Pathways to Mutagenesis in vivo and in Stem Cells

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DESCRIPTION (provided by applicant): The importance of somatic mutation in the genesis of cancer and other diseases is undisputed. However, the extent to which loss of heterozygosity (LOH), as a consequence of mitotic recombination, contributes to the frequency of spontaneous mutation has been under-appreciated. Using a mouse model that is heterozygous at Aprt we have previously reported that in vivo spontaneous mutation frequencies at Aprt can approach 10-4 and that up to 80 percent of these events can be due to mitotic recombination. The global mutation frequencies, in fact, are even higher given that all loci between the point of crossover and the reporter locus are also affected, and that mitotic recombination can occur between all autosome homologs. The goals of this program are to further exploit our Aprt heterozygous model and to develop additional Aprt-derived models to ask questions regarding local events at sites of double strand breaks, and to determine whether different pathways to mutation/repair are preferred in different isogenic cell types. An emphasis will be placed on embryonic stem (ES) cells and adult stem cells since they have requirements beyond those of somatic cells for preserving the integrity of their genomes. We have already reported that ES cells suppress mutation and mitotic recombination by as much as 100 fold compared with isogenic mouse embryo fibroblasts (MEFs), one mechanism to protect their genomes. We and others have also shown that ES cells are hypersensitive to DNA damage, and that they lack a G1 checkpoint, presumably facilitating death and removal of cells that have acquired a mutational burden. This represents a second mechanism by which ES cells protect their genomes. We have identified the signaling pathway that is compromised in ES cells after DNA damage, reconstituted it, and shown that reestablishment of a G1 arrest after challenge protects the cells from apoptosis. We wish to ask whether adult stem/progenitor cells behave more like somatic cells or ES cells with respect to cell cycle regulatory mechanisms and mechanisms to suppress mutation. The role of adult stem cells in the genesis of tumors is an important question that is not yet resolved. We will use our Aprt null mice and intestinal epithelium to ask whether mutations in the mouse gut arise exclusively in the crypt base, site of the progenitor cells or whether they also arise elsewhere within the crypt. This experiment should provide evidence for or against the "top down" model of colon cancer that has been proposed.