ASSAYS FOR DNA METHYLATION

THE OVERALL GOALS OF THIS SBIR CONTRACT ARE TO DEVELOP NEW METHODS FOR QUANTIFYING DNA CYTOSINE METHYLATION, AND TO APPLY THESE METHODS TO THE STUDY OF METHYLATION ALTERATIONS IN NEOPLASTIC TRANSFORMATION. METHYLCYTOSINES ARE LOCATED IN REGULATORY REGIONS SUCH AS PROMOTERS, AND HYPOMETHYLATION IN THESE REGIONS ARE AMONG THE EARLY EVENTS IN CARCINOGENESIS. THE SPECIFIC AIMS ARE TO: 1) SYNTHESIZE ANTIGENS FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES SPECIFIC FOR 5-METHYLCYTOSINE, 2) PRODUCE MONOCLONAL ANTIBODIES REACTIVE WITH METHYLCYTOSINE, AND 3) TO APPLY THESE ANTIBODIES TO MODEL SYSTEMS WHICH HAVE ALTERATIONS IN DNA METHYLATION WHICH CORRELATE TO TRANSFORMATION. THESE METHODS SHOULD CONTRIBUTE TO KNOWLEDGE RELATING DNA METHYLATION TO CARCINOGENESIS AND METASTASIS. THE ANTIBODIES WILL BE EMPLOYED TO MEASURE GLOBAL CHANGES IN GENOMIC DNA METHYLATION BY MULTIPARAMETER FLOW CYTOMETRY AND AT HIGH RESOLUTION BY ANTIBODY-BLOCKED PCR AND LCR. THESE ANTIBODIES BASED METHODS WILL BE DEVELOPED IN PHASE I. PHASE II WILL BE CONCERNED WITH APPLICATIONS OF THESE METHODOLOGIES AND EXPANDED TO STUDIES IN TRANSFORMED CELL LINES AND RODENTS. THE REAGENTS AND TECHNIQUES TO BE DEVELOPED WILL HAVE IMMEDIATE COMMERCIAL APPLICATION TO CANCER DIAGNOSIS AND ENVIRONMENTAL TOXICOLOGY.