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First published online 16 January 2007
doi: 10.1242/jcs.03348

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Wnt/β-catenin mediates radiation resistance of Sca1⁺ progenitors in an immortalized mammary gland cell line

Mercy S. Chen^1,*, Wendy A. Woodward^2,*, Fariba Behbod¹, Sirisha Peddibhotla¹, Maria P. Alfaro¹, Thomas A. Buchholz² and Jeffrey M. Rosen^1,

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, M638a Houston, TX 77030-3498, USA
² Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA

View larger version (22K): [in this window] [in a new window]   Fig. 1. CDβgeo Sca1+ cells are capable of self-renewal and expansion. (a) CDβgeo cells were stained with FITC-conjugated antibody against Sca1, and analyzed by flow cytometry. Two populations can be distinguished based on Sca1 fluorescence: Sca1+ 13.1%; and Sca1- 67.4%. (b) Cells were sorted into Sca1+ and Sca1- populations directly into 96-well plates containing growth-factor-reduced Matrigel at 500 cells/well, and clones were counted after 10 days. The Sca1+ cells gave rise to 47±14 colonies in Matrigel, whereas the Sca1- produced no colonies. Colonies were stained with Crystal Violet and counted by two independent researchers. (c) CDβgeo Sca1+ population give rise to 75±4.2% Sca1+ and 24±4.1% Sca1- cells. (d) CDβgeo Sca1- cells remained mostly Sca1- after culture, and gave rise to 94±1.5% Sca1- cells and only 6±1.45% Sca1+ cells.

View larger version (94K): [in this window] [in a new window]   Fig. 2. CDβgeo cells can self-renew in suspension culture and express putative stem cell markers. (a) Bright-field image. Approximately 1 in 600-1000 CDβgeo cells forms a mammosphere. Bar, 100 µm. (b-e) CDβgeo mammospheres were cytospun onto glass slides, and immunostained for CD49f (b, red. Bar, 20 µm), TIE-2 (c, red. Bar, 20 µm), K14 (d, red. Bar, 50 µm), and keratin 6 (e, green. Bar, 50 µm). All nuclei were counterstained with DAPI (blue).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Stabilized β-catenin expression enriches for stem/progenitor cells by increasing the number of mammospheres. CDβgeo cells transduced with GFP (control), β-catenin, or β-engrailed were FACS sorted into Sca1+ and Sca1- populations and grown in suspension at a density of 20,000 cells per well for 14 days. The mammospheres were passaged once after 7 days, and the secondary mammospheres were counted using a Leica dissecting microscope. GFP control Sca1+ vs Sca1- *P<0.02; GFP Sca1+ vs β-catenin Sca1+, **P<0.008. The efficiency of mammosphere formation is calculated as the number of mammospheres per the number of plated cells. Data were collected from three individual experiments performed in triplicate.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Radiation does not decrease colony formation from Sca1+ cells in Matrigel. (a) 500 Sca1- or Sca1+ cells treated with either 0 or 2 Gy were sorted using flow cytometry into 96-well plates containing 10 µl of Matrigel. Representative images of wells containing 500 cells are shown. Bars, 2 mm. Sca1+ 0 Gy vs 2 Gy, P<0.07 by two-tailed t-test. (b) CDβgeo cells were irradiated at either 0 or 2Gy, sorted into Sca1+ and Sca1- populations. Sorted cells were stained with 7-AAD and pyronin Y to discriminate G0 from different stages within G1(see Materials and Methods). There is no obvious difference in the cell cycle profiles between the Sca1+ and Sca1- population, at either 0 or 2 Gy. Cell cycle was analyzed using flow cytometry. Statistics were assessed on FlowJo version 4, Tree Star, Inc. (c) Significant differences in proliferation 144 hours following irradiation. MTT assay was performed on the sorted Sca1+ and Sca1- cells after irradiation for 144 hours at 0 (sham), 2, 4 and 6 Gy. Following irradiation, the sorted cells were serum starved (0.1% adult bovine serum) for 48 hours, and then stimulated to proliferate with serum (5% adult bovine serum). See Materials and Methods for details. The bars indicate average OD (absorbance) and the error bars are the standard deviation of three replicate OD measurements within each group. There were statistically significant differences in growth rate between the Sca1+ and Sca1- cells at 2 Gy and 4 Gy, *P<0.0001, **P<0.04, two-tailed t-test. (d) Growth properties of Sca1+ and Sca1- cells at 0 and 4 Gy. Cells were directly sorted into 6-well plates at 50,000 cells per well, and the medium was replaced every 48 hours with fresh medium (plus 5% adult bovine serum). Cells were counted every 48 hours using a hemocytometer. The graph shows the growth curve of 0 Gy Sca1+ (blue), 0 Gy Sca1- (red), 4 Gy Sca1+ (yellow), 4 Gy Sca1- (green). Each data point represents the mean ± s.e.m. of three experiments in triplicate. The different cell culture conditions may account for the differences in the kinetics of recovery.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Radiation induces more DNA damage foci in Sca1- cells. (a) Sca1+ and Sca1- cells were sorted directly onto glass slides following irradiation at 4 Gy and immunostained with anti--H2AX (red). Nuclei were stained with DAPI (blue). Bars, 5 µm. (b) There were significantly more DNA-damage foci in the Sca1- population (4 Gy Sca1+ vs Sca1-, *P<0.0001). A minimum of 200-300 cells were counted in each group.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Differences in β-catenin level and localization between Sca1+ and Sca1- subpopulations. (a) Sca1+ and Sca1- subpopulations were stained with non-phosphorylated β-catenin antibody and analyzed by flow cytometry with an Alexa Fluor 488 secondary antibody against β-catenin. Comparison of the fluorescence intensity of Alexa Fluor 488 from Sca1+ and Sca1- cells (in blue) with that from IgG control (in red). (b) Immunostaining for non-phosphorylated β-catenin in Sca1+ and Sca1- cells after 0 Gy (sham irradiation) and 4 Gy. β-catenin is visualized in green, and the nuclei are stained with DAPI (blue). Bars, 5 µm. Images were captured by deconvolution microscopy using a Zeiss AxioVert S100 TV microscope and a DeltaVision restoration microscopy system (Applied Precision, Inc.). For high-resolution deconvolved images, captured raw images were deconvolved with the DeltaVision constrained iterative algorithm. (c) β-Catenin regulates survivin expression following irradiation. CDβgeo cells were transduced with control vector, β-catenin, or β-engrailed, and then irradiated at 0 or 2 Gy. Cells were harvested after 24 hours, FACS sorted into Sca1+ and Sca1- subpopulations, and RT-PCR was performed using the ABI real-time PCR system. β-gal control Sca1+, 0 Gy vs 2 Gy, *P<0.02; 0 Gy GFP Sca1+ vs 0 Gy β-cat Sca1+, **P<0.03, β-cat Sca1+, 0 Gy vs 2 Gy, ***P<0.04. Data were obtained from five individual experiments performed in triplicate.

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