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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA109821-02
Application #
6911501
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Arya, Suresh
Project Start
2004-08-01
Project End
2009-05-31
Budget Start
2005-08-01
Budget End
2006-05-31
Support Year
2
Fiscal Year
2005
Total Cost
$232,880
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
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

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