Project Abstract:

Title: Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation.

PI: William F. Morgan.

This is a “glue grant” to work in collaboration with Drs David L. Springer from Pacific Northwest National Laboratory (PNNL) and John H. Miller from Washington State University, Tri-cities (WSU) on their DOE Low Dose grant funded last year. The goal of their application also titled “Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation” is to purify, isolate and identify the factor(s) responsible for perpetuating chromosomal instability in GM10115 cells. This collaborative program evolved from studies in the Morgan laboratory demonstrating that genomically unstable cells secrete/shed a soluble factor(s) into the culture medium that generate DNA double strand breaks leading to cytogenetic change and ultimately apoptosis. We have shown that these factors elicit a cytotoxic Death Inducing Effect (DIE) when parental GM10115 cells are exposed and hypothesize these factors cause the persistent chromosomal instability that characterize our unstable clones.

Currently the DOE Low Dose Program funds Dr. Springer to complete the biochemical characterization of DIE, identify candidate DIE factors and estimate relative abundance of these factors using mass spectrometry approaches. Under the auspices of this same grant the DOE Low Dose Program is also funding Dr. Miller to integrate data generated by Dr. Springer with existing knowledge of cellular signaling pathways to guide studies whereby candidate factors involved in DIE will be investigated and develop a rank-ordered list of factors from DIE medium according to the likelihood that they are DIE factor(s). These will be prioritized for analysis in this glue grant based on abundance changes and cellular signaling pathways that involve these factor(s) from the published literature.

This “glue grant” is to provide proof of principal that candidate factors identified in the currently funded project do indeed modulate DNA damage, genomic instability and cytotoxicity in parental GM10115 cells. We will test for DIE activity after the various medium manipulations performed by Dr. Springer and verify experimentally the abundance changes he identifies. We will test the hypothesis that factors with altered abundances in medium from unstable clones are responsible for DIE by adding that compound to fresh medium and investigating instability and cytotoxicity. Alternatively, compounds missing or significantly reduced in unstable medium will be investigated by inactivating candidate factors with immunoneutralizing antibodies and determine the effect on instability and cytotoxicity.

We have a unique opportunity to uncover the mechanism for delayed radiation induced genomic instability. We have the radiation induced unstable clones, and we have demonstrated that they exhibit DIE. DIE is a robust assay so the evaluation of candidate compounds is straightforward and unambiguous. The DOE Low Dose Program is currently funding investigators with the expertise and technologies to drive the studies described in this application. Our hypothesis that radiation induced instability is due to secreted/shed soluble factors represents a paradigm shift in thinking about the mechanism of these non-targeted effects. Successfully accomplishing the goals proposed would permit rational evaluation of these responses in risk assessment and their implications for radiation-exposed individuals.

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