Complete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), a phenomenon known as anoikis. Besides the induction of pro-apoptotic events, activation of survival pathways was observed in detached intestinal epithelial cell lines, providing a transient apoptosis protection. However, nothing is known about molecular mechanisms protecting primary CEC from anoikis. In this study intact CEC crypts were isolated and kept in suspension, a condition which leads to the loss of cell-cell anchorage and induces anoikis. To reconstitute cell-cell contacts, cells were centrifuged to form cell aggregates. Induction of apoptosis was assessed by caspase-3 activity assay; activation of survival pathways was analyzed by Western blot. Immediately after loss of cell anchorage a rapid activation of survival proteins was observed before active caspase-3 could be detected. Src hyperactivation significantly contributed to transient protection from anoikis in CEC because its inhibition reversed the protecting effect of re-establishment of cell contacts. Basal levels of active Src in CEC from patients with inflammatory bowel disease were markedly reduced compared to control patients. These results demonstrate that loss of cell anchorage activates survival pathways in primary human CEC providing transient anoikis protection. Src is an important mediator of this mechanism and therefore constitutes a key regulatory molecule coordinating survival signals mediated by cell adhesion in primary human CEC. (C) 2009 Elsevier Inc. All rights reserved.