Investigating the effects of expressing APE1 human population variants in cellular systems.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multi-functional mammalian protein which has recently been shown to possess the ability to endonucleolytically cleave single-stranded RNA and abasic RNA. Several population variants of APE1 (L104R, E126D and D148E) are known to exist in the human population. L104R and E126D have been linked to Amyotrophic Lateral Sclerosis while D148E has been linked to various cancers. The exact molecular mechanisms which correlate these variants with human disease are currently unknown. Recent evidence has shown that the in vitro endoribonuclease activities of these variants are different from the wild-type APE1 protein. Here, we hypothesize that the altered endoribonuclease activity of APE1 population variants may be associated with phenotype changes leading to disease pathogenesis. The goal of this thesis was to determine whether APE1 population variants can cause an altered phenotype when expressed in prokaryotic (Origami™ (DE3) cells) and eukaryotic systems (HeLa cervical cancer and HepG2 hepatoma cancer cell lines). Subsequently, these changes were to be linked to altered endoribonuclease activity of these variants. Using two separate assays, it was shown that the L104R and E126D variants possess enhanced cytotoxicity to Origami™ (DE3) cells. This correlates with their distinct endoribonuclease activity demonstrated in vitro. The D148E variant, which had lost endoribonuclease activity, had no effect on colony formation and growth of Origami™(DE3) cells. Interestingly, this study also showed that, when over-expressed, the L104R and E126D variants are capable of causing enhanced growth in the mammalian HepG2 cells. Preliminary microarray and quantitative real time polymerase chain reaction experiments were conducted in an attempt to understand the mechanism for the L104R-induced cell growth in HepG2 cells. Unfortunately, the results were inconclusive. In summary, this thesis has demonstrated a solid correlation between having distinct endoribonuclease act