This project will determine whether the mechanism by which arsenate induces mitotic effects on the Anaphase Promoting Complex (APC) mediate arrest. We hypothesize that arsenite inhibits the APC and subsequently mitotic progression. The APC orchestrates progress through the cell cycle and mitosis by targeting specific mitotic substrates for proteasomal degradation via ubiquitination.
Specific aim 1 will determine whether arsenite treatment inhibits APC substrate degradation necessary for mitotic exit.
In Specific aim 2 we will immunoprecipitate (IP) the APC and identify its subunits in order to investigate arsenite binding to APC subunits using radiolabeled Na[73As]O2. Determination of arsenite binding requires resolution of APC subunits using SDS-PAGE, examination of protein banding pattern using Sipro-Red and fluorescence imaging, and subsequent autoradiography to elucidate bands incorporating radiolabeled arsenite. Western blot analysis of SDS-PAGE resolved APC subunits will be used to identify specific APC subunits. Once arsenite association is determined, the proteins will be excised from the gel and identity confirmed by mass spectrometry.
Specific aim 3 will determine whether arsenite inhibits APC ubiquitin ligase activity. Isolated APC will be used in an ubiquitin conjugating assay with radiolabeled APC substrates treated with or without arsenite. Reaction products will be resolved on SDS-PAGE and autoradiography will be used to determine the degree of substrate laddering due to ubiquitin conjugation. Identification of the molecular targets of arsenite is important for chemotherapeutic drug development.
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| Taylor, B Frazier; McNeely, Samuel C; Miller, Heather L et al. (2008) Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization. Toxicol Appl Pharmacol 230:235-46 |
| McNeely, Samuel C; Belshoff, Alex C; Taylor, B Frazier et al. (2008) Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest. Toxicol Appl Pharmacol 229:252-61 |
| McNeely, Samuel C; Taylor, B Frazier; States, J Christopher (2008) Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent. Toxicol Appl Pharmacol 231:61-7 |
| Taylor, B Frazier; McNeely, Samuel C; Miller, Heather L et al. (2006) p53 suppression of arsenite-induced mitotic catastrophe is mediated by p21CIP1/WAF1. J Pharmacol Exp Ther 318:142-51 |
| McNeely, Samuel C; Xu, Xiaogiang; Taylor, B Frazier et al. (2006) Exit from arsenite-induced mitotic arrest is p53 dependent. Environ Health Perspect 114:1401-6 |
