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. 2013 Aug 12;2(8):e62.
doi: 10.1038/oncsis.2013.22.

Suppression of radiation-induced DNA double-strand break repair by MyD88 is accompanied by apoptosis and crypt loss in mouse colon

X Y Lai  1 , L J Egan
Affiliations

Suppression of radiation-induced DNA double-strand break repair by MyD88 is accompanied by apoptosis and crypt loss in mouse colon

X Y Lai et al. Oncogenesis. .

Abstract

Intestinal microbes promote the injurious effects of radiation on those tissues. However, the molecular factors mediating this effect are largely unknown. In this work, we explored the effects of orally administered antibiotics and MyD88, a key adapter molecule in toll-like receptor signaling, on molecular and cellular responses of mouse colon to radiation. Results show that oral antibiotics lowered radiation-induced colonic damage by protecting epithelial cells against radiation-induced apoptosis, leading to increased survival of crypts. MyD88 deficiency partially phenocopied the effects of oral antibiotics on apoptosis and crypt survival, suggesting that colonic microbes exert their injurious effects in part via that molecule. Analysis of DNA double-strand breaks, the primary genotoxic lesions induced by radiation, showed that their early induction in mouse colon was unaffected by MyD88. However, MyD88 deficiency resulted in the later disappearance of DNA double-strand breaks. Loss of DNA double-strand breaks was accompanied by the evidence of increased activation of both the non-homologous end-joining and homologous recombination pathways of DNA repair in MyD88-deficient mice. These results show that colonic microbes and MyD88 regulate DNA double-strand break repair in irradiated mouse colon, effects which exert significant control over radiation-induced apoptosis and crypt survival.

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Figures

Figure 1
Figure 1
Antibiotics attenuate the GI radiation syndrome by promoting the survival of colonic epithelial crypts. (a) Daily consumption of water and food (b) in antibiotic-treated and control mice in the 4.5 days following the indicated doses of whole-body radiation. (c) Body weight at baseline and 4.5 days following 14 Gy whole-body radiation in antibiotic-treated and control mice. (d) H&E-stained sections of colon at baseline and 4.5 days post 14 Gy whole-body radiation in antibiotic-treated and control mice. (e) Results of the colon crypt microcolony assay at 4.5 days following whole-body radiation in antibiotic-treated and control mice. (f) Representative PCNA immunofluorescence of colon sections, at baseline and 4.5 days following 14 Gy whole-body radiation in antibiotic-treated and control mice. Blue is DAPI nuclear staining. Red is PCNA staining. Quantification of PCNA staining at 4.5 days post radiation in antibiotic-treated and control mice. (g) Assessment of PCNA staining by crypt cell position at 4.5 days post radiation. In all panels, means±standard error are shown, **P<0.01 antibiotic-treated versus control mice.
Figure 2
Figure 2
Antibiotics protect the colonic epithelial cells located in the stem and progenitor cell zone against radiation-induced apoptosis. (a) Representative images of H&E-stained colon sections in antibiotic-treated and control mice at baseline and 4.5 h after 14 Gy radiation. (b) Representative images of DAPI and TUNEL stained sections of colon in antibiotic-treated and control mice at baseline and 4.5 h after 14 Gy radiation. Blue is DAPI nuclear staining and TUNEL is green. Quantification of TUNEL-positive cells at baseline and 4.5 h post 14 Gy radiation. (c) Assessment of TUNEL positivity by crypt cell position. In all panels, means±standard error are shown, **P<0.01 antibiotic-treated versus control mice.
Figure 3
Figure 3
MyD88 deficiency decreases the radiation-induced GI syndrome through increasing the survival of colon crypts. (a) Body weight at baseline and 4.5 days following 14 Gy whole-body radiation in MyD88-deficient and wild-type mice. (b) Daily consumption of food and water (c) in MyD88-deficient and wild-type mice in the 4.5 days following 14 Gy whole-body radiation. (d) H&E-stained sections of colon at baseline and 4.5 days post 14 Gy whole-body radiation in MyD88-deficient and wild-type mice. (e) Results of the colon crypt microcolony assay at baseline and 4.5 days following 14 Gy whole-body radiation in MyD88-deficient and wild-type mice. (f) Representative PCNA immunofluorescence of colon sections, at baseline and 4.5 days following 14 Gy whole-body radiation in MyD88-deficient and wild type mice. Quantification of PCNA staining at baseline and 4.5 days post 14 Gy radiation. Blue is DAPI nuclear staining. Red is PCNA staining. In all panels, means±standard error are shown, *P<0.05, **P<0.01 MyD88-deficient versus wild-type mice.
Figure 4
Figure 4
MyD88 deficiency decreases radiation-induced apoptosis in mouse colon following whole-body radiation. (a) Representative images of H&E-stained colon sections in MyD88-deficient and wild-type mice 4.5 h after 14 Gy whole-body radiation. (b) Representative images of consecutive DAPI, TUNEL and γ-H2ax-stained sections of colon in MyD88-deficient and wild-type mice 4.5 h after 14 Gy radiation. Blue is DAPI nuclear staining, TUNEL is green and γ-H2ax is red. In the merged image, yellow indicates colocalization of TUNEL-γ-H2ax double-positive nuclei. (c) Quantification of TUNEL-positive cells at baseline and 4.5 h post 14 Gy radiation. In all panels, means±standard error are shown, **P<0.01 MyD88-deficient versus wild-type mice.
Figure 5
Figure 5
MyD88 deficiency results in differentiated γ-H2ax and p-s1981-Atm kinetics in mouse colon following whole-body radiation. (a) Representative γ-H2ax foci immunofluorescence of colon sections, at the indicated times following 14 Gy whole-body radiation in MyD88-deficient and wild-type mice. Quantification of γ-H2ax foci and their time course following 14 Gy radiation. (b) Representative p-s1981-Atm foci immunofluorescence of colon sections, at baseline and 4.5 h following 14 Gy radiation in MyD88-deficient and wild-type mice. (c) Quantification of p-s1981-Atm foci at baseline and 4.5 h post 14 Gy radiation. Blue is DAPI nuclear staining. Red is γ-H2ax or p-s1981-Atm staining. In both panels, means±standard error are shown, **P<0.01 MyD88-deficient versus wild-type mice.
Figure 6
Figure 6
MyD88 deficiency results in enhanced DNA repair in mouse colon following whole-body radiation. (a) Representative Ku 70–80 immunofluorescence of colon sections, at baseline and 4.5 h following 14 Gy whole-body radiation, in MyD88-deficient and wild-type mice. Quantification of Ku 70–80 foci at baseline and 4.5 h post 14 Gy radiation. (b) Representative Rad51 immunofluorescence of colon sections, at baseline and 4.5 h following 14 Gy whole-body radiation, MyD88-deficient and wild-type mice. Quantification of Rad51 foci at baseline and 4.5 h post 14 Gy radiation. Blue is DAPI nuclear staining. Red is Ku 70–80 or Rad51 staining. In both panels, means±standard error are shown, **P<0.01 MyD88-deficient versus wild-type mice.
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