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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 The human carcinoma cell line, HCT-116, and the human colorectal adenoma cell line, SW620, were obtained from the American Type Culture Collection (Manassas, VA) and grown as recommended. HCT-116 cells were grown in McCoy’s medium and SW620 cells in DMEM in a humidified atmosphere at 37°C with 5% CO2. All medium was supplemented with 10% FBS (fetal bovine serum) and antibiotics (1000 U/mL penicillin and 1000 μg/mL streptomycin). Migration and Invasion Assays HCT-116 and SW620 cells were serum starved for 48 h before seeding at a density of 1 x 10 5 cells/well. Uncoated Boyden chambers were used to assess the effect of retinol on cell migration while Matrigel coated Boyden chambers (Becton Dickinson, Franklin Lakes, NJ) were used to determine the ability of retinol to inhibit cell invasion through a basement membrane. To measure cell migration, the upper portion of the chambers contained 0 (ethanol vehicle control), 0.1, 1, or 10 μM retinol. Retinol was prepared as 10 mM stock in 100 % ethanol. All treatments, including control, received equal volumes of ethanol vehicle and all retinol manipulations were performed in the dark. A filter containing pores 8 μm in diameter separated the cells from a lower chamber containing 10% FBS, which served as a chemoattractant. After 8 h all cells were removed from the upper chamber using a cotton swab. Cells that had migrated 45

through the membrane were fixed in methanol prior to staining with propidium iodide. The bottom of the filter was examined microscopically and the number of stained cells present in ten random fields of view were counted with a 20X objective on an Olympus upright fluorescence microscope to determine cell migration as described (96). To examine the effect of retinol on cell invasion, the upper portion of the chambers contained 0 (ethanol vehicle control), 1, or 10 μM retinol while the lower portion, separated by an 8 μm pore-sized filter coated with Matrigel, contained 20 ng/ml hepatic growth factor (HGF) (Sigma-Aldrich, St. Louis, MO) and 10% FBS, which served as chemoattractants. Cell invasion was measured after 24 h. All cells remaining in the upper portion of the chamber were removed. Cells that had migrated through the membrane were fixed in methanol prior to staining with propidium iodide and quantified, as described above. To determine if the effects of retinol on cell invasion required the transcription, particularly RAR-mediated transcription, or translation of new genes, cells were treated with 2 mg/ml actinomycin D, 10 μg/ml cycloheximide, or 10 μM of a RAR pan- antagonist (AGN193109, Allergan, Irvine, Ca), with or without 1 or 10 μM retinol added to the upper portion of Matrigel-coated Boyden chambers. Twenty-four h later, cell invasion was quantitated as described above. To confirm the role of MMP-2 and –9 in colon cancer invasion, MMP activity was blocked using specific antibodies against MMP-2 (#AB809, Chemicon, Temecula, CA) and MMP–9 (#AB19016, Chemicon, Temecula, CA). Control cells were incubated with a non-specific IgG antibody (#SC-2027, Santa Cruz Biotechnology, Santa Cruz, CA). HCT-116 and SW620 cells were plated in Matrigel-coated Boyden chambers as 46

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