Biological Sciences Division
Now It's Molecular: New Understanding of Radiation Effects on Cells
Scientists find altered mitochondria proteins in cells showing genomic instability
Results: In recent proteomics studies on radiation effects on cells, new molecular data indicates that dysfunctional mitochondria cause elevated reactive oxygen species in the offspring of cells that survived radiation exposure. These results bring molecular-based evidence to a previously cell-based area of radiation research. The results appear in the June 2008 issue of Radiation Research.
Why it matters: Mitochondria are the power generators in cells that oxidize food molecules to produce chemical energy. Reactive oxygen species (ROS) are a natural byproduct of oxygen metabolism and have important roles in cell signaling. However, environmental stress, such as radiation exposure, can cause ROS levels to increase through a mechanism that can be passed on to the daughters of cells that survive radiation exposure. Some of these cell lines with persistently high levels of ROS also exhibit sporadic changes in their DNA.
This chart shows protein abundance changes in unstable cells relative to genomically stable controls. Seventy-four mitochondria were identified using mass spectrometry-based proteomics, and 12 were identified as significantly up- or down-regulated in at least one unstable cell line. The results clearly show that the proteins profile changes associated with elevated ROS are different between the two cell lines. Enlarged View
Radiation can also cause genomic instability that can be expressed in the progeny of irradiated cells as increased mutations, chromosomal changes and other genetic alterations. Genomic instability is thought to play a role in cancer development. Scientists do not yet understand the mechanisms of radiation-induced genome instability; however, in the case of cell lines with persistently elevated oxidative stress it could result from ROS-induced DNA changes.
Methods: Previous work at the University of Maryland on radiation-induced genome instability in Chinese hamster ovary cell lines showed that some of these unstable cells had persistently elevated levels of ROS likely caused by dysfunctional mitochondria. To further investigate this, scientists from Pacific Northwest National Laboratory, Washington State University and the University of Maryland performed quantitative high-throughput mass spectrometry proteomics studies on samples enriched in mitochondrial proteins from three unstable cell lines and their stable, unirradiated parental cell line. Out of several hundred identified proteins, they collected sufficient data on 74 mitochondrial proteins to test for statistically significant differences in the abundance between unstable and stable cell lines.
Two of the unstable cell lines investigated in this study had elevated ROS levels. The cell line with the highest level of ROS had eight down-regulated mitochondrial proteins-all associated with the tricarboxylic acid cycle, also called the Krebs cycle. The cell line with the second-highest ROS had five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation and electron transport. Therefore, molecular data from these two lines complement the cellular data in suggesting that the persistently elevated ROS results from dysfunctional mitochondria. The profiles of significantly altered mitochondrial proteins are completely different for the two cell lines, which suggests that different types of dysfunction at the molecular level give rise to similar cellular phenotypes (see figure). The third cell line was harder to interpret because both the cellular and molecular data are more ambiguous, therefore no conclusions were drawn for it.
What's next: More work is needed to establish a mechanistic relationship between persistently elevated ROS and genome instability. The researchers plan to investigate the mitochondrial proteome in genetically stable cell lines with elevated ROS and unstable cell lines with normal levels of oxidative stress. They are also applying the mitochondrial profiling approach in other areas of radiation research such as non-targeted bystander effects where cells that were not exposed to radiation receive signals from irradiated cells and respond as if they have been irradiated.
Acknowledgments: This research was supported by the U.S. Department of Energy's Low Dose Program within the Office of Biological and Environmental Research. The research team members are David Springer, Bill Morgan and Jonathan Peters, PNNL; John Miller and Shuanshuang Jin, WSU Tri-Cities; and Austin Yang, Yunhu Wan and Umut Aypar, University of Maryland.
Reference: Miller JH, S Jin, WF Morgan, A Yang, Y Wan, JS Peters, and DL Springer. 2008. "Profiling mitochondrial proteins in radiation-induced genome-unstable cell lines with persistent oxidative stress by mass spectrometry." Radiation Research 169(9):700-706.