Our broad, long-term objective is to understand on a molecular level the mechanism of action of anticancer drugs with an overall goal of improving current chemotherapy and identification of novel targets and drugs. The role that Bcl-2 proteins play in microtubule inhibitor-induced apoptosis is of special interest. Bcl-2 proteins are subject to complex regulation, and one of the most prominent post-translational modifications is the phosphorylation of Bcl-xL and Bcl-2 occurring in response to microtubule inhibitors. However, the role of these modifications and the kinase(s) responsible have remained largely obscure, despite their potential importance as a mechanistic link between microtubule dysfunction and apoptosis induction. Preliminary studies using KB-3 cells have indicated that vinblastine-induced phosphorylation of Bcl-xL and Bcl-2 are coordinated events catalyzed by the same kinase and that their dephosphorylation correlates with apoptosis initiation. Our hypothesis is that Bcl-xL/Bcl-2 phosphorylation is a key event controlling apoptosis induction by antimitotic drugs and is catalyzed by a novel kinase activated in response to microtubule dysfunction.
In Specific Aim 1, the sites of phosphorylation in Bcl-xL and Bcl-2 in response to vinblastine treatment of KB-3 cells will be identified by protein purification, trypsin digestion, and mass spectrometry analysis.
In Specific Aim 2, the role of Bcl-xL/Bcl-2 phosphorylation in vinblastine-induced apoptosis will be evaluated by expressing phosphorylation-defective or phosphorylation-mimic molecules and examining cellular sensitivity to vinblastine.
In Specific Aim 3, mechanistic studies will be performed to determine whether phosphorylation affects Bcl-xL or Bcl-2 subcellular localization or protein/protein interactions.
In Specific Aim 4, the vinblastine-activated Bcl-xL/Bcl-2 kinase will be purified and characterized, with a long-term goal of understanding its function and regulation in the cellular response to microtubule damage. This study will provide novel insight into the role of Bcl-xL/Bcl-2 phosphorylation in the mechanism of action of vinblastine and other antimitotic drugs and provide important basic information on regulatory mechanisms of Bcl-2 protein function. These findings in turn will aid in the search for superior chemotherapeutic drugs, new drug targets, and methods to overcome tumor cell resistance to these agents.
|Kothari, Anisha; Hittelman, Walter N; Chambers, Timothy C (2016) Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells. Cancer Res 76:3553-61|
|Alford, Sarah E; Kothari, Anisha; Loeff, Floris C et al. (2015) BH3 Inhibitor Sensitivity and Bcl-2 Dependence in Primary Acute Lymphoblastic Leukemia Cells. Cancer Res 75:1366-75|
|Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E et al. (2014) Identification of a mitotic death signature in cancer cell lines. Cancer Lett 343:232-8|
|Eichhorn, Joshua M; Alford, Sarah E; Sakurikar, Nandini et al. (2014) Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival. Exp Cell Res 322:415-24|
|Eichhorn, Joshua M; Kothari, Anisha; Chambers, Timothy C (2014) Cyclin B1 overexpression induces cell death independent of mitotic arrest. PLoS One 9:e113283|
|Eichhorn, J M; Alford, S E; Hughes, C C et al. (2013) Purported Mcl-1 inhibitor marinopyrrole A fails to show selective cytotoxicity for Mcl-1-dependent cell lines. Cell Death Dis 4:e880|
|Pawlowska, Monika; Chu, Rong; Fedejko-Kap, Barbara et al. (2013) Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use. Drug Metab Dispos 41:414-21|
|Eichhorn, J M; Sakurikar, N; Alford, S E et al. (2013) Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death. Cell Death Dis 4:e834|
|Chu, Rong; Terrano, David T; Chambers, Timothy C (2012) Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration. Biochem Pharmacol 83:199-206|
|Sakurikar, Nandini; Eichhorn, Joshua M; Chambers, Timothy C (2012) Cyclin-dependent kinase-1 (Cdk1)/cyclin B1 dictates cell fate after mitotic arrest via phosphoregulation of antiapoptotic Bcl-2 proteins. J Biol Chem 287:39193-204|
Showing the most recent 10 out of 17 publications