Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the UK and has a poor 60% 5-year survival rate. The Wnt signalling pathway is fundamental for homeostasis of the intestinal epithelium and its deregulation drives development of CRC and induces DNA damage. Histone-2AX (H2AX) is a component of the nucleosome whose phosphorylated form, γH2AX, is a marker of DNA damage. Objectives: Using a well-characterised inducible CRC mouse model of early Wnt deregulation, and established Apc-deficient driven tumour and ex vivo organoid models, we have assessed whether the spontaneous DNA damage generated in these models can be targeted using 111In-anti-γH2AX-TAT (RH2AX), a radio-labelled antibody targeting γH2AX. Methods: Deletion of the Apc gene was effected in the intestine of VilCreERApcfl/fl and Lgr5CreERApcfl/fl models by intraperitoneal or oral induction with tamoxifen. γH2AX immunohistochemical (IHC) characterisation of intestines were performed as well as γH2AX whole mount immunofluorescent analysis on organoids derived from them. RH2AX, an anti-γH2AX antibody conjugated to the cell-penetrating peptide TAT to allow cellular internalisation and nuclear localisation, was used in these models as an imaging agent SPECT/CT imaging and biodistribution studies were conducted after oral induction of VilCreERApcfl/fl and intravenous injection of RH2AX. γH2AX and Lgr5 FACS analysis were carried out on intestinal crypt cells of VilCreERApcfl/fl mice expressing Lgr5-EGFP reporter. Results: Intestinal Apc deficiency increased DNA damage levels in the small intestine of both dysplastic (VilCreERApcfl/fl) and tumour CRC mouse (Lgr5CreERApcfl/fl) models. Apc-deficiency-associated DNA damage is most likely generated through WNT signalling pathway activation and, more specifically, by c-Myc transcription. For the first time, we demonstrated that intestinal dysplasia can be identified through in vivo SPECT imaging, using low SA RH2AX treatment. Low SA RIC treatment in intestinal dysplasia increased the DNA damage levels in healthy and Apc-deficient small and large intestines, increased proliferation in the Apc-deficient tissue and resulted in variable levels of apoptosis depending on the tissue. Conclusion: These findings together indicate that DNA damage is induced by Apc-deficiency, and that there is the possibility to exploit the endogenously-increased DNA damage signal, γH2AX, to attract the RH2AX for in vivo imaging of intestinal dysplasia. This could help diagnose early stages of CRC to provide patients with the appropriate treatment sooner and increase their survival.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:742847 |
| Date | January 2017 |
| Creators | Konstantinou, Maria |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/111847/ |
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