV preferentially is associated with particular malignancies or represent geographical polymorphism remains controversial.This review summarized the literature on sequence variation in EBV genes,focusing on LMP-1,EBNA-1,and BZLF-1 and their distribution by geography and disease.

Investigação da LMP1 do EBV e a coinfeçcão do HPV em lesões genitais de pacientes infectados ou não pelo HIV / Investigation of the LMP1 EBV and co-infection by HPV in genital lesions of patients infected or not by HIV

INTRODUÇÃO: Vários estudos têm demonstrado associação do vírus Epstein-Barr (EBV) com neoplasias malignas, inclusive genitais, em que o papilomavírus humano (HPV) é o principal vírus associado às neoplasias epiteliais benignas e malignas. OBJETIVO: Investigar a presença do EBV e do HPV em lesões genitais de ambos os sexos, em pacientes soropositivos (grupo A) ou não (grupo B) para o vírus da imunodeficiência humana (HIV). MATERIAL E MÉTODO: Selecionados 126 pacientes e 135 lesões anogenitais, sendo 67 pacientes (53 por cento) e 75 lesões (56 por cento) no grupo A e 59 pacientes (47 por cento) e 60 lesões (44 por cento) no grupo B, para análise imuno-histoquímica (IHQ) por meio dos anticorpos monoclonais antiproteína latente de membrana 1 (LMP1) e HPV (DAKO®). RESULTADOS: A análise mostrou que o número total de lesões com imunopositividade para o HPV e para a LMP1 foi maior no grupo A (32 e 35, respectivamente) quando comparado ao B (16 e seis, respectivamente). A análise estatística (nível de significância de 5 por cento) mostrou que as proporções para o HPV não são estatisticamente significativas (z = 1,93; valor p = 0,053). Entretanto, para a LMP1, a diferença (47 por cento no grupo A e 10 por cento no B) é significativa (z = 4,60; valor p = 4,2×10-6). Do mesmo modo, a associação HPV-LMP1 (21 por cento no grupo A e 7 por cento no B) também mostrou diferença estatisticamente significativa (z = 2,38; valor p = 0,017). CONCLUSÃO: Esses resultados indicam a possibilidade de sinergismo da infecção pelo EBV e a coinfecção EBV-HPV em lesões epiteliais genitais, particularmente em pacientes soropositivos para o HIV. Entretanto, investigações com metodologia de maior especificidade e sensibilidade são necessárias para a verificação da real participação do EBV na patogênese de lesões epiteliais genitais.

INTRODUCTION: Several studies have demonstrated the association between Epstein-Barr virus (EBV) and malignant neoplasias, including genital lesions, in which the human papillomavirus (HPV) is the main virus associated with both benign and malignant epithelial neoplasias. OBJECTIVE: Investigate the presence of EBV and HPV in genital lesions in HIV-infected patients (group A) or HIV non-infected patients (group B) from both genders. MATERIAL AND METHOD: We selected 126 patients and 135 anogenital lesions, comprising 67 patients (53 percent) and 75 lesions (56 percent) from group A and 59 patients (47 percent) and 60 lesions (44 percent) from group B, to histopathological and immunohistochemical analyses through latent membrane protein 1 (LMP1) monoclonal antibodies and HPV (DAKO®). RESULTS: The analysis showed that the total number of lesions with immunopositivity for HPV and for LMP1 was higher in group A (32 and 35 respectively) in comparison with B (16 and six respectively). Statistical analysis (significance level of 5 percent) showed that the proportions for HPV are not statistically significant (z = 1.93; value p = 0.053). However, the difference (47 percent in group A and 10 percent in B) is significant for LMP1 (z = 4.60; value p = 4.2×10-6). Similarly, the association of HPV and LMP1 (21 percent in group A and 7 percent in B) also showed a significant statistical difference (z = 2.38; value p = 0.017). CONCLUSION: The results demonstrated the possibility of synergism between EBV infection and EBV-HPV co-infection in genital epithelial lesions, mainly among HIV-infected patients. However, further investigations with a more specific and sensitive methodology are required in order to assess the real influence of EBV on the pathogenesis of genital epithelial lesions.

