Nephrotoxicity is a major dose-limiting factor of cisplatin chemotherapy. In addition to its cytotoxic effect on cancer cells, cisplatin induces cell death in the kidney proximal tubule cells. We previously reported that cisplatin-induced kidney cell death was dependent on Cdk2 activity since Cdk2 inhibition protected kidney cells both in vitro and in vivo. However, it was not known whether inhibition of Cdk2 activity specifically in the kidney proximal tubule would be sufficient to protect against the nephrotoxic effect of cisplatin in vivo. Using two transgenic mouse strains with inducible p21-GFP or DN-Cdk2-GFP specifically in the kidney proximal tubules, we showed that inhibition of Cdk2 activity in the kidney proximal tubule was sufficient to protect kidney at the functional and morphological level. Using AS-Cdk2, we found that Cdk2 phosphorylated Bcl-xL in response to cisplatin, and with mass spectrometry, we determined that the phosphorylation occurred at three previously unreported sites—Ser 72, Ser 73, and Ser 74—with Ser 73 phosphorylation being the highest incidence.
The overall theme of this thesis project was to explore the role of cisplatin-induced Cdk2 phosphorylation of Bcl-xL. We showed that the expression of the phosphomimetic Bcl-xL at Ser 73 (S73D-Bcl-xL) was sufficient to induce cell death. In contrast, expression of the phosphodefective Bcl-xL (AAA-Bcl-xL) did not induce cell death in cultured kidney proximal tubule cells, and protected them from cisplatin-induced cell death. Cell death induced by S73D-Bcl-xL was not due to its reduced affinity towards Bax. S73D-Bcl-xL formed high-molecular-weight oligomers at the mitochondria, suggesting that mitochondrial pore-formation could be a possible mechanism of S73D-Bcl-xL induced cell death.
Since 15 kDa- and 12 kDa- cleavage products of Bcl-xL were detected in cells expressing S73D-Bcl-xL and in cells treated with cisplatin, we examined whether these cleavage products participated in cell death. Abolishing the caspase cleavage sites in S73D-Bcl-xL did not attenuate cell death since mitochondrial clustering, oligomer formation, caspase activation and nuclear fragmentation were still detected. We conclude that Cdk2 phosphorylation of Bcl-xL at Ser 73 is a mechanism whereby cisplatin induces kidney cell death, and that the cleavage of Bcl-xL was dispensable for this cell death pathway.
|Advisor:||Price, Peter M.|
|Commitee:||Chambers, Timothy, Megyesi, Judit K., Shmookler Reis, Robert, Storrie, Brian|
|School:||University of Arkansas for Medical Sciences|
|Department:||Physiology and Biophysics|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/11(E), Dissertation Abstracts International|
|Keywords:||Apoptosis, Cell cycle, Cell death, Cisplatin nephrotoxicity|
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