[PubMed] [Google Scholar] 14. and position.5 Interestingly, among resistant neuroblastomas, differences in the sensitivity to atRA can also be significant, and the mechanism underlying this variability is unknown. Thus, there remains a need to elucidate the mechanisms that mediate the differences observed in the inhibition of neuroblastoma tumorigenicity by selective agents in order to develop new therapeutic approaches for this disease. Peroxisome proliferator-activated receptor-/ (PPAR/) dynamically Crotamiton heterodimerizes with retinoid X receptor (RXR) by ligand activation, and regulates expression of target genes that modulate cell proliferation, differentiation, inflammation, and lipid and glucose homeostasis.6 The role of PPAR/ in carcinogenesis remains controversial as some studies suggest that activating PPAR/ promotes tumorigenesis, while others suggest the opposite.6,7 Recently, several studies have revealed that over-expression of PPAR/ inhibits tumorigenesis in different types of human cancer types Crotamiton including breast, colon, and testis.8C10 These studies are similar to results showing that knockdown of PPAR/ promotes tumorigenesis in human colon cancer models, an effect that is mediated in part by Crotamiton inhibition of induction of terminal differentiation.11C13 This is of interest to note because it is firmly established that PPAR/ promotes terminal differentiation in multiple cell types.14,15 Induction of terminal differentiation is one approach used for cancer therapy,16,17 including neuroblastoma patients.18 This is particularly true for high-risk neuroblastoma patients and can be accomplished by treatment with retinoic acid to induce terminal differentiation of neuroblastoma cells.19C24 It was recently hypothesized that atRA may promote tumorigenesis by alternatively activating PPAR/, rather than retinoic acid receptors, based on the ratio of cellular retinoic acid binding protein-II and fatty acid binding protein-5.25 However, this hypothetical mechanism is not consistently observed26C28 and should be carefully re-evaluated.6,7,29 Since previous studies revealed that PPAR/-induced differentiation inhibits tumorigenesis,6,7,14 the hypothesis that over-expressing and/or activating PPAR/ modulates tumorigenesis of human were treated daily with either vehicle control (0.02% dimethylsulfoxide) or GW0742 (2.5 mg/kg/ day). After 2C3 weeks, mice were euthanized and tumors were excised and weighed. A tumor section was SLC4A1 fixed in 10% phosphate buffered formalin overnight and then switched to 75% ethanol. The remaining tumors were snap frozen in liquid nitrogen for protein or mRNA analysis. Fixed tumor sections were processed for hematoxylin-eosin (H&E) staining and examined by a pathologist as previously described.9,10 2.7 |. Immunohistochemistry Expression and localization of SOX2 in ectopic xenograft tumors was determined by immunohistochemistry using 3,3-diaminobenzidine (DAB) as a substrate and counterstained by hematoxylin as previously described.9 Twenty fields per sections and two sections per tumor sample were analyzed. Relative expression was determined by normalizing the intensity of DAB to hematoxylin signals using ImageJ software (Version 1.47c). 2.8 |. Transient over-expression and activation of RAR in neuroblastoma cells Cells were transiently transfected with 10 g of a pSG5-hRAR plasmid using Lipofectamine LTX reagent (Invitrogen, Grand Island, NY) following the manufacturers recommended procedures. Transfected cells were treated with or without 10 M atRA for 24 h. Over-expression of RAR was confirmed by quantitative Western blot analysis, and the expression of SOX2 and the RAR target gene was determined by qPCR and/or Western blot analysis. 2.9 |. Luciferase assays To identify the cis-regulatory element responsible for PPAR/-dependent expression in human neuroblastoma cells, three luciferase reporter gene constructs driven by the human promoter were generated. Three deletion fragments of promoter were amplified by PCR. Primers were designed to contain overhanging sites for restriction enzymes promoter-luciferase reporter constructs or the control plasmid (pGL3-basic vector) using Lipofectamine LTX reagent following the manufacturers recommended procedures. Cell lysate were prepared in passive lysis Crotamiton buffer. Luciferase and -galactosidase activities were assessed using Luciferase and Beta-Glo assay system (Promega Corp., Madison, WI), respectively, following the manufacturers recommended procedures. Relative luciferase activity was normalized to -galactosidase activity. 2.10 |. Chromatin immunoprecipitation (ChIP)-qPCR Putative promoter were identified using the ConSite program.33 To confirm occupancy of promoter, ChIP-qPCR was.