↵1-1 RT-PCR analysis was done as follows: total RNA from about 1 × 10⁶ cells was extracted by the acid guanidinium thiocyanate-phenol-chloroform protocol described by Chomczynski and Sacchi3; 1 μg RNA together with 250 ng of oligo (dT)₁₅, primer was diluted in a.d. to a final volume of 14 μl, denatured by heating up to 70°C for five minutes and immediately chilled on ice. To each reaction, 6 μl RT mixture containing 4 μl 5× buffer, 2 pmol each of dATP, dCTP, dGTP and dTTP, and 200 units Moloney-murine leukaemia virus reverse transcriptase, was added (all reagents from Promega, Wisconsin, USA). For cDNA synthesis all samples were incubated at 37°C for 60 minutes. The reaction was stopped by heating the sample to 80°C for two minutes; 100 ng cDNA obtained was amplified by 50 cycles with 1 unit Taq polymerase (Promega). The reaction conditions were: denaturation, 60 seconds at 95°C; annealing, 60 seconds each at 63°C (cycle 1-3), 59°C (cycle 4-6), and 56°C (cycle 7-50); and extension, 45 seconds at 72°C. The oligonucleotide primers used were: TTC TTC CCT GTC CAA CCT CTG TGC (sense) and TCA TCT TCC CCT CCA TCA TCA CCA (antisense).4 PBMC of a healthy individual served as a positive control.

↵1-2 Constitutive expression of FasL protein was determined using two different monoclonal antibodies, NOK- 1 (Pharmingen, San Diego, California, USA) and Hl1 (Alexis, Läufelfingen, Switzerland). For detection of FasL expression, 0.5 × 10⁶ cells were fixed with paraformaldehyde, permeabilised with a buffer containing 0.05% saponin and 1% bovine serum albumin and stained with 1 μg of the respective specific monoclonal antibody or a relevant isotype matched negative control antibody for 30 minutes at 4°C. In the case of staining with NOK- 1, cells were incubated for 20 minutes at 4°C with a secondary fluorescein isothiocyanate (FITC) labelled rabbit anti-mouse antibody (Dako, Vienna, Austria; dilution 1 in 10). Cells were washed and immediately analysed by flow cytometry for their specific fluorescence signals. Mean specific fluorescence intensities (MFI) were calculated as the ratio of mean fluorescence intensity achieved with the specific antibody/isotype matched control antibody. A ratio ⩾ 1.5 was considered positive. The mean value of MFI for three independent experiments is given.

↵1-3 Time kinetics (l-3 days' stimulation) were performed and values are given for day 3. Tumour necrosis factor (TNF) α and interferon (IFN) γ were purchased from R&D Systems (Minneapolis, Minnesota, USA). Flow cytometric analysis was performed using the NOK- 1 monoclonal antibody.

↵1-4 For detection of Fas expression 0.5 × 10⁶cells were stained with 1 μg of a specific FITC labelled anti-Fas monoclonal antibody (UB2, Immunotech, Marseille, France) or an isotype matched control. The mean value of MFI for three independent experiments is given.

↵1-5 Cells were incubated with the CH11 monoclonal antibody (250 ng/ml) for 24 hours and the proportion of apoptotic cells was determined using the propidium iodide assay.4 Even after 72 hours' incubation, there was only a very small increase in the percentages of apoptotic cells (e.g. in the SNU-1 cell line the increase was from 3% (control) to 5% (CH11)).