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Chair(s): Hauer-Jensen, Martin
(MS042) Absence of the Smad3 gene product increases radiation resistance of bone marrow stromal cells.
Greenberger, Joel*,1, Zhang, Xichen1, Hricisak, Lauren1, Epperly, Michael1, 1 Radiation Oncology/Medicine, Pittsburgh, PA, USA
ABSTRACT- Irradiation of cells leads to increased synthesis of TGF. Binding of TGF to the TGF receptor results in activation of a signal transduction pathway, which goes through the Smad3 gene product to facilitate a nuclear transcriptional response. Absence of the Smad3 gene in vivo has been shown by Flanders, et al. to decrease ionizing irradiation-induced skin fibrosis. To investigate the role of the Smad3 gene product in bone marrow stromal cell biology, we isolated clonal bone marrow stromal cell lines from both Smad3+/+ and Smad3-/- mouse long term bone marrow cultures (LTBMCs). Smad3-/- cells were smaller in size than Smad3+/+ cells and had a shorter in vitro doubling time of 24 hours compared 48 hours for Smad3+/+ cells. Smad3-/- cells reached a saturation density of 15.3 ± 0.96 x 105 cells compared to 3.76 ± 0.14 x 105 cells for Smad3+/+ cells in 25cm2 tissue culture flasks. Irradiation survival curves utilized doses ranging from 0 to 800 cGy. Irradiated cells from each line were plated in 4-well tissue culture plates and stained with crystal violet 7 days later. Colonies of greater than 50 cells were counted and analyzed by linear quadratic and single hit, multi-target models. Smad3+/+ cells were more sensitive to irradiation, D0 of 0.97 ± 0.01 Gy compared to 1.51 ± 0.16 Gy for Smad3-/- cells (p=0.0374). Smad3+/+ cells exhibited increased apoptosis at 24 hour following 5 or 8 Gy irradiation with 15 or 43% of cells undergoing apoptosis compared to less than 1% for the Smad3-/- cells. Growth of Smad3+/+ cells in the presence of SB-431542 (inhibitor of the TGF receptor) increased radiation resistance and decreased apoptosis to levels comparable to that of Smad3-/- cells. The data indicate that the TGF receptor pathway plays an important role in radiation sensitivity of clonal bone marrow stromal cell lines.
Key words: Smad3, TGFbeta, radioresistance
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