To examine the role of these pathways in the regulation of DR4 or DR5 function, genes induced or repressed by recombinant human TRAIL (rhTRAIL) and DR5-selective rhTRAIL variants were determined in a colon cancer cell model using cDNA microarray technology. In this report we show that the immediate www.selleckchem.com/products/z-vad-fmk.html early gene, Egr-1, is constitutively expressed in colon cancer cells and further induced in response to rhTRAIL by both DR4 and DR5. Furthermore, we show that the short isoform of c-FLIP controls the activity of the DR5 receptor, but not of DR4. The constitutively expressed Egr-1 inhibits TRAIL-mediated apoptosis, probably by driving constitutive c-FLIP expression. Materials and methods Cell culture and treatments Colo205 cells were obtained from American Tissue Culture Collection (ATCC).

HCT15 and HCA7 cells were a kind gift from Professor L Egan (National University of Ireland, Galway). Colo205 and HCT15 cells were maintained in RPMI-1640 medium and HCA7 in DMEM medium, both media supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 50Uml�C1 penicillin and 50mgml�C1 streptomycin at 37��C, 5% CO2 in a humidified incubator. Cells were seeded at 2 �� 105cellsml�C1 at 1 day before treatment. To induce apoptosis, cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114�C281, Triskel Therapeutics, Groningen, The Netherlands) DR5-selective mutants D269H, D269H/E195R, agonistic DR4 or DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland), recombinant human TNF (PromoCell, Heidelberg, Germany) or agonistic anti-Fas antibody (clone CH-11, MBL International, Woburn, MA, USA) at the concentration and times specified in the figure legends.

All reagents were from Sigma-Aldrich (St Louis, MD, USA) unless otherwise stated. Cell viability assay Cell viability was monitored using 3-(4, 5-dimethylthiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. After treatment, MTT (0.5mgml�C1) was added to cells and incubated for 3h at 37��C. The reaction was stopped by addition of an MTT stop solution of 20% SDS in 50% dimethyl formamide. The purple formazan precipitate generated was allowed to dissolve for 1h on an orbital shaker. The colour intensity GSK-3 was measured at 550nm on a Wallac Victor 1420 Multilabel counter (PerkinElmer Life Sciences, Waltham, MA, USA). Cell viability was expressed relative to the absorbance of untreated cells, which was taken as 100% viable. Cell death assay Cell death was monitored by labelling of phosphatidyl serine externalised on the surface of apoptotic cells with Annexin-V-FITC (IQ Corporation, Groningen, The Netherlands) or by haematoxylin and eosin staining of cytospins.