Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of proregeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human participants following laser rejuvenation treatment and compared them with mice with enhanced wound-induced hair neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel-/-mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36 alpha. Consistent with RNase L's known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36-dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L-activated caspase-1 restrains the proregenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA-seq and spatial transcriptomic profiling, we confirmed OAS & IL-36 genes to be highly expressed at the site of wounding and elevated in Rnasel-/- mouse wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional trade off that tempers immune hyperactivation during viral infection at the cost of inhibiting regeneration.

Mammalian tissue injury response is usually characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcriptome screening in human subjects after laser rejuvenation treatment and cross-referenced these transcripts to those found in mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We find the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit a remarkable regenerative capacity with elevated WIHN (n=10, p<0.0001) through enhanced IL-36α (n=3, p<0.01). Consistent with the known role of RNase L to stimulate caspase-1, we find that pharmacologic inhibition of caspases promotes regeneration (n=3-4, p<0.001) in an IL-36-dependent manner. This occurs by a negative feedback loop, where RNase L activated caspase-1 inhibits the pro-regenerative dsRNA-TLR3 pathway through the cleavage of TRIF (n=7, p<0.05). Additionally, these responses are not limited to skin but extend to other organs, such as the colon (n=4, p<0.05), suggesting that suppression of regeneration is a fundamental characteristic of epithelial wound healing. Taken together, this work suggests that RNase L functions as a regeneration repressor gene in a functional tradeoff that limits immune hyper-activation during viral infection at the cost of also inhibiting regeneration.

Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called wound-induced hair follicle neogenesis (WIHN), we identified skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multiomic analysis and single-cell RNA sequencing in murine skin, we show that bacterially induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved and coherent physiologic context in which bacterially induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This unexpected proregenerative modulation of metabolism by the skin microbiome in both mice and humans suggests important methods for enhancing regeneration after injury.

Supplementary data contain Supplementary Table 1-3 and Supplementary Figures 1-11.

Abstract Chronic and low-grade inflammation associated with persistent bacterial infections has been linked to colon tumor development; however, the impact of transient and self-limited infections in bacterially driven colon tumorigenesis has remained enigmatic. Here we report that UshA is a novel genotoxin in attaching/effacing (A/E) pathogens, which include the human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and their murine equivalent Citrobacter rodentium (CR). UshA harbors direct DNA digestion activity with a catalytic histidine–aspartic acid dyad. Injected via the type III secretion system (T3SS) into host cells, UshA triggers DNA damage and initiates tumorigenic transformation during infections in vitro and in vivo. Moreover, UshA plays an indispensable role in CR infection–accelerated colon tumorigenesis in genetically susceptible ApcMinΔ716/+ mice. Collectively, our results reveal that UshA, functioning as a bacterial T3SS-dependent genotoxin, plays a critical role in prompting transient and noninvasive bacterial infection–accelerated colon tumorigenesis in mice. Significance: We identified UshA, a novel T3SS-dependent genotoxin in A/E pathogens that possesses direct DNA digestion activity and confers bacterially accelerated colon tumorigenesis in mice. Our results demonstrate that acute and noninvasive infection with A/E pathogens harbors a far-reaching impact on the development of colon cancer. This article is highlighted in the In This Issue feature, p. 1

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL‑36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL‑36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL‑36 cytokines in human atopic dermatitis skin and in IL‑36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL‑36 responses represent a key mechanism and potential therapeutic target against allergic diseases.

Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1β to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.

Environmental factors that enhance regeneration are largely unknown. We hypothesized that skin bacteria modulate regeneration. Here, we assessed low, medium, and high levels of bacterial burden in wound healing and Wound Induced Hair follicle Neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers indeed correlated with bacterial counts, being lowest in germ free (GF) (fold= -17.9, n=13, p=1.9X10-6), intermediate in conventional specific pathogen free (SPF), and highest even in mice infected with pathogenic Staphylococcus aureus (fold= 3.3, n=12, p=7.5X10-5). We identified IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling as necessary and sufficient for bacteria to promote regeneration. Finally, in a small clinical trial, we found that a topical broad-spectrum antibiotic slowed skin wound healing. These results demonstrate a novel role for IL-1β to control morphogenesis and counter conventional notions that infection inhibits regeneration with a need for full sterility of small wounds.

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment and cross-referenced these transcripts to those found in mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We find the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity, with elevated WIHN (n=10, p<0.0001) through enhanced IL-36α (n=3, p<0.01). Consistent with the known role of RNase L to stimulate caspase-1, we find that pharmacologic inhibition of caspases promotes regeneration (n=3 versus 4, p<0.001) in a novel IL-36-dependent manner (n=4, n.s.= not significant). Additionally, these responses are not limited to skin but extend to other organs, such as the colon (n=4, p<0.05), suggesting that suppression of regeneration is a fundamental characteristic of epithelial wound healing. Taken together, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.

