Investigating the roles of CDA and DCTD in cancer nucleotide metabolism

Thesis

Nucleotides play essential roles in cellular function, including as components of the nucleic acids DNA and RNA. Cancer cells have elevated proliferation and DNA replication, necessitating a reliable supply of nucleotides from the nucleotide de novo synthesis and salvage pathways. These have consequently been exploited as targets for nucleotide analogue chemotherapy. Resistance to chemotherapy can arise from enzymes in the synthesis and salvage pathway, including upregulation of the pyrimidine salvage enzyme cytidine deaminase (CDA). CDA functionally overlaps with the salvage enzyme deoxycytidylate deaminase (DCTD), and it is uncertain whether either enzyme is the principal deaminase and whether they deaminate cytidine nucleotides derived from nucleotide synthesis, in crosstalk between the synthesis and salvage pathways. I investigated these queries by setting up and optimising targeted HPLC-MS/MS systems for nucleic acid composition and nucleotide pool analysis, using them to measure isotopically labelled nucleotide precursor incorporation into cell lines and observe effects of CDA and DCTD siRNA knockdown (KD). The nucleoside analysis platform I developed is capable of sensitive analysis of nearly 20 nucleosides, whereas the free nucleotide analysis platform has analyte sensitivity comparable to or lower than those reported in literature and can detect most nucleotide phosphates without sample purification steps. The KD and pulse-chase experiment provided evidence that CDA and DCTD are mutually redundant in deaminated 2’-deoxycytidine nucleotides and validates previous reports that DCTD has low activity against cytidine monophosphate. Additionally, there was no impact of CDA and DCTD KD on L-glutamine labelling of cytidine nucleotides, suggesting that de novo synthesis-derived cytidine nucleotides are not dephosphorylated and deaminated in significant amounts. It was also observed that nucleotide phosphate interconversion occurs at much shorter timeframes than one hour.

Taken together, the studies in this thesis, using targeted and sensitive MS analytical methods, support the theory of redundancy of the CDA and DCTD, with implications for their roles in resistance to cancer chemotherapy as well as in other disease states.

Authors: Spingardi, P

• DOI: 10.5287/ora-jnk6e6ram
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cancer; metabolism; nucleotide
• Subjects: Cancer; Metabolism