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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

Necrosis assays. Trypan

Necrosis assays. Trypan blue exclusion assays were performed to measure cell death. Briefly, an aliquot of the cells harvested for the growth curve assays was pelleted by centrifugation, resuspended in 0.5 mL HBSS, and incubated with an equal volume of 0.4% trypan blue solution (Sigma-Aldrich, St. Louis, MO) for five minutes at room temperature before counting with a hemocytometer. Blue cells were scored as necrotic. Flow Cytometry Analysis. To determine if growth inhibition was due to cell cycle arrest and to confirm the absence of apoptosis through lack of a sub-G1 peak, cells were seeded on 60 mm dishes at a density of 3 x 10 5 (HCT15 and SW620) or 2 x 10 5 (HCT-116 and WiDr) cells per dish to provide 50-60% confluence at the time of harvest. To synchronize, cells were plated in serum-free medium for 24 h and treated with 1 (HCT-15) or 3 μg/mL (HCT- 116, SW620, WiDr) aphidicholin for an additional 24 hr. The following day, the cells were washed with PBS and treated with fresh FCS-supplemented media containing 0, 1, and 10 μM retinol. Cells were harvested, fixed in 70% ethanol overnight and stained with 40 μg/mL propidium iodide as described previously (Ormerod). At least 10,000 cells were analyzed per sample using a FACSCalibur machine (Becton Dickinson, San Jose, CA). DNA content was determined using Modfit software version 3.0 (Verity Software House, Inc, Topsham, ME). 19

RESULTS Growth of ATRA-resistant colon cancer cells is inhibited by retinol. The ability of retinol to inhibit cell growth was examined in three ATRA-resistant human colon cancer cell lines HCT-116 (42), SW620 (53), and WiDr (39). HCT-15, an ATRA-sensitive cell line, was chosen to serve as a positive control for the inhibitory effects of ATRA on colon cancer cell growth (54). Serum concentrations of retinol range from 0.5 to 2 μM (55). Therefore, 0.1 μM was selected to represent a sub-physiological, and 1 μM a physiological, concentration of retinol. The highest level, 10 μM retinol, was used as a pharmacological, but potentially therapeutically relevant, concentration. There is very little ATRA (4-14 nM) in the serum (56,57). ATRA levels were chosen to match the concentrations of retinol used and to reflect ATRA levels commonly found in the literature (39-41,58). After 96 h of treatment, the growth of HCT-15 cells was inhibited by ATRA (Figure 2.1A), as expected. In addition, HCT-15 cell growth was also inhibited by retinol in a dose-responsive manner. Cells treated with 10 μM retinol exhibited the largest degree of inhibition to 36.7 ± 7.8 % of control. HCT-116 cell growth was inhibited slightly by 0.1 and 1 μM retinol and this decrease was not significant when compared to the same concentrations of ATRA (Figure 2.1B). However, HCT-116 cell growth was significantly inhibited by 10 μM retinol (37.5 ± 9.2 % of control) when compared to 10 μM ATRA (74.3 ± 4.7 % of control). SW620 and WiDr cell growth was significantly inhibited by treatment with 0.1 and 1 μM retinol for 96 h when compared to ATRA (Figure 2.1C and D) indicating that physiological levels of retinol 20

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