The overall goals of this SBIR are to develop new methods for quantifyingDNA cytosine methylation, and to apply these methods to the study ofmethylation alterations in neoplastic transformation. Methylcytosines arelocated in regulatory regions such as promoters, and hypomethylation inthese regions are among the early events in carcinogenesis.The specific aims of the proposal are to: 1) synthesize antigens for theproduction of monoclonal antibodies specific for 5-methylcytosine, 2)produce monoclonal antibodies reactive with methylcytosine, and 3) to applythese antibodies to model systems which have alterations in DNA methylationwhich correlate to transformation.These methods should contribute to knowledge relating DNA methylation tocarcinogenesis and metastasis. The antibodies will be employed to measureglobal changes in genomic DNA methylation by multiparameter flow cytometryand at high resolution by antibody-blocked PCR and LCR.These antibodies based methods will be developed in Phase I. Phase II willbe concerned with applications of these methodologies and expanded tostudies in transformed cell lines and rodents. The reagents and techniquesto be developed will have immediate commercial application to cancerdiagnosis and environmental toxicology.
