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Title: X-linked inhibitor of Apoptosis (XIAP) in colorectal cancer models
Author: Connolly, Kathryn C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Two methods of XIAP down regulation were considered; transient knock down using antisense (AS) oligonucleotides (OGNs) and stable down regulation by generating short hairpin RNA expression cells. AEG35156 (Aegera Inc), a 2nd generation mixed backbone AS OGN, binds specifically to the sense XIAP and mRNA strand, stimulating its breakdown and preventing translation into protein. A clinical phase I trial of AEG35156 was undertaken in patients with advanced cancer, in Manchester and Edinburgh. The maximum tolerated dose was 96 mg/m2/day of AEG35156, using a 7 day continuous infusion regimen. Dose limiting toxicities were all abnormal laboratory values, reported at higher dose levels (125mg/m2/day and 160mg/m2/day). In laboratory studies, a panel of colorectal cell lines (Colo205, HT29, SW620, HCT15, HCT116) have been characterised according to p53, MLH1 and XIAP status. The HCT116 cell line is mismatch repair deficient (MLH1-) but has a normal functioning p53. It expresses XIAP at levels similar to other members of the cell line panel and is up regulated when compared to normal tissue. A method of transient transfection of XIAP AS (AEG35156) in vitro was developed which achieved 81% down regulation of XIAP mRNA using the AEG35156 compound. Down regulation of XIAP mRNA was also seen with the missense (MS) OGN, AEG35187. Cytotoxicity experiments showed no significant therapeutic benefit of AS over MS. Using short hairpin (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones were developed. XIAP mRNA levels were established by RT-PCR, the 4 X (XIAP knockdown) clonal cell lines showing 82-92% reduction in XIAP mRNA when compared to the 4 L (luciferase control) cell lines. Immunoblot analysis showed a 63-89% reduction in XIAP protein in X cell lines compared to L. A colorectal cancer model has been developed using isogenic cell lines which differ only in their XIAP status. The XIAP deficient cell lines show a 2-fold increase in sensitivity to rhTRAIL and 20% increase in sensitivity to radiotherapy. There was a >2-fold increase in sensitivity to paclitaxel and docetaxel. All 8 shRNA cell lines can be established in vivo, the L8 and X23 cell lines showing a similar growth pattern. XIAP knockdown is maintained at the mRNA level for 26 days after implantation, though the down regulation is less than that seen in vitro. However, treatment of X23 and L8 xenografts with docetaxel showed no significant different effect on growth.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available