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.
|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|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be