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Cell Transforming Bystander Signals

Thomas Weber, Principal Investigator

Funding Agency: U.S. Department of Energy, Low Dose Radiation Research Program

EYFP Bystander Colony.
(Top) Illustration of the bystander transformation assay. JB6 cells transfected with membrane-localized enhanced yellow fluorescent protein (EYFP-membrane) are mixed with sham exposed or irradiated JB6 cells post irradiation, and the co-culture is maintained for 14 days. On day 14, total and EYFP-expressing colonies are counted to derive the bystander-specific transformation response, representing the total number of fluorescent colonies induced by co-culture with irradiated or sham exposed cells (bottom). View full image.

Radiation carcinogenesis is complex and may involve paracrine or intercellular signaling activities broadly referred to as the bystander effect. Radiation-induced bystander signals with the potential to influence carcinogenesis responses have been observed in a number of experimental model systems. However, bystander signal identity and modes of action remain unclear. The Cell Transforming Bystander Signals project team at Pacific Northwest National Laboratory (PNNL) is addressing these knowledge gaps. Specifically, researchers are working to define the role of annexin A2 and osteopontin, paracrine signals produced by irradiated cells that are firmly linked to human carcinogenesis, in the transformation response induced in bystander cells by low dose radiation.

Low dose radiation induces the expression of annexin II in a dose- and time-dependent manner.
Annexin A2 is observed as a soluble paracrine factor in the medium of cells exposed to low dose radiation. Acutely following radiation exposure, a low molecular weight form of annexin A2 (p23) is observed, while the full length product (p36) predominates at later time points. View full image.

We have demonstrated that irradiated cells secrete a soluble factor that induces the transformation of non-irradiated bystander cells. Annexin A2 was identified by mass spectrometry as an abundant paracrine signal in our model. Importantly, annexin A2 is believed to play an important role in the etiology of oxidative stress-induced cancers and also functions as a prosurvival factor. Further, annexin A2 is linked to the activation of matrix metalloproteinases (MMPs) that regulate the release of ligands associated with tumor promoting activities from the plasma membrane. Osteopontin has been identified as a novel substrate for MMPs and, consistent with the link between annexin A2 and MMP activity, osteopontin secretion also specifically increases in response to low dose radiation exposure in an apparent MMP-dependent fashion. Osteopontin treatment alone is sufficient to induce cell transformation, suggesting that the annexin A2-osteopontin pathway is a likely candidate for the observed transforming bystander signal. Examination of this pathway is broadly relevant, because annexin A2 and osteopontin are widely implicated in the etiology of human cancers. Because annexin A2 and osteopontin have also been implicated as prosurvival factors, the results of this work may have cross-cutting implications in the radioadaptive response.

Annexin II-osteopontin pathway in clonogenic survival and the transformation response to low dose radiation.
To examine the role of annexin A2 in clonogenic survival and the transformation response to low dose radiation, researchers at PNNL are using JB6 cells transfected with annexin A2 short interfering RNA (siRNA or shRNA). Illustrated are the pooled clones derived from transfection of JB6 cells with annexin A2 shRNA or empty vector, demonstrating selective knockdown of annexin A2 by Western blot (top) and epifluorescence microscopy (bottom, Annexin A2 is green and nuclei are blue). View full image.

Using a co-culture model in which irradiated cells are mixed with non-irradiated cells that have been transfected with green fluorescent protein to discriminate the two populations, we are studying the total and bystander-specific transformation response as a function of low doses of radiation (0.3 – 10 cGy) and dose rate, determining how long the cells must be in communication for a transformation response to occur, and defining critical time points associated with the secretion of transforming paracrine signals. To determine the role of annexin A2 in the bystander-specific transformation response, we have developed an annexin A2 knockdown model. Studies are currently underway to improve our understanding of annexin A2 signaling in low dose radiation responses using this model.

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