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Fluoropyrimidine Sensitivity in TP53 Knockout Colon Cancer Cell Line

Avram Rago, Phillip Buckhaults, PhD, Carolyn Banister, PhD

TP53 is the most frequently mutated gene across all cancers. Its normal function is to activate DNA repair, arrest growth at the G1/S growth checkpoint, and perhaps most significantly induce apoptosis in cells with DNA damage. When TP53 is mutated, cells can no longer under this programmed cell death, leading to aberrant growth. Because TP53 is so frequently mutated and is a strong driver of many breast and colorectal cancers, it is a good candidate for targeted therapies. Previous work has shown that TP53 knockout cell lines are much more sensitive to F10, a novel chemotherapeutic that is a polymer of the active metabolite of 5-Fluorouracil, FdUMP. Here, we devise a novel approach to measure the effects of F10 on TP53 knockout RKO cells. Using CRISPR-Cas9, we knocked out TP53 and OR1C1, the latter being dummy gene giving us a functional TP53 wild type cell line. These cells were then grown in media containing 5µM F10.  After purifying DNA from each time point, we ran a qPCR across the TP 53 locus. In WT cells, we generated a 1.6kb product. In TP53KO cells, we generated a ~200kb product. By using a much shorter extension time, we were able to isolate the knockout product and therby quantify how much of the TP53KO cells were present, normalized to LINE PCR. This allowed us to quantify relative cell death of TP53KOs in F10. Further work will be done to further streamline this process and explore whether or not patterns we have seen are generalizable in more cell lines.

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