Construction of co-expression plasmid pBudCE4.1-LMP-1-LMP-3 and its expression in vitro / 重庆医科大学学报

Objective:To construct the mammalian CO-expression plasmid pBudCE4.1-LMP-1-LMP-3,and to detect the expression of the plasmid in vitro.Methods:Fragments of LMP-1 gene and Fragments of LMP-3 gene were gained from the Company,and were constructed respectively into the plasmid vector Puc57,The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes.fragments of LMP-1 gene were constructed firstly into the plasmid vector pBudCE4.1,fragments of LMP-3 gene were constructed into the plasmid vector pBudCE4.1-LMP-1 after iden tification with nucleotide sequencing and enzymes.MSCs cell line was transfected with this Co-expression plasmid using lipofectin reagent.according to the transfect situation,the MSCs were divided into 5 groups,the non-transfected group(Group A),the group transfected by empty vector(Group B),the group transfected by LMP-1(Group C),the group transfected by LMP-3(Group D)and the group transfected both LMP-1 and LMP-3(Group E).the expression of LMP-1 and LMP-3 were detected by RT-PCR and Western blot technique.Results:The plasmids Puc57-LMP-1、Puc57-LMP-3 and pBudCE4.1-LMP-1-LMP-3 were obtained successfully and verified by nucleotide sequencing and enzymes.After transfection with this mammalian Co-expression plasmid,the LMP-1 and LMP-3 molecules were expressed in MSCs cells.The results of RT-PCR and Western Blot were measured with the grey value.To the expression of mRNA and protein of LMP-1,the diferences between groups A、B and groups C、D、E were significant(P0.05);To the expression of mRNA and protein of LMP-3,the diferences between groups A、B and groups C、D、E were significant(P0.05).Conclusion:the constructed mammalian Co-expression plasmid pBudCE4.1-LMP-1-LMP-3 can express LMP-1 and LMP-3 molecules in vitro at the same time.

Expression of LMP-1,NET-1,and VEGF in non-keratin nasopharyngeal carcinoma and its clinical significance / 中国肿瘤生物治疗杂志

Objective:To investigate the expression of latent membrane protein-1(LMP-1),NET-1,and vascular endothelial growth factor(VEGF)in non-keratin nasopharyngeal carcinoma(NK-NPC)and its clinical significance.Methods :Sixty biopsy specimens from pathologically-confirmed NK-NPC patients,who were treated in the Affilitated Hospital of Nantong University from May 1999 to May 2003,were included in the present study.Using immunohistochemical techniques (Envison two-step),we examined the expression of LMP-1,NET-1 and VEGF protein in the specimens.The relationship between their expression and elnicopathological parameters and the prognosis was anayzed.Ten specimens of chronic nasopharyngitis served as control.Results:(1)The positive rates of LMP-1,NET-1 and VEGF in NK-NPC were significantly higher than those in the chronic nasopharyngitis(P0.05).(3)NET-1 and VEGF expression in NK-NPC specimens were positively correlated with lymph node metastasis and distant metastasis(P

The influence of siRNA targeting LMPI gene on expression of AP-1 and its related factors / 中国癌症杂志

Background and purpose:LMP1 was one of the protein encoded by EBV latent gene,which was found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.In human epithelial cells,LMP1 alters many functional properties that are involved in tumor progression and invasions.In this study we investigated the influence of LMP1 silence on AP-1 signal transduction pathway and its downstream factors involved with cell transformation,proliferation and apoptosis.Methods:The chemically synthetic siRNA targeting LMP1 was transfected into EBV positive gastric carcinoma epithelial cell line by lipofectamine 2000 at 50 nmol/L final concentration.The protein expression of c-Jun,JunB and CDK4 was tested by,Western blotting.The mRNA of c-Jun,surviving,CDK4 and MMP9 mRNA were tested by RT-PCR.The expression of survivin were tested by immunohistochemistry.Results:Compared with the cell control,CDK4 mRNA was up regulated(P

Expression of LMP-1、EBNA-2 in the Follicular Epithelium Neoplasm of the Thyroid / 医学研究杂志

0.05);LMP-1 and EBNA-2 in papillary thyroid carcinoma had no correlation.Conclusion Some thyroid papillary carcinomas may be related to EB virus infection,the relationship has yet to be further studied.

