Microtubule targeting agents (MTAs) are an important class of chemotherapeutics which are widely used in the treatment of various cancers. It was thought that they exert their cytotoxicity in mitosis; however increasing evidence suggests that MTAs also act in interphase, potentially relevant to their clinical mode of action. Recently, it was demonstrated that vincristine induced death in primary acute lymphoblastic leukemia (ALL) cells in both mitosis and G1 phase. Based on this model, it was hypothesized that MTAs are able to disrupt interphase microtubule functions which are necessary for the survival of primary ALL cells in G1 phase. The goal of this dissertation was to further characterize MTA-mediated G1 phase cell death. First, systematic studies were undertaken comparing primary ALL and HeLa cells treated with three different MTAs, namely the microtubule stabilizer paclitaxel and two microtubule destabilizers, vincristine, and eribulin. Destabilizers induced death in G1 phase of primary ALL cells at concentrations ≥ 100 nM but induced death in mitosis at lower concentrations. Paclitaxel induced mitotic death at all tested concentrations in primary ALL cells. In contrast, HeLa cells underwent mitotic death in response to all three drugs regardless of concentration. Thus interphase death was both drug type- and cell type-restricted and depended on complete depolymerization of interphase microtubules. Combination studies in which G1 phase primary ALL cells were treated with vincristine, and the CDK4/6 inhibitor, palbociclib, demonstrated that palbociclib induced G1 phase arrest and caused cells to become refractory to vincristine. These studies indicated that primary ALL cells actively cycling through G1 phase are sensitive to microtubule destabilizers but arrested cells are insensitive, implying a key role for G1 phase microtubules in successful cell cycle advance. Finally, experiments comparing cell death mechanisms in G1 phase and M phase, through investigations into activation status of Bcl-2 family proteins and caspase-3, indicated that mitotic cell death is controlled through intrinsic apoptosis while G1 phase is controlled through distinct mechanism(s). Overall, these studies indicate that MTAs are able to exert their cytotoxicity in primary cancer cells in two distinct ways. A model summarizing the findings and highlighting the advances made is presented.
|Advisor:||Chambers, Timothy C.|
|Commitee:||Davidson, Mari, Eoff, Robert, Kelly, Thomas, Mackintosh, Samuel G.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/2(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Molecular biology|
|Keywords:||Microtubule targeting agents, Acute lymphoblastic leukemia, Cytotoxicity|
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