[Epstein-Barr virus (Herpesviridae: Lymphocryptovirus) types 1 and 2 and other viral markers in patients with nasopharyngeal carcinoma in two geographically and ethnically distinct regions of Russia].

INTRODUCTION: The discovery of two types of Epstein-Barr virus (EBV) (EBV-1 and EBV-2) that have different biological properties stimulated the search for neoplasms associated with each type of the virus. The aim of the work is to study the nature of the association of nasopharyngeal cancer (NPC) with EBV-1 and EBV-2, serological activity for each viral type and the concentration of EBV DNA in the blood plasma of two gender, age and ethnic groups of NPC patients that represent geographically and climatically different regions of Russia,. MATERIALS AND METHODS: In the blood plasma of patients with NPC and other non- EBV associated tumors of oral cavity (OTOCEBV-) from the North Caucasian (NCFD) and Central (CFD) Federal Districts of Russia, the types of EBV and the concentration of viral DNA were determined using respectively «nested¼ and real time PCR; titers of IgG and IgA antibodies to viral capsid antigen (VCA) were measured in indirect immunofluorescence assay. RESULTS: The blood plasma samples testing showed that NPC and OTOCEBV- patients were infected with both types of EBV in approximately equal proportions. In two groups of NPC patients infected with one of the virus types only, EBV-1 or EBV-2, respectively, no statistically significant differences were found between the geometric mean values of IgG and IgA anti-EBV antibody titers and viral DNA concentrations in blood plasma. The distribution of virus types was not affected by either patient gender or ethnogeographic origin. The difference was found only between age groups: EBV-2 dominated in NPC patients up to 60 years, and EBV-1 was prevalent in patients over 60 years. CONCLUSION: The lack of the predominance of one of EBV types in NPC patients that are the representatives of different ethnic groups from geographically and climatically different regions, suggests that none of these factors play an important role in the NPC carcinogenesis. Evidently, each type of EBV, EBV-1 or EBV-2, if the necessary conditions arise, are able to exhibit its oncogenic potential to initiate tumor development.

[Epstein-Barr virus (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) in Kalmyks and Slavs living in Russia: virus types, LMP1 oncogene variants, and malignancies].

INTRODUCTION: The discovery of the Epstein-Barr virus types (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4) (EBV) - EBV-1 and EBV-2, which have different transforming abilities in vitro, stimulated the study of their prevalence in populations in order to elucidate the relationship with malignant neoplasms.The aims of the work are to study the prevalence of EBV-1 and EBV-2 among representatives of 2 ethnic groups of Russia, Kalmyks and Slavs, sequencing analysis of the LMP1 oncogene in virus isolates, and analysis of the correlation between virus types and the incidence of certain forms of tumors. MATERIALS AND METHODS: DNA samples were isolated from the biological material of oral swabs obtained from ethnic Kalmyks of the Republic of Kalmykia (RK) (n = 50) and Slavs, residents of the Moscow Region (MR) (n = 40). DNA samples were used to amplify EBV DNA, followed by determination of its concentration per 1 cell of washout, amplification of the LMP1 oncogene in viral samples, their sequencing, and determination of LMP1 protein variants. RESULTS: It has been established that with the same burden of EBV among representatives of both ethnic groups in the Kalmyk group, the ratio of persons infected with transforming and non-transforming types of the virus was almost the same (EBV-1 - 51%; and EBV-2 - 49%). Meanwhile, in the group of Slavs the transforming EBV-1 type virus dominated (80.6%). The predominance of EBV-1 type in representatives of the Slavs correlated with increased incidence of certain forms of tumors in the population of the MR when compared with similar values in the population of the RK, where both types of the virus were prevalent. Differences between the compared rates of cancer incidence were not statistically significant. Analysis of viral isolates showed a similar set of LMP1 variants in both ethnic groups. CONCLUSION: In order to establish the influence of EBV types on the incidence of malignant tumors, additional studies involving representatives of various ethnic groups from different geographical regions are needed.

EPSTEIN-BARR VIRUS AND NASOPHARYNGEAL CARCINOMA: VIRAL MARKERS FOR DIAGNOSTICS AND ASSESSMENT OF CLINICAL STATUS OF PATIENTS.

