Mitochondrial ROS and radiation induced transformation in mouse embryonic fibroblasts

Abstract

Manganese superoxide dismutase (SOD2) is a nuclear encoded and mitochondria localized antioxidant enzyme that converts mitochondria derived superoxide to hydrogen peroxide. This study investigates the hypothesis that mitochondria derived reactive oxygen species (ROS) regulate ionizing radiation (IR) induced transformation in normal cells. Mouse embryonic fibroblasts (MEFs) with wild type SOD2 (+/+), heterozygous SOD2 (+/-), and homozygous SOD2 (-/-) genotypes were irradiated with equitoxic doses of IR, and assayed for transformation frequency, cellular redox environment, DNA damage, and cell cycle checkpoint activation. Transformation frequency increased ( approximately 5-fold) in SOD2 (-/-) compared to SOD2 (+/+) MEFs. Cellular redox environment (GSH, GSSG, DHE and DCFH-oxidation) did not show any significant change within 24 h post-IR. However, a significant increase in cellular ROS levels was observed at 72 h post-IR in SOD2 (-/-) compared to SOD2 (+/+) MEFs, which was consistent with an increase in GSSG in SOD2 (-/-) MEFs. Late ROS accumulation was associated with an increase in micronuclei frequency in SOD2 (-/-) MEFs. Exit from G(2) was accelerated in irradiated SOD2 (+/-) and SOD2 (-/-) compared to SOD2 (+/+) MEFs. These results support the hypothesis that SOD2 activity and mitochondria generated ROS regulate IR induced transformation in mouse embryonic fibroblasts.

Figure 1. Cellular ROS levels and plating efficiency in SOD2 genotype mouse embryonic fibroblasts

Asynchronously growing exponential cultures of SOD2 wild type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mouse embryonic fibroblasts (MEFs) were analyzed for (A) SOD2 protein and actin levels, upper two panels; bottom panels represent SOD activity analyzed by native gel-electrophoresis; flow cytometry measurements of (B) DHE and (C) DCFH-fluorescence; (D) plating efficiency; mean ± SE; * p < 0.05 vs. SOD2 (+/+).

Figure 2. Increased transformation frequency in irradiated SOD2 (−/−) compared to SOD2 (+/+) MEFS

Clonogenic MEFs were seeded on 30 Gy irradiated hamster fibroblasts feeder cells, and at 24 h post-plating monolayer cultures were irradiated with equitoxic doses of ionizing radiation (dose rate, 0.83 Gy/min): 6.8 Gy for SOD2 (+/+); 4.4 Gy for SOD2 (+/−); 5.6 Gy for SOD2 (−/−) MEFs. Cells were continued in culture for 6–8 weeks; (A) representative microscopy pictures of Giemsa-stained monolayers containing Type II and III foci; transformation frequency of Type II (B) and Type III (C) foci calculated as the ratio of transformed foci relative to the number of viable cells.

Figure 3. SOD2 activity suppressed late ROS accumulation in irradiated MEFs

Asynchronously growing exponential MEFs were irradiated with 5 Gy and harvested at indicated time for measurements of (A & B) cellular ROS levels by flow cytometry and (C & D) GSH and GSSG by biochemical assay. Fold-change was calculated relative to time matched un-irradiated cultures for each cell types: (A) DHE-fluorescence, (B) DCFH-fluorescence. * p< 0.05 vs. 24 h; €, p< 0.05 vs. SOD2 (+/+) at 72 h. Data represent mean ± SEM, n=3.

Figure 4. SOD2 activity suppressed radiation induced late DNA damage in MEFs

Asynchronously growing exponential MEFs were irradiated with 5 Gy and harvested at indicated times for measurements of DNA damage: (A) representative microscopy pictures showing micronuclei (MN) and nucleoplasmic bridge (NPB): line arrows indicate MN and solid arrows indicate NPB; (B) percentage of micronuclei bearing bi-nucleated cells (MNBNCs); *, p < 0.05 vs. SOD2 (+/+) at 24 h, and €, p < 0.05 vs. SOD2 (+/+) at 72 h post-IR. (C) Immunoblotting of γH2AX in 5 Gy irradiated MEFs.

Figure 5. G₂-exit is accelerated in irradiated SOD2 (+/−) and SOD2 (−/−) compared to SOD2 (+/+) MEFs

Asynchronously growing exponential MEFs were irradiated with 5 Gy and harvested at indicated time for flow cytometry measurements of DNA content. Representative DNA histograms are shown in (A); (B) fold-change in G₂ was calculated relative to 0 h un-irradiated control for individual cell types; the percentage of G₂ in un-irradiated MEFs was as follows: SOD2 (+/+), 22%; SOD2 (+/−), 19%; SOD2 (−/−), 17%. *, p<0.05 vs. SOD2 (+/+) MEFs. (C) SOD2 (−/−) MEFs were infected with AdEmpty and AdSOD2, and irradiated with 5 Gy at 72 h post-infection. Cell cycle phase distributions analyzed at indicated times, and fold change in G₂ calculated. $, p<0.05 vs. AdEmpty infected cells.