The Synergistic Effect of LMP1 and IRF7 on the Expression of TAP1 in DG 75 Cell Line

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.

The Synergistic Effect of LMP1 and IRF7 on the Expression of TAP1 in DG 75 Cell Line

In this study, the author explored the role of interferon regulatory factor 7 (IRF7) and latent membrane protein 1 (LMP1) on the regulation of antigen presenting molecules in B-lymphoblastoid cell lines. First, the author observed the endogenous expression of IRF7 and LMP1 in paired EBV-positive B-lymphoblastoid cell lines, Sav I and Sav III, which represent EBV type I and type III latency, respectively. The Sav I cell, which does not express LMP1, showed very low levels of endogenous IRF7 in the cytoplasm. However, Sav III cells, which express large amounts of LMP1, contained high levels of endogenous IRF7 in both the cytoplasm and the nucleus. The expression of surface MHC class I antigen was 7.8-fold higher in Sav III compared with Sav I cells when measured by fluorescence activated cell sorter (FACS) analysis. To understand whether IRF7 is involved in regulation of MHC I and TAP1, LMP1 or IRF7 were expressed by cotransfection in DG75 cells. Levels of TAP1 protein were up-regulated by LMP1 and IRF7 alone, and by LMP1 co expression with IRF7, the expression level was highest after co-transfection of LMP1 with IRF7. TAP1 promoter activity was also up-regulated to 2.4, 2.0, 3.2-fold by LMP1, by IRF7, and by LMP1 plus IRF7, respectively. Surface expression of MHC class I antigen was up-regulated by LMP1 alone and LMP1 plus IRF7, but not by IRF7 alone. These results suggest that IRF7 induces the expression of TAP1, but not MHC class I antigen and that LMP1 and IRF7 have additive effects on the expression of TAP1 protein.

Expression of STAT3 and its relation with LMP1 expression in human nasopharyngeal carcinoma / 西安交通大学学报(医学版)

Objective To study the expression and activation of signal transducer and activator of transcription 3(STAT3) in human nasopharyngeal carcinoma (NPC) tissues and its relation with the expression of latent membrane protein 1(LMP1) encoded by Epstein-Barr virus.Methods The expressions of LMP1,STAT3 and phosphated STAT3(p-STAT3) in 45 cases of NPC and 27 cases of chronic nasopharyngitis were studied by immunohistochemical method.Correlation between protein expressions was analyzed.Results The positive rates of LMP1,STAT3 and p-STAT3 in NPC were significantly higher than those in chronic nasopharyngitis(P0.05).Conclusion There are overexpression and abnormal activation of STAT3 protein in NPC tissues.LMP1 may play a role in the activation of STAT3.

The prevalence and deleted mutations of LMP1-EVB gene UCNT-NPC patients

The study was performed on 20 couples samples of nasopharyngeal carcinoma patients in K hospital in Hanoi. The findings indicated that: the prevalence of LMP1-EBV gene in peripheral blood of healthy persons was 96.7% (29/30 positive cases); the prevalence of LMP1-EBV gene in 20 UCNT nasopharyngeal carcinoma patients was 100% (20/20 positive cases); the prevalence of LMP1-EBV gene in 20 patients with other head and neck tumors was 100% (5/5 positive cases). On electrophoresis, 90% biopsies of nasopharyngeal carcinoma patients had deletion in LMP1 gene, while LMP1-EBV gene in peripheral blood of these patients, there were only 10% having deletion mutations.

Evaluation of EBV-LMP1 as prognostic indicator of nasopharyngeal carcinoma in Indonesian patients.

