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(PP207) X-ray microbeam bystander studies with human mammary epithelial cells and fibroblasts.
Blakely, Eleanor *,1, Schwarz, Richard1, Thompson, Al2, Bjornstad, Kathleen1, Chang, Polly1, 4, Rosen, Chris1, Sudar, Damir1, Romano, Raquel3, Parvin, Bahram3, 1 Life Sciences Division, Berkeley, CA, USA2 Advanced Light Souce Division, Berkeley, CA, USA4 Biosciences Division, Menlo Park, CA, USA3 Computational Research Division, Berkeley, CA
ABSTRACT- We are using the unique synchrotron-based source of a 12.5 keV X-ray Microbeam line 10.3.1 at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory (LBNL) to quantitatively characterize low-dose responses of low-LET, radiation-induced bystander effects in a novel tissue-like model of human mammary epithelial cells (HMEC), or normal human fibroblast cells (HFC), or in a third scenario with both cells together in a co-culture system. In our experiments, cultures grown in microwell slide chambers are irradiated with precise stripes of dose up to 100 m wide. Samples are processed for the expression of radiation-induced protein markers with fluorescent immunohistochemistry in a time course from 10 minutes to several hours after exposure. Using fluorescence microscopy on a high-precision-controlled microscope stage and fiducial marked references, the physical locations of the dose stripes are mapped exactly to the location of the biological responses. Computer-based fluorescent analysis of radiation-induced signals has revealed statistically significant differences in the broadening of the effects of the dose stripes to neighboring unirradiated cells with time after exposure for doses below 10 cGy. This involvement of cells not in the irradiated field represents a radiation-induced bystander effect that can be quantitatively evaluated. The intensity of the fluorescence is dose dependent and the fluorescence signal decreases more slowly with time after high-dose (100 cGy) exposure than after lower doses (≤25 cGy). Results from a rapid time course study show that radiation-induced signals in the bystander cells are observed within 10 min but is still significant 3 hrs after exposure. Using the Gene array coupled with the quantitative RT-PCR validation approach, we have obtained evidence demonstrating cell-type specificity in the constitutive expression of genes, as well as dose-dependent Xray-induced genes, known to be involved in ATM/ATR damage responsive pathways and cell adhesion family of genes suggesting cell-type specificity in mechanisms of cell communication. This work was supported by the U. S. DOEs Low Dose Radiation Research Program under Contract No. DE-AC03-76SF00098.
Key words: bystander effects, human mammary epithelial cells, human fibroblasts, X-ray microbeam
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