2019DarweeshOOPhD.pdf (5.62 MB)
Investigation Of The Molecular Mechanism Of Paclitaxel-Induced Apoptosis In Cultured-Human Cells
thesisposted on 2019-07-31, 11:44 authored by Omeed O. Darweesh
Paclitaxel (Taxol) is a microtubule-targeting drug. It binds to the β-tubulin subunit of microtubules and interferes with microtubules disassembly in cell division. This prevents microtubule-kinetochore attachments and causes activation of the spindle assembly checkpoint (SAC). Prolonged activation of the SAC induces mitotic arrest by inhibiting the inactivation of Cdk1, and results in mitochondrial-mediated cell death. It is known that the key effector pro-apoptotic proteins, Bax and Bak, commit mitotically arrested cells to undergo apoptosis by inducing mitochondrial outer membrane permeabilization. However, the molecular signal that links SAC activation to the induction of apoptosis remains far from clear. In this thesis, I show that both Bak and Bax are required for Taxol-induced apoptosis in HeLa cells as siRNA-mediated depletion of either Bak or Bax individually or in combination caused a significant decrease in Taxol-induced apoptosis. My data also demonstrate that Bak and Bax form a complex in the mitochondria with Mitochondrial Permeability Transition Pore components such as VDAC and ANT2 in the Taxol-arrested mitotic cells. Furthermore, I show here that inactive Cdk1 exists as two independent complexes with Bak and Bax in normal healthy interphase cells. In Taxol-arrested cells, a complex comprising activated Cdk1, cyclin B1, Bak and Bax is present in the mitochondrial membrane. In an in vitro assay, I demonstrate a direct interaction between recombinant Cdk1 and Bax. In functional assays, I show that the phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-xL is dependent on the activated Cdk1/Bak and activated Cdk1/Bax complexes suggesting that Bak and Bax clearly mediate the transfer of activated Cdk1 to the MOM. My results lead me to predict that the Bax/Cdl1/cyclin B1 complex is the cytoplasmic signal that links SAC activation to the mitochondrial-mediated cell death.
Supervisor(s)Patel, Raj; Prigent, Sally
Date of award2019-06-25
Author affiliationDepartment of Molecular and Cell Biology
Awarding institutionUniversity of Leicester