Differential expression of apoptosis-related proteins along the crypt-villus axis following radiation in mouse intestinal epithelial cells

Intestinal epithelium is one of the most proliferating tissues in the mammalian and these epithelial cells are thought to be sensitive to radiation. Exposure of abdominal part to high dose radiation leads to gastrointestinal tract (GIT) injury which is one of serious problems in accidental exposure and also in radiation therapy. However, the mechanism(s) is not fully understood and its treatment is unknown. Stem cells in the crypt differentiate into mature cells and migrate upward from the base of the crypt toward the villus tip. Differentiated cells rapidly lose their proliferative ability and then undergo apoptosis. Radiation breakdowns a dynamic equilibrium of cell proliferation and differentiation in the intestine. In order to study the mechanisms of GIT injury by radiation, we investigated the expression of apoptosis-related proteins in mouse intestinal epithelial cells along the crypt-villus axis following radiation. Epithelial cells were sequentially isolated cells from the villus tip to the crypts of mouse small intestine by the modified Weiser method. This method allowed us to obtain cell fractions along the crypt to villus tip. Western blot analysis showed that proteins of the proliferating cell nuclear antigen (PCNA), the Bax and the Bcl2 were constitutively expressed in the crypt and their levels were reduced toward the villus axis. On the other hand, active form of caspase 3 and cytochrome c were accumulated in the villus tip. Radiation increased levels of Bax, active form of caspase 3, and cytochrome c with concomitant reduced levels of PCNA and Bcl2 in the crypt. In contrast, radiation did not affect the caspase 3 level in the villus tip. The p53 and p21 proteins were not detected in intestinal epithelial cells without radiation. However, radiation increased the levels of these proteins toward the crypt but not in the villus. On the other hand, the p27 protein was constitutively expressed in the crypt but not in villus, and radiation decreased its level in the crypt. Immuno-histochemistry of paraffin section of the small intestine also showed that numbers of p53 and p21 positive cells were increased by radiation in the crypt. Our results suggest that the cell proliferation and response to radiation is differentially regulated in the crypt from the villus; p53 may play an important role in the crypt.ＡＡＣＲ Ａｎｎｕａｌ Ｍｅｅｔｉｎｇ 200