Scott, Bradley James2013-11-082013-11-0820092009Source: Dissertation Abstracts International, Volume: 71-05, Section: B, page: 2988.http://hdl.handle.net/10393/29841http://dx.doi.org/10.20381/ruor-13166Resistance to traditional modes of cancer therapy is a major obstacle facing cancer patients. The untreated HCT116 cell line was subcloned and each clone was assessed for its response to several drugs and X-radiation. One clone HCT116-K was resistant to cisplatin/etoposide/topotecan but sensitive to XR while another HCT116-2 was sensitive to cisplatin but resistant to XR. HCT116-10, a clone that responded to drugs and radiation in a manner similar to parental HCT116 cells was used as a control clone. Comparing gene expression between HCT116-K, or HCT116-2, vs. HCT116-10, we were able to identify several genes whose expressions were altered in clones that were resistant/sensitive to various insults. Each identified gene's expression was then examined in a panel of HCT116 clones to determine whether the expression observed in the resistant/sensitive clone was outside the normal expression distribution within the population. One gene APM2 was significantly upregulated in HCT116-K. Upon follow-up, APM2, was found to promote cisplatin resistance when overexpressed in sensitive HCT116 clones. Furthermore, silencing APM2, using sequence specific siRNAs, in a panel of cell lines encompassing all combinations of p53 status and MMR proficiency (HCT116-K, HCT116, SW620, MCF7, PC-3, and OV2008) resulted in sensitization regardless of these two factors. In addition, silencing APM2 stably, using shRNAs, also resulted in the sensitization of cells to cisplatin. More importantly, cisplatin inhibited the growth of APM2 silenced tumour xenografts (HCT116-K or OV2008 cells) significantly better than it inhibited the growth of xenografts carrying non-targeting control shRNAs. Additionally, APM2 was found to be localized to the cytoplasm and membrane. Further investigation revealed that HCT1 16 cells secrete APM2 to the extracellular space representing a possible mechanism by which APM2 promotes cellular resistance to cisplatin, by binding to and carrying cisplatin out of the cell. These findings represent a novel strategy that could be exploited to overcome cisplatin resistance in patients regardless of p53 status or ability to perform MMR.174 p.enHealth Sciences, Pharmacology.Thesis