Research Highlights
November 2004
Mechanisms of 3D intercellular signaling in mammary epithelial cells in response to low dose, low-LET radiation: Implications for the radiation-induced bystander effect
Pacific Northwest National Laboratory scientists are developing methods to monitor living cells in real time and characterize responses to low-dose radiation to ultimately understand the influence of radiation-induced bystander effects (RIBE) on long-term growth and survival of cells grown in three-dimensional (3D) cultures. These methods include standardizing 3D cell culture systems and using novel high-speed confocal microscopy in situ to visualize the transmission of RIBE signaling in a 3D human mammary epithelial system.
This is important because previous in vitro observations in vitro of cells in two-dimensional (2D) culture could have very little relevance as compared to 3D systems that more closely mimic reality. Researchers are using human mammary epithelial cells grown on extra-cellular matrix material to generate either complex or simple structures. Using these 3D cell systems in combination with PNNL's newly developed, high-speed confocal microscope enables the real-time 3D visualization of cell signaling.
The confocal microscope monitors signals in bystander cells following exposure to low doses of low-linear energy transfer (LET) radiation. Cell survival, growth, cell differentiation, radiation-induced activation of signaling pathways, and changes in patterns of gene induction will be determined as a function of both dose and the proportion of cell population irradiated. Bystander-induced changes in cell signaling in this 3D system will be compared to bystander effects in simple tissue culture systems.
The PNNL scientists are also using flow cytometry to analyze structures and determine whether there are long-term survival consequences from low-dose radiation exposure. Cells are tagged with fluorescent proteins to differentiate the irradiated from the non-irradiated cells. Because of the relatively low numbers of cells in the experimentally generated 3D cell structures, the scientists were unsure if this type of analysis would be appropriate; however, it indeed enabled them to identify and quantify the two cell populations that comprised the 3D structure. The results are shown here.
Fluorescence Activated Cell Sorter (FACS) analysis of mammary cell 3D structures containing fluorescent and non-fluorescent cells. Structures were formed from a mixture of 50% fluorescent cells. After 1 week in culture the structures were dissociated and analyzed by FACS. These results are shown in the middle panel where the right-hand peak represents the cells expressing red fluorescent protein (compare to the left panel with all fluorescent cells and the right panel with no fluorescent cells). The relative proportion of fluorescent cells in the week-old structures was 35%.
Shown here is an acinus, which is the 3D structure that is formed by MCF10A breast cells grown on extracellular matrix. It is comprised of cells expressing a gene for monomeric red fluorescent protein. The use of this fluorescent dye resulted in fully viable cells that were able to form 3D structures on matrigel. This particular structure is also stained with a green dye (Syto 13) that delineates the cell nucleus. This acinus is fully mature with all the cell nuclei at the structure periphery.
The scientists have also been investigating intercellular communication via gap junctions by introducing a dye into the cells, using a scrape-loading technique, and then recording the progressive spread of the dye over time using the confocal microscope. One such study is shown below. In the absence of the gap junction inhibitors Lindane and AGA, a progressive expansion of the dye (shown in aqua pseudo color) occurs from the acellular, scraped area to cells more distal to the scrape.
This study of bystander-induced signaling in 3D systems will give new insight for understanding the importance of RIBE in low-dose risk assessment. The work is one of four current PNNL projects funded by DOE's Office of Biological and Environmental Research's Low Dose Radiation Research Program. The PNNL team members are Lee K. Opresko, Marianne B. Sowa-Resat, and William B. Chrisler. The confocal microscope was developed by H. Steven Wiley, Marianne B. Sowa-Resat, Kenneth Perrine, and Derek Hopkins.
Three-Dimensional Images of Mammary Cell Structures - Cells are labeled with syto 13 (green dye) and are expressing low levels of red flourescent protein