Anti-cancer functions and mechanisms of a pRb2/p130 peptide fragment

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 known 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, in which the half maximal inhibition concentration of the Spa310 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 concentrations, 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. Initially, co-immunoprecipitation experiments indicated a weak binding between Spa310 peptide and Cdk2. However, further studies indicated that this weak binding was 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. Based on the fact that three classic Cdk phosphorylation sites are present in Spa310, I hypothesized that Spa310 serves as a substrate for Cdk1, 2, 4 and 9. This hypothesis was later proven. In addition, 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, information derived from and the approach used in this research can be applied to design and develop more potent and specific Cdk2 peptide inhibitors that have the potentials to work as powerful anti-cancer reagents.