Mol Carcinog 2002 Mar;33(3):137-45
Hergenhahn M; Soto U; Weninger A; Polack A; Hsu CH; Cheng AL; Rosl F
Division of Genetic Alterations in Carcinogenesis, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system.
These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome.
Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene.
Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin.
Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-beta (TGF-beta) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells.
TPA treatment also induced luciferase mRNA with similar kinetics.
Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-beta-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin.
Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin.
Protein binding to the
complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin.
Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis.
The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription.
Copyright 2002 Wiley-Liss, Inc.