Metabolic regulation and anticancer drug resistance in the yeast Saccharomyces cerevisiae

Cancer is a malignant neoplastic tissue. The pyrimidine analogue 5-fluorouracil is an anticancer drug used against several types of cancer. It owes its specificity against neoplasms due to the higher rate of nutrient uptake, RNA and DNA synthesis, and in particular the higher rate of nucleobase scavenging and salvage of neoplastic cells. However, tumours can develop resistance to 5-fluorouracil and other anticancer drugs, rendering the therapy useless. Due to genetic variation, humans can also be inherently more or less sensitive to 5-fluorouracil and other anticancer drugs such as the purine analogue mercaptopurine. In this thesis I have investigated the mechanisms of resistance to 5-fluorouracil and the model purine analogue 6-N-hydroxylaminopurine in the budding yeast Saccharomyces cerevisiae. The mechanisms found to confer resistance in S. cerevisiae to 5-fluorouracil and 6-N-hydroxylaminopurine included both deregulation of the de novo synthesis of nucleotides, and detoxification of non-canonical nucleotides. I proceeded to study genetic interactions between genes involved in resistance to both 5-fluorouracil and 6-N-hydroxylaminopurine by using plasmid overexpression in yeast strains knocked out for different resistance genes and other genes involved in nucleotide metabolism. HAM1 is a yeast gene coding for a nucleotide pyrophosphatase, the overexpression of which we found to confer resistance to both 5-fluorouracil and 6-N-hydroxylaminopurine. HAM1 has a human homologue called ITPA. We proceeded to test if a known genetic polymorphism in ITPA could help predict toxicity in patients receiving a chemotherapeutic regimen including 5-fluorouracil. Our results suggest that loss of ITPA function may lead to increased sensitivity to 5-fluorouracil. We have also investigated the mechanism of transcriptional regulation by the yeast Rph1p protein, a zinc finger transcription factor which also contains a jmjC domain with histone demethylase activity. Rph1p functions downstream of the RAS-cAMP-PKA pathway and several other nutrient sensing pathways. We found, surprisingly, that the histone demethylase activity of Rph1p is largely dispensable for its role in transcriptional regulation