Study On The Adaptive Response Of Lymphocyte Nucleus Bridge Induced By Low Dose γ - Ray And The Mechanism Of Action Of AKT

ObjectiveAdaptive response (radiation adaptive response) refers to a cell or organism was previously irradiated with low doses of radiation to stimulate the cells or the body to resist the induced damage and protect them within a certain time interval followed by a high dose radiation. The advanced low doses of ionizing radiation can enhance the immune system function, inhibit the tumor growth and metastasis, and reduce the incidence of cancer. It’s significant for tumor therapy and radiological protection.Current studies about adaptive response mainly include cytogenetic damage, gene mutations, and cell survival as the biological endpoint. Studies have shown that cell genomics indexes — nucleoplasmic bridges (NPB) derived from the dicentric chromosome. And there was a strong correlation between the number of NPB and the dicentic chromosome plus centic ring (die+r) number in the metaphases at the same lymphocyte cultures. Our previous study showed that there is a dose-response relationship between the yields of NPB and the irradiated dose, so NPB is expected to become a new ionizing radiation biomarkers. Therefore, this study uses NPB as an indicator to explore low-dose ionizing radiation-induced adaptive response in human peripheral blood lymphocytes, to explore the range of the conditioning dose and the effective conditioning dose.At present, the mechanism of the adaptive response was not fully understood; there are DNA damage repair, cell cycle regulation, cell apoptosis, cell signaling pathways regulation involved in. AKT (protein kinase B) is a cell regulatory factor to regulate many reaction of in cells, such as cell growth, glucose metabolism and cell proliferation. PI3K/AKT pathway is widely present in the cell, which is a signal transduction pathway involved in cell proliferation, growth and differentiation. Adaptive response may be related to cell survival through phosphorylation the related AKT downstream target molecules. Therefore, this study is to explore the changes of AKT on translational level and the AKT phosphorylation levels in the adaptive response.Materials and methodsThree human peripheral blood samples were collected and irradiated with 0,20,50, 75,100,150 and 200 mGy (dose-rate is 25 mGy/min) of 60Co γ-rays, and after 6 hours, the samples were irradiated again with 2 Gy (dose-rate of 1 Gy/min) of 60Co γ-rays. The cytokinesis-block micronucleus assay was carried out to analyze the NPB and micronuclei (MN) in binucleated cells by optical microscope.Healthy human peripheral blood samples were collected and irradiated with the explored optimal conditioning dose. The blood samples were put in 37℃ incubator for 6 hours for repairing, then lymphocytes were isolated from the blood and the cell proteins were extracted. The AKT and p-AKT (Ser473) expression changes on translational level were analyzed by Western blot method.Results1. Within the dose range of 0-200 mGy, the yields of NPB and MN increased with enhanced doses of γ-rays. The dose-response relationship between the yields of NPB (or MN) and the irradiated dose followed a linear model. The equations were as follows: NPB y= 0.0059x+0.0001 (R2= 0.963, p<0.0\), where y is NPB per cell induced byirradiated dose, x is the irradiated dose in Gy. MN:y= 0.0620x+0.0073 (R2= 0.972, p<0.01), where y is MN per cell induced by irradiated dose of x in Gy.2. When the dose-rate was 25 mGy/min, the yields of NPB (or MN) significantly reduced if the sample A and B were irradiated with low dose γ-rays (75-100 mGy) 6 hours before 2 Gy irradiation. The differences between the experimental group and control group was statistically significant (p<0.05). For sample C, the 100 mGy radiation may reduce the yields of MN (p<0.05) compared to the control group. For the three samples, when the conditioning dose is 100 mGy, previous low dose radiation can reduce the yields of NPB and MN which manifested the largest reduction effect.3. Compared with the non-irradiated control group, AKT expression level enhanced on the translational level when irradiated with the conditioning dose, and the AKT phosphorylation activity at Ser473 point increased.Conclusion1. There is a liner relationship between the yields of NPB and the irradiated doses, so NPB is expected to become a new ionizing radiation biomarkers.2. Low dose of 60Co γ-rays (25 mGy/min,75-100 mGy) could induce the adaptive response with NPB in human peripheral blood lymphocytes, and the the optimal conditioning dose of 100 mGy.3. The AKT expression enhanced on the protein level during the adaptive response occuring, and the AKT phosphorylation activity increased at Ser473 point, so the adaptive response may be related to this.