Poly(ADP-ribose) polymerase-1 is a survival factor for radiation-exposed intestinal epithelial stem cells in vivo

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme mediating the cellular response to DNA strand breaks. It plays a critical role in genomic stability and survival of proliferating cells in culture undergoing DNA damage. Intestinal epithelium is the most proliferative tissue in the mammalian body and its stem cells show extreme sensitivity to low-level genotoxic stress. We investigated the role of PARP-1 in the in vivo damage response of intestinal stem cells in crypts of PARP-1-/- and control mice following whole-body gamma-irradiation (1 Gy). In the PARP-1-/- mice there was a significant delay during the first 6 h in the transient p53 accumulation in stem cells whereas an increased number of cells were positive for p21(CIP1/WAF1). Either no or only marginal differences were noted in MDM2 expression, apoptosis, induction of or recovery from mitotic blockage, or inhibition of DNA synthesis. We further observed a dose-dependent reduction in crypt survival measured at 4 days post-irradiation in control mice, and this crypt-killing effect was significantly potentiated in PARP-1-/- mice. Our results thus establish that PARP-1 acts as a survival factor for intestinal stem cells in vivo and suggest a functional link with early p53 and p21(CIP1/WAF1) responses.

Figure 1

Profiles of p53, p21 and MDM2 expression and of the fractions of apoptotic, mitotic and [³H]TdR-incorporating cells in small intestinal crypts of PARP-1^–/– (continuous curves) and wt (dashed curves) mice following 1 Gy γ-irradiation. Cell positions are counted from the base of the crypt. The stem cells are concentrated at cell position 4–5. Each measure was determined as the average value for 50 half crypts taken from groups of at least four animals. Irradiated groups of mice were sacrificed at 40 min, 1 h, 2 h, 4 h 40 min and 6 h post-irradiation. (a) p53 positive cells; (b) p21 positive cells; (c) MDM2 positive cells; (d) apoptotic cells; (e) mitotic cells; (f) cells positive for [³H]TdR-incorporation. The range of cell positions where a statistically significant difference (P < 0.05) was found between PARP-1^–/– and wt mice is indicated by a horizontal bar.

Figure 1

Profiles of p53, p21 and MDM2 expression and of the fractions of apoptotic, mitotic and [³H]TdR-incorporating cells in small intestinal crypts of PARP-1^–/– (continuous curves) and wt (dashed curves) mice following 1 Gy γ-irradiation. Cell positions are counted from the base of the crypt. The stem cells are concentrated at cell position 4–5. Each measure was determined as the average value for 50 half crypts taken from groups of at least four animals. Irradiated groups of mice were sacrificed at 40 min, 1 h, 2 h, 4 h 40 min and 6 h post-irradiation. (a) p53 positive cells; (b) p21 positive cells; (c) MDM2 positive cells; (d) apoptotic cells; (e) mitotic cells; (f) cells positive for [³H]TdR-incorporation. The range of cell positions where a statistically significant difference (P < 0.05) was found between PARP-1^–/– and wt mice is indicated by a horizontal bar.

Figure 2

Crypt survival curves for PARP-1^–/– and wt mice measured 4 days post-irradiation. A marked reduction in crypt survival was observed in the PARP-1^–/– mice (filled symbols) as the dose was raised. Means and the standard errors of the means are indicated. Statistically significant differences are indicated by asterisks (P < 0.05).