An in vivo model of mitotic cell death and Ras/MAPK signaling

Full text: We have created the first and only existing tissue-model of mitotic cell death using the nematode C. elegans. We are able to measure radiation sensitivity in C. elegans by microscopically scoring the percentage of radiation-induced abnormal vulvae. We have found that these abnormalities are due to the death of the vulva cells after their third (and final) division, consistent with post-mitotic cell death. In C. elegans the Ras/MAPK signaling pathway is primarily responsible for the development of the hermaphrodite vulva, and is highly conserved to the mammalian Ras/MAPK pathway. We began by studying the effects of radiation on worm strains with mild loss-of-function (lof) mutations in components of the Ras/MAPK pathway. While the mutant strains that we studied have no abnormalities in normal vulva development, we found that all were radiosensitive, with increased radiation-induced vulval abnormalities as compared to wild-type worms. We therefore wanted to see if overexpression of the Ras/MAPK pathway would confer radioresistance in our system, so we irradiated a gain-of-function (gof) EGFR mutant worm strain. We found that this strain was radioresistant, with less radiation-induced vulval abnormalities than wild-type worms. We have concluded that the Ras/MAPK pathway protects against mitotic cell death in C. elegans. We wanted to better understand the downstream effectors of Ras/MAPK signaling that facilitate protection from mitotic cell death. Since mitotic cell death is due to DNA damage, we hypothesized that worm strains with mutations in the DNA damage response pathway should also be sensitive to mitotic cell death. We have begun analyzing worms with mutations in cell cycle checkpoint genes and DNA damage sensor genes, and have found that all of the strains tested thus far are highly radiosensitive. We plan to genetically cross gain-of-function Ras/MAPK mutants and loss-of-function checkpoint or damage response mutants, and determine the linearity of these pathways