| Popis: |
Much of the information on carcinogenesis in the intestine derives from studies based on the small intestine, yet the majority of the mutations leading to cancer occur in the large intestine. To overcome some of these issues, the use of a large bowel model and in particular the primary murine colonocytes system was preferred. The objectives were to better understand the importance of p53 and related proteins in the regulation of growth and responses to DNA damage in these cells. In order to investigate both p53 dependent and independent death pathways we exposed primary colonocytes to cisplatin. The initial study showed that the role of p53 in preventing entry into the S-phase following colonocytes DNA damage appears crucial whereas its role in apoptosis seems redundant. This suggests that p53-mediated apoptosis and growth arrest are neither a major nor a unique protector of colonic epithelia cells against mutation following DNA damage. Furthermore, the nuclear translocation of endogenous p73α in response to DNA damage in primary colonocytes is highly suggestive of a functional pro-apoptotic role for p73α in these cells, within the context of p53-independent apoptosis. Moreover, in the context of p53 deficiency, p73α activation is highly suggestive of its involvement in the p53-independent pathway of apoptosis in these cells. Consequently, it seemed plausible that other “early” genetic events could affect the colonic epithelium to lead to cancer. As cancer appears due to abnormal growth control it was of interest to investigate a possible involvement of Hedgehog genes in colorectal neoplasia. Therefore, the second part of this thesis focused on investigating the pattern of expression of Hedgehog signalling pathway members in normal colon versus colonic lesions including hyperplastic polyp, adenoma and adenocarcinoma of the colon. Results showed that Hedgehog signalling pathway members are expressed in normal colonic epithelium with sonic hedgehog (Shh) at the top of the crypts and a few basally located cells, patched in the neuroendocrine cells and smoothened at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of Shh, patched and smoothened was upregulated with an increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cell in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralising anti-Shh antibody. Results illustrated that exogenous Shh promotes cell proliferation in colonocytes while anti-Shh inhibits proliferation, suggesting that sonic hedgehog is required during proliferation of epithelial cells in vitro. Therefore, the results suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo. |
