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IFITM6 Expression Is Increased in Macrophages of Tumor-Bearing Mice

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IFITM6 Expression Is Increased in Macrophages of Tumor-Bearing Mice 531-536.qxd 20/12/2010 01:11 ÌÌ ™ÂÏ›‰·531 ONCOLOGY REPORTS 25: 531-536, 2011 531 IFITM6 expression is increased in macrophages of tumor-bearing mice JEONG HYE HAN, SUNYI LEE, YUN SUN PARK, JEONG SU PARK, KUN-YONG KIM, JONG SEOK LIM, KI SOOK OH and YOUNG YANG Department of Biological Science, Sookmyung Women's University, Seoul 140-742, Republic of Korea Received August 31, 2010; Accepted October 13, 2010 DOI: 10.3892/or.2010.1092 Abstract. The family of interferon-induced transmembrane and parasitic infections activate natural killer cells and protein (IFITM) genes consists of IFITM1, 2, 3, 5, and 6. macrophages. They increase the recognition of infected cells They encode cell surface proteins that modulate cell-cell or tumor cells by up-regulating antigen presentation. Binding adhesion and cell differentiation. In a previous study, we of IFNs to their receptors results in phosphorylation of showed that IFITM1 is involved in the immune escape and JAK1/2 and TYK2. Activated JAK1/2 and TYK2 activate metastasis of gastric cancer cells. In this study, we determined STAT1/2 through phosphorylation. In turn, IFNs induce the the difference in expression of IFITM family genes in tumor- expression of several genes to execute their function (4). bearing mice. IFITM1 and 6 were found to be significantly Among these genes are the IFITM family of genes. IFITM1, increased. IFITM6 gene expression was increased only in the 2, and 3 have been experimentally identified (5), and bio- spleen of tumor-bearing mice but not in the bone marrow, informatic analysis has further revealed IFITM5 and IFITM6 lymph node, or thymus. IFITM6 expression was induced in (6). The IFITM family proteins encode two putative trans- various macrophages, including splenic, thioglycollate-elicited, membrane domains. IFITM1, also known as 9-27 and Leu13, and bone marrow-derived macrophages, but not in T cells. was first identified as an IFN-inducible gene (7,8). IFITM1 Lipopolysaccharides (LPS) also increased IFITM6 expression induces the homotypic adhesion of cells (9), and IFITM1- 24 h after administration, and Toll-like receptor 1, 2, 3, 4, expressing tumor cells show resistance to natural killer cell and 9 agonists stimulated IFITM6 expression. These findings cytotoxicity and an increase in the invasiveness of gastric imply that the increase in IFITM6 expression may be involved cancer cells (10). IFITM1 also inhibits extracellular signal- in macrophage functions of tumor-bearing mice. regulated kinase-mediated p53 phosphorylation, which results in the stabilization of p53 that, in turn, results in anti- Introduction proliferative activity (11). IFITM1, 2, and 3 are differentially expressed during development of primordial germ cells (5). Protective immune function is impaired early in cancer IFITM6 expression appears to be much more restricted to development, and this dysfunction progresses to metastatic macrophages (12). disease. The nature of and molecular mechanisms underlying Previously, we showed that IFITM1 has an immune immune dysfunction are not clearly defined. Potential escape function in gastric cancer cells. Little is known about mechanisms of impaired immune function in cancer include the function of IFITM2, 3, 5, and 6 in tumor-bearing mice. In defects in antigen recognition, costimulation, and removal of this study, we investigated changes in the expression of the target using natural killer cells activated by interferons IFITM family genes in the spleens of tumor-bearing mice. (IFNs) (1). Although type I IFN-· and -ß were originally The expression of IFITM6 was increased. Thus, we focused thought to be mainly antiviral agents, recent studies have on whether IFITM6 is involved in tumor immune functions. shown their importance as immunomodulators (2), whereas the immunomodulatory activity of IFN-Á has long been Materials and methods appreciated (3). IFNs released by lymphocytes against viral Reagent. Lipopolysaccharide (Escherichia coli, serotype 055:B5) was purchased from Sigma-Aldrich (St. Louis, MO, _________________________________________ USA). The mouse Toll-like receptor (TLR) 1-9 Agonist kit was purchased from Invivogen (San Diego, CA, USA). Murine Correspondence to: Dr Young Yang, Department of Life Science, IFN-Á and IL-4 were purchased from Peprotech (Seoul, Korea). Sookmyung Women's University, Seoul 140-742, Republic of Korea Phorbol-12-myristate-13-acetate (PMA) and ionomycin were E-mail: [email protected] purchased from Sigma-Aldrich. Key words: interferon-induced transmembrane protein, macro- Cell cultures. The mammary carcinoma cell line 4T1 derived phages, breast cancer, lipopolysaccharides, Toll-like receptor from Balb/c mice and the murine macrophage cell line RAW 264.7 were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Daegu, Korea) supplemented with 10% 531-536.qxd 20/12/2010 01:11 ÌÌ ™ÂÏ›‰·532 532 HAN et al: IFITM6 IS INCREASED IN MACROPHAGES fetal bovine serum (Hyclone, South