Involvement of p53 in the S-phase Checkpoint during Nucleotide Deficiencies

摘要: INVOLVEMENT OF P53 IN THE S-PHASE CHECKPOINT DURING NUCLEOTIDE DEFICIENCIES By Cortney L. Heyer, B.S. A dissertation submitted in partial fulfillment of the requirements for degree Doctor Philosophy at Virginia Commonwealth University. University, 2011 Advisor: Richard G. Moran, Ph.D. Professor, Department Pharmacology and Toxicology Several classes antimetabolites have been developed treatment cancer, including numerous inhibitors nucleotide biosynthesis. N-(phosphonacetyl)-L-aspartate (PALA) hydroxyurea (HU) are two that inhibit biosynthesis; PALA inhibits de novo pyrimidine synthesis HU conversion ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Due similar mechanisms, it was thought cancer cells would respond similarly treatment. However, studies this revealed strikingly different responses either or HCT116 cells. cytoprotective S-phase arrest activated upon while failed activate checkpoint, resulting p53-dependent apoptosis. The checkpoint effector kinase, Chk1, not significantly phosphorylated during due a failure recruit ATR, upstream chromatin sites. post-translational modifications p53, phosphorylation serines 46 392, suggested promotes accumulation transcriptionally active p53 does not. ChIP analysis showed bound pro-apoptotic promoters, therefore activating apoptosis To gain more insight into these differential cellular responses, we tandem-affinity purification (TAP) tagged cell line which TAP tag inserted Cterminus endogenous genetic locus through homologous recombination. This technology allows with its protein binding partners expression levels. accumulated promoters response DNA damage untagged suggesting did interfere normal functions p53. Using mass spectrometry, can identify We also determine variable pattern on drug-stabilized responsible promoting versus arrest. then exploit identified proteins development new chemotherapeutic agents.