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. 2011 Jan;175(1):57-65.
doi: 10.1667/RR2349.1. Epub 2010 Nov 8.

SOD2-mediated effects induced by WR1065 and low-dose ionizing radiation on micronucleus formation in RKO human colon carcinoma cells

Jeffrey S Murley  1 , Yasushi Kataoka, Richard C Miller, Jian Jian Li, Gayle Woloschak, David J Grdina
Affiliations

SOD2-mediated effects induced by WR1065 and low-dose ionizing radiation on micronucleus formation in RKO human colon carcinoma cells

Jeffrey S Murley et al. Radiat Res. 2011 Jan.

Abstract

RKO36 cells exposed to either WR1065 or 10 cGy X rays show elevated SOD2 gene expression and SOD2 enzymatic activity. Cells challenged at this time with 2 Gy exhibit enhanced radiation resistance. This phenomenon has been identified as a delayed radioprotective effect or an adaptive response when induced by thiols or low-dose radiation, respectively. In this study we investigated the relative effectiveness of both WR1065 and low-dose radiation in reducing the incidence of radiation-induced micronucleus formation in binucleated RKO36 human colon carcinoma cells. The role of SOD2 in this process was assessed by measuring changes in enzymatic activity as a function of the inducing agent used, the level of protection afforded, and the inhibitory effects of short interfering RNA (SOD2 siRNA). Both WR1065 and 10 cGy X rays effectively induced a greater than threefold elevation in SOD2 activity 24 h after exposure. Cells irradiated at this time with 2 Gy exhibited a significant resistance to micronucleus formation (P < 0.05; Student's two-tailed t test). This protective effect was significantly inhibited in cells transfected with SOD2 siRNA. SOD2 played an important role in the adaptive/delayed radioprotective response by inhibiting the initiation of a superoxide anion-induced ROS cascade leading to enhanced mitochondrial and nuclear damages.

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Conflict of interest statement

Conflict of Interest Notification: Dr. David J. Grdina is a paid consultant to Pinnacle Biologics on potential novel uses of amifostine.

Figures

FIG. 1
FIG. 1
Effect of WR1065 exposure on SOD1 and SOD2 enzymatic activity in RKO36 cells. Cells were exposed for 30 min to either 40 μM or 4 mM WR1065 and then assayed for SOD activity either 30 min or 24 h later. P values above the bar compare the treatment groups with the corresponding unirradiated control cells. Each experiment was performed three times, and the error bars represent the SEM.
FIG. 2
FIG. 2
Effect of SOD2 siRNA transfection of RKO36 cells on subsequent SOD2 activity measured 24 h after exposure to either 40 μM or 4 mM WR1065 for 30 min. Background SOD2 activities were also determined for non-thiol-treated control cells transfected with either negative control (NC) siRNA or SOD2 siRNA. Each experiment was repeated three times, and error bars represent the SEM. P values comparing cells treated with WR1065 to their respective negative controls or SOD2 siRNA-transfected controls are presented.
FIG. 3
FIG. 3
Effect of SOD2 siRNA transfection on micronucleus formation in RKO36 cells irradiated with 2 Gy 24 h after exposure to 40 μM or 4 mM WR1065. The percentage of micronuclei was determined in non-pretreated cells (Control), mock-transfected cells (MT), cells transfected with negative control (NC) siRNA, and cells transfected with SOD2 siRNA. Comparisons of micronucleus percentages of non-pretreated RKO36 cells irradiated with 2 Gy and of selected mock-transfected (MT) and SOD2 siRNA irradiated experimental groups exposed to 40 μM and 4 mM WR1065 are identified with resulting P values. Each experiment was repeated three times, and error bars represent the SEM.
FIG. 4
FIG. 4
Effect of 10 cGy X rays on the induction of SOD2 activity in RKO36 cells as a function of time after irradiation. P values compare treatment groups with untreated control cells. Each experiment was repeated three times, and error bars represent the SEM.
FIG. 5
FIG. 5
Effect of 10 cGy X rays on micronucleus induction in RKO36 cells by a 2-Gy challenge dose as a function of the time between radiation doses. Comparisons of micronucleus frequencies in unirradiated control cells and cells irradiated with either 10 cGy or 2 Gy only are presented. The resulting P values are included above the corresponding treatment groups. All experiments were repeated three times, and error bars represent the SEM.
FIG. 6
FIG. 6
Effect of SOD2 siRNA transfection on micronucleus formation in RKO36 cells irradiated with 10 cGy, 2 Gy and 10 cGy followed 24 h later with 2 Gy. Cells were either not transfected, transfected with negative control (NC) siRNA, or transfected with SOD2 siRNA 24 h prior to irradiation. Comparisons of micronucleus frequencies induced by 10 cGy and followed 24 h later with 2 Gy in nontransfected or SOD2 siRNA-transfected cells were compared to the frequency of micronucleus formation induced by 2 Gy alone in nontransfected cells. The resulting P values are included above the corresponding treatment groups. All experiments were repeated three times, and error bars represent the SEM.
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