Wound Induced Hair follicle Neogenesis (WIHN) is a rare adult organogenesis model where stem cells form de novo hair follicles following full-thickness wounding. As wounds inevitably contact the skin microbiota, it is important to understand the role of the skin microbiome in WIHN. To do so, we modified bacterial burdens and tested WIHN. We used 3 levels of microbial burden to measure WIHN: For minimal bacteria loads, we used germ-free (GF) mice, applied antibiotic ointment (Neosporin) or frequent cage changes of standard specific pathogen free (SPF) mice housing. For intermediate bacterial loads and as a baseline comparator, we used standard SPF mice housing. For maximal bacteria loads, we injected each of the top three strains of SPF mice skin commensal bacteria to the wound bed early during wounding. We found that GF mice (fold= -14.4, n=5, p=3.5X10-5), mice treated with antibiotic (fold= -7.9, n=5, p=8.4X10-5) or mice in clean cages (fold= -2.8, n=4, p=0.015) have lower regeneration capacity respectively and lower stem cell marker expression. Increase commensal bacteria loads can enhance SPF mice regeneration capacity (fold=3.3, n=6, p=7.5X10-5) and also promote stem cell marker expression. Mice deficient in Myd88 (fold=-29.6, n=7, p=8.3X10-7) and IL1R deficient mice (fold=-9.4, n=5, p=0.4X10-4) have poor regeneration capacity, and are resistant to the ability of exogenous bacterial to enhance WIHN. Keratinocytes and Myeloid cell-specific Myd88 deficient mice have similar regeneration capacity to wild type mice. Taken together, these results demonstrate that commensal and exogenous bacterial burden enhance regeneration. This is mediated through IL1R- Myd88 signaling, but not in keratinocytes or myeloid cells. Future studies will define the cell type and cytokine responsible for activation of Myd88 and WIHN during commensal exposure.

Wound-induced hair follicle neogenesis (WIHN) is a phenomenon that occurs in adult mammalian skin, where fully functional hair follicles are regenerated in the center of large full-thickness excisional wounds. Although originally discovered over 50 years ago in mice and rabbits, within the last decade it has received renewed interest, as the molecular mechanism has begun to be defined. This de novo regeneration of hair follicles largely recapitulates embryonic hair development, requiring canonical Wnt signaling in the epidermis, however, important differences between the two are beginning to come to light. TLR3 mediated double stranded RNA sensing is critical for the regeneration, activating retinoic acid signaling following wounding. Inflammatory cells, including Fgf9-producing gamma-delta T cells and macrophages, are also emerging as important mediators of WIHN. Additionally, while dispensable in embryonic hair follicle development, Shh signaling plays a major role in WIHN and may be able to redirect cells fated to scarring wounds into a regenerative phenotype. The cellular basis of WIHN is also becoming clearer, with increasing evidence suggesting an incredible level of cellular plasticity. Multiple stem cell populations, along with lineage switching of differentiated cells all contribute towards the regeneration present in WIHN. Further study of WIHN will uncover key steps in mammalian development and regeneration, potentially leading to new clinical treatments for hair-related disorders or fibrotic scarring.

When compared to animals across other phyla, mammals have restricted regeneration and more fibrosis. This limited regenerative capacity may reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover the programs governing regeneration in mammals, we investigated Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in adult mammals. Through comprehensive screening of transcripts associated with WIHN—as well as after rejuvenation lasers in human subjects--, we found that the endoribonuclease RNase L associates with regeneration/rejuvenation, but actually functions as a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity, with increased WIHN (n=10, p<0.0001) and accelerated wound healing following injury (n=3, p<0.05). This is mediated through the production of IL-36α, which is increased in Rnasel-/- mice, enhances WIHN when added exogenously (n=3, p<0.01), and is required for WIHN given its absence in IL36R-/- mice (n=3, p<0.01). Consistent with the known role of RNase L to stimulate caspase-1 signaling, we find that in wild type mice, pharmacologic inhibition of caspases promotes regeneration in an IL-36-dependent manner. These responses are not limited to skin, but occur following intestinal injury as well, suggesting that suppression of regeneration is a general attribute of mammalian wound healing. Taken together, this work suggests a therapeutic strategy to uncover latent regenerative capacity and promote functional response to injury.

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment. We searched for overlapping transcripts also associated with Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in mice. We found the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity and accelerated wound healing following injury, through the production of IL-36α. Consistent with the known role of RNase L to stimulate caspase-1 signaling, we find that pharmacologic inhibition of caspases promotes regeneration in a novel IL-36-dependent manner. We also find a gene set of inner root sheath keratins, such as KRT71, that is associated with poor wound healing in mouse and man, and decreases with caspase inhibition. Finally, these responses are not limited to skin, but occur following intestinal injury as well, suggesting that suppression of regeneration is a general characteristic of epithelial wound healing. In all, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