Logan, UT, USA), and 1 day previously. For the non-contact co-culture experiments, the cells were maintained at 37˚C in a humidified atmosphere a plate of six 3.0-μm pore size transwells (Corning, NY, of 5% CO2. USA) was used. The thioglycollate-elicited macrophages were cultured in the transwell chamber, and the 4T1 cells were Syngeneic tumor model. Eight-week-old and 12-week-old cultured in the bottom of the plate. Balb/c mice were purchased from Samtako Biokorea (Osan- City, Korea). All mice were housed in a 12-h lights-on/12-h RNA extraction and reverse transcription-PCR. The total lights-off cycle with free access to standard rodent food and cellular RNA from tissues and cells was extracted using the clean water at animal care facilities in Sookmyung Women's RNAiso plus kit (Takara, Shiga, Japan). A total of 3 μg RNA University. Cultured 4T1 cells were harvested with 0.05% were transcribed with 0.5 μg of random primer using reverse trypsin-EDTA (Invitrogen, Carlsbad, CA), washed with 1X transcriptase (Fermentas, Ontario, Canada) at 42˚C for 1 h. phosphate-buffered saline (PBS), and resuspended at a The following primer sequences used for the amplification: IFITM1, forward primer (5'-GGAGCAGCAAGAGGTG concentration of 1x106 cells/ml in PBS. A total of 200 μl 4T1 cells were administered to the 12-week-old Balb/c mice by GTTG-3') and reverse primer (5'-GATGTTCAGGCACTTG subcutaneous injection. On day 15 after tumor inoculation, all GCGG-3'); IFITM2, forward primer (5'-TCTTGTCCACC mice were sacrificed, and spleens, thymuses, tumors, lymph AATGCCGGG-3') and reverse primer (5'-AACCACATC nodes, and bone marrows were collected. All animal GCCCACCATCT-3'); IFITM3, forward primer (5'-CAA experiments were performed as approved by the Ethics GCCTTCATCACCGCTGC-3') and reverse primer (5'-GGG committee of our university. CTCCAGTCACATCACCC-3'); IFITM5, forward primer (5'-TCCATCATCCCGCAAGGCTG-3') and reverse primer Isolation of macrophages from various tissues in Balb/c mice. (5'-ATGGGGGCACCAATGTCCAC-3'); IFITM6, forward Thioglycollate (TG) medium (Difco Laboratories Inc., MI, primer (5'-GGTTAAGAGGGATCC-3') and reverse primer USA) was prepared according to a previous study (15). The TG (5'-CTTTGACAGTGCATG-3'); IL-1ß, forward primer medium was intraperitoneally administered, and thioglycollate- (5'-ACAGATGAAGTGCTCCTTCCA-3') and reverse primer elicited macrophages (TG-macrophages) were obtained from (5'-GTCGGAGATTCGTAGCTGGAT-3'); IL-2, forward Balb/c mice using peritoneal lavage four days after the primer (5'-CTGCCACCTAAGTGTGGGCT-3') and reverse injection of TG (15). The harvested cells were washed and primer (5'-GTACATGCCTGCAGGACTTGAGG-3'); IL-6, incubated in DMEM supplemented with 10% fetal bovine forward primer (5'-GAAATGAGAAAAGAGTTGTGC-3') serum (Hyclone), penicillin, and streptomycin. and reverse primer (5'-ATTGGAAATTGGGGTAGGAAG-3'); Bone marrow cells were isolated from Balb/c mouse tibias IFN-Á, forward primer (5'-CTTATTGGGACAATCTC-3') and and femurs of the hind legs using DMEM containing 10% reverse primer (5'-GCCAGATTATCTCTTTCTAC-3'); TNF-·, fetal bovine serum (Hyclone) and then cultured in DMEM forward primer (5'-ATGAGCACAGAAAGCATGA-3') and supplemented with 10% L929-conditioned medium (LCM) reverse primer (5'-TACAGGCTTGTCACTCGAATT-3'); as a macrophage colony-stimulating factor. After one day, ß-actin, forward primer (5'-GTGGGGCGCCCCAGGCA the cells were washed three times with PBS to remove non- CCA-3') and reverse primer (5'-CTCCTTAATGTCACGCAC adherent cells and then incubated in DMEM with LCM to GAT-3'). PCR was performed with 30 cycles of amplifi- obtain bone marrow-derived macrophages for three more cation. Each amplification cycle consisted of 30 sec of days. Then, the medium was replaced with fresh DMEM denaturation at 94˚C, 30 sec of annealing at 55˚C, and 30 sec with 10% FBS without LCM, and the cells were incubated of extension at 72˚C; and PCR was performed on a PTC-100 for 24 h. from MJ Research Inc. (Waltham, MA, USA). Amplification Splenocytes were obtained from spleens of Balb/c mice. of ß-actin was used as the control. PCR products were Spleens were ground up using rubber of 1 ml syringe, and separated on a 1% agarose gel and photographed. the resulting cells were passed through a cell strainer (pore diameter 70 μm; BD Falcon, MA, USA) to remove debris. The Results cells were then incubated in 2 ml of RBC lysis buffer for 2 min Expression pattern of the IFITM family in lymphoid organs to remove red blood cells (Sigma-Aldrich). The cells were of tumor-bearing mice washed twice with PBS and then incubated in RPMI-1640 . To determine the function of the IFITM medium supplemented with 10% FBS, penicillin, and strepto- family in tumors, the 4T1 mouse mammary tumor cell line was mycin for 3 h to allow adherence. inoculated into syngeneic Balb/c mice, and spleens were prepared two weeks after 4T1 injection. The size of the spleen Treatment with LPS and Toll-like receptor agonists in vivo and was greatly increased in tumor-bearing mice (Fig. 1A). The in vitro. Twelve-week-old Balb/c mice were randomly divided expression pattern of the IFITM family in spleens was into three groups.
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