Evidence mounts for the hypothesis that epigenetics may be involved in the initiation and perpetuation of radiation-induced genomic instability. Historically, scientists believed the only effects of ionizing radiation exposure were due to direct DNA damage in target cells. We now observe, however, that the harmful effects of radiation show up in both nontargeted cells and in the offspring of irradiated cells well past exposure.1
Among others, observed damage includes genomic instability, ploidy alterations, micronucleus formation, and gene mutation. Although numerous studies have successfully identified these phenomena, the exact mechanisms responsible for genomic instability remain unclear. Genomic instability, however, cannot be explained by physical DNA damage alone. As a result, researchers have turned to searching for epiginetic mechanisms.
One research team from the Department of Radiation Oncology at the University of Maryland in Baltimore used proteomic techniques to investigate the contribution of oxidative stress — which has been implicated in other studies — to radiation-induced DNA damage.2 Because phenotypic instability is a chief feature of cancer cells, which are resistant to apoptosis, learning more about how they maintain survivability under environmental stresses such as oxidation could lead to improved treatments.
The Baltimore researchers were refining research studies preformed by a team from the Department of Radiology, University of California, San Francisco. Their work showed a link between chromosomal instability, oxidative stress, and mitochondrial dysfunction in the GM10115 cell line.3 This team looked at the difference between the proteomes of irradiated clone LS12 and its parent, GM10115. The LS12 clone has a well-known unstable phenotype with oxidative stress, which led the Baltimore team to investigate mitochondrial protein expression changes in comparison with GM10115 parent cells.
The Baltimore team compared the two proteomes using quantitative mass spectrometry analysis and stable isotope labeling to increase the accuracy of both peptide and protein quantification. They also used free-flow electrophoresis purification to produce a higher percentage of mitochondrial proteins than in previous proteomic efforts.2
Their data show that LS12 cells have decreased levels of mitochondrial Cyt c, which would contribute to electron leakage and could explain the decrease in complex IV activity observed by scientists in other studies. These disturbances to the electron transport chain result in oxidative stress. Rather than proceeding through the normal cell death cycle, however, LS12 cells exhibit proteins that are either up- or downregulated, which would protect against apoptosis.
Significant changes included 3-mercaptopyruvate sulfur transferase (MST) upregulation. In addition to protecting against oxidative stress, MST contributes to redox homeostasis by mediating cysteine degradation.2
Another of these protective proteins is Lon protease. An enzyme featured in the tricarboxylic acid cycle, aconitase is vulnerable to oxidation and accumulates during oxidative stress. It has been hypothesized that Lon protease degrades damaged aconitase and other proteins, preventing the accumulation of damaged proteins that can compromise mitochondrial function.2
Tyrosine 3-monooxygenase is downregulated. Tyrosine 3-mono-oxygenase has been shown to play a role in inflammatory responses, DNA replication, DNA damage mitogenic and cell survival signaling, cell-cycle regulation, transcriptional activity, and apoptosis. The lack of tyrosine 3-monooxygenase would contribute to cell survival.
The fourth significantly upregulated protein is tumor necrosis factor-associated protein 1 (TRAP1) — a protein similar to heat-shock proteins. Cells that overexpress TRAP1 exhibit a phenotype resistant to H2O2-induced DNA damage and apoptosis.
The team hypothesizes that the initial electron transport chain dysfunction leads to increased oxidative stress, triggering Cyt c release to induce apoptosis. This promotes mitochondrial dysfunction and creates a pro-oxidant environment. LS12 cells have adaptations via the expression of antiapoptotic and antioxidant proteins, which contribute to their survival.2
They also discovered that mitochondrial dysfunctions manifest not only as altered protein levels and enzyme activities, but also as altered gene expression. By evaluating mRNA and microRNA (miR) expression levels, the team also showed changes in expression for mitochondrial and cellular redox homeostasis pathways. This study suggests that epigenetic mechanisms regulating mitochondrial function are involved in radiation-induced genomic instability in the LS12 cell line.2 Further work on other clonal cell lines may show differences in mechanisms.
1. Aypar, U., Morgan, W.F., and Baulch, J.E. (2011) ‘Radiation-induced genomic instability: are epigenetic mechanisms the missing link?’ International Journal of Radiation Biology, 87 (2), (pp. 179-191)
2. Thomas, S.N., et al. (2012) ‘Quantitative proteomic analysis of mitochondrial proteins reveals prosurvival mechanisms in the perpetuation of radiation-induced genomic instability‘, Free Radical Biology & Medicine, 53 (3), (pp. 618-628)
3. Limoli, C.L., (2000) ‘Chromosomal instability induced by heavy ion irradiation‘, International Journal of Radiation Biology, 76 (12), (pp. 1599-1606)