Wound care is a major public health burden, frequently in patients with diabetes, pressure ulcers, prolonged immobilization, and venous insufficiency. To find better wound treatments, we and others have shown the importance of the innate immune response in coordinating the earliest events after wounding. To determine the role of neutrophils in wound healing, we performed full-thickness punch biopsies on mouse back skin and measured neutrophil recruitment by FACS analysis. We find that shortly after wounding, neutrophils are rapidly recruited to the wound site, where they can prevent infection and facilitate repair. To study neutrophil function, we sought to use antibody-mediated cell depletion. In the literature, the intraperitoneal injection of an antibody recognizing the GPI-anchored protein Lymphocyte antigen 6 complex, locus G (Ly6G) is commonly used for neutrophil depletion. Here, we show that the published efficacy for this depletion in the literature is erroneously successful when antibodies recognizing overlapping Ly6G epitopes are selected for flow cytometry analysis proof of depletion, a common error since the "distinct" clones 1A8 and REA526 actually are identical. Additionally, large full-thickness excisional wounds on mice dramatically alter neutrophil dynamics. After wounding, neutrophils substantially increase in skin, but not in the blood, spleen, or liver. Interestingly, wounding decreases the true ability of anti-Ly6G-mediated neutrophil depletion in skin, liver, spleen, and blood—despite the fact that blood neutrophil levels are not significantly changed with wounding. These data suggest that localized skin wounding likely induces a systemic dynamic flux of neutrophils from the blood to sites of skin damage and previous antibody-mediated neutrophil depletion studies should be interpreted with appropriate limitations.

How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.

Nuclear factor kappa B (NF-κB)-mediated transcription is an important mediator for cellular responses to DNA damage. Genotoxic agents trigger a 'nuclear-to-cytoplasmic' NF-κB activation signaling pathway; however, the early nuclear signaling cascade linking DNA damage and NF-κB activation is poorly understood. Here we report that Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) is a key NF-κB regulator in genotoxic stress-initiated signaling pathway. Sam68 deficiency abolishes DNA damage-stimulated polymers of ADP-ribose (PAR) production and the PAR-dependent NF-κB transactivation of anti-apoptotic genes. Sam68 deleted cells are hypersensitive to genotoxicity caused by DNA damaging agents. Upregulated Sam68 coincides with elevated PAR production and NF-κB-mediated anti-apoptotic transcription in human and mouse colon cancer. Knockdown of Sam68 sensitizes human colon cancer cells to genotoxic stress-induced apoptosis and genetic deletion of Sam68 dampens colon tumor burden in mice. Together our data reveal a novel function of Sam68 in the genotoxic stress-initiated nuclear signaling, which is crucial for colon tumorigenesis.

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.

Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Previously we reported that Src-associated-substrate-during-mitosis-of-68kDa (Sam68/KHDRBS1) is pivotal for DNA damage-stimulated NF-κB transactivation of anti-apoptotic genes (Fu et al., 2016). Here we show that Sam68 is critical for genotoxic stress-induced NF-κB activation in the γ-irradiated colon and animal and that Sam68-dependent NF-κB activation provides radioprotection to colon epithelium in vivo. Sam68 deletion diminishes γ-irradiation-triggered PAR synthesis and NF-κB activation in colon epithelial cells (CECs), thus hampering the expression of anti-apoptotic molecules in situ and facilitating CECs to undergo apoptosis in mice post whole-body γ-irradiation (WBIR). Sam68 knockout mice suffer more severe damage in the colon and succumb more rapidly from acute radiotoxicity than the control mice following WBIR. Our results underscore the critical role of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon tissue and whole animal and reveal the pathophysiological relevance of Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic DNA damage. https://doi.org/10.7554/eLife.21957.001 Introduction Nuclear factor kappa B (NF-κB) plays a crucial function in a variety of human disorders, in particular inflammatory diseases and cancers (Hayden and Ghosh, 2008; Scheidereit, 2006; Sun et al., 2013; Vallabhapurapu and Karin, 2009; Wan and Lenardo, 2010; Wu and Miyamoto, 2007). Accumulating evidence highlights an important role of NF-κB signaling pathway in cellular responses to various genotoxic stresses and DNA damage-stimulated NF-κB signaling cascade in the nucleus that leads to NF-κB activation has been recently revealed (McCool and Miyamoto, 2012; Miyamoto, 2011). In particular, ataxia telangiectasia mutated (ATM), inhibitor of NF-κB kinase gamma subunit (IKKγ), protein inhibitor of activated STATy (PIASy), and poly (ADP-ribose) polymerase 1 (PARP1) were reported to be indispensible for genoxic stress-induced NF-κB activation (Huang et al., 2003; Li et al., 2001; Mabb et al., 2006; Piret et al., 1999; Stilmann et al., 2009). Moreover, we recently revealed that Sam68/KHDRBS1 (Src-associated substrate during mitosis of 68 kDa/KH domain containing, RNA binding, signal transduction associated 1, encoded by KHDRBS1 gene), a versatile single-strand nucleic acid binding protein (Lukong and Richard, 2003; Richard, 2010), is an important molecule in orchestrating genotoxic stress-initiated NF-κB signaling in the nucleus (Fu et al., 2016). Specifically, Sam68 is essential for DNA damage-triggered PARP1 activation and the subsequent polymers of ADP-ribose (PAR) synthesis (Fu et al., 2016). Sam68 deletion dampens the PAR-dependent NF-κB signaling and transcription of an array of anti-apoptotic genes, thus sensitizing Sam68-deficient mouse embryonic fibroblasts (MEFs) and colon epithelial cells (CECs) in culture to genotoxicity caused by DNA-damaging agents (Fu et al., 2016). The levels of Sam68, PAR, NF-κB activation, and anti-apoptotic molecules B-cell lymphoma-extra large (Bcl-XL) and X-linked inhibitor of apoptosis protein (XIAP) are elevated and positively correlated in colon tumors compared to adjacent normal tissue derived from either the tumor-laden Apcmin716/+ mice or human colon cancer patients. Moreover, downregulation of Sam68 substantially sensitizes human colon cancer cells to spontaneous and genotoxic stress-induced cell death and retards colon tumor burden in Apcmin716/+ mice (Fu et al., 2016). These findings suggest that upregulated Sam68 is crucial in orchestrating DNA damage-initiated NF-κB activation signaling in cultured cells and conferring the PAR-dependent NF-κB activation to respond to the intrinsic DNA damage frequently occurred in cancerous cells. However, the in vivo impact of physiological Sam68 levels on extrinsic genotoxic stress-induced NF-κB signaling and activation in normal cells at the organ and even the whole animal levels has not been fully understood. Radiotherapy and chemotherapy are extensively used in current-day cancer treatments. It has been recognized that γ-irradiation induced DNA damage triggers rapidly-proliferating tumor cells to undergo apoptosis; whereas non- and slowly-dividing cells rarely die of γ-irradiation. Of note, CECs in colon crypts are among the most rapidly dividing cells in the body, which makes them susceptible to γ-irradiation-induced cell death. Indeed, colon tissue injury remains one of the major adverse effects of radiotherapy, when γ-irradiation is employed to treat colon cancer and other intra-abdominal cancers (Egan et al., 2004). Although greater antitumor effects could be produced by higher doses of γ-irradiation, the tolerance of patients to the acute side effects caused by γ-irradiation to the colon limits the administered dose (Egan et al., 2004). Given the essential role of DNA damage-induced NF-κB transactivation of anti-apoptotic genes in cell fate determination post genotoxic stresses, it will be extremely important to understand genotoxic stress-induced NF-κB activation signaling pathway not only in cancerous cells but also in normal cells under pathophysiological conditions. Besides the recently revealed critical function of Sam68-dependent NF-κB activation to overcome intrinsic DNA damage for the development and survival of colon cancer (Fu et al., 2016), whether Sam68-dependent NF-κB signaling is crucial in normal colon epithelium in response to extrinsic γ-irradiation remains elusive. We therefore examined the hypothesis that Sam68-dependent NF-κB activation offers an anti-apoptotic response in the γ-irradiated colon epithelium in vivo hence providing radioprotection to the organ and the animal following whole-body γ-irradiation (WBIR). Results Sam68 confers genotoxic stress-induced NF-κB signaling in the