Reconstitution of a G1 checkpoint and protection from apoptosis in embryonic stem cells
Vontz Center for Molecular Studies, University Cincinnati College Medicine, OH, Cincinnati
Mutation frequency at heterozygous loci such as Aprt and Tk in somatic cells in vivo approaches 10-4. Up to 80% of these events are due to loss of heterozygosity (LOH) as a consequence of mitotic recombination. Such a high mutation frequency, although detrimental to the individual, is sustained in somatic cells, but eventually results in somatic disease. Such a high mutation frequency cannot be tolerated by germ cells or stem cells since they give rise to all of the cells in the body as well as to the germ cells of succeeding generations. There are at least two operative mechanisms in germ cells to ensure that they maintain their genomic integrity. One mechanism is to suppress spontaneous mutation, and we have previously demonstrated that spontaneous mutation, including LOH due to mitotic recombination, is diminished by about 100-fold in embryonic stem (ES) cells compared with somatic cells. A second mechanism is to facilitate apoptosis in order to clear the ES cell population of cells that have sustained a mutational burden. Others have shown that ES cells are hypersensitive to DNA damage and that they lack a G1 checkpoint. Absence of a G1 checkpoint would allow cells with damaged DNA to enter S phase unrepaired and result in further DNA damage and consequent apoptosis. We have now identified a signaling pathway that is compromised in ES cells and that results in the inability to arrest in G1 after DNA damage. We have reconstituted this pathway and demonstrated that its restoration reestablishes the G1 checkpoint. Consistent with the model, flow cytometry shows that the cells are now protected from apoptosis, an attribute that is not advantageous to the stem cell population since it will impede cleansing the population of cells that have incurred mutations.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 998 (Signaling pathways - Part III).