Id1 Interacts and Stabilizes the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) in Nasopharyngeal Epithelial Cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells

Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development. In the last decades, numerous evidences showed that polyploidization also involves in either an early step or a critical promoting factor in tumorigenesis. In this study, I investigated whether ploidy influences the response of cancer cells to genotoxic agents. Using a novel polyploid system, Hep3B-T cells, generated by prolonged nocodazole treatment of Hep3B cells followed by mitotic slippage and re-replication of the DNA, the genotoxicity of the new tetraploid cells was compared with its diploid counterpart. I found that tetraploidization increased the cell volume without affecting the cell cycle distribution and doubling time. In spite of the centrosome overduplication due to polyploidization, the majority of Hep3B-T cells underwent bipolar mitosis. Polyploidization sensitized cells to genotoxic agents inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, I found that more gamma-H2AX foci, a molecular marker present on sites of DNA damage, were induced by radiation in Hep3B-T cells than in Hep3B cells. Furthermore, similar increase in radiosensitivity occurred in primary tetraploid fibroblasts in comparison with normal diploid fibroblasts. These results raise an implication for chemotherapy of which some cancer cells may be sensitized to genotoxic agents by a preceding step that induces polyploidization

Regulation and function of LMP1 in epithelial cells

published_or_final_versionAnatomyDoctoralDoctor of Philosoph

Epstein–Barr Virus Hijacks DNA Damage Response Transducers to Orchestrate Its Life Cycle

The Epstein–Barr virus (EBV) is a ubiquitous virus that infects most of the human population. EBV infection is associated with multiple human cancers, including Burkitt’s lymphoma, Hodgkin’s lymphoma, a subset of gastric carcinomas, and almost all undifferentiated non-keratinizing nasopharyngeal carcinoma. Intensive research has shown that EBV triggers a DNA damage response (DDR) during primary infection and lytic reactivation. The EBV-encoded viral proteins have been implicated in deregulating the DDR signaling pathways. The consequences of DDR inactivation lead to genomic instability and promote cellular transformation. This review summarizes the current understanding of the relationship between EBV infection and the DDR transducers, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), and discusses how EBV manipulates the DDR signaling pathways to complete the replication process of viral DNA during lytic reactivation

Id1 inhibits LMP1 ubiquitination in epithelial cells.

<p><i>A</i>, Ubiquitination status of B95.8 LMP1 wild type and CTARs mutants in epithelial cells. HEK293 cells were transiently transfected with plasmids as the figure shown. <i>In vivo</i> ubiquitination assay was performed as described in “Experimental Procedures” MG132 was added and cells were incubated for 6 hours before cell harvest. The extract was immunoprecipitated with mouse monoclonal anti-LMP1 (S12) antibody and subjected to Western blot with anti-HA antibody to detect the polyubiquitin chain of LMP1 and its mutants. <i>B</i>, Id1 overexpression reduced LMP1 ubiquitination. HEK293 cells were transiently transfected with plasmids as the figure shown. MG132 was added to the cells and incubated for 6 hours before cell harvest. Immunoprecipitation was performed with anti-FLAG antibody and subjected to Western blot with anti-HA antibody to detect the polyubiquitin chain. <i>C</i>, Id1 binds to LMP1 and reduces LMP1 ubiquitination. HEK293 cells were cotransfected either with wild type (WT) B95.8 LMP1 or B95.8 LMP1Δ194–233, with or without FLAG-Id1. Cells extracts were prepared and subjected to immunoprecipitation followed by Western blot analysis as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021176#pone-0021176-g004" target="_blank">Fig. 4B</a>.</p

Upregulation of LMP1 level in epithelial cells with Id1 overexpression.

<p><i>A</i>, The levels of LMP1 protein could be detected after overexpressing Id1 in C666-1 cells. C666-1 cells were transiently transfected either with vector alone or FLAG-Id1, and were lysed 48 hours post-transfection. Protein were transferred to PVDF membrane and subjected to Western blot analysis with anti-LMP1 and anti-FLAG antibody. <i>B</i>, Increase of LMP1 protein levels in HONE1-2117 LMP1 stable cells. HONE1-2117 LMP1 cells were transiently transfected with FLAG-Id1. Cells were examined by Western blot analysis for LMP1 (left panel) and RT-PCR for LMP1 expression with specific primers as described under “Experimental Procedures” (right panel). <i>C</i>, Id1 upregulated LMP1 in a dose-dependent manner. Increasing amount of Id1-HA plasmid was transfected into HONE1-2117 LMP1 which stably expressed LMP1. Western blotting was performed 48 hours post transfection. The amount of plasmids used are indicated on the figure in µg. Protein extracts were subjected to Western blot with anti-LMP1 and anti-HA antibody. Actin was used as loading control. <i>D</i>. Id1 knockdown was followed by decrease of LMP1 protein level. HONE1-2117 LMP1 was transiently transfected with either scramble shRNA or Id1 specific shRNA. Cells lysates were subjected to Western blotting analysis with anti-LMP1 and anti-Id1 antibody (left panel). RT-PCR was performed with specific primers to detect LMP1 and ID1 gene transcripts. GADPH was used as loading control. <i>E</i>. Immunofluorescence examination of HONE1-2117 LMP1 cells transiently transfected with FLAG-Id1. HONE1-2117 LMP1 cells were transfected with FLAG-Id1 plasmid and immunostained with anti-LMP1 (green), anti-FLAG (Id1, Red) antibody and counterstained with Hoechst 33258 to locate the nucleus (see “Experimental Procedures for further details). Higher expression of LMP1 was observed in HONE1-2117 LMP1 cells overexpressing Id1.</p