γ-irradiated colon To assess the in vivo impact of Sam68 on genotoxic stress-induced NF-κB signaling and transactivation, Khdrbs1+/− (Sam68 heterozygote) and Khdrbs1−/− (Sam68 knockout) mice were subjected to a sublethal dose of WBIR and we examined the γ-irradiation-initiated NF-κB activation signaling cascade in the derived colons at defined times post WBIR (Figure 1A). As expected, vigorous PAR production, as illustrated by immunofluorescence staining on colon tissue sections, occurred in Khdrbs1+/− colon 20 min post WBIR; whereas such an acute response was almost abolished in the colon from Khdrbs1−/− mice (Figure 1B). In support, immunoblot analyses showed that robust PAR chair formation in whole cell lysates of CECs from Khdrbs1+/− mice at 20 min post WBIR, which was markedly tempered in Khdrbs1−/− mice post WBIR (Figure 1C). These results suggest that Sam68 is crucial for facilitating genotoxic stress-induced PAR production in the γ-irradiated colon tissue in vivo. Consistently, WBIR-induced p65 phosphorylation, one of the biochemical hallmarks of NF-κB activation, was profound in the CECs from γ-irradiated Khdrbs1+/− mice, but was substantially tempered in Khdrbs1−/− animals (Figure 1D). Moreover, WBIR triggered nuclear translocation of p65, as assayed by immunohistostaining and subcellular fractionation, in Khdrbs1+/− CECs; whereas p65 nuclear accumulation was greatly attenuated in the γ-irradiated Khdrbs1−/− cells (Figure 1E–F). Of note, the levels of PAR, total p65, and nuclear accumulated p65 were comparable in CECs derived from mock-irradiated Khdrbs1+/− and Khdrbs1−/− animals (Figure 1B–F), suggesting that Sam68 deletion does not affect the physiological PAR synthesis and NF-κB signaling in the colon without any stimulation. In contrast, Sam68 deletion almost abolished genotoxic stress-triggered PAR formation and the signaling events that lead to NF-κB activation in the colon following WBIR (Figure 1B–F). Consistent with our recent report that Sam68 plays a key role in DNA damage-initiated PAR synthesis and the PAR-dependent NF-κB signaling in the isolated and in vitro cultured CECs (Fu et al., 2016), these results further support the crucial function of Sam68 in genotoxic stress-triggered PAR production and signaling to NF-κB activation in the γ-irradiated colon from whole animals in vivo. Figure 1 Download asset Open asset Sam68 deletion diminishes genotoxic stress-induced NF-κB signaling in the γ-irradiated colon. (A) A schematic of the experimental timeline for the impact of Sam68 deletion on DNA damage-induced NF-κB signaling pathway in γ-irradiated mice. Khdrbs1+/− and Khdrbs1−/− mice subjected to a sublethal dose (6.5 Gy) of whole body γ-irradiation (WBIR) or mock irradiation were euthanized at the indicated periods post WBIR, followed by the analyses as indicated. (B) Immunofluorescence micrographs of PAR in colon tissue collected from Khdrbs1+/− and Khdrbs1−/− mice at 20 min following WBIR or mock irradiation, with nuclei counterstained by DAPI. Scale bar, 25 μm. (C and D) Colon epithelial cells (CECs) were isolated from Khdrbs1+/− and Khdrbs1−/− mice at the indicated periods post WBIR, and whole cell lysates were derived and immunoblotted (IB) for indicated proteins, with β-actin as a loading control. p-p65, phosphorylated p65. (E) Immunofluorescence micrographs of p65 in colon tissue collected from Khdrbs1+/− and Khdrbs1−/− mice at 60 min post WBIR. Scale bar, 50 μm. (F) CECs were collected from Khdrbs1+/− and Khdrbs1−/− mice as treated in (E) and nuclear fractions were derived and IB for indicated proteins. Caspase-3 (Casp3) and PARP1 served as loading controls and cytosolic and nuclear markers, respectively. https://doi.org/10.7554/eLife.21957.002 Sam68 is critical for anti-apoptotic gene transcription in the γ-irradiated colon It has been well established that NF-κB mediates the gene transcription of a panel of anti-apoptotic molecules in cells following genotoxic stress (Fu et al., 2016; Kim et al., 2005; Stilmann et al., 2009). We therefore assessed the impact of Sam68 on γ-irradiation-induced expression of NF-κB target gene Bcl2l1, which encodes B-cell lymphoma-extra large (Bcl-XL). As illustrated by digoxigenin-labeled messenger RNA (mRNA) in situ hybridization, Bcl2l1 mRNA levels were elevated in colon tissue sections derived from whole-body γ-irradiated Khdrbs1+/− mice (Figure 2A). In contrast, WBIR-induced transcription of Bcl2l1 was substantially tempered in the γ-irradiated Khdrbs1−/− colon tissue (Figure 2A). In line with the nearly abolished γ-irradiation-initiated NF-κB signaling cascade in the colon of Khdrbs1−/− mice post WBIR (Figure 1B–F), these results demonstrate that Sam68 deletion suppresses the inducible transcription of NF-κB target genes in the colon in situ following WBIR. Mirroring the robust transcription of Bcl2l1 triggered by γ-irradiation (Figure 2A), Bcl-XL protein levels were also induced in the colon tissue derived from Khdrbs1+/− mice at 4 hr post WBIR (Figure 2B). In striking contrast, WBIR-induced Bcl-XL upregulation was diminished in the colon derived from the whole-body γ-irradiated Khdrbs1−/− animals (Figure 2B). Moreover, these results were further supported by immunoblot of Bcl-XL and another anti-apoptotic protein B-cell lymphoma 2 (Bcl2), encoded by the NF-κB target gene Bcl2, in the CEC lysates isolated from the whole-body γ-irradiated mice (Figure 2C). Hence Sam68 is essential for genotoxic stress-induced and NF-κB-mediated expression of anti-apoptotic genes in the γ-irradiated colon epithelium. Figure 2 Download asset Open asset Sam68 is pivotal for NF-κB-mediated anti-apoptotic gene expression in the γ-irradiated colon. (A) Colon tissue sections derived from Khdrbs1+/− and Khdrbs1−/− mice at 4 hr post whole body γ-irradiation (WBIR) or mock irradiation were stained by in