Forty eight cases of nasopharyngeal carcinoma patients who had been treated with radiotherapy were analysed for the expression of EBER in the nucleus of the tumor cells by in situ hybridization technique. EBER was expressed in 69% of the cases. Histologic specimens positive for EBER were then examined for the expression of EBV-LMP1 by immunohistochemistry, which showed 68% positivity. Clinical staging of 48 cases and radiation response of 32 cases were collected. Data of up to 2 years of survival of 16 cases were obtained through postal and phone communication. Statistical analysis showed that the Working Formulation histologic classification of NPC correlated significantly with radiation response. The expression of EBV-LMP1 was correlated significantly with the survival of the patients. Thus EBV-LMP1 which has been shown to possess influence on lymphoid and epithelial cells, clinicopathologically proved to have a function as prognostic indicator.

Apoptosis regulated by (-)-epigallocatechin-3-gallate in nasopharyngeal carcinoma cells using apoptosis cDNA expression array / 中国药理学通报

AIM: To investigate the mechanism of apoptosis induced by (-)-epigallocatechin-3-gallate (EGCG) and the intervenient effect of EGCG on apoptosis inducted by Epstein-Barr (EB) virus latent membrane protein 1 (LMP1). METHODS: The established doxycycline regulated nasopharyngeal carcinoma cell line pTet-on-LMP1 HNE2 were exposed to 0.6 mg·L-1 Dox and 100 mg·L-1 EGCG for 24 h. Total RNAs were extracted from cells hybridized with the Atlas apoptosis cDNA expression array membrane containing mainly apoptosis related genes. RESULT: EGCG regulated several apoptosis related genes, they were either up- or down-regulated. CONCLUSION: EGCG induced apoptosis might be mediated through some specific genes and signal transduction pathways, and the presence U of EGCG showed some intervenient effect on LMP1 induced cell apoptosis.

Detection of Epstein-Barr virus in the inflammatory and neoplastic uterine cervical lesions / 대한세포병리학회지

The prevalence of Epstein-Barr virus(EBV) in the uterine cervix was investigated to define the possible etiologic role in cervical carcinogenesis. The viral genotyping and LMP-1 30bp deletion were also studied. The materials included 169 uterine cervical swabs(152 within normal limits, 12 atypical squamous cells of uncertain significance, 3 low grade intraepithelial lesions, and 2 high grade squamous intraepithelial lesion) and 104 uterine cervical tissues obtained from hysterectomy specimens(32 carcinoma in situ, 9 microinvasive squamous cell carcinomas, 37 invasive squamous cell carcinomas, 7 adenocarcinomas, 7 adenosquamous carcinomas, and 12 cervicitis). EBV detected by PCR for EBNA-1 was positive in 52(56.5%) of 92 invasive and noninvasive cervical carcinomas, and 80(48.8%) of 164 inflammatory or normal cervices. The viruses detected in carcinomas were all type A, and LMP-1 30bp deletion form was more frequent in premalignant and malignant cervical lesions than in nonneoplastic cervices. From the above results, it may be concluded that EBV is one of common viruses detected in uterine cervix of Korean women, and type A virus and LMP-1 30bp deletion form may have a role in cervical carcinogenesis.

Genotype of Epstein-Barr Virus and Comparative Genomic Hybridization Analysis of NK/T Cell Lymphoma

