Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Room: Exhibit Hall
Bianca Davis - University of Missouri- Columbia
Co-Author(s): Maram Essawy, University of Minnesota, Minneapolis, MN; Dr. Colin Campbell, University of Minnesota, Minneapolis, MN
Common cancer treatment utilizes medications that create DNA damage in cancer cells, but these cells customarily form a resistance to the drugs. Investigating the techniques the cells use to overcome DNA damage would lead to better treatments. DNA-Protein Crosslinks (DPCs) are a type of DNA damage often created by cancer drugs and form when proteins are covalently attached to the DNA backbone. This damage inhibits processes such as replication and transcription, thus toxic to the cell by inducing mutagenesis and cell death. They can be created from common antitumor drugs, ultraviolet (UV) radiation, reactive oxygen species, and endogenous aldehydes. Nuclear excision repair (NER) and homologous recombination (HR) are two of the main processes the cell can use to repair the DPCs. The actions the cells take to choose between repair pathways are unknown. This project seeks to develop cells that have defects in numerous DNA repair genes at once. These cells would allow for determining how specific changes affect the repair pathways chosen. To create this model method, an siRNA transfection targeting XPC protein was performed in HEK293T wildtype (WT) cells to generate a NER repair deficiency. Afterward, duplicate cell plates were either used for clonogenic assays or DPC insertion. Clonogenic assays involved exposure to 0 J/m2 or 10 J/m2 of UV followed by incubation at 37°C for nine days to allow colony formation. DPC transfection affixed the DPCs onto the DNA in the cells, cut the DNA into two fragments (one containing the DPC and one unassociated with the DPC), removed the DPCs, and performed qPCR targeting each fragment. The western blot results visually confirmed a reduction in XPC expression following the siRNA transfection. NER deficient cells are sensitive to UV radiation and unable to repair the damage, and the clonogenic assay verifies that effect. The UV-exposed nontransfected plates showed a higher percent survivability in colony count than UV-exposed transfected plates compared to their respective control groups, and the difference between treatment groups’ survivability is significantly significant. Therefore, the assay was able to confirm the generation of NER deficient cells. Finally, a qPCR analysis was used to experimentally determine if there was a loss of NER in siXPC cells, and the results suggest a decrease in percent DPC repair compared to WT cells. Future work involves more fully developing the model by performing additional qPCR analysis so the results reach significance and testing other siRNA concentrations. Future experiments will be conducted to explore additional repair pathways that the cell uses and those pathways’ processes. In addition, the model can be used to understand the role of proteasomes, ubiquitination, and SPRTN protease in repair. Learning about these roles and their implications in DPC repair can lead to targeted cancer treatment that can avoid resistance.
Funder Acknowledgement(s): HLB R25 grant support: R25HL088728; MU’s MARC Fellows Program via grant number T34 GM 136493 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH).
Faculty Advisor: Dr. Colin Campbell, firstname.lastname@example.org
Role: This was an independent project preformed by me with the assistance of Maram Essawy and Dr. Colin Campbell. All experiments were conducted by me, and the results figures were also developed by me.