DOI
https://doi.org/10.25772/4ZF5-2439
Defense Date
2011
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmacology & Toxicology
First Advisor
Richard Moran
Abstract
Several classes of antimetabolites have been developed for the treatment of cancer, including numerous inhibitors of nucleotide biosynthesis. N-(phosphonacetyl)-L-aspartate (PALA) and hydroxyurea (HU) are two antimetabolites that inhibit nucleotide biosynthesis; PALA inhibits de novo pyrimidine synthesis and HU inhibits the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Due to the similar mechanisms, it was thought that cancer cells would respond similarly to HU and PALA treatment. However, studies in this dissertation revealed strikingly different responses to either HU or PALA treatment in HCT116 cells. A cytoprotective S-phase arrest was activated upon HU treatment while PALA treatment failed to activate the S-phase checkpoint, resulting in p53-dependent apoptosis. The checkpoint effector kinase, Chk1, was not significantly phosphorylated during PALA treatment due to a failure to recruit ATR, the upstream kinase, to chromatin sites. The post-translational modifications of p53, phosphorylation of serines 46 and 392, suggested that PALA treatment promotes the accumulation of a transcriptionally active p53 while HU does not. ChIP analysis showed that p53 bound to pro-apoptotic promoters, therefore activating p53-dependent apoptosis during PALA treatment. To gain more insight into these differential cellular responses, we developed a tandem-affinity purification (TAP) tagged p53 cell line in which a TAP tag was inserted into the C-terminus of the endogenous p53 genetic locus through homologous recombination. This technology allows purification of p53 with its protein binding partners at endogenous expression levels. The tagged p53 accumulated and bound to promoters in response to DNA damage similar to the untagged p53, suggesting that the TAP tag did not interfere with the normal cellular functions of p53. Using mass spectrometry, we can identify the different p53 protein binding partners in response to PALA or HU treatment. We can also determine the variable pattern of post-translational modifications on different drug-stabilized p53 and determine which modifications are responsible for promoting apoptosis versus cytoprotective arrest. We can then exploit the identified proteins and post-translational modifications in the development of new chemotherapeutic agents.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
May 2011