Davies, A.; Ottewell, P.D.; McNamara, A.V.; Watson, A.J.M.; Jenkins, J.R.
April 2003
Gut;Apr2003 Supplement 1, Vol. 52, pA36
Academic Journal
Background and Aims: Etoposide (VP16) induced stress in the mouse small intestine displays a dose and time dependent apoptotic response. Apoptosis only occurs in the stem cell compartment of the crypts and is Bax and p21 independent, but p53 dependent. Apoptosis is absent in the villi. We exploited the differential response to VP16 along crypt-villus axis to identify novel p53 dependent genes that are either pro-apoptotic or protective against cell death. Methods: cDNA array analysis was performed in the p53 +/+ and -/- mouse total epithelium of the small intestine (10 mg/kg VP16 exposure for 4.5 h). Data were analysed using GeneSpring software (Silicon Genetics), which generated a list of >2-fold p53 dependent gene expression changes. Laser capture microdissection (LCM) was used to isolate pure crypt and pure villus epithelial cell populations, and real time PCR (QPCR) performed to detect differential gene expression along the crypt-villus axis. Protein expression was analysed by Western blot and immunohistochemistry techniques. Results: Presenilin 2 (PS2) and baculoviral lAP repeat 3 (BIRC3) were identified as VP16 induced p53 dependent genes by array analysis. Western blot analysis of total epithelium showed an upregulation of PS2 and BIRC3 protein levels in a p53-dependent manner. QPCR analysis of LCM isolated p53-wildtype crypts and villi revealed a differential gene expression of PS2 along the crypt-villus axis: a decrease of gene expression in the crypts, but an increase in the villus. Our initial histopathological observations support these data. BIRC3 expression was downregulated in both the crypt and villus. Conclusions: BIRC3 does not appear to regulate apoptosis in intestinal epithelium. The differential mRNA expression of PS2 along the crypt-villus axis may indicate a pro-apoptotic role for Iow PS2 expression in the crypts, while providing apoptotic protection in the villus following VP16-induced stress.


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