Kwan Chen
B.S. Northwestern University, 2005

3rd Year Graduate Student

Advisor: Bruce Demple

Department: Pharmacological Sciences

Graduate Program: Genetics

Title:  The contribution of various DNA damages to cell toxicity

Abstract:

         DNA within our cells can be damaged in numerous ways. Chemical damages include alkylation, oxidation, and hydrolysis, all of which can originate from normal metabolism. Exposure to UV radiation or environmental toxins can also cause DNA damage. At a cellular level, such damages can lead to cell cycle arrest, apoptosis, or mutations that modify gene expression. Repair mechanisms such as base excision repair (BER) have evolved to reduce the effects of DNA damage on cell survival. BER is the major repair pathway for small single-base lesions. At least 10,000 such lesions are repaired through BER in each human cell per day. To initiate BER, a DNA glycosylase detects and removes a damaged nucleobase, leaving behind an apurinic/apyrimidinic (AP) site. AP endonuclease (APE1) nicks the sugar-phosphate backbone at the 5’ end of the AP site. DNA polymerase ß replaces the proper missing nucleotide. Finally, DNA ligase completes the repair by joining the sugar-phosphate backbone with a new phosphodiester bond.

         Mice with single genetic knockouts of the DNA glycosylases exhibit little or no phenotypic change compared to wild-type animals. Uracil glycosylase (UNG) deficient mice have a 100-fold increase in uracil misincorporation during cell replication, yet are healthy for the first 12 months of life. 8-oxoG glycosylase (OGG1) deficient mice have a three-fold increase in 8-oxoG. However, the accumulation of 8-oxoG is not associated with tumorigenesis (Barnes & Lindahl, 2004). In contrast with glycosylase knockouts, genetic knockout of APE1, DNA polymerase ß, or DNA ligase 1 in mice is embryonic lethal (Xanthoudakis, Smeyne, Wallace, & Curran, 1996) (Sugo, Aratani, Nagashima, Kubota, & Koyama, 2000)(Bentley et al., 1996). The vital role of Ape1 depends on its DNA repair activity and is observed even at the cellular level; severe APE1 knockdown reduces cell proliferation and promotes apoptosis (Fung & Demple, 2005). It appears that the presence of unrepaired AP sites is more toxic to the cell than are damaged nucleobases. We do not know, however, what types of damage have greater toxic effects to APE1-deficient cells. One of the goals is to understand the contributions that different damaged bases have towards cell toxicity.

         We are using several approaches to study the contribution that various types of DNA damage have on cell toxicity. One method we are using to determine the contribution of oxidative damage on DNA is incubating U2OS and HeLa cell lines under 21% O2 and 1% O2. Cells that have been grown under hypoxic conditions should have lower levels of reactive oxygen species and decreased numbers of nucleobases damaged by ROS. In a preliminary experiment using quantitative PCR, we have shown that cells grown under 1% O2 have between a 2- to 4-fold decrease in the level of the mRNA transcripts of the UNG2 (uracil-DNA glycosylase) and APE1 genes compared to cells grown under 21% O2. I am replicating this experiment, and will check this observation with western blotting to assess the Ung2 and Ape1 protein levels. I will continue to explore whether changes in O2 affect the proliferation rate of Ape1-deficient cells. Future studies include knocking down DNA glycosylases such as UNG2 and OGG1 and quantifying the contribution of glycosylases to cell proliferation in Ape1-deficient cells.

Publications:
(pre-MSTP publications indicated with an *)

*Zhou CC , Chen KY , Breslow JL. (2008). Activation of pregnane X receptor induces hypercholesterolemia and accelerates atherosclerosis in mice. ATVB. Jun; 28(6):E32

*Wolfrum S , Teupser D , Tan M , Chen KY , Breslow JL. (2007). The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappa B target genes. PNAS. Nov. 20; 104(47):18601-18606. PMCID 2141823

*Windler-Hart SL, Chen KY, Chenn A. (2005). A cell behavior screen: identification, sorting, and enrichment of cells based on motility. BMC Cell Biol. 6:14.