The etiological role of the Epstein-Barr virus (EBV) in the development of an undifferentiated histological variant of nasopharyngeal carcinoma (uNPC) found for the first time in regions with a high incidence of this pathology, the Southern provinces of China and the countries of Southeast Asia, and later in the rest of the world, has served as a basis for the widespread use of EBV serological markers for the diagnosis of this form of tumor. In recent years, the use of a test based on the quantitative determination of the EBV DNA concentration in the blood plasma of uNPC patients for early detection and monitoring of the disease has become widespread in endemic regions. In non-endemic regions, such studies virtually have not been carried out, and moreover, the comparative evaluation of the significance of two viral markers, serological and EBV DNA load in the bloodstream of uNPC patients, for diagnostics and evaluation of the therapeutic effect was not investigated. The aim of this study was to compare the clinical value of two serological markers and plasma EBV DNA load in uNPC patients from non-endemic region (Russia). The obtained results indicate that IgA antibodies to the viral capsid antigen (IgA/VCA) and plasma EBV DNA concentration can be successfully used for the diagnosis of uNPC, while IgG/VCA antibodies have no practical significance as an uNPC marker. In addition, it was found that plasma EBV DNA load is more sensitive marker of uNPC than IgA/VCA titers because DNA copy numbers reflect more accurately the effect of the therapy and the clinical state of patients at the stages of remission or relapse. It was shown for the first time that in the non-endemic region the simultaneous evaluation of IgA/VCA antibody levels and the plasma EBV DNA loads are the most effective markers for the diagnostics of uNPC. However, we believe, that it is more practical to use IgA/VCA antibody levels for uNPC screening, and plasma EBV DNA copies - for monitoring of the disease.

Epstein-Barr virus biomarkers for nasopharyngeal carcinoma in non-endemic regions.

The Epstein-Barr virus (EBV) plays a key role in the development of undifferentiated nasopharyngeal carcinoma (uNPC). In uNPC endemic regions EBV-specific antibodies and plasma EBV DNA load are used as markers for the early detection of uNPC and monitoring of the disease. In non-endemic regions, such studies were practically not conducted. The aim of this study was to compare the clinical significance of EBV serological markers and plasma EBV DNA levels for uNPC patients in a non-endemic region, Russia. The results obtained indicate that both viral capsid antigen/immunoglobulin A (VCA/IgA) antibodies and plasma EBV DNA copies can effectively be used for nasopharyngeal carcinoma (NPC) diagnosis. Besides, plasma EBV DNA load was found to be a more sensitive marker of uNPC than VCA/IgA antibody titres, as it reflected the effect of the therapy in stages of remission and relapse of the disease more precisely. Our study, for the first time, demonstrates that the simultaneous use of plasma EBV DNA loads and VCA/IgA antibody levels are indispensable markers for uNPC in non-endemic regions: a serological marker can be more effectively used for NPC screening, but EBV DNA copies are better for monitoring the disease. However, both markers turned out to be practically unsuitable for assessing the clinical status of patients. Serological markers did not correlate with any signs of the tumour process estimated by tumour, node and metastasis (TNM) classification and the plasma EBV DNA loads correlated only with the size of the pathologically altered lymph nodes (N). Additional study is required to confirm these findings.

STRUCTURAL AND FUNCTIONAL CHARACT ERISTICS OF THE LMP1 ONCOGENE IN PATIENTS WITH TUMORS ASSOCIATED AND NOT ASSOCIATED WITH THE EPSTEIN-BARR VIRUS.

Epstein-Barr virus (EBV) - the etiological agent of a number of human benign and malignant tumors including infectious mononucleosis (IM), Burkitt lymphoma (BL), Hodgkin (HL) and non-Hodgkin (NLH) lymphomas, nasopharyngeal carcinoma (NPC), and many other tumors. Latent membrane protein 1 (LMPl) encoded by the gene of the same name (LMP I) is the main oncoprotein of EBV. LMP1 is a transmembrane protein capable of activating many signaling pathways and transcription factors of the cells, which leads to its transformation. Molecular analysis of LMP1 of various clinical origins identified many variants with different types of amino acid mutations that influence its biological activity. Since the role of LMPl in the development of NPC is still not fully understood, it is important to find out how LMPl samples from patients with EBV-associated form of NPC differ from those of patients with other tumors also located in the oral cavity (OTOC), but not associated with this virus. Unlike most investigations conducted in endemic regions, the present work is intended to compare the genetic structure and the transforming activity of LMPl variants from NPC and OTOC patients has been carried out in a non-endemic region of Russia, where NPC is rarely diagnosed. The obtained data show structural and functional similarities of LMP1 variants in the two groups of patients and, accordingly, a genetic relationship of EBV strains persisting in these patients. Our work suggests that in non-endemic regions any EBV strain with any structure of LMP1 may become the etiologic agent of NPC. However, based on modem concepts, the cancer can occur only if EBV-infected persons have a unique pattern of HLA associated with a high sensitivity to the development of NPC combined with exposure to harmful environmental factors (chemical or physical carcinogens) and lifestyle.

[Epstein-Barr Virus LMP1 oncogene variants in cell lines of different origin].

