PROBES TO DETECT EBV INTEGRATION IN HUMAN CELLS

THIS PROJECT WILL DEVELOP AN ASSAY TO DETECT INTEGRATED FORMS OF EPSTEIN-BARR VIRUS (EBV) DNA IN THE GENOMES OF CELLS TRANSFORMED BY THIS VIRUS. THE ASSAY WILL UTILIZE SOUTHERN BLOTTING AND POLYMERASE CHAIN REACTION METHODOLOGY TO IDENTIFY THE PRESENCE OF VIRUS-CELL JUNCTION FRAGMENTS INDICATIVE OF EBV INTEGRATION. THE MODEL SYSTEM WILL UTILIZE A SERIES OF PERMANENT HUMAN B-CELL LINES DERIVED FROM THREE SEROPOSITIVE HEALTHY DONORS PRODUCED BY IN VITRO INFECTION WITH EBV AND BY SPONTANEOUS OUTGROWTH OF LATENTLY INFECTED LYMPHOCYTES PRESENT IN PERIPHERAL BLOOD. A TOTAL OF 120 TO 150 MONOCLONAL CELL LINES WILL BE GENERATED. A RECONSTRUCTION EXPERIMENT USING A MIXTURE OF DNA'S FROM A BURKITT'S LYMPHOMA CELL LINE CONTAINING A SINGLE INTEGRATED COPY OF EBV DNA AND VARYING NUMBERS OF AN EBV DNA CONTAININGCIRCULAR PLASMID MOLECULES WILL BE USED TO DETERMINE THE LIMITS OF SENSITIVITY OF THE ASSAY. THE ASSAY WILL THEN BE APPLIED TO A SUBSET OF THE PERMANENT HUMAN B-CELL LINES DESCRIBED ABOVE. THE PHASE I STUDIES WILL PROVIDE THE BASIS FOR PHASE II RESEARCH IN WHICH THE REMAINING CELL LINES GENERATED BY PHASE I WILL BE EXAMINED AS WELL AS CELLS DERIVED FROM PATIENTS WITH EBV-RELATED MALIGNANCIES. THE PRODUCTION OF REAGENTS TO ASSESS EBV INTEGRATION IN A KIT CONFIGURATION ISPLANNED.