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053 NUCLEAR EXPRESSION OF E2F4 INDUCES P53-DEPENDENT APOPTOSIS OF NORMAL HUMAN INTESTINAL EPITHELIAL CRYPT CELLS BUT NOT IN COLON CANCER CELLS H Garneau, MC Paquin, L Alvarez, C Lussier, N Rivard The E2F transcription factors controls cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells stimulated by growth factors.
Department of Anatomy and Cell Biology, University of Sherbrooke, Sherbrooke, Quebec
AIMS OF THE STUDY: To investigate the endogenous expression of E2F4 in colon cancer cells and the impact of constitutive nuclear expression of E2F4 in normal intestinal epithelial cells.
METHODS: Expression and localization of E2F4 and of survival regulatory proteins were analyzed by Western blot and immunofluorescence. Adenoviruses expressing fusion proteins between GFP and either E2F4 wild-type (E2F4wt) or E2F4-NLS containing a strong NLS from SV40LgT were used to infect non immortalized human intestinal epithelial cells (HIEC). Caspase activities were analyzed with fluorogenic substrates. Cell death was analyzed by trypan blue exclusion assay and electron microscopy.
RESULTS: 1- Examination of living cells revealed that HIEC-expressing E2F4-NLS acquired a fibroblastic morphology few hours after infection, in contrast to non-infected epithelioid HIEC or HIEC-expressing E2F4wt. 2- Three days after infection, HIEC-expressing E2F4-NLS died and detached. An induction of p53 (5.5-fold), phospho-serine 15/p53 (4-fold), PUMA (3.5-fold), FAS (2-fold), BAX (2-fold) and RIP (4-fold) was also observed in HIEC-expressing E2F4-NLS comparatively to cells-expressing GFP. 3- Caspase-3, 8 and 9 activities were enhanced by respectively 12, 3 and 2-fold in HIEC-expressing E2F4-NLS. 4-The lentiviral infection of a shRNA which specifically knocked-down p53 expression significantly inhibited cell death induced by E2F4-NLS expression. 5- In contrast to normal crypt cells, endogenous E2F4 in colonic cancer cells (Caco-2, HCT116, DLD-1 and LOVO) was overexpressed and constitutively nuclear even in absence of exogenous growth factors and independently of p53 gene status, suggesting that the growth factor-dependency of E2F4 nuclear localization is lost during colon cell transformation. In addition, the level of apoptosis in these cells was negligible.
CONCLUSIONS: Persistent localization of E2F4 into the nucleus of normal human crypt cells induced apoptosis in a p53-dependent manner. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but also may accelerate cell death. However, colonic cells acquiring mutations in p53 locus or in other genes regulating cell survival (INK4A/ARF, ATM/ATR, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of E2F4.