Dissertation/Thesis Abstract

Bcl-2 proteins in cancer cell survival and mitotic death
by Eichhorn, Josh M., Ph.D., University of Arkansas for Medical Sciences, 2013, 132; 3610331
Abstract (Summary)

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and while this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. We used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti- apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown, phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that the disabling of anti-apoptotic Bcl-2 protein function by phosphorylation plays a key role in regulation of mitotic death. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins in more detail, we used a combined knockdown/overexpression strategy in HeLa cells to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. The results provide novel insight into the molecular mechanisms of compensation exhibited by pro-survival Bcl-2 family proteins.

Indexing (document details)
Advisor: Chambers, Timothy C.
Commitee: Haun, Randy S., Higuchi, Masahiro, Raney, Kevin D., Wahls, Wayne P.
School: University of Arkansas for Medical Sciences
Department: Biochemistry and Molecular Biology
School Location: United States -- Arkansas
Source: DAI-B 75/05(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Cellular biology, Biochemistry, Oncology
Keywords: Apoptosis, Bcl-2 proteins, Cancer, Cdk1, Microtubule inhibitors, Mitotic arrest, Mitotic death
Publication Number: 3610331
ISBN: 978-1-303-70045-3
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