Targeting uracil exclusion mechanisms for development of anti-viral and anti-cancer therapies.

摘要

Uracil has the potential to be deleterious when introduced into DNA and because of this therapeutic approaches targeting viruses and cancer cells have been developed to increase the likelihood of uracil formation in DNA. However, organisms have evolved very efficient mechanisms to prevent uracil formation/retention in DNA. Two of the most well characterized mechanisms involve the enzymes uracil DNA glycosylase (UDG) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase). In an attempt to further understand the roles of UDG and dUTPase in viruses and tumor cells, as well as determine whether these enzymes could possibly be targets of future chemotherapy, a protein transduction technique and a small interfering RNA (siRNA) approach were utilized. An inhibitor protein of UDG (UGI) coupled to the protein transduction domain of the human immunodeficiency virus type 1 Tat protein (TaT-UGI) was able to efficiently transduce multiple cell types and inhibit cellular UDG activity. UDG activity was decreased approximately 94.4 +/- 4.1% in U-937 cells, 29.6 +/- 5.6% in HeLa cells, 69.9 +/- 5.0% in HT29 cells, and 78.9 +/- 1.0% in SW620 cells. On the other hand, a double stranded siRNA synthesized against domain 3 (nucleotides 339 to 357) of human dUTPase (siRNA-dut3) was able to diminish cellular dUTPase activity in all cell types examined. dUTPase activity was decreased approximately 50.0 +/- 0.9% in HeLa and HT29 cells and approximately 27.0 +/- 11.0 in SW620 cells. While TaT-UGI decreased cellular protein concentration, dUTPase levels, and doubling times in cells treated for 72 hours with the highest concentration of TaT-UGI, siRNA-dut3 had no other statistically significant effects on the cells, other then decreasing dUTPase levels, when compared to non-treated controls. These results demonstrate that TaT-UGI and siRNA-dut3 can be used to decrease UDG and dUTPase activities, respectively, in human cells and that such an approach could be useful either alone or in combination with established therapies for anti-viral and/or cancer chemotherapy. Moreover, these reagents can be used to elucidate the functions/roles of UDG and dUTPase in normal cells and viral homeostasis so that rational therapeutic approaches can be developed targeting these enzymes.