It is well known that the Epstein-Barr virus (EBV) is a widespread infection in the human population. Typically, infection occurs in early childhood without serious consequences for infected people. At the same time, a secondary infection with an additional EBV strain occurs quite often. During the in vitro cultivation of peripheral blood lymphocytes from persons infected with multiple strains of the virus, only one of these strains with higher transforming potential becomes dominant, while the others are eliminated. Under certain conditions, such a highly transforming EBV strain apparently is able to be the etiologic agent of EBVassociated diseases. To find out the range of highly transforming EBV strains prevalent among Russians, cell lines from patients with EBV-associated and non-associated tumors, as well as healthy individuals, were established. The structural analysis of the latent membrane protein 1 gene (LMP1), a key oncogene of the virus, isolated from established cell lines and peripheral blood lymphocytes of blood donors was carried out, and data obtained were compared with the respective data for LMP1 isolates, amplified from cell lines established from African and Japanese patients with Burkitt's lymphoma. The data obtained show a genetic relationship between Russian LMP1 isolates regardless the fact whether they come from patients with tumors or healthy individuals and differ significantly from LMP1 isolates from Burkitt's lymphoma patients. Thus, the results of the study suggest that in nonendemic region for EBV-associated pathology, Russia, any strain of EBV with any structure of LMP1 with concomitant effect of additional factors may become an etiologic agent for EBV-associated neoplasia.

[Molecular biological properties of the Epstein-Barr virus LMP1 gene: structure, function and polymorphism].

Recent studies indicate that the latent membrane protein 1 (LMP1) encoded by the same name gene of the Epstein-Barr virus (EBV) plays an extremely important role in the pathogenesis of a number of malignant neoplasia. Specifically, LMP1 has the ability to transform human B-lymphocytes in vivo and in vitro and rodent fibroblasts (Rat-1) in vitro. The introduction of the latter into athymic mice leads to tumor development. In addition, expression of the oncoprotein has been often found in EBV-associated tumors at the DNA and constantly at the RNA levels. Having pleiotropic effects, LMP1, participates in the transmission and activation of multiple intracellular signals. It is also involved in the inhibition of key tumor suppressors, has significant influence on proliferation, apoptosis and morphological alteration of the infected cells finally resulting in their transformation. General characteristics of EBV and LMP1 gene as well as functional activity of the encoded LMP1 protein and a brief description of human pathologies associated with the virus have been discussed in this review. The questions concerning the polymorphism LMP1 in EBV-associated pathologies have been also analyzed in details.

[The LMP1 oncogene sequence variations in patients with oral tumours associated or not associated with the Epstein Barr].

The role of the Epstein-Barr virus (EBV), a ubiquitous lymphotropic human herpesvirus type 4, in the etiology of nasopharyngeal carcinoma (NPC) is not fully understood. The mechanism of NPC carcinogenesis, associated with the virus, is also not clear. The objective of present investigation was to carry out comparative analysis of the structure of an LMP1 oncogene of EBV in viral isolates obtained from patients with two types of tumors of the oral cavity: (a) associated (i.e., NPC) and (b) not associated (other tumors of the same anatomical region, OTOC) with EBV. Comparative analysis of C-terminal regions of LMP1 variants that was based on a sequence analysis of LMP1 from tumor, blood and throat washing samples of NPC and OTOC patients showed that all structural characteristics of LMP1 in both groups of patients were genetically similar, and differences found between compared parameters were statistically insignificant. Thus, for the first time it has been revealed that in NPC and OTOC patients in Russia genetically related EBV strains with structurally similar LMP1 variants are persisting that are likely to reflect a polymorphism of the virus circulating in population. The findings allow us to suggest that in non-NPC-endemic regions of the world, which include Russia, the risk of NPC development does not depend on the EBVstrain and its variant of LMP1 so much, but mostly from the genetic predisposition of infected persons to the disease and the exposure to other, as yet unknown agents.

Functionally significant mutations in the Epstein-Barr virus LMP1 gene and their role in activation of cell signaling pathways.

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively activated analog of the tumor necrosis factor receptor TNF-R1. LMP1 serves as a viral oncogene able to transform human B-lymphocytes and rodent fibroblasts via activation of numerous cellular signal cascades. Two specific motifs within LMP1 are responsible for interaction of this viral protein with the receptor protein beta-TrCP/HOS SCF of the ubiquitin ligase E3 complex, playing an important role in degradation of numerous cellular proteins including NF-kappaB inhibitor IkappaBalpha. In this study, we demonstrate for the first time the importance of point mutations affecting HOS-recognizing motifs of LMP1 for activation of NF-kappaB, AP1, and PI3K/Akt signaling pathways. It has also been shown that rat fibroblast cell lines (Rat-1) expressing different HOS mutants of LMP1 produce different amounts of reactive nitrogen species. Our data confirm the hypothesis that point mutations in the C-terminal region of the LMP1 cytoplasmic domain can influence the transforming potential of the Epstein-Barr virus.

Human oncogenic viruses: hepatitis B and hepatitis C viruses and their role in hepatocarcinogenesis.

