1N NaOH. FasL-induced cell death The murine neuroblastoma cell line Neuro-2A FasL has been transfected to produce soluble murine FasL (Rensing-Ehl et al, 1995) and is used as a source of active FasL as previously definitely described (Peduto-Eberl et al, 2000). Briefly, the collection of supernatants of Neuro-2A FasL cells was performed in a medium containing less than 0.5% FCS and the FasL titre of the preparations was controlled using murine 497 glioblastoma cells, which are responsive to FasL. Supernatant from neo vector Neuro-2A cells were used as control for FasL-containing supernatants. HT-29 and SW480 cells were maintained in DMEM 4.5gl?1 glucose supplemented with 10% FCS.

Cells were grown to half confluence, washed to eliminate any ET-1 in the culture medium for the initial period of exposure to effectors and incubated at 37��C with ET-receptor antagonists (bosentan (Actelion, Basel, Switzerland), BQ123 or BQ788 (both from RBI, Natick, MA, USA) at the indicated concentrations) in the presence or absence of FasL-containing supernatant and of 10% FCS for 24 or 48h. For some experiments performed with bosentan, exogenous ET-1 was added at increasing concentrations from 10?13 to 10?7M, together with FasL-containing supernatant for 24h. For experiments performed with the caspase inhibitor zVAD-fmk (Bachem, Bubendorf, Switzerland), cells were preincubated with the inhibitor at the indicated concentrations and time, prior to the addition of bosentan and/or FasL-containing supernatant and incubation was continued for 24h.

As a control for the specificity of the FasL induction of apoptosis, cells were exposed to FasL-containing conditioned medium from Neuro 2A cells in the presence of 50��gml?1 of the FasL- binding Fas-Fc-IgG fusion protein (Alexis Corporation, Taufaliugeu, Switzerland) to deplete specifically FasL from conditioned medium and bosentan. After 24h, apoptosis was evaluated in adherent cells. Cell death evaluation Apoptosis was quantified as previously described (Egidy et al, 2000c; Peduto-Eberl et al, 2000) using the Cell Death Detection ELISAPLUS (Roche, Rotkreuz, Switzerland), a photometric enzyme-linked immunoassay for quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes), according to the supplier’s instructions. Increase in absorbance at 405nm is proportional Batimastat to apoptosis, and the enrichment in dead cell proportion (apoptosis index) was calculated as the ratio of absorbance of treated cells/absorbance of untreated cells. Flow cytometric analysis HT-29 cells were detached by incubation with EDTA (1mgml?1) at 37��C for 10min, essentially as previously described (Peduto-Eberl et al, 1999).