THE PURPOSE OF THIS PHASE I PROPOSAL IS TO DEVELOP A MAMMALIAN CELL CULTURE ANEUPLOIDY ASSAY USING THE CAK MOUSE CELL LINE.

THE PURPOSE OF THIS PHASE I PROPOSAL IS TO DEVELOP A MAMMALIAN CELL CULTURE ANEUPLOIDY ASSAY USING THE CAK MOUSE CELL LINE. WHILE A GREAT NUMBER OF SHORT TERM ASSAYS HAVE BEEN DEVELOPED TO DETECT COMPOUNDS WHICH INDUCE EITHER GENE MUTATIONS OR CHROMOSOMAL DAMAGE, LITTLE RESEARCH HAS FOCUSEDON THE DEVELOPMENT OF SHORT TERM ASSAYS TO DETECT ANEUPLOIDY. THE DEVELOPMENT OF A MAMMALIAN CELL CULTURE ASSAY TO DETECT ANEUPLOIDY WOULD PLAY A CRUCIAL ROLE IN DETECTING CHEMICALS WHICH MAY CAUSE EITHER BIRTH DEFECTS OR CANCER IN HUMANS. THE CAK LINE IS EXTREMELY USEFUL FOR THISPURPOSE SINCE IT IS NEAR DIPLOID LINE, AND A SELECTIVE SYSTEM CAN BE DEVELOPED IN WHICH ANEUPLOIDY CAN BE DETECTED USING RESISTANCE TO 2,6-DIAMINOPURINE (DAP). THIS ELIMINATES THE NEED TO DETECT ANEUPLOIDY BY COMPARING THE DISTRIBUTION OF CHROMOSOMES IN TREATED AND UNTREATED CELLS. A CLONE OF CAK CELLS, CAK-B3 TOY(R)-13, IS HETEROZGOUS FOR A PHYSICAL MARKER ON CHROMOSOME 8, WHICH CARRIES THE ADENINEPHOSPHORIBOSYL TRANSFERASE GENE. THE MAJOR GOAL OF THIS PROJECT IS TO ESTABLISH A CAK LINE WHICH IS HETEROZYGOUS FOR BOTH APRT AND FOR THE PHYSICAL MARKER ON CHROMOSOME 8. THE GENE CODING FOR APRT WOULD THEN BE LINKED TO A CHROMOSOME WHICH CAN BE EASILY IDENTIFIED IN METAPHASE PREPARATIONS. THE CLONE WHICH IS HETEROZYGOUS FOR BOTH THE PHYSICAL MARKER ON CHROMOSOME 8 AND FOR APRT ACTIVITY CAN BE USED TO SCREEN CHEMICALS EITHER FOR THEIR ABILITY TO INDUCE MUTATIONS OR INDUCE ANEUPLOIDY. RESISTANT CELLS TO DAP (PRESUMABLY APRT) WILL BE EXAMINED FOR LOSS OF THE CHROMOSOME CARRYING THE APRT+ ALLELE WHICH IS A DEMONSTRATION OF ANEUPLOIDY. THIS IS IN CONTRAST TO INDUCTION OF MUTATIONS TO APRT- IN WHICH BOTH THE PHYSICALLYNORMAL AND ABNORMAL CHROMOSOME 8 WOULD BE PRESENT IN RESISTANT CELLS. THE SUCCESSFUL DEVELOPMENT OF A RELATIVELYINEXPENSIVE, RAPID SCREENING TEST FOR THESE END-POINTS (ANEUPLOIDY, TUMOR PROMOTION) WOULD BE A SIGNIFICANT ADDITION TO CURRENT "IN VITRO" SCREENING BATTERIES AND COULD SIGNIFICANTLY LESSEN THE OVERALL COST (TO BOTH THE GOVERNMENT AND PRIVATE SECTOR) OF DETERMINING THE SAFETY OF NEW CHEMICALS AND DRUGS. THUS, THE ASSAY DEVELOPED UNDERTHIS PROJECT WILL BECOME A VALUABLE RESOURCE ASSET TO THE NIH, NIEHS, AND OTHER GOVERNMENT AGENCIES INVOLVED IN PROTECTING HUMAN HEALTH.