![]() These findings suggest DIM stimulates an ATM-driven DDR-like response. Total protein levels remain unchanged ( Fig. High levels of the six phosphoproteins were maintained for ≥24 h ( Fig. CHK1 is usually phosphorylated by ATR rather than ATM ( 34), but some studies indicate a role for ATM in CHK1 phosphorylation. DIM also stimulated phosphorylation of CHK1 on S345. Increased phosphorylation of ATM and NBS1, a key substrate, was observed after a 1-min exposure to DIM, followed by phosphorylation of p53, BRCA1, and CHK2 ( Fig. 4 B), suggesting DIM can “hyperactivate” ATM. With combined DIM+radiation, at 30 min after radiation, the combination gave greater phosphorylation of ATM and several substrates than either agent alone ( Fig. These events were blocked by a selective ATM inhibitor (KU55933) ( 33), suggesting the pathway is ATM driven. Subsequently, phosphorylation was observed on two ATM substrates (BRCA1 and CHK2) at their ATM sites. Phospho-ATM levels were elevated at 24 h after TBI, consistent with the idea that radiation causes ongoing oxidative stress in normal tissues ( 28, 29), because most DSBs in irradiated cells are repaired in 2–3 h ( 30, 31).ĭIM alone caused rapid activation of ATM, indicated by phosphorylation on S1981, in 184A1 cells ( Fig. Similar results were observed for DIM administered 24 h before, 1 h before, or 10 min after irradiation. Under different conditions, DIM caused ATM phosphorylation and enhanced radiation-induced phosphorylation in normal rat kidney, liver, and brain ( Fig. Phospho-ATM levels 24 h after irradiation and appeared to be greater after treatment with DIM+radiation than either agent alone. In contrast, in normal mouse kidney tissue, basal phospho-ATM levels were low, and the levels were increased by DIM and/or radiation ( Fig. No increases in phospho-ATM were observed whether DIM was given 4 or 24 h before or 10 min after radiation. ATM was constitutively phosphorylated in MDA-MB-231 tumors and was not further activated by DIM and/or radiation (20 Gy) ( Fig. We tested the ability of DIM to activate ATM in vivo, using phospho-ATM (S1981) as a marker of activation ( 26, 27). In C57BL/6 mice, five treatments with DIM significantly attenuated the reductions in red blood cells, white blood cells, and platelets due to lower doses of TBI (2–6 Gy) ( Table S1). DIM similarly protected C57BL/6 mice against TBI, indicating that protection is not species specific. When the first DIM dose was given 24-h before TBI (13 Gy), a lower daily dose of DIM (7.5 mg/kg) yielded 55% 30-d survival ( P < 0.001), suggesting that if one DIM dose is given before exposure, radioprotection is achieved with a much lower dose. Although control animals died by day 10, the 30-d survival rates were 60% ( P < 0.001 vs. 1 A shows dose-dependent protection of Sprague–Dawley (SD) rats given daily injections of DIM for 14-d starting 10 min after TBI (13 Gy). injection for convenience, because preliminary studies showed DIM was most effective against total body irradiation (TBI) when given in multiple once-daily doses. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.ĭIM can be given to mice by gavage at 250 mg/kg with no toxicity and wide tissue distribution ( 7). In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. DIM also caused activation of ATM in rodent tissues. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. DIM (3,3′-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. ![]()
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