University of Louisville
Arsenite Induced Disruption of Mitosis
Institution
University of Louisville
Faculty Advisor/ Mentor
J. Christopher States
Abstract
Arsenite is a known carcinogen that causes skin, bladder, and liver cancer. However, the mechanism of carcinogenesis is unknown. Arsenite (AsO2 - ) delays entry into mitosis by activation of the G2 checkpoint. Arsenite also induces a mitotic delay in anaphase and is aneuploidogenic in normal diploid human cells (p53(+)). In contrast, arsenite induces mitotic arrest and apoptosis in p53 deficient cells (p53(-)). We hypothesize that arsenite, in the presence of p53, induces the inactivation of cyclinB/cdc2 by cdc2 phosphorylation, leading to the derepression of the mitotic exit network. Cdc2, cdc2-P and cyclin B levels were analyzed as the cells progressed from G2 through mitosis to determine whether the p53-dependent G2 checkpoint pathway plays a role in releasing cells from arsenite induced mitotic arrest. TR9-7 cells are a model human fibroblast line in which p53 expression is regulated exogenously in a tetracycline-off system. TR9-7 cells, expressing and not expressing p53, were synchronized in G2 and released into media containing and not containing sodium arsenite (NaAsO2, 5µM). Western blot analyses were used to monitor whether there were any arsenite-induced differences in the proteins expressed and if these changes were p53 dependent. These analyses showed that arsenite stabilization of cyclin B level is p53 independent. Arsenite stabilizes the phosphorylation of Cdc2 in both p53(+) and p53(-) cells even when cyclin B has diminished. Cdc2-P levels are also stabilized early in p53(+) and p53(-) cells not treated with arsenite yet diminish after the 12th hour release from Hoechst 33342. These results are consistent with the inactivation of cyclinB/cdc2 allowing cells to exit mitosis and escape arsenite induced mitotic arrest.
Arsenite Induced Disruption of Mitosis
Arsenite is a known carcinogen that causes skin, bladder, and liver cancer. However, the mechanism of carcinogenesis is unknown. Arsenite (AsO2 - ) delays entry into mitosis by activation of the G2 checkpoint. Arsenite also induces a mitotic delay in anaphase and is aneuploidogenic in normal diploid human cells (p53(+)). In contrast, arsenite induces mitotic arrest and apoptosis in p53 deficient cells (p53(-)). We hypothesize that arsenite, in the presence of p53, induces the inactivation of cyclinB/cdc2 by cdc2 phosphorylation, leading to the derepression of the mitotic exit network. Cdc2, cdc2-P and cyclin B levels were analyzed as the cells progressed from G2 through mitosis to determine whether the p53-dependent G2 checkpoint pathway plays a role in releasing cells from arsenite induced mitotic arrest. TR9-7 cells are a model human fibroblast line in which p53 expression is regulated exogenously in a tetracycline-off system. TR9-7 cells, expressing and not expressing p53, were synchronized in G2 and released into media containing and not containing sodium arsenite (NaAsO2, 5µM). Western blot analyses were used to monitor whether there were any arsenite-induced differences in the proteins expressed and if these changes were p53 dependent. These analyses showed that arsenite stabilization of cyclin B level is p53 independent. Arsenite stabilizes the phosphorylation of Cdc2 in both p53(+) and p53(-) cells even when cyclin B has diminished. Cdc2-P levels are also stabilized early in p53(+) and p53(-) cells not treated with arsenite yet diminish after the 12th hour release from Hoechst 33342. These results are consistent with the inactivation of cyclinB/cdc2 allowing cells to exit mitosis and escape arsenite induced mitotic arrest.