situ hybridization with in vitro synthesized anti-sense probe targeting Bcl2l1 mRNA (purple dots as indicated by triangles), with Bcl2l1 mRNA sense probe as a negative control. Scale bar, 100 μm. (B) Immunofluorescence micrographs of Bcl-XL (encoded by Bcl2l1) in colon tissue collected from mice treated as in (A), with nuclei counterstained by DAPI. Scale bar, 200 μm. (C) Colon epithelial cells were isolated from mice treated as in (A) and whole cell lysates were derived and immunoblotted (IB) for indicated proteins, with β-actin as a loading control. https://doi.org/10.7554/eLife.21957.003 Sam68-deleted colon epithelial cells are more sensitive to whole-body γ-irradiation The balance between severe DNA damage-triggered programmed cell death and genotoxic stress-induced NF-κB-mediated anti-apoptotic transcription is pivotal for cell fate determination in cellular responses to DNA-damaging agents (Fu et al., 2016; Kim et al., 2005; Stilmann et al., 2009). We barely detected the cleavage of Caspase-3, one well-established biochemical hallmark for apoptosis, in the colon tissue derived from Khdrbs1+/− mice at 8 hr post WBIR (Figure 3A), as supported by the evidence that WBIR triggered profound NF-κB activation signaling (Figure 1) and expression of anti-apoptotic molecules Bcl-XL and Bcl2 (Figure 2) in Sam68-sufficient CECs. In contrast, Caspase-3 cleavage was substantially augmented in the γ-irradiated Khdrbs1−/− colon (Figure 3A), which correlates with the diminished NF-κB signaling in the nucleus (Figure 1) and inefficient anti-apoptotic gene expression (Figure 2) in the absence of Sam68. Moreover, immunoblot analyses of the CEC lysates further ascertained that the elevation in cleaved Caspase-3 and cleaved PARP1, another known biochemical hallmark for apoptosis, occurred in the whole-body γ-irradiated Khdrbs1−/− mice, but not Khdrbs1+/− controls (Figure 3B). Such an inverse correlation between NF-κB-mediated anti-apoptotic gene expression and DNA damage-triggered apoptosis underscores the crucial function of Sam68 in genotoxic stress-induced NF-κB signaling and transactivation in the γ-irradiated colon epithelium. Consistently, far more apoptotic cells, as assayed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), were observed in the colon tissue sections in situ from Khdrbs1−/− mice post WBIR, compared to those from Khdrbs1+/− controls (Figure 3C–D). The amount of apoptotic cells on the colon tissue sections from the mock-γ-irradiated animals was comparable, regardless of Sam68 presence (Figure 3C–D). These results thus demonstrate that Sam68 deletion expedites CECs to undertake apoptosis in vivo, in parallel to the substantially dampened NF-κB signaling and anti-apoptotic gene expression caused by genotoxic stress, in the mice subjected to WBIR. Figure 3 Download asset Open asset Sam68 deletion sensitizes colon epithelial cells to undergo apoptosis in the γ-irradiated mice. (A) Immunofluorescence micrographs of cleaved Caspase-3 (c-Casp3) in colon tissue collected from Khdrbs1+/− and Khdrbs1−/− mice at 8 hr post whole body γ-irradiation (WBIR) or mock irradiation, with nuclei counterstained by DAPI. Lu, lumen; Scale bar, 200 μm. (B) Colon epithelial cells were isolated from mice treated as in (A) and whole cell lysates were derived and immunoblotted (IB) for indicated proteins, with β-actin as a loading control. The full-length and cleaved PARP1 are indicated by a black triangle and a red triangle, respectively. (C) Micrographs of TUNEL staining in colon tissue collected from mice treated as in (A), with nuclei counterstained by DAPI. Scale bar, 100 μm. (D) Relative cells with TUNEL staining from four random fields, as in (C), were quantified. Data are representative of at least two independent experiments. Results in (D) are expressed as mean and s.e.m. n.s., non-significant difference and **p<0.01, ***p<0.001 by Student’s t tests. https://doi.org/10.7554/eLife.21957.004 Sam68 is crucial for the NF-κB-mediated radioprotection in the colon of γ-irradiated animals Previous studies reveal that the intestine and the colon are hypersensitive to radiotoxicity (Barlow et al., 1996; de Murcia et al., 1997; Gannon et al., 2012) and that NF-κB signaling pathway executes an important protective function in the γ-irradiated colon (Egan et al., 2004). To assess the impact of Sam68 on the radiodamage to the colon tissue, we examined the morphology of the colon from mice, relative to mock-treated controls, by gross dissection and histological staining. Indeed, the colonic morphology, length, and structure of mock-irradiated Khdrbs1+/− and Khdrbs1−/− mice were indistinguishable, suggesting that Sam68 is dispensable for mouse colon development (Figure 4A–E). Fourteen days post WBIR, the colons in Khdrbs1+/− mice were comparable to those from mock-irradiated animals in morphology and length (Figure 4A–C). In contrast, the γ-irradiated Khdrbs1−/− mice, compared to Khdrbs1+/− controls, suffered more severe and widespread damage to the colon, with substantially shortened colon lengths (Figure 4A–C). Moreover, histological analyses showed more severe crypt shrinkage, more goblet cell depletion, and less crypt survival in the colon derived from Khdrbs1−/− mice than in those from Khdrbs1+/− controls following WBIR (Figure 4D–E). Consequently, far fewer Khdrbs1−/− mice survived the sublethal dose of WBIR, compared to Khdrbs1+/− controls (Figure 4F), demonstrating that Sam68 deletion promotes the mice to be hypersensitive to radiotoxicity. Consistent with the reported crucial role of NF-κB signaling for providing radioprotection to the colon epithelium (Egan et al., 2004), our results emphasize that Sam68 executes a key function in genotoxic stress-induced NF-κB signaling and transactivation of a panel of anti-apoptotic genes, thus conferring radioprotection