Inhibition by uridine but not thymidine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: Evidence for the involvement of RNA perturbation

Abstract

The epithelia from the crypts of the intestine are exquisitely sensitive to metabolic perturbation and undergo cell death with the classical morphology of apoptosis. Administration of 40 mg͞kg 5-f luorouracil (5-FU) to BDF-1 p53؉͞؉ mice resulted in an increase in p53 protein at cell positions in the crypts that were also those subjected to an apoptotic cell death. In p53؊͞؊ mice apoptosis was almost completely absent, even after 24 hr. 5-FU is a pyrimidine antimetabolite cytotoxin with multiple mechanisms of action, including inhibition of thymidylate synthase (TS), which gives rise to DNA damage, and incorporation into RNA. The inhibition of TS can be increased by coadministration of folinic acid and can be abrogated by administration of thymidine. The incorporation of 5-FU into RNA is inhibited by administration of uridine. p53-Dependent cell death induced by 5-FU was only inhibited by administration of uridine. Uridine had no effect on the apoptosis initiated by 1 Gy of ␥-radiation. Although thymidine abrogated apoptosis induced by the pure TS inhibitor Tomudex, it had no effect on 5-FU-induced apoptosis, and coadministration of folinic acid did not increase apoptosis. The data show that 5-FU-induced cell death of intestinal epithelial cells is p53-dependent and suggests that changes in RNA metabolism initiate events culminating in the expression of p53.

Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo

“…We have con®rmed here the results of earlier studies showing that 5FU caused apoptosis in the small intestinal epithelium in vivo and that the drug targeted the highly proliferative early transit cells at cell positions 6 ± 8 (Figures 2 and 4) (Ijiri andPotten, 1983, 1987;Pritchard et al, 1997). These cell positions of 5FU-induced apoptosis in the small intestine are di erent from those observed after 1 or 8 Gy of gradiation, where cells at positions 4 ± 6 were preferentially deleted by apoptosis (Potten, 1977;Merritt et al, 1994Merritt et al, , 1995.…”

“…The di erence in locus of cell death may re¯ect our recent ®nding that 5FU appears not to be inducing a p53-dependent death by induction of DNA damage but instead that perturbations in RNA metabolism are occurring . Despite the nature of the primary lesions induced, both 5FU and 1 Gy of g-irradiation depend upon the expression of p53 for the deaths observed over a 24 h time period (Clarke et al, 1994;Merritt et al, 1994Merritt et al, , 1997Pritchard et al, 1997). Surprisingly then, we observed no profound e ect of the loss of the bax gene on either 5FU-or g-irradiation-induced apoptosis (Figures 2 and 3, Table 1).…”

“…The data from the irradiated animals was unequivocal (Figure 3). This is profoundly di erent from the results reported when p53 null animals were used (Clarke et al, 1994(Clarke et al, , 1997Merritt et al, 1994Merritt et al, , 1997Pritchard et al, 1997Pritchard et al, , 1998. As discussed earlier, both these stimuli for death require p53, which is expressed strongly 4.5 h after irradiation, so that a mechanism other than a p53-directed transcription of bax (Miyashita and Reed, 1995) must be responsible for the apoptosis of these epithelial cells.…”

“…We have previously determined the timecourse and dose response of 5FU-and g-irradiation-induced apoptosis in murine intestinal crypts from BDF1 mice (Potten, 1977;Pritchard et al, 1997;Potten and Grant, 1998). With increasing dose of 5FU, we found a plateau in the amount of apoptosis observed over a 24 h period after 40 mg/kg.…”

The relationship between ionizing radiation-induced apoptosis and stem cells in the small and large intestine

“…It is more likely that this apoptosis is indicative of the cell number homeostatic mechanisms operating to remove essentially healthy cells. This is further suggested by the observations that late apoptosis occurs in p53 knockout animals, while the earlier apoptosis is absent, suggesting that the late apoptosis does not involve the DNA damage recognition and damage response processes involved in the early (3-6 h) cell death, which are completely p53 dependent (Merritt et al, 1997;Pritchard et al 1997). The low level of mitotic activity seen 12 h after 1 Gy may be partially explained by circadian rhythms in mitosis (Qiu et al 1994).…”