The mitotic checkpoint gene Mad2 has been shown in metazoans to be an important part of the mitotic checkpoint apparatus through biochemical experiments. Mad2 is a component of the machinery which arrests a cell prior to the metaphase to anaphase transition if all the chromosomes are not attached to the mitotic spindle apparatus. The main goal of the research being proposed is to determine if complete or partial loss of Mad2 function in mammalian cells leads to a loss of mitotic checkpoint control, genomic instability and/or tumor progression either under normal growth conditions or when the checkpoint apparatus is challenged with mitotic spindle inhibitors. It has not yet been demonstrated whether the loss of any component of the mitotic checkpoint mechanism in vertebrates leads to chromosome instability or cellular transformation. Mice and human cells that have one copy of the Mad2 gene inactivated by gene targeting will be further analyzed with respect to defects in the mitotic checkpoint pathway. It will be determined whether such cells can arrest appropriately during mitosis when the mitotic spindle is inhibited, and if not, the consequences of this failure on whole chromosome loss rates and tumorigenic properties. In addition, the dosage of Mad2 will be decreased in both mouse and human cells by the introduction of dominant negative Mad2 alleles, or conditional inactivation of the second Mad2 allele. These cells, likely to have a more severe mitotic checkpoint defect, will also be analyzed for changes in chromosome stability and alterations in tumorigenic properties. In this way, the role of the mitotic checkpoint pathway in cancer initiation and/or progression in mammals will be assessed.