Chronic infections caused by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) are the main risk factors for the development of hepatocellular carcinoma (HCC) in humans. Both viruses cause a wide spectrum of clinical manifestations ranging from healthy carrier state to acute and chronic hepatitis, liver cirrhosis, and HCC. HBV and HCV belong to different viral families (Hepadnoviridae and Flaviviridae, respectively); they are characterized by different genetic structures. Clinical manifestations of these viral infections result from the interaction between these viruses and host hepatocytes (i.e. between viral and cell genomes). Proteins encoded by both viruses play an important role in processes responsible for immortalization and transformation of these cells. Chronic inflammation determined by host immune response to the viral infection, hepatocyte death and their compensatory proliferation, as well as modulation of expression of some regulatory proteins of the cell (growth factors, cytokines, etc.) are the processes that play the major role in liver cancer induced by HBV and HCV.

Epstein-Barr-virus-associated gastric cancer in Russia.

The present investigation was carried out to estimate the prevalence of EBV-associated cases among gastric carcinoma (GC) patients of Russia and the Republics of the former Soviet Union (FSU). With this aim, formalin-fixed paraffin-embedded blocks from 206 gastric carcinomas obtained from patients of the Cancer Research Center, Moscow, were investigated by EBV-encoded RNA-1 (EBER-1) in situ hybridization applied to paraffin sections. As a result, 18 GC cases (8.7%) revealed uniform EBER-1 expression restricted to the carcinoma cells. Hybridized signals not detected in non-neoplastic gastric epithelium. EBV involvement was significantly more frequent among males, especially in tumors of less differentiated types (moderately differentiated tubular adenocarcinomas and poorly differentiated solid adenocarcinomas) and located in the upper stomach (cardia and middle). Most EBV-positive GCs were characterized by strong lymphoid-compartment involvement. Our findings concerning the distribution of EBV-positive GCs by sex, site and hystological type are similar to those in Japan, however, EBV-positive rate of GC cases in Russia is higher than in Japan and lower than in USA.

Metastatic capacity of Burkitt's lymphoma cell lines in nude mice.

Burkitt's lymphoma (BL) cell lines estimated in a previous study as having a high, low and no tumourigenicity (7) were intravenously (i.v.) injected into preirradiated (480 rad) nude mice. BL cell lines with a high tumourigenic potential produced metastatic tumours in the brain, spinal cord, bone marrow, stomach and kidney, but did not disseminate into the lung, liver, ovary and spleen. The survival time of the tumour bearing animals ranged from 2 to 10 weeks. The majority of mice i.v. injected with highly tumourigenic BL cell lines showed paresis or paralysis of the hind legs. This was associated with the presence of neoplastic nodules either in the brain and/or in the spinal cord. In animals with metastasis to the stomach and kidney progressive cachexia was observed. The described experimental model of metastatic BL tumours in nude mice can effectively be used for the in vivo study of new therapeutic molecules such as monoclonal antibodies coupled or not to substances, toxic to tumour cells. This model can also be useful for the identification and analysis of homing properties of BL cells and their implication in BL pathogenesis.

Burkitt's lymphoma cell lines reveal different degrees of tumorigenicity in nude mice.

A quantitative in vivo assay for evaluating the tumorigenicity of Burkitt's lymphoma (BL) cell lines in nude mice is described. It is based on the dose-response kinetics of BL cell lines in pre-irradiated (480 rad) nude mice following the s.c. injection of 4 different cell doses. This model system was used to estimate the xenografting potential of 26 BL cell lines derived from BL patients of different geographic and ethnic origins, as well as lymphoblastoid cell lines (LCLs) established by Epstein-Barr virus (EBV) immortalization of normal B lymphocytes. The results indicate that most BL cell lines are tumorigenic, but LCLs fail to produce progressively growing tumours in nude mice. However, BL cell lines revealed individual degrees of tumorigenicity and accordingly could be divided into 4 groups with high, moderate, low or no tumorigenicity. Preliminary attempts to correlate the xenografting phenotype of BL lines with other characteristics indicate that: (1) the aberrations of chromosome 1 are more often encountered in cell lines with high and moderate tumorigenicity; (2) EBV-positive BL lines do not reveal a higher tumorigenic phenotype in comparison with EBV-negative ones; (3) cell lines carrying translocations t(8;22) and t(2;8) might fall more frequently in the group of lines with high and moderate tumorigenicity; and (4) when LCLs grow in nu/nu mice, rejection always occurs and is associated with massive tumour necrosis. These findings suggest that the tumorigenicity of BL cell lines in immunosuppressed animals is not related with EBV, but with certain chromosomal abnormalities (BL-specific and non-specific) indicating that this in vivo model system can be instrumental for the identification of other factors or stages involved in BL development.