The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells

• Published in: Molecular carcinogenesis Vol. 33; no. 3; pp. 137 - 145
• Main Authors: Hergenhahn, Manfred, Soto, Ubaldo, Weninger, Annette, Polack, Axel, Hsu, Chih-Hung, Cheng, Ann-Lii, Rösl, Frank
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 01.03.2002
• Subjects: Anticarcinogenic Agents - pharmacology; AP-1 protein; BZLF1 mRNA; BZLF1 protein; Carcinogens - antagonists & inhibitors; chemoprevention; curcumin; Curcumin - pharmacology; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EBV reactivation; Epstein-Barr virus; Gene Expression Regulation, Viral - drug effects; Genes, Reporter; Herpesvirus 4, Human - drug effects; Herpesvirus 4, Human - genetics; Herpesvirus 4, Human - metabolism; Humans; Kinetics; luciferase; luciferase induction; Luciferases - analysis; Luciferases - genetics; Promoter Regions, Genetic; Raji DR-LUC cells; RNA, Viral - biosynthesis; Tetradecanoylphorbol Acetate - antagonists & inhibitors; Trans-Activators - biosynthesis; Trans-Activators - genetics; Transcription Factors - metabolism; Transcriptional Activation - drug effects; Tumor Cells, Cultured; Viral Proteins; Virus Latency
• ISSN: 0899-1987; 1098-2744
• DOI: 10.1002/mc.10029

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‐β (TGF‐β) 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‐β‐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. © 2002 Wiley‐Liss, Inc.