Ionizing radiation of cells results in a prolongation of the G2 phase of the cell cycle in all eukaryotic cells examined including yeast, plants, invertebrates and mammalian cells. Since some details of the biochemical mechanisms which induce mitosis have recently been elucidated, it is our intent to use this information to explore the mechanisms underlying this G2 delay. Recent research has identified the increase in mitotic cyclins as a trigger for the exit of cells from G2 into M phase, These cyclins will be the target of our investigation. There are at least two mammalian mitotic cyclins, cyclin A and B. Both of these increase in amount at the G2/M boundary and the increase has been shown to be required for the induction of mitosis in some systems. Hence alterations in the expression of these molecules could influence progression through the cell cycle. Our preliminary results have indicated that cyclin B expression is inhibited after irradiation. This effect occurs through two different mechanisms. First, when cells are irradiated in S phase, although they enter G2, they do not accumulate the cyclin B mRNA. Cyclin A mRNA levels rise at the same time as in the controls. Second, G2 delay is also known to occur after irradiation in G2 phase when cyclin B mRNA has already begun to rise. Irradiation at this time in the cell cycle results in a marked fall in cyclin B protein levels, again cyclin A protein levels are unaffected. Thus, through effects both at the protein and the RNA levels radiation results in decreases in cyclin B but not cyclin A. Thus, these experiments have indicated that reductions in the levels of cyclin B could be a factor leading to the G2 delay seen after ionizing radiation. The experiments which we propose will define the levels of regulation of cyclin B at both the RNA and protein levels. Whether the effects on the RNA are regulated at the level of transcription or message stability will be examined; similarly the effects on the protein will be analyzed for translational controls and for effects on protein turnover regulated via ubiquitination. The effects of the gene and protein sequence on the stability of the message and protein will be investigated. Cdc2 kinase levels after irradiation will also be studied. This information will be important for two reasons. First this research will investigate at the molecular level a universal response of eukaryotic cells to radiation damage which may be related to cellular radiosensitivity. Second, by studying the mechanisms through which radiation perturbs the triggering of mitosis, we may uncover further information about this process under normal circumstances.
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