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Mechanisms of Three-Dimensional Intercellular Signaling in Mammary Epithelial Cells in Response to Low Dose, Low Linear Energy Transfer (LET) Radiation: Implications for the Radiation-Induced Bystander Effect

Lee Opresko and Marianne Sowa, Principal Investigators

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

Micronuclei, indicated by arrows, are present in 3-D structures following irradiation (3-D reconstruction).

Current studies on cell physiology and reaction to stimuli usually are done using cells cultured on two-dimensional surfaces. This geometry greatly simplifies the methodology used to observe and propagate cells; however, it places great constraints on how one can investigate intercellular communication and stress responses. Researchers at Pacific Northwest National Laboratory (PNNL) are developing the tools necessary to analyze cell signaling in a three-dimensional (3-D) environment. We have designed and assembled a high-speed confocal microscope that can simultaneously acquire two-color images at speeds up to 30 frames per second. This provides the capability to perform near-real-time fluorescent resonance energy transfer (FRET) and ratiometric analysis of confocal images.

3-D mammary cell structures.
Effect of radiation on 3-D mammary cell structures. View full image.

Using this novel imaging tool, the team investigating the Mechanisms of Three-Dimensional Intercellular Signaling in Mammary Epithelial Cells in Response to Low Dose, Low Linear Energy Transfer (LET) Radiation is visualizing radiation-induced bystander signaling in 3-D cell cultures in situ . It is well established that radiation-induced bystander effects (RIBE) can be evoked in vitro using cells in two-dimensional culture; however, entirely different types or levels of signaling may be occurring in three dimensions. Using a model 3-D mammary epithelial cell system, we are investigating the mechanism of low LET RIBE. Confocal capabilities are necessary to allow 3-D reconstruction of signal propagation as well as to permit the analysis of very localized portions of cells. The combination of our 3-D cell system and our newly developed, high-speed confocal microscope will enable the real-time, 3-D visualization of cell signaling.

We are also developing tools to monitor signals in living cells in real time so we can characterize the response patterns initiated by different stimuli. We will obtain quantitative information on the spatial and temporal characteristics of signals that propagate from one cell to another. We will follow signaling initiated in intact structures and monolayers by various doses of low LET X-ray radiation (0.1 – 10cGy) as well as by the localized delivery of radiomimetic agents. Understanding such processes is crucial to predicting how tissues respond to injury. Signal propagation could either be mediated by gap junctions or by diffusible factors that are released from the initially stimulated cell; we will determine the relative contribution of each mode of signal transmission. We will also determine whether responses by the target cell and the surrounding cells are rapid and transient in nature or quite slow and long lasting. To observe such responses requires a technology capable of capturing an image quickly and repeatedly as well as for extended periods of time. The long-term consequences of the low LET RIBE in both 2- and 3-D culture will be examined by mixing irradiated and non-irradiated cells. The effect of dose and the proportion of irradiated cells on cell survival and growth, cell differentiation, activation of signaling pathways, and the pattern of gene induction will be determined.

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