Comprehensive analyses of Epstein-Barr virus(EBV) -dependent cellular gene expressions responsible for the viral oncogenesis

本研究的目的是通过利用能够从细胞中根除EBV附加体的独特分子，即，显性阴性EBNA 1（DNE 1; Mol. Ther.，11（4）：578-90，2005）。我们通过DNE 1转导制备了EBV阳性和阴性细胞系的同基因对，并比较了它们的恶性程度。同时用多细胞因子测定和基因芯片系统全面评估这些细胞对中细胞基因表达的改变，从而阐明EBV在恶性转化中的作用。研究结果如下：1.腺病毒载体介导的DNE 1转导在几天内成功地从大多数自然EBV阳性的T/NK淋巴瘤和上皮肿瘤细胞系中根除EBV附加体.与亲代携带EBV的T/NK肿瘤细胞相比，DNE 1产生的EBV缺失的T/NK细胞显示出对其恶性表型的显著抑制，例如倍增时间延长和锚定非依赖性生长潜力的丧失。在部分EBV失活的T/NK细胞中，病毒基因组的缺失也导致细胞死亡.与亲代携带EBV的T/NK肿瘤细胞相比，EBV丢失的T/NK细胞显示出一些细胞因子如白细胞介素-9的产生显著降低。这些结果表明，T/NK细胞中的EBV依赖性恶性表型以及可能还有上皮细胞中的EBV依赖性恶性表型，如B细胞恶性肿瘤中的EBV依赖性恶性表型，至少部分与EBV依赖性恶性表型的上调相关（即，自分泌机制）和/或某些细胞因子基因的下调。我们正计划探索细胞因子基因控制的机制和对EBV肿瘤发生的特异性影响。

The aim of this study was to identify the cellular genes closely related to or responsible for Epstein-Barr virus (EBV)-induced oncogenesis in naturally EBV-positive T/NK and epithelial tumor cells, by utilizing a unique molecule capable of eradicating EBV episomes from cells, i.e., dominant-negative EBNA1 (DNE1; Mol. Ther., 11(4):578-90, 2005). We prepared isogenic pairs of EBV-positive and-negative cell lines by DNE1 transduction and compared their malignant grade. Concomitantly alterations of cellular gene expression in those cell pairs were comprehensively assessed with the multi-cytokine assay and GeneChip microarray system, thereby elucidating the role(s) of EBV in malignant conversion. The results obtained are as follows.1. Adenovirus vector-mediated transduction of our DNE1 successfully eradicated EBV episomes from the majority of naturally EBV-positive T/NK lymphoma and epithelial tumor cell lines in a few days.2. The EBV-lost T/NK cells produced by DNE1 showed a striking suppression of their malignant phenotypes, such as prolongation of doubling time and loss of anchorage-independent growth potential, compared with parental EBV-harboring T/NK tumor cells. In a part of the EBV-lost T/NK cells, loss of viral genome also brought about cell death.3. The EBV-lost T/NK cells showed significantly decreased production of some cytokines, such as interleukin-9, compared with the parental EBV-harboring T/NK tumor cells. Conversely, the production levels of a few other cytokines were also found to be upregulated in the EBV-lost T/NK cells.These results indicate that the EBV-dependent malignant phenotypes in T/NK cells and perhaps also in epithelial cells, as in B-cell malignancy, are associated at least partly with upregulation (i.e., the autocrine mechanism) and/or downregulation of certain cytokine genes. We are planning to explore precisely the mechanisms of cytokine gene control and specific effects on EBV oncogenesis.