dUTPase and Uracil-DNA Repair in Cancer Chemotherapy

Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (dTMP) (Fig. 1A). This reaction represents the sole de novo source of dTMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents.

Previous studies suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme dTTP pool depletion observed following TS inhibition. Although depletion of dTTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage (Fig. 1B). Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate simultaneously eliminating dUTP pools for use during replication and repair, and providing substrate (dUMP) for the TS reaction. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis.

Recently, we have developed a model system using the yeast S. cerevisiae in which we have modulated the levels of dUTPase and uracil-DNA glycosylase and analyzed the effects of antifolate treatment. Through a series of genetic and biochemical-based analyses including viability, nucleotide pool analysis, FACS analysis, and pulsed field gel electrophoresis, we demonstrate that aberrant uracil misincorporation and excision plays a dominant role in antifolate toxicity and that dTTP pool depletion alone does not mediate extreme lethality in yeast. Our data suggest that the relative cellular expression of dUTPase and uracil-DNA glycosylase can have profound effects on the ability of cells to respond to antifolates. We are now pursuing these studies in mammalian cells to understand the role of uracil misincorporation vis a vis other cellular events that are also believed to be mediators of lethality induced by inhibition of TS.

dUTPase

Much of the research in our laboratory focuses on the characterization of dUTPase and its role in cellular responses to TS directed chemotherapy. Nuclear and mitochondrial isoforms of human dUTPase exist that arise through alternative use of the first two exons. These proteins are identical except for a short region in their N-termini. In addition, only the nuclear form of dUTPase is phosphorylated; however, to date the functional significance of this phosphorylation has not been elucidated. In cell culture models, DUT-N is tightly regulated to coincide with replication whereas DUT-M expression is regulated in a constitutive manner. We are currently investigating the regulation of these genes at the transcriptional as well as post-transcriptional levels.

Development of monoclonal antibodies against dUTPase (DUT415) in our lab has allowed the extensive characterization of dUTPase expression in normal and neoplastic tissues by immunohistochemistry (Fig. 2). We have found that in agreement with cell culture models, nuclear expression of dUTPase in normal human tissues is proliferation dependent and cytoplasmic dUTPase is evident in normal tissues with high mitochondrial content (Fig. 2A-C). Interestingly, in human cancers we found that dUTPase expression varies dramatically in both amount and intracellular localization (Fig.2D-F). In a retrospective study of tumor specimens from 20 metastatic colon cancer patients who had received 5-FU and leucovorin, we found that nuclear dUTPase expression by immunohistochemistry was associated resistance to chemotherapy, shorter time to progression, and a shorter median survival. Larger patient studies on the prognostic impact of dUTPase expression on drug response are ongoing in our laboratory in collaboration with the laboratory of Dr. Heinz-Josef Lenz (USC/Norris Comprehensive Cancer Center).