ABSTRACT
Epstein-Barr virus (EBV) can establish latent infection and has been associated with various human cancers. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein that is expressed in all EBV-associated malignant tissues. The N- and C-terminal domains of EBNA1, which are connected by internal glycine/alanine-rich short repeat sequences of various sizes, show sequence divergence across EBV strains isolated from around the world. At least five subtypes have been described, according to the amino acid at residue 487: P-ala, P-thr, V-val, V-pro, and V-leu. Whether the variations of EBNA-1 contribute to the pathogenesis of EBV or simply reflect the geographical distribution of EBV remain to be investigated. Furthermore, the cell effects conferred by EBNA1 subtypes that differ from that of the B95.8 prototype, which belongs to the P-ala subtype, remain to be elucidated. In this study, PCR was amplified with the full-length V-val EBNA1 gene from the CG3 cell line, an EBV-carrying lymphoblastoid cell line derived from a Taiwanese chronic myeloid leukemia patient. Plasmids expressing His-tagged EBNA1 fusion proteins in E. coli were constructed and used to raise antibodies in rabbit. The V-val EBNA1 gene was then cloned into a eukaryotic expression vector and successfully expressed in the transfected cultured cells. Expression of V-val EBNA1 rendered 293 cells able to undergo serumindependent cell proliferation, providing them with anti-apoptotic abilities, which are two characteristics of cancer cells. These data suggested that use of EBNA1 originally derived from tumor cells, rather than the more commonly utilized prototype, when investigating the potential role of EBNA1 in the oncogenesis of EBV-associated malignancies, is crucial.
Subject(s)
Cell Survival , Epstein-Barr Virus Nuclear Antigens/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/virology , Animals , Apoptosis , Callithrix , Cell Line, Tumor , Cell Proliferation , Culture Media, Serum-Free , Escherichia coli/metabolism , Genetic Variation , HEK293 Cells , Humans , Polymerase Chain Reaction , Polymorphism, Genetic , Protein Structure, Tertiary , Rabbits , Recombinant Fusion Proteins/chemistryABSTRACT
The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput .OH-generating system (Fe2+ concentration >1.0 microM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade .OH-generating system (Fe2+<0.1 microM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.
