Epstein-Barr virus latent membrane protein does not inhibit differentiation and induces tumorigenicity of human epithelial cells

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.

Section: Discussionmentioning

confidence: 96%

Human Monocytic Cell Lines Transformed In Vitro by Epstein-Barr Virus Display a Type II Latency and LMP-1-Dependent Proliferation

Masy

Adriaenssens²,

Montpellier

et al. 2002

“…Another EBV latent membrane protein, LMP1, has been shown to induce tumorigenicity and transformation of primary rodent fibroblasts and to inhibit differentiation of a squamous carcinoma cell line, SCC12F, in organotypic culture (16). However, LMP1-expressing HaCaT cells formed welldifferentiated tumors in SCID mice and terminal differentiation in suspension culture was not inhibited by LMP1 (39). In addition, LMP1 HaCaT cell tumors appeared slowly 6 to 8 weeks postinjection, in contrast to LMP2A-expressing HaCaT cells, which formed aggressive metastatic neoplasms 1 to 2 weeks after inoculation.…”

Section: Discussionmentioning

confidence: 99%

Epstein-Barr Virus LMP2A Transforms Epithelial Cells, Inhibits Cell Differentiation, and Activates Akt

Scholle

Bendt

Raab‐Traub

2000

262

249

The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase-Akt pathway. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus of the familyGammaherpesviridae. Primary infection occurs in the oropharyngeal epithelium, which is permissive for virus replication. Trafficking B lymphocytes are subsequently infected by progeny virions that go on to establish a lifelong latent infection in the B-cell compartment with periodic reactivation and reinfection of the oropharyngeal epithelium, virus release, and transmission of virus via the salivary route (27). EBV is associated with a number of human malignancies, such as Burkitt's lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, and the epithelial cell malignancy nasopharyngeal carcinoma (NPC) (27,42,(44)(45)(46)56). In EBV latency, only a subset of genes is expressed. Different types of latency can be distinguished according to the latent gene products expressed. In type I latency, characteristic of Burkitt's lymphoma, only the EBV nuclear antigen 1 (EBNA1), transcripts from the BamHI-A region of the genome (10), and the EBERs, small, nonpolyadenylated RNAs with an unknown function, are expressed. In B-cell lines immortalized by EBV in vitro, all latent genes are expressed, which include EBNA1 through -6, BamHI-A transcripts, EBERs, and three latent membrane proteins, LMP1, LMP2A, and LMP2B (latency III). The pattern of gene expression is more restricted in NPC, with expression of EBNA1, EBERs, BamHI-A transcripts, and the latent membrane proteins (latency II) (27).LMP2A and -2B arise from two different promoters, and transcription proceeds across the fused ...

“…In addition, LMP1 has been shown to induce the transformation of certain established rodent fibroblast cell lines, including Rat-1 and BALB/c 3T3 (2,33,49). Furthermore, LMP1 induces the morphological transformation of the RHEK-1 cell line (12) and the tumorigenicity of epithelial cell lines in severe combined immunodeficient mice (18,36).…”

mentioning

confidence: 99%

LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene

Yang¹,

Sham²,

Ng³

et al. 2000

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-Epstein-Barr virus (EBV) is a human gammaherpesvirus commonly carried in the majority of the human population. It is the causative agent for proliferative diseases such as infectious mononucleosis and oral hairy leukoplakia in AIDS patients. It is also implicated in a variety of human malignancies that include Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, nasal T-cell lymphoma, and immunoblastic lymphomas in posttransplant and AIDS patients (39). The virus often adopts latent forms in EBV-associated cancers. In nasopharyngeal carcinoma and Hodgkin's lymphoma, type II latency gene expression was observed. During this type II latency, only three viral proteins are routinely detected: EBV nuclear antigen 1 (EBNA1) and two latent membrane proteins, LMP1 and LMP2. Among the three EBV genes, the LMP1 gene is the only one implicated in cell immortalization and transformation. It is one of the viral genes required for B-cell immortalization, together with the EBNA2, EBNA3a, and EBNA3c genes (8,15,22,23,48). In addition, LMP1 has been shown to induce the transformation of certain established rodent fibroblast cell lines, including Rat-1 and BALB/c 3T3 (2, 33, 49). Furthermore, LMP1 induces the morphological transformation of the RHEK-1 cell line (12) and the tumorigenicity of epithelial cell lines in severe combined immunodeficient mice (18, 36).Although EBV is an important oncogenic virus in human, the LMP1 genes appeared to act differently from oncogenic genes of other DNA oncogenic viruses, including simian virus 40 (SV40) large T antigen, adenovirus E1A and E1B, and human papillomavirus (HPV) E6 and E7. There is little evidence suggesting that the LMP1 gene can function similarly to these viral oncogenes or even a cellular oncogene such as ras or myc. Though LMP1 transforms several immortalized rodent fibroblast cell lines and participates in B-cell immortalization, a single LMP1 gene has not been shown to induce continuous proliferation of any primary cell culture. Similarly, although it cooperates with a number of EBV genes in inducing B-cell proliferation, there is no report on the cooperativity of the LMP1 gene with another cellular oncogene in transforming primary cells.Protein sequence analysis of LMP1 revealed at least three domains: an amino-terminal cytoplasmic domain of 20 amino acid residues, a six-transmembrane domain of 185 amino acid residues, and a carboxy-terminal cytoplasmic domain of 200 amino acid residues (24). The six-transmembrane domain was shown to be required to form cytoplasmic membrane patches. The oligomerization of LMP1 in the cytoplasmic membrane may mimic that of other membrane receptor molecules induced by ligand-receptor interactions, resulting in constitutive activation of the LMP1 signaling pathway (14). The C-terminal cytoplasmic region was shown to interact with two families of proteins, TRAF (tumor necrosis factor [TNF] receptor-associated factor) and TRADD (TNF receptor-assoc...