NEW ISOLATION TECHNIQUE TO STUDY APOPTOSIS IN HUMAN INTESTINAL EPITHELIAL-CELLS

Intestinal epithelial cells derive from stem cells at the base of the crypt and migrate along the crypt-lumen axis. Their life is terminated as they reach the luminal surface where they detach and are shed. Int estinal epithelial cells show evidence of apoptosis in the region of s hedding, and cell death is thought to resemble a form of apoptosis cal led detachment-induced cell death, or anoikis, Human intestinal epithe lial cells die rapidly in vitro due to loss of anchorage during isolat ion, making primary culture of these cells a goal that has not yet bee n reached. However, the molecular mechanisms underlying this process o f anoikis are largely unknown. In this study, a. novel protocol for th e rapid, temperature-controlled isolation of highly purified human col onic epithelial cells from surgical specimens is described. Using this method, early molecular events of anoikis in nontransformed epithelia l cells were studied. Intestinal epithelial cells were isolated at the beginning of the apoptotic cascade, before the activation of caspase 3 family members and cleavage of poly(ADP-ribose) polymerase and DNA f ragmentation. Elucidating the molecular mechanisms of detachment-induc ed cell death may facilitate the establishment of long-term primary cu ltures of human intestinal epithelial cells and enhance our understand ing of homeostasis in the intestinal epithelium.