to the colon in the whole-body γ-irradiated mice. Figure 4 Download asset Open asset Sam68 is essential for the NF-κB-mediated radioprotection in vivo. (A and B) Representative photographs of colons in (A) and collected from (B) Khdrbs1+/− and Khdrbs1−/− mice at 14 days post whole body γ-irradiation (WBIR) or mock irradiation. Scale bars, 5 mm (A) and 1 cm (B), respectively. (C) The length changes in the colons derived from Khdrbs1+/− (n = 3) and Khdrbs1−/− (n = 3) mice at 14 days post WBIR or mock-irradiation, normalized to mock-irradiated controls. (D) Hematoxylin and eosin staining of colon tissue sections collected from mice treated as in (A). Scale bar, 100 μm. (E) Percentage of crypt survival in (D) was quantified. (F) Kaplan-Meier analysis of the survival rate in Khdrbs1+/− (n = 25) and Khdrbs1−/− (n = 17) mice following WBIR. p<0.0001 by Gehan-Breslow-Wilcoxon test. Results in (C and E) are expressed as mean and s.e.m. n.s., non-significant difference and **p<0.01, ***p<0.001 by Student’s t tests. https://doi.org/10.7554/eLife.21957.005 Discussion Herein, we report that Sam68 is critical for γ-irradiation-initiated NF-κB signaling and anti-apoptotic transcription in the colon in vivo and that Sam68-dependent NF-κB activation executes a protective function to the colon epithelium in the whole-body γ-irradiated animals. Sam68 deletion substantially dampens the γ-irradiation-initiated signaling cascade essential for NF-κB activation, which includes PAR synthesis, p65 phosphorylation, and p65 nuclear translocation, in the colon derived from mice at various time periods post WBIR. As a consequence, γ-irradiation-induced expression of NF-κB target genes, in particular Bcl2l1 encoding the anti-apoptotic protein Bcl-XL, is remarkably tempered in the colon epithelium from Khdrbs1−/− mice, compared to Khdrbs1+/− controls. These results are consistent with our prior report that Sam68 deletion diminishes the genotoxic stress-induced NF-κB signaling and NF-κB-mediated anti-apoptotic gene expression in the cultured MEFs and CECs in vitro (Fu et al., 2016). Moreover, WBIR fosters Khdrbs1−/− CECs to undertake apoptosis in situ in the colon from Khdrbs1−/− mice, but not Khdrbs1+/− controls, which also mirrors our prior report that Khdrbs1−/− CECs are hypersensitive to γ-irradiation and other genotoxic stresses in culture (Fu et al., 2016). Our results generated from whole-body γ-irradiated animals, along with our previous reports in the cultured cells, further support the physiological relevance of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon epithelium in response to genotoxic stresses. As elucidated previously (Fu et al., 2016), knockdown/knockout of Sam68 substantially sensitizes human colon cancer cells to undergo spontaneous apoptosis and retards colon tumor development in Apcmin716/+ mice, which highlights the critical role of Sam68-dependent NF-κB transactivation in the cellular responses to the intrinsic DNA damage that occurs frequently in the rapidly-dividing/proliferating cancer cells. We show here that Khdrbs1−/− mice suffer more severe damage in the colon and succumb rapidly from acute radiotoxicity than their Khdrbs1+/− controls post the extrinsic DNA damage challenge by WBIR. These results, extending additional support to the reported key role of NF-κB in providing radioprotection to the colon epithelium (Egan et al., 2004), highlight the pathophysiological relevance of the Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic/environmental DNA damage. Elevation in Sam68 protein levels has been proposed as a prognostic marker in multiple cancers (Chen et al., 2012; Liao et al., 2013; Song et al., 2010; Zhang et al., 2009), although the exact function of Sam68 in these cancers remains obscure. We recently revealed that Sam68 plays a crucial role in controlling DNA damage-induced PARP1 activation and PAR production; hence Sam68 deficiency dramatically dampens the PAR-dependent NF-κB signaling and DNA repair pathways initiated by DNA damage (Fu et al., 2016; Sun et al., 2016). As a key early signaling regulator that converges at the proxy of the DNA damage-triggered signaling cascade in the nucleus, Sam68 could provide a novel target for cancer therapeutics. In support of this notion, manipulation of Sam68 sensitizes colon cancer to DNA damage-triggered apoptosis in human colon cancer cell lines and retards colon tumor burden in Apcmin716/+ mice (Fu et al., 2016). Besides its crucial role in cancer cells to overcome the frequently-occurred intrinsic DNA damage, our results here demonstrate that Sam68-dependent NF-κB transactivation is pivotal for normal cells in the colon epithelium by executing an important physiological function to prevent the radiodamage to the colon caused by extrinsic/environmental γ-irradiation. The levels of Sam68 proteins in both normal and cancerous colon tissues could be a potential biomarker to facilitate the optimization of the administered dose of γ-irradiation, when employed as a single therapy or combined with other means for cancer treatment, in order to achieve superior outcomes via an elegant balance between the antitumor effects to tumor tissue and the acute side-effects to normal tissue caused by γ-irradiation. Materials and methods Mice and ethics statement Request a detailed protocol All animal experiments were performed according to protocol number MO16-H285, approved by the Johns Hopkins University’s Animal Care and Use Committee and in direct accordance with the NIH guidelines for housing and care of laboratory animals. Khdrbs1−/− mice and their gender-matched littermate Khdrbs1+/− mice were produced using heterozygous breeding pairs, as previously described (Fu et al., 2016). Mice were maintained in a specific pathogen-free facility and fed autoclaved food and water ad libitum. Whole-body γ-irradiation Request a detailed protocol Whole-body γ-irradiation (WBIR) in mice was performed as previously described (Sun et al., 2016). The γ-irradiated mice were sacrificed at indicated time points post WBIR for the indicated analyses, and the mortality and survival of mice were also monitored post γ-irradiation. Antibodies and reagents Request a detailed protocol Antibodies used were: Sam68 (RRID: AB_631869) and p65 (RRID: AB_632037) from Santa Cruz Biotechnology (Dallas, TX); β-actin (RRID: AB_476744) from Sigma-Aldrich (St. Louis, MO); PAR (RRID: AB_2572318) from Trevigen (Gaithersburg, MD); PARP1 (RRID: AB_2160739), phospho-p65 (RRID: AB_330570), Bcl-2 (RRID: AB_1903907), and cleaved Caspase-3 (RRID: AB_2341188) from Cell Signaling Technology (Danvers, MA); Bcl-XL (RRID: AB_1949733) from GeneTex (Irvine, CA). 