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Emert-Sedlak, Lori A (2005) MEDIATION OF CHEMOTHERAPY-INDUCED APOPTOSIS BY THE LYSOSOMAL PROTEASE CATHEPSIN D. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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One of the most common hallmarks of cancer is dysregulation of cellular apoptotic processes. A comprehensive knowledge of the underlying mechanisms of the apoptotic machinery is vital for the identification of new drug targets and the development of innovative agents that stimulate the cell death process in cancer cells. Studies have shown that the lysosomal protease cathepsin D is important in the extrinsic apoptotic pathway stimulated by the death receptor ligands for TNFR1 and FAS, as well as by oxidative stress and the protein kinase C inhibitor staurosporine. To date, the role of cathepsin D in the chemotherapy-induced apoptotic pathway has not been characterized. This project examined the role of the lysosomal protease cathepsin D in chemotherapy-induced apoptosis of HeLa and U937 cells. The data demonstrated that following stimulation of U937 cells with the chemotherapy drug VP-16, cathepsin D was released into the cytosol approximately 4 hours after drug treatment. This release was selective for cathepsin D, as cathepsin B and the lysosomal markers LAMP and â-hexosaminidase were not released into the cytosol following VP-16 treatment. Inhibitors of caspases andcathepsin D had no effect on cathepsin D release, demonstrating that cathepsin D release occurred independently of caspase and cathepsin D activities. Downregulation of cathepsin D expression in U937 and Hela cells using siRNA was found to inhibit cell death resulting from a variety of stimuli, including death receptor ligands, oxidative stress, PKC inhibitors, and importantly, chemotherapy drugs. In addition, U937 and HeLa cells expressing cathepsin D siRNA exhibited delayed cytochrome c release and caspase-3 activation following VP-16 treatment. Moreover, isolated mitochondria from wild-type U937 cells released cytochrome c in response to cytosolic extracts that were treated with cathepsin D, suggesting that cathepsin D acts on a cytosolic factor to induce cytochrome c release. Inhibition of caspases had no impact on cytochrome c release provoked by cathepsin D-cleaved cytosolic extract, demonstrating that caspases are not mediators of cathepsin D-induced cytochrome c release. Taken together, these results demonstrate that cathepsin D is an important component of the apoptotic pathway and that it acts via an intermediary cytosolic factor to promote cytochrome c release and caspase activation during chemotherapy-induced apoptosis.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Emert-Sedlak, Lori
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeFranco, Dondod1@pitt.eduDOD1
Committee MemberJohnson, Daniel Ejohnsond@pitt.eduJOHNSOND
Committee MemberYalowich, Jackyalowich@server.pharm.pitt.eduYALOWICH
Committee MemberZhang, Linzhanglx@upmc.eduLIZ22
Committee MemberYin, Xiaomingxmyin@pitt.eduXMYIN
Date: 25 July 2005
Date Type: Completion
Defense Date: 27 May 2005
Approval Date: 25 July 2005
Submission Date: 1 June 2005
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Pharmacology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: apoptosis; caspases; cathepsin D; cell death; chemotherapy; cytochrome c; lysosomes; proteases; VP-16
Other ID:, etd-06012005-221616
Date Deposited: 10 Nov 2011 19:46
Last Modified: 19 Dec 2016 14:36


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