Uncoupling p53 functions in radiation-induced intestinal damage via PUMA and p21

Abstract

The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.

Figure 1. p53 or p21 deficiency led to accelerated death and crypt regeneration following WBI

A) Validation of mice genotypes by PCR (left) and Western blotting using intestinal mucosal extracts 24 h after IR (right). B) The survival of WT, PUMA KO, p21 KO, PUMA/p21 DKO, and p53 KO mice following 15 Gy WBI. PUMA KO vs. WT, p < 0.0001; DKO vs. WT, p = 0.0174; p21 KO vs. WT, p = 0.0405; and p53 KO vs. WT, p < 0.0001. C) Mice with indicated genotypes were treated with 15 Gy WBI and received 100 mg/kg BrdU by IP injection 2 h prior to sacrifice. Representative images of regenerated crypts identified by BrdU staining (brown) after 72 hr (magnification 100×). D) Quantitation of regenerated crypts 72 h after IR in mice with the indicated genotypes from 6–8 complete circumferences. Values are means ± SEM; n = 3 or more in each group. * indicates p < 0.05, and ** indicates p < 0.01 compared to WT.

Figure 2. p53 or p21 deficiency led to non-apoptotic cell death in the crypts following IR

A) Mice with the indicated genotypes were treated with 15 Gy WBI and sacrificed at 72 h. Cell death in the crypts was assessed by TUNEL and active caspase-3 staining. Representative images are shown (magnification 400×). B) TUNEL- or active caspase-3-positive cells were counted in 10 400× fields. When crypt structure was largely absent, the area below the villi was considered to be the crypt region. Values are means ± SEM; n = 3 in each group. ** indicates p < 0.01 compared to WT. C) Representative images and quantification of double IF staining for TUNEL (green) and pan-cytokeratin (red) 72 h after 15 Gy (magnification 400×). Values are means ± SEM; n = 3 in each group. ** indicates p < 0.01 compared to WT. D) Quantitation (positive cells/400× field) of active caspase-3 (Cas3*, left) and TUNEL (right) in the crypts of WT and p21 KO mice at 0, 4, 24, 48, 72, and 96 h after 15 Gy. Values are means; n = 3 in each group.

Figure 3. p21 deficiency did not affect PUMA-dependent crypt apoptosis induced by IR

Mice with the indicated genotypes were treated with 15 Gy WBI and sacrificed after 4 and 24 h. Apoptosis was analyzed by TUNEL and active caspase-3 staining. A) Representative images of TUNEL IF staining in the crypts (magnification 600×). B) Quantitation of TUNEL-positive cells by counting at least 100 crypts. Values are means ± SEM; n = 3 in each group. * indicates p < 0.05, and ** indicates p < 0.01 compared to WT. C) Representative images of active caspase-3 staining in the crypts (magnification 600×). D) Quantitation of caspase-3-positive cells by counting at least 100 crypts. Values are means ± SEM; n = 3 in each group. ** indicates p < 0.01 compared to WT.

Figure 4. p21 deficiency compromised IR-induced G1/S checkpoint and DNA repair in the intestinal crypts

Mice with the indicated genotypes were treated with 15 Gy WBI and sacrificed at indicated times. A) Proliferation in the intestinal crypts 0, 4, or 24 h after irradiation was assessed by BrdU staining. Upper, representative images are shown (magnification 400×). Lower, Quantitation of BrdU-positive cells from at least 100 crypts. Values are means ± SEM; n = 3 in each group. * indicates p < 0.05, and ** indicates p < 0.01 compared to WT at the same time point. # indicates p < 0.01 compared to WT 0 h. B) Representative images of γ-H2AX foci in the crypts of WT and PUMA KO mice 4 h after IR (magnification 400×). C) Quantitation of γ-H2AX foci in the crypts of WT and PUMA KO mice at 0, 0.5, 1, 2, 4, and 24 h post-IR, time points not drawn in scale. Values are means; n = 3 in each group. D) Quantitation of nuclear p21 expressing cells in 100 intestinal crypts from WT and PUMA KO mice at 0, 4, 24, and 48 h after IR. Values are means ± SEM; n = 3 in each group. *** indicates p < 0.001 compared to WT.

Figure 5. p53 or p21 deficiency led to compromised G2/M checkpoint, replication stress and persistent DNA damage in the intestinal crypts following IR

Mice with the indicated genotypes were treated with 15 Gy WBI and sacrificed at 48 and 72 h. Cell proliferation and mitosis were analyzed by Ki67 and phospho-H3 (pH3) staining, respectively. The presence of DNA double strand breaks and single stranded DNA at the replication forks were analyzed by phospho-γ-H2AX (γ-H2AX) and phospho-RPA32 (p-RPA32), respectively. A) Quantitation of γ-H2AX and Ki67 double positive cells at 48 and 72 h after IR from 10 400× fields in the area below the villus. B) Quantitation of double γ-H2AX and pH3 positive cells at 48 and 72 h after IR from 10 400× fields in the area below the villus. C) Representative pictures of double IF staining for p-RPA32 and Ki67 (magnification 400×). Dashed circles indicate cells stained positive for both markers. D) Quantification of p-RPA32 (upper), or p-RPA32 /Ki67 double positive cells (lower) in (C). Values are means ± SEM; n = 3 in each group. ** indicates p < 0.01 compared to WT.

Figure 6. p53 or p21 deficiency increased aberrant mitoses and genome instability in the intestinal crypts after IR

Mice with the indicated genotypes were treated with 15 Gy WBI and sacrificed at the indicated times. Mitoses were analyzed on H&E stained sections. A) Examples of normal and aberrant mitoses before or 24 h after IR (magnification 600×) are indicated by an arrow and asterisks, respectively. B) Quantitation of normal and aberrant mitoses by counting at least 100 crypts before or 24 h after IR. Values are means ± SEM; n = 3 in each group. * indicates p < 0.05, ** indicates p < 0.01 compared to WT. C) Quantitation of aberrant mitoses in 20 regenerated crypts 72 h after IR. Values are means ± SEM; n = 3 in each group. ** indicates p < 0.01 compared to WT. D) A model of p53-mediated responses in IR-induced intestinal damage and protection. PUMA deficiency coupled with p21 induction, but not p53 deficiency, facilitates intestinal stem cell survival and regeneration. The p21-dependent mechanisms suppress genome instability and non-apoptotic cell death following radiation.