After a further wash, cells were suspended in PBS containing 2% FCS and analysed on a FACScan flow cytometer (Becton-Dickinson and Co., San Jose, CA, USA). To label dead cells, 0.5��gml?1 propidium iodide was added to the cells before analysis. Irrelevant monoclonal IgG antibodies (at equivalent concentrations) were used as isotype http://www.selleckchem.com/products/Roscovitine.html controls. Determination of p42/p44 MAP kinase/extracellular signal regulated kinase (ERK), FLIP and caspase-8 by Western blotting Cells were grown in the presence of FCS, deprived of serum for 24h and exposed or not to ET-1 for 10min. Cell cultures were extracted using 0.1% Triton X-100 in the presence of a cocktail of protease inhibitors (Roche, Rotfzreuz, Switzerland) and submitted to SDS electrophoresis.

Following transfer, the membrane was probed using monoclonal antiphosphorylated p42/p44 antibody (clone E10) (New England Biolabs, Bioconcepts, Alschwill, Switzerland) as previously described (Egidy et al, 2000c). Alternatively, cell extracts were exposed following blotting to anti-caspase-8 (New England Biolabs), anti-FLIP (a kind gift of J Tschopp, Lausanne, Switzerland) or anti ��-SMA (Sigma, Buchs, Switzerland) monoclonal antibodies. Calculations of results Each experiment was repeated at least three times unless otherwise stated. Means and s.d. were calculated. Statistical significance was assessed using an unpaired two-tailed Student’s t-test. RESULTS Expression of the ET-1 system in human colon and colon carcinoma cells The presence of immunoreactive ET-1 was assessed in human normal colon (Figure 1A, upper panel) and colon carcinoma (Figure 1A, lower panel).

Cancer cells, but not normal epithelial cells, ubiquitously expressed ET-1. The expression of the mRNAs of the components of the ET system in HT-29 and SW480 human colon carcinoma Batimastat cells was evaluated using RT�CPCR. ETA and ETB receptors, ECE-1 and PPET-1 mRNAs confirmed the presence of all the transcripts of the ET system in these cells (Figure 1B). Figure 1 Imunnohistochemical localisation of ET-1 and expression of the mRNAs of the ET-1 pathway in human colon carcinoma cells. (A) ET-1 immunoreactivity is ubiquitously localised in tumour cells of colon carcinoma (lower panel), but not in nontumoral epithelial … Confluent cultures of SW480 (4��104cellscm?2) and HT-29 (5��104cellscm?2) cells secreted ET-1, either in the absence or presence of FCS (Figure 1C). The presence of cell membrane and functional ET-1 receptors in SW480 and HT-29 cells was evaluated using 125I-ET-1 binding (Figure 1D) and the phosphorylation of ERK, a known intracellular mediator of ET-1 (Figure 1E).