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Title: | Quantification of the Activities of the Anti-Apoptotic Proteins BCL-2 and BCL-XL |
Other Titles: | Quantification of the Activities of BCL-2 and BCL-XL |
Authors: | Fiebig, Aline |
Advisor: | Andrews, David |
Department: | Biochemistry |
Keywords: | anti-apoptotic;proteins;BCL-2;BCL-XL |
Publication Date: | May-2003 |
Abstract: | Apoptosis is the process by which organisms eliminate excess, damaged or hazardous cells in a controlled manner. This process is controlled by the Bcl-2 family of proteins. Bcl-2 and Bcl-XL are anti-apoptotic paralogues that can replace CED-9, the sole homologue in C. elegans. It has therefore been assumed that Bcl-2 and Bcl-XL are replaceable and functionally identical. However, evidence in some mammalian cells indicates that this may not be the case. The purpose of this project was to exhaustively compare the anti-apoptotic activities of Bcl-2 and Bcl-XL in one cell type. As Bcl-2 and Bcl-XL have been found to localize to the ER and the outer mitochondrial membrane, we also determined whether subcellular location affects the function of these proteins differently. MCF-7 breast cancer cells were stably transfected with Bcl-2 and Bcl-XL alternatively targeted to the ER or mitochondria, and exposed to various doses of doxorubicin; PARP cleavage was measured using quantitative Western blotting as an indication of apoptosis to obtain EC₅₀ values in the different cell lines. The levels of both Bcl-2 and Bcl-XL affected anti-apoptotic activity; specific degradation of both proteins was noted at higher doses of doxorubicin. Nevertheless, the results indicated clearly that there was a difference between Bcl-2 and Bcl-XL. Using EC₅₀ values Bcl-XL mutants were at least 8 times more protective than Bcl-2 mutants. Furthermore, most of the cleavage products of PARP in Bcl-XL expressing clones were due to non-caspase-7 proteases, a pattern not seen with Bcl-2. Bcl-2 and Bcl-XL localized to mitochondria were most effective, while cytosolic and ER localized Bcl-XL were less effective, and Bcl-2 at these sites did not inhibit apoptosis. |
URI: | http://hdl.handle.net/11375/22684 |
Appears in Collections: | Digitized Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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fiebig_aline_a_2003May_masters.pdf | 23.5 MB | Adobe PDF | View/Open |
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