ABSTRACT
Gastric cancer is the most common cancer in Japan and infection with Epstein-Barr virus (EBV) is responsible for about 10% of gastric cancers worldwide. Although EBV infection may be involved at an early stage of gastric carcinogenesis, the mechanisms underlying its involvement remain unknown. To investigate the role of EBV in gastric carcinogenesis, we performed proteomic analyses of an EBV-infected gastric carcinoma cell line NU-GC-3 (EBV(+)) and its uninfected control (EBV(-)). 2-DE was combined with MS to identify differentially expressed proteins. We found that EBV infection upregulated one of the phosphorylated heat shock protein 27 kDa (HSP27). The phosphorylated HSP27 isoform which increased in EBV(+) cells can be induced by both heat shock and arsenite. The increase of phosphorylated HSP27 in EBV(+) cells was reduced by treatment with the phosphoinositide 3-kinase (PI3K) inhibitors (LY294002 and wortmannin). In addition, we found increased levels of phosphorylated Akt in EBV(+) cells. These findings suggest that EBV infection upregulates the phosphorylation of HSP27 via the PI3K/Akt pathway. Thus, activation of the PI3K/Akt pathway may contribute to the establishment of a malignant phenotype in EBV-infected gastric carcinomas.
Subject(s)
Cell Transformation, Viral/physiology , Gene Expression Regulation, Viral , HSP27 Heat-Shock Proteins/biosynthesis , Herpesvirus 4, Human/physiology , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , Stomach Neoplasms/virology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/virology , Amino Acid Sequence , Androstadienes/pharmacology , Arsenites/pharmacology , Cell Line, Tumor , Chromones/pharmacology , Electrophoresis, Gel, Two-Dimensional , Female , HSP27 Heat-Shock Proteins/analysis , HSP27 Heat-Shock Proteins/genetics , Heat-Shock Proteins , Hot Temperature , Humans , Male , Middle Aged , Molecular Chaperones , Molecular Sequence Data , Morpholines/pharmacology , Neoplasm Proteins/analysis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Isoforms/analysis , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , RNA, Viral/genetics , RNA, Viral/physiology , Signal Transduction/physiology , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Up-Regulation , Viral Matrix Proteins/genetics , Viral Matrix Proteins/physiology , WortmanninABSTRACT
To investigate the role of Epstein-Barr virus (EBV) in epithelial tumors, we compared the expression pattern of cellular genes in the EBV-infected gastric carcinoma cell line, NU-GC-3, and its uninfected control. Subtractive suppression hybridization (SSH) was combined with high-density DNA array screening to identify differentially expressed genes. We have discovered that EBV infection upregulated a truncated variant of human basic hair keratin 1 (hHb1-DeltaN), a gene that had previously been identified in metastatic breast carcinoma. We verified the differential expression of hHb1-DeltaN in 3 independent EBV-positive and -negative NU-GC-3 clones by Northern blotting. We further verified the EBV-dependent upregulation of hHb1-DeltaN in 3 other carcinoma cell lines (AGS, TWO3 and DLD1) by RT-PCR. Inhibition of CpG methylation by 5-Aza-CdR induced hHb1-DeltaN mRNA expression in the EBV-negative clones but did not alter the expression in the EBV-positive clones. The expression of hHb1-DeltaN protein was detectable by immunofluorescence and Western blotting in EBV-positive but not in EBV-negative NU-GC-3 clones after proteasome inhibitor (MG132) treatment. hHb1-DeltaN protein formed fibrous structures in the cytoplasm and accumulated in distinct nuclear bodies in the euchromatic areas of the cell nucleus. We suggest that the unstable hHb1-DeltaN protein may inhibit some of the functions of the keratin cytoskeleton and/or interfere with transcription regulation. It also may establish a link between EBV and the low differentiated or anaplastic status of the carcinomas that carry the virus.
