Abscopal effect of low-LET γ-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response

Abstract

Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.

Figure 1.

Photographs of specially designed cerrobend shield (22 cm diameter and 1.2 cm thickness) used to encase the body of the mice, exposing only 1 cm diameter of the lower abdomen.

Figure 2.

(a) Venn diagrams showing numbers of genes significantly upregulated, with or without application of stringent criteria (⩾2-fold) in nontargeted heart tissues of mice exposed to 2 Gy, 10 Gy or fractionated ionizing radiation (FIR; 2 Gy per day for 5 days (2 Gy × 5)). Numbers outside each circle indicate total upregulated genes in response to a specific dose, those inside greater part of the circles indicate selectively upregulated in that particular dose, those in two overlapping circles indicate upregulated commonly under both conditions and those in the center indicate upregulated in all three doses. (b-d) Histograms showing expression levels of nuclear factor kappa B (NF-κB) pathway molecules that were significantly (>2-fold) upregulated in nontargeted heart after (b) 2Gy, (c) 10 Gy, or (d) 2 Gy x 5. In all, 22 genes were commonly upregulated (highlighted as black bars) after all three dose and fractionation regimens of radiation.

Figure 3.

(a) Representative electrophoretic mobility shift assay (EMSA) autoradiograms (A–C) showing nuclear factor kappa B (NF-κB) DNA-binding activity in nontargeted heart tissues of mice exposed to 2 Gy, 10 Gy or fractionated ionizing radiation (FIR; 2 Gy per day for 5 days (2 Gy × 5D)). Specificity of NF-κB DNA-binding activity D). Nuclear protein was incubated in the absence (lane 1) or presence (lanes 2, 3 and 4) of increased concentrations of homologous unlabeled competitor and probed with [γ-³²P]-ATP-labeled NF-κB-specific oligonucleotide. Identification of NF-κB subunits by supershift assay (E). The addition of antibodies directed against potential components of NF-κB complex resulted in supershift when antibody of p65 was used. (b) Immunoblots showing total and phosphorylated p38 and extracellular signal-regulated kinase-1/2 (ERK1/2) expression in nontargeted heart tissues of mice exposed to 2 Gy, 10 Gy or FIR (2 Gy × 5). (c) Histograms showing densitometry analysis of total and phosphorylated p38 and ERK1/2. The experiments were repeated at least three times and the group-wise comparisons were made using analysis of variance (ANOVA).

Figure 4.

(a) Representative photomicrographs (× 40 magnifications) of hematoxylin and eosin (H&E) staining, p65 and cMYC levels in nontargeted heart in mice exposed to 2 Gy, 10 Gy or 2 Gy × 5 (2 Gy per day for 5 days). Histograms showing immunohistochemical-intensity of p65 and cMyc in nontargeted tissue. (b) Representative photomicrographs of total cell population (4’,6-diamidino-2-phenylindole (DAPI)) and DNA fragmentation in nontargeted heart in mice exposed to 2 Gy, 10 Gy or 2 Gy × 5. Each tissue section was analyzed for fluorescence intensity in at least 10 areas. Histograms showing fluorescein intensity in nontargeted heart tissue in mice exposed to varying doses of low-abdomen-focused radiation.