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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 colorectal carcinoma cell line, HCT-116 was obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM in a humidified atmosphere at 37°C with 5% CO2. Media were supplemented with 10% FBS (fetal bovine serum) and antibiotics (1000 U/mL penicillin and 1000 �μg/mL streptomycin). Animal Studies This animal study was performed in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory animal under assurance number A4107-01 and with the University of Texas IACUC, protocol number 05060201. Metastasized liver tumors were generated by intrasplenic injection of 2 x 10 6 HCT-116 cells in 50 μl of magnesium and calcium free HBSS into female BALB/cAnNCr-nu/nu mice (Strain code 01B70, NCI-Frederick, Frederick, Maryland). Purified AIN-76 rodent diet was modified to contain different concentration of retinyl palmitate and irradiated (Research Diets, Inc). This modified AIN-76 diet contains 2,400 (control-meets National Research Council (NRC) mouse vitamin A requirement (177)), 12,000, 25,000, 50,000, 100,000 or 200,000 IU vitamin A/kg diet as retinyl palmitate. Diet compositions are described in Table 5.1. Diets were stored at 4 °C in black painted containers. Diets and water were consumed ad libitum. Fresh diets were 101

provided every other day and feed consumption was recorded at this time. The mice were weighed weekly. Chemotherapy experiment: After tumor cell injection, mice (n=15 per group) aged 6 to 8 weeks were randomly assigned to diets containing 2,400, 12,000, 25,000, 50,000, 100,000 or 200,000 IU vitamin A/kg diet as retinyl palmitate. These diets provided 1, 5, 10, 20, 40 and 80 X the daily vitamin A requirement of mice. Diets were supplied immediately after surgery and consumed for five weeks, until sacrifice. Chemoprevention experiment: Mice (n=14 per group) between aged 6 to 8 weeks were fed either the 2,400 IU vitamin A/kg control diet or a diet containing 200,000 IU vitamin A/kg diet as retinyl palmitate for four weeks before surgery. After surgery, the mice consumed the same diets for five more weeks prior to sacrifice to examine liver metastases. Immunohistochemistry Livers were extracted, fixed with 10% buffered formaldehyde and paraffin- embedded. Cytokeratin (CK) 20 (sc-17113, Santa Cruz Biotechnology, Santa Cruz, CA), a protein expressed in human goblet cells and enterocytes (178), was used to detect metastasized human colon cancer cells in paraffin-embedded liver sections. For the chemotherapy experiment, two random slides were stained with CK 20 from each liver to determine the presence of microscopic liver metastasis of colon cancer cells. If both 102

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