Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation
Funding Agency: U.S. Department of Energy, Low Dose Radiation Research Program
Evidence of the Death-Inducing Effect. A: Within 30 minutes of adding media conditioned by unstable clones to parental GM10115 cells, a significant number of γH2AX foci are seen in recipient cells, which is indicative of induced DNA double strand breaks. B: After 24 hours of growth in media conditioned by unstable clones, many parental GM10115 cells have micronuclei. C: Evidence of apoptosis by Annexin V staining, note the micronuclei. D: Evidence of apoptosis by TUNEL assay.
There is compelling evidence from in vitro tissue culture studies, in vivo animal models, irradiated human subjects, and radiotherapy patients that a variety of biological effects occur outside the radiation field and to the progeny of irradiated cells. An understanding of these non-targeted mechanisms of radiation damage is needed to reliably assess the health risks from low-level exposure to ionizing radiation. The Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation project team at Pacific Northwest National Laboratory (PNNL) in collaboration with John Miller at Washington State University is studying the mechanisms of non-targeted radiation damage using a cell model developed by Bill Morgan at the University of Maryland. Through this project, we have the opportunity to apply state-of-the-art proteomic and bioinformatic tools to study the mechanisms of radiation-induced genomic instability.
After irradiation of human-hamster hybrid GM10115 cells, Dr. Morgan et al. isolated several chromosomally unstable clones that have persistently elevated levels of oxidative stress and DNA damage. This condition normally leads to programmed cell death but in these cell lines appears to drive continual chromosomal rearrangement. Furthermore, media conditioned by the chromosomally unstable clones is cytotoxic to parental GM10115 cells - a phenomenon termed the death-inducing effect (DIE). We hypothesize that the clones release pro-apoptotic factors into their growth medium as a consequence of ongoing DNA damage. Whereas the clones are able to block the effects of these factors, they are fatal to the parental line. Using proteome-wide, high-throughput mass spectrometry we are working to characterize biochemically the soluble factor or factors released upon radiation exposure of GM10115 cells with a focus on those responsible for the DIE.
Preliminary data from our laboratory suggests that the DIE factor or factors is a protein. We are working to confirm this hypothesis, to identify the protein size range, and to determine whether DIE activity is sensitive to oxidation and/or proteolysis. We are using mass spectrometry to characterize the function of DIE proteins (e.g., ligand, receptor, and/or protease) and to estimate the proteins' relative abundances. We will integrate our mass spectrometry data with existing pathway information and implement METACORE and CellDesigner to identify signaling pathways that involve candidate DIE factors and to model the observed protein abundance changes in DIE media relative to controls. After identifying a candidate factor or factors, we will obtain proof of principal that the candidates modulate cytotoxicity in parental GM10115 cells.
The identification and biochemical characterization of DIE factors (1) will give us insight into unstable GM10115 clones' persistently elevated levels of oxidative stress and DNA damage, (2) will help us understand how unstable cells tolerate cytotoxic factors released into their medium, and (3) is a critical step in understanding the role of the DIE in radiation-induced genomic instability of GM10115 cell lines. Importantly, elucidating the mechanisms of radiation-induced genomic instability in GM10115 cell lines may have far-reaching implications, because genomic instability is probably the most significant non-targeted effect of radiation exposure for cancer risks in humans.