Dissertation/Thesis Abstract

Involvement of cyclin-dependent kinase 2 in cisplatin-induced cell death pathways
by Hodeify, Rawad, Ph.D., University of Arkansas for Medical Sciences, 2011, 195; 3454560
Abstract (Summary)

The aim of the work in this thesis was to study the involvement of cyclin-dependent kinase 2 (Cdk2) in cisplatin cytotoxicity. The first studies were designed to confirm that the expression of p21 Cdk inhibitor and dominant-negative Cdk2 (DN-Cdk2) protect from cisplatin nephrotoxicity in vivo. Two transgenic mouse strains were created that expressed either p21-GFP or DN-Cdk2-GFP under the control of a testosterone-inducible (KAP2) promoter specific to the kidney proximal tubule. We next studied the localization of the transgenes and whether their induction can protect the whole kidney from damage associated with cisplatin. When female p21-GFP or DN-Cdk2-GFP transgenic mice were implanted with testosterone, the expression of the transgenes was restricted to the kidney proximal tubules, and induction of transgenes protected the whole kidney from damage associated with cisplatin. We next examined the role of Cdk2 in cisplatin cytotoxicity in TKPTS cells, using an inactive mutant Cdk2-F80G that protected from cytotoxicity. When active, this mutant binds modified ATP analogues to specifically label Cdk2 substrates. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We concluded that cisplatin induces both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The inactive Cdk2-F80G protected from both pathways, but when it is activated by cyclin A, caspase-independent cell death predominated. Our next goal was to identify Cdk2 substrates involved in cisplatin cytotoxicity. We found that an 18 kDa protein identified by mass spectrometry as p21WAF1/Cip1 was phosphorylated by Cdk2 after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. We created a phosphomimic p21S78D and we showed that it was an inefficient inhibitor of Cdk2 and was inefficient at protecting from cisplatin cytotoxicity. We concluded that p21 is a Cdk2 substrate during cisplatin cytotoxicity and Cdk2-dependent phosphorylation of p21 limits the effectiveness of p21 to inhibit Cdk2.

Indexing (document details)
Advisor: Price, Peter M.
Commitee: Megyesi, Judit, Nowak, Grazyna, Price, Peter M., Reis, Robert J.S., Storrie, Brian
School: University of Arkansas for Medical Sciences
Department: Interdisciplinary Biomedical Sciences
School Location: United States -- Arkansas
Source: DAI-B 72/08, Dissertation Abstracts International
Subjects: Molecular biology, Cellular biology
Keywords: Apoptosis, Cdk2, Cell death, Cisplatin, P21, Phosphorylation
Publication Number: 3454560
ISBN: 978-1-124-64314-4
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