1. ¹Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, China
2. ²Laboratory of Molecular Technology SAIC-Frederick, National Cancer Institute, Frederick, Maryland, USA
3. ³St Vincent's Clinical School, Faculty of Medicine, The University of New South Wales, Sydney, Australia

Correspondence: Dr Lun-Quan Sun or Professor Ya Cao, Faculty of Medicine, University of New South Wales, Sydney, Australia. E-mail: lun-quan.sun@unsw.edu.au Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, 410078, PR China. E-mail: ycao98@publics.cs.hn.cn

Received 23 September 2004; Published online 1 April 2005.

Abstract

The latent membrane protein (LMP1) encoded by Epstein–Barr virus (EBV) has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. RNA-cleaving DNA enzymes are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. In this study, we explore the potential of using DNAzymes as a therapeutic approach to EBV-associated carcinomas by targeting the LMP1 gene. In all, 13 different phosphorothioate-modified "10–23" deoxyribozymes (DNAzymes) were designed and synthesized against the LMP1 mRNA and transfected into B95-8 cells, which constitutively express the LMP1. Fluorescence microscopy was used to examine the cellular uptake and distribution in B95-8 cells. As demonstrated in Western blots, three out of 13 deoxyribozymes significantly downregulated the expression of LMP1 in B95-8 cells. These DNAzymes were shown to markedly inhibit B95-8 cell growth compared with a disabled DNAzyme and untreated controls, as determined by an alamarBlue Assay. It was further demonstrated that these DNAzymes arrested the B95-8 cells in G0/G1 using flow cytometry. Interestingly, the active DNAzymes could also downregulate the expression of Bcl-2 gene in treated cells, suggesting a close association between the LMP1 and Bcl-2 genes and their involvement in apoptosis. This was further confirmed with the result that the DNAzymes could induce the release of cytochrome c from mitochondria, which is the hallmark of the apoptosis. The present results suggest that the LMP1 may present a potential target for DNAzymes towards the EBV-associated carcinoma through cell proliferation and apoptosis pathways.