p53, a key regulator in gemcitabine-induced Epstein-Barr virus lytic activation in EBV associated gastric carcinoma

Abstract

Epstein-Barr virus (EBV), a dsDNA human gamma herpes virus, is associated with the development of malignancies including EBV-associated gastric carcinoma (EBVaGC) and constant presence of the viral genome in EBVaGC suggests the applicability of novel EBV-targeted therapies. An antiviral nucleoside drug, ganciclovir (GCV), is effective only in the context of the viral lytic cycle in the presence of EBV-encoded thymidine kinase (TK)/protein kinase (PK) expression. For the treatment of EBVaGC, gemcitabine was identified as a candidate for combination with GCV by screening of the Johns Hopkins Drug Library. In this study, it was circumscribed that pharmacological induction of EBV-TK or -PK by gemcitabine in EBVaGC-originated tumor cells might be useful for the combination treatment with GCV in vitro and in vivo. Gemcitabine induced EBV lytic activation via ataxia telangiectasia-mutated (ATM)/p53 genotoxic stress pathway, which was evaluated using an EBVaGC mouse model and a [125I] fialuridine (FIAU)-based lytic ctivation imaging system. To determine the precise molecular mechanism of how p53 is involved in lytic activation in EBVaGC, I screened several EBVaGC cell lines, SNU-719, YCCEL1 and NCC-24, in which gemcitabine could induce lytic activation except in NCC24. NCC-24 has two-point mutations in the DNA binding sites of p53. Wild type TP53 transfection restored gemcitabine-induced lytic activation in NCC-24. Role of p53 status in lytic activation was also evaluated in in vivo gemcitabine-GCV combination therapy. It was found that gemcitabine-GCV combination treatment was effective in SNU-719. To determine how p53 is associated with gemcitabine-induced EBV lytic activation, interaction of p53 with EBV Z promoter (Zp) region and proteins capable of binding to it were investigated. p53 protein binding to ZID region of Zp is verified by ChIP and EBV Zp reporter assays in SNU-719, but not in NCC-24. ZID domain does not have p53 binding site but have Sp1 and MEF2D binding sites. Formation of p53 and Sp1 complex and p53 binding to EBV Zp via Sp1 in gemcitabine-induced lytic activation were shown by IP and ChIP only in SNU-719, but not in Sp1 knockdowned SNU-719 and NCC-24. Therefore, p53 binding ability is a key factor for Zp activation by gemcitabine. Together, p53 is a key determining factor in gemcitabine-induced EBV lytic activation and this viral enzyme-targeted anti-tumor strategy may provide a new therapeutic approach for EBVaGCs.