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    Anti-cancer Functions and Mechanisms of a pRb2/p130 Peptide Fragment

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    Genre
    Thesis/Dissertation
    Date
    2009
    Author
    Sun, Ang
    Advisor
    Waring, Richard B.
    Committee member
    Giordano, Antonio, MD
    Feitelson, Mark
    Sang, Nianli
    Department
    Biology
    Subject
    Biology, Molecular
    Cdk2
    Kinase
    Peptide
    Prb2/p130
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/2481
    
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    DOI
    http://dx.doi.org/10.34944/dspace/2463
    Abstract
    The spacer region of pRb2/p130 was reported to be able to inhibit the kinase activity of Cdk2. The region responsible for the inhibitory effect was further narrowed down to a 39-amino-acid sequence, which was named as Spa310. In this dissertation, the anti-cancer functions and mechanisms of Spa310 were studied. The synthesized Spa310 peptide was able to inhibit the kinase activities of Cdk2/Cyclin E/A complexes. In vitro kinase assays showed the inhibition occurred in a dose-dependent manner. The half maximal inhibition concentration of the Spa310 in the kinase assay was 1.67mM. In addition, it has been shown that Spa310 peptide is able to inhibit the kinase activities of both Cdk2/Cyclin E and Cdk2/Cyclin A. Intra-cellular distribution study using fluorescein-labeled Spa310 peptide showed that Spa310 was able to localize to the nuclei of A549 cancer cells. Some data indicated the endoplasmic reticulum might play a role in transporting Spa310 peptide from cytoplasm to the nucleus. At high concentration, the treatment of Spa310 peptide was able to arrest cells at the G0/G1 phase of the cell cycle and reduce the growth of xenografted tumors in nude mice. Further studies indicated Spa310 peptide is not a specific inhibitor for Cdk2/Cyclin E/A. It is also able to inhibit the kinase activities of Cdk1/Cyclin B, Cdk4/Cyclin D and Cdk9/Cyclin T/K. Result of a binding assay using GST-Spa310 and in vitro transcribed/translated Cdk2 did not support a direct binding between Spa310 and Cdk2. Additionally, GST-Spa310 was unable to bind to the in vitro transcribed/translated Cyclin E. At first, co-immunoprecipitation experiments indicated a weak binding between Spa310 peptide and Cdk2. However, later this weak binding was proven to be unspecific and only occurred when the concentration of Spa310 peptide was high. Thus, the hypothesized mechanism of the inhibitory effect of Spa310 was not supported. After noticing three classic Cdk phosphorylation sites present in Spa310, it was proven that Spa310 is a substrate for Cdk1, 2, 4 and 9. Results of kinase assays supported the inhibitory effect of Spa310 on the different Cyclin-dependent kinases was resulted from a substrate-competitive mechanism. Although the data generated from this study does not support Spa310 is a potent peptide inhibitor for the Cdks, knowledge gained from and the approach used in this research can be applied to design and develop more potent and specific Cdk2 peptide inhibitors, which have their potentials to work as powerful anti-cancer reagents.
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