NK/T cell lymphoma is a distinct clinicopathologic entity which is more prevalent in Asia than in America and Europe and is highly associated with Epstein-Barr virus (EBV) infection. Although the clinicopathologic features of the tumor have been clearly defined, genetic changes and roles of virus associated with the development and progression of tumor have not been well studied. In this study, we carried out polymerase chain reaction (PCR) for EBNA-3B, EBNA-3C, and LMP-1 30 bp deletion to investigate EBV subtype and variants in tumor tissue and performed comparative genomic hybridization (CGH) to screen chromosomal imbalances using frozen tissues from 7 patients with nasal-type NK/T cell lymphoma and 1 patient with blastic NK cell lymphoma. Of 6 cases infected with EBV, there were EBV type 1 in six, LMP-1 30 bp deletion variant in four, and LMP-1 40 bp deletion variant in one. Frequent chromosomal imbalances included deletions at 1p31-pter (4), 12q23-q24 (3), and 17p (4), and gains at 2q (5), 10q (3), and 13q34-qter (4). Blastic NK cell lymphoma displayed deletions of 9q, 7q, and 6q, similar to that of nasal-type NK/T-cell lymphoma. With these results, we assumed that candidate genes in these imbalanced chromosomal loci would provide the clue for further molecular studies to identify putative tumor suppressor genes or proto-oncogenes associated with pathogenesis of this neoplasm.

Expression of p53 Protein and Proliferating Cell Nuclear Antigen in Epstein - Barr Virus-associated Gastric Adenocarcinoma / 대한암학회지

PURPOSE: Recently, it has been reported that Epstein-Barr virus (EBV) is associated with some gastric cancers. But EBVs role in EBV-associated gastric carcinomas (EBVaGCs) has not been fully elucidated. This study was undertaken to evaluate the characteristics of EBVaGCs and to compare those with non-EBVaGCs. MATERIALS AND METHODS: EBV infection was studied using paraffin-embedded tissue blocks of 119 cases of gastric adenocarcinomas by in situ hybridization for EBV-encoded small RNAs (EBERs). In EBVaGCs and non-EBVaGCs, molecular characteristics were evaluated by immunohistochemical staining for latent membrane protein (LMP)-1, p53 protein, and proliferating cell nuclear antigen (PCNA). RESULTS: EBERs were detected in 12 cases (10.1%) of 119 gastric adenocarcinomas. LMP-1 was negative in all carcinomas tested, p53 protein was positive in 7 cases (58.3%) of 12 EBVaGCs and in 51 (47.7%) of 107 non-EBVaGCs, the difference between two groups being not significant. Mean PCNA index was 38.2+-26.1% in EBVaGCs and 22.8 +- 20.0% in non-EBVaGCs. The index was significantly higher in the former than in the latter. CONCLUSION: These results suggested that neoplastic progression in EBVaGCs was implicated with high expression of PCNA, but not consistently with overexpression of p53 protein or LMP-1.

Deletion within LMP-1 Oncogene in Hodgkin's Disease in Korea

LMP (latent membrane protein)-1 protein is one of the Epstein-Barr viral proteins and it is the most crucial one for the transforming activity. It is known to show considerable variation in its nucleic acid sequence and some biologic difference is reported to be associated with the variation. Twenty four cases of the EBV-associated Hodgkin's disease cases were searched for the 30-bp deletion within the C terminal intracytoplasmic domain of LMP-1 oncogene, one of the well-known genetic variation, by PCR and Southern blot using selected sets of primers and probes. The strain of the virus was also determined with PCR. Each case was positive both on LMP-1 immunostaining and in situ hybridization for EBER (Epstein-Barr encoded RNA). Deletion within LMP-1 oncogene was identified in 22 cases (92%), of which 5 cases showed wild form as well as a deleted form of LMP-1 at the same specimens. In seven cases showing the non-deleted form, pure or mixed with a deleted form, the distribution of sex and age was similar to that of the deleted form-only-group, but there was a slight tendency for a higher stage at presentation (4 of the 7 cases presented with stage IV). Those seven cases comprised of 4 cases of nodular sclerosis (NS), 2 cases of mixed cellularity (MC) and a case of lymphocyte depletion subtype while there were 9 and 12 cases of NS and MC among all the examined cases, respectively. Two cases with both a deleted form and the non-deleted form of LMP-1 showed type I and II strain of the virus while all the others contained only type of the. In conclusion, the rate of deletion in LMP-1 oncogene in our series was higher than that reported in western countries and there was a slight tendency for higher stages in cases detecting mixed deleted and non-deleted forms of LMP-1 than in cases a of deleted from of LMP-1.