Versatile Mutation Assay Platform

DESCRIPTION (provided by applicant): DNA mutation is a primary mechanism by which cancers arise. Alterations of genetic material have also been implicated in diseases such as atherosclerosis, and processes such as aging. Thus, there is an important need for sensitive analytical methods which facilitate the study of mutagenesis, as well as the identification of chemical or physical agents that can mutate DNA. This research project will result in the development and validation of several novel and efficient assays for identifying and studying mutagens and their effects on DNA. The innovative mutation scoring platform that we will devise differs from currently available methods in many important ways, especially in regards to its extreme versatility. This is a characteristic that stems from our careful choice of a target locus (phosphatidylinositol glycan-class A gene; pig-a) coupled with the numerous reagents that are available for studying the mutant phenotype: glycosyl-phosphatidylinositol (GPI) anchor deficiency. Tremendous utility exists for at least three assay configurations, each of which will be developed over the course of Phase I and Phase II investigations. The first tier assay will be a high throughput in vitro platform that serves as an extremely rapid and efficient mutagen screening tool; the second tier in vitro assay will facilitate mutation spectra analyses. Thirdly, an in vivo mutation assay will be compatible with every mammalian species, including man. The basis of these assays is related to the key role that the pig-a gene product plays in the biosynthesis of glycosyl-phosphatidylinositol (GPI) anchors. Mutations giving rise to non-functional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype which can be measured via high throughput instrumentation. This assay platform will be applicable across in vitro and in vivo models. This is an important characteristic of the proposed assay system, as it provides for a true bridging biomarker, one that will allow investigations to extend their investigations from tissue culture to whole animal models. The experiments proposed herein extend our promising preliminary work in important ways. Our Phase I feasibility research has been designed around the lymphoblastoid mouse cell line L5178Y, together with a complementary in vivo model, CD-1 mice. Ultimately, successful completion of Phase I and Phase II will allow us to supply pharmaceutical and chemical companies with important tools that help them more efficiently eliminate mutagens from their new product development processes, thus decreaseing drug production costs and potential risk to consumers. Furthermore, if the system proves compatible with human blood specimens, myriad other research activities will benefit as methods developed to study laboratory animal models could be translated to the investigation of real- world human exposure scenarios. It is well known that DNA damage is a precursor to the development of cancer and other significant diseases. It is, therefore, in the interest of public health to reduce the occurrence of mutagenic chemicals in the environment, in our drugs, and from our workplaces. This research project will optimize and validate a powerful in vitro and in vivo methods for detecting mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds.