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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

MATERIALS AND METHODS

MATERIALS AND METHODS Tissue Culture Three human colorectal adenoma cell lines, HCT-15, SW620, and WiDr, and one human colon carinoma cell line, HCT-116, were obtained from the American Type Culture Collection (Manassas, VA) and grown as recommended. HCT-15 cells were grown in MEM, HCT-116 in McCoy’s medium, and SW620 and WiDr cells in DMEM in a humidified atmosphere at 37°C with 5% CO2. All medium was supplemented with 10% FCS (fetal calf serum) and antibiotics (1000 U/mL penicillin and 1000 μg/mL streptomycin). The experiment was repeated with each cell line grown in either supplemented DMEM or McCoy’s medium. Medium type did not affect cell growth. Cells were seeded in 12-well culture dishes at a density of 1 x 10 4 cells per well. The following day the medium was removed and replaced with medium containing 0, 0.1, 1 or 10 μM ATRA or all-trans-retinol (Sigma, St Louis, MO). All retinoids were prepared as 10 mM stocks in 100 % ethanol. All treatments, including control, received equal volumes of ethanol vehicle and all retinoid manipulations were performed under subdued lighting. All treatments were performed in duplicate. Cells were harvested using trypsin and counted via hemocytometer every 24 h for four days. Retinoid Extraction and HPLC Analysis. To examine retinol metabolism, cells were seeded in 60 mm dishes at the following densities to yield 60 to 80% confluency and maximum HPLC detection 15

sensitivity at the time of harvest: 5 x 10 5 cells/dish for 24 h, 2.5 x 10 5 cells/dish for 48 h, 1 x 10 5 cells/dish for 72 h, and 5 x 10 4 cells/dish for 96 h. Twenty-four hours after plating, cells were treated with 0, 1, and 10 μM retinol for 24, 48, 72 or 96 h. Sixteen hours before harvest, the culture medium was removed and replaced with medium containing 5% FCS and 50 nmol/L [ 3 H]retinol (specific activity = 52.5 Ci/mmol). Cells and medium were harvested 2, 4, 8, and 16 h after the addition of label as described previously (50). A control of labeling medium without cells was also incubated for 16 h. F9 murine teratocarcinoma cells, treated with 1 μM ATRA for 48 hr and incubated with 50 nM [ 3 H]-retinol for 16 hr were used as a positive control for 4-oxoretinol production (17). Retinoids were extracted and separated using a Waters Millennium HPLC system as described previously (51). Cell Transfection and CAT Assays. To examine the possibility that an undetected metabolite of retinol was activating RAR/RXR-mediated transcription, all cell lines were transiently transfected with pRARE-CAT (generously provided by Dr. Dianne Soprano, Temple University, Philadelphia, PA). Cells were seeded on to 12-well plates at a density of 1.75 X 10 5 cells/well, and incubated overnight in FCS-supplemented medium. The following day cells were transfected using Lipofectamine 2000 (Promega, Madison, WI) according to the manufacturer’s protocol with 1 μg of pRARE-CAT and 0.5 μg of pSV-β-gal. Twenty-four hours later the transfection medium was removed and the cells were treated with fresh medium containing 0, 1, and 10 μM ATRA or retinol. The cells were 16

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