4',6-diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich. Immunofluorescence staining Request a detailed protocol Immunofluorescence staining on colon tissue sections was performed as we did previous (Fu et al., 2016). Briefly, after euthanizing mice, the entire colons were excised under aseptic conditions and frozen in optimal cutting temperature (O.C.T.) media (Tissue-Tek, Elkhart, IN) or embedded in paraffin (Sigma-Aldrich). Tissue sections (5-micron) were cut, collected on coated slides, fixed in paraformaldehyde, washed with PBS, and blocked with appropriate sera in PBS. After incubating with appropriate antibodies, sections were washed and incubated with fluorescence dye-conjugated second antibodies and 1 µg/ml of DAPI (Sigma-Aldrich). Stained sections were washed and mounted under a coverslip using Fluoro-gel with Tris Buffer (Electron Microscopy Sciences, Hatfield, PA) and examined using an Axio Observer fluorescence microscope (Zeiss, Oberkochen, Germany). Isolation of primary colonic epithelial cells Request a detailed protocol Colonic epithelial cells (CECs) were isolated from mice as previously described (Fu et al., 2016; Hodgson et al., 2015). Subcellular fractionation Request a detailed protocol Subcellular fractionation was performed by differential centrifugation as previously described (Wan et al., 2007; Wier et al., 2012). Immunoblot Request a detailed protocol Immunoblot assays were conducted as previously described (Fu et al., 2013; Hodgson et al., 2015). In brief, cells were harvested and lysed on ice by 0.4 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate, 1 × complete protease inhibitor cocktail [Roche Applied Science, Indianapolis, IN]) for 30 min. The lysates were centrifuged at 10,000 × g at 4°C for 10 min. The protein-normalized lysates were separated by SDS-PAGE under reduced and denaturing conditions. The resolved protein bands were transferred onto nitrocellulose membranes and probed by the Super Signaling system (Thermo Scientific) according to the manufacturer's instructions, and imaged using a FluorChem E System (Protein Simple, Santa Clara, CA). mRNA in situ hybridization Request a detailed protocol Digoxigenin (DIG)-labeled probes were employed to visualize Bcl2l1 mRNA encoding Bcl-XL in colon tissues, as previously described (Hobbs et al., 2015). Briefly, Bcl2l1 gene specific sequence was first ligated to the pCRII-TOPO Vector (Life Technologies). The antisense and sense complementary RNA probes specific for Bcl2l1 mRNA were transcribed using a Lig'n Scribe Kit (Life Technologies), and then labeled with DIG using a DIG RNA labeling kits (Roche Applied Science) according to the manufacturer’s instructions. The mRNA in situ hybridization on frozen colon tissue sections was performed using adapted methods from Gu and Coulombe (2007). Briefly, colon tissues were post-fixed in 4% paraformaldehyde/PBS for 20 min, followed by proteinase K digestion at 37°C for 6 min and re-fixed in 4% paraformaldehyde/PBS, then acetylated by 0.25% acetic anhydride in 0.1 M triethanolamine (10 min). Hybridization solution containing 3 μg of each denatured DIG-labeled probe was mixed with the samples for overnight incubation at 65°C. The next day, slides were rinsed and incubated in the HSW solution (50% formamide, 0.5 × standard sodium citrate, 0.1% Tween-20) for 30 min at 65°C. The slides were then washed in the HSW solution (2 × 20 min) at 65°C, 2 × standard sodium citrate, 0.1 × standard sodium citrate at 37°, respectively. The slides were switched to blocking solution (10% normal goat serum [NGS] in PBST) for 1 hr, followed by an incubation in alkaline phosphatase (AP)-conjugated sheep anti-DIG-antibody (Roche Applied Science), 1:2000 diluted in PBST/1% NGS overnight at 4°C in the dark. To visualize the mRNA in situ hybridization signal, tissues were washed with PBST (3 × 2 hr) and NTMT (0.1 M NaCl, 0.1 M Tris-HCl [pH7.9], 50 mM MgCl2, 0.1% Tween-20) for 10 min, and incubated in BM purple AP-substrate (Roche Applied Science) containing 0.5 mg/ml levamizole overnight, then stopped the reaction by washing in PBS. Hybridized tissues are mounted in crystal/mount media in preparation for microscopy. TUNEL assays Request a detailed protocol Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in situ on colon tissue sections were carried out using a DNA fragmentation Image Kit (Roche Applied Science), according to the manufacturer's instructions. Histology Request a detailed protocol Histological analyses were carried out as we did previously (Fu et al., 2016). In brief, the excised entire colons were embedded in paraffin. Tissue sections (5-micron) were cut, deparaffinised, rehydrated, and stained with hematoxylin and Eosin (H and E) staining and stained sections were washed and mounted under a coverslip and examined under light microscopy (Zeiss). The crypt survival assays (Lai and Egan, 2013) were employed to evaluate the radio-sensitivity of the colon post whole-body γ-irradiation in mi