Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.


Mice
The procedures for all animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. The methods were carried out in "accordance" with the approved guidelines. Mice 6-12 weeks old were used. The PUMA +/+ and PUMA -/littermates on C57BL/6 background (F10) were generated from heterozygote intercrosses. The previously described Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice 1 were crossed with PUMA -/mice to generate Lgr5-EGFP/PUMA +/mice. The Lgr5-EGFP/PUMA +/+ and Lgr5-EGFP/PUMA -/littermates were generated by Lgr5-EGFP/PUMA +/intercrosses. Genotyping was performed as previously described for PUMA 2 and for Lgr5 1 . The mice were housed in micro-isolator cages in a room illuminated from 7:00 AM to 7:00 PM (12:12-hour light/dark cycle) and were allowed access to water and chow ad libitum.
For TBI models, mice were irradiated at dose 15 Gy at a rate of 76 cGy/min in a 137 Cs irradiator (Mark I; JL Shepherd and Associates, San Fernando, CA, USA). Mice were injected intraperitoneally (i.p.) with 2 mg/kg of CHIR99021 (Cat# C-6556, LC Laboratories, Woburn, MA) 4h before radiation or 1 mg/kg of SB415286 (Cat# 1617, Tocris bioscience, Ellisville, MO) 28h and 4h before radiation. Mice were sacrificed to collect small intestines for histology analysis and western blotting at 4, 24 and 96 h after radiation. All mice were injected i.p. with 100 mg/kg of BrdU (Cat# 858811, Sigma-Aldrich) before sacrifice. Three or more mice were used in each group.
Abdominal irradiation (ABI) was used for survival studies and administered in the form of X-ray with a clinical grade linear accelerator (Varian Medical Systems, Palo Alto, CA). For ABI experiments, a 3-cm wide radiation band was used to deliver the required doses at a rate of 600 Monitor Units (146 cGy)/min to anesthetized animals in groups of 10-15 per run 3 .

Small intestinal crypt and cell isolation, culture and treatment
The mouse crypts was isolated and cultured as previously described 4,5 . Similar results were obtained from at least three independent experiments using two or more donor mice, and triplicate wells were included in each experiment. A total of 500 crypts were mixed with 50 µl and B27 supplement (Cat# 12587-010, Invitrogen, Grand Island, NY)] was added. 2.5 µM CHIR99021, 2.5 µM, SB415286, 400 ng/ml bFGF or 100 ng/ml IGF-1 6 was added as needed.
For passage, enteroids were removed from Matrigel and mechanically dissociated into single crypt domains and then transferred to fresh Matrigel. Passage was performed every 5 days with a 1:3 split ratio. Ad-PUMA-GFP 7 was added to matrigel mixtures (0.02 µl/ 50µl/), and medium (0.2 µl/500 ul) in 24-well culture plates (0.04 µl/mL) at the time of plating with or without CHIR9921. Crypts were irradiated at 5 Gy 24h after plating. Enteroid growth was quantified 6 days after radiation in primary culture and 4 days after radiation in passaged culture. The cultured crypts were harvested 4 h or 24 h after radiation, fixed with 10% formalin and mixed with 2% agarose and then processed. Sections (5 µm) from paraffin-embedded crypt enteroids were subjected to immunostaining. Protein and RNA were isolated form cultured crypts Intestines were cut longitudinally in HBSS, contents rinsed, cut into 1-inch pieces, transferred to EBSS/1 mM EGTA/1% HEPES (Life Technologies, NY/Sigma-Aldrich, MO/Mediatech, VA) and minced. Tissue was then transferred to a tube and incubated for 5 min at room temperature.
After an EBSS wash, the tissue was treated three times with a cocktail containing 1mg/mL Growth Factor Reduced (GFR), phenol-red free Matrigel TM (BD Bioscience, CA) and plated in tissue culture dishes at low density (~50,000 cells/well of 48-well plate). After solidification at 37C for 30 min, 500L of media was added to each well (See JMC Table). For the first 24 hours, the media included Y-27632 to prevent anoikis. Subsequently, cultured cells were treated with 2.5 µM CHIR99021 3 days after plating in Matrigel and irradiated at 5 Gy 24h after CHIR99021 treatment. Enteroid growth was quantified 10 days after radiation. The cells cultured in Matrigel were harvested 24 h after radiation, fixed with 10% formalin and mixed with 2% agarose and then processed. Sections (5 µm) from paraffin-embedded enteroids were subjected to immunostaining. Protein and RNA were isolated form cultured cells following digestion of the Matrigel with Cell Recovery Solution 24 h after radiation.
Human adult intestinal crypts were isolated as previously described 9 . Fresh tissues were obtained with appropriate IRB approval from the UCLA Department of Pathology Translational Pathology Core Laboratory. Isolated crypts were mixed with 50 ul Matrigel and plated in 24well plates. Culture medium included 50% conditioned media from cultured myofibroblast and 50% Advanced DMEM/F12 supplemented with 2 mM GlutaMax, 10 mM Hepes, 1 mM N-Acetylcysteine, 50 ng/ml EGF, 100 ng/ml Noggin, 1 µg/ml R-spondin 1,100 ng/ml FGF10, 10 uM Y27632, 1% N2 supplement and B27 supplement 9 . Passage was performed every 4 days with a 1:3 split ratio. 2.5 µM CHIR99021 was added as needed. One day after plating, crypts were irradiated at 5 Gy. Enteroid growth was quantified 6 days after radiation. RNA was isolated from cultured crypts following digestion of the Matrigel with Cell Recovery Solution 24h after radiation.

Western blotting
Total protein was prepared from freshly isolated small intestine 5 and cultured crypts, separately, western blotting was performed as previously described 5  Detailed sequences for real-time PCR are found in supplementary tables (Table S1 and S2).

Immunohistochemistry (IHC) and immunofluorescence (IF)
Slides were deparaffinized, rehydrated, and treated 3% hydrogen peroxide. Antigen retrieval was performed by boiling the sections for 10 minutes in 0.1 M Citrate Buffer Antigen Retrieval Solution (pH 6.0). Nonspecific antibody binding was blocked using 15% goat serum for 30 minutes. For active caspase 3 IHC, the slides were then incubated with rabbit polyclonal anti- The crypt microcolony assay was used to quantify stem cell survival by counting regenerated crypts in BrdU-stained cross sections 4 days after radiation as described previously 11 (15). The regenerated crypts contained 5 or more BrdU positive cells with a lumen. At least three mice were used in each group and the data from BrdU staining was reported as means ± SD. Table   Table S1: Mouse primers used for RT-PCR analysis