Is there a role for the Fas-/Fas-Ligand pathway in chemoresistance of human squamous cell carcinomas of the head and neck (SCCHN)?

Cell lines and culture conditions 

Jurkat leukaemia cells (DSMZ, Braunschweig, Germany), HT-29 human colon adenoma cells (ATCC, Rockville, USA) and the HaCaT keratinocyte cell line17 were chosen as positive controls for Fas expression and function. The following SCCHN cell lines were used: UM-SCC-11B, -17, and -22B,18 UD-SCC-1, -2, -3, -4, -5, -6, -7A, -7B and -7C,19 UT-SCC-920 as well as HLaC79,21 the 8029NA line and it’s platinum-resistant subline 8029DDP.22 The Jurkat cell line was maintained in RPMI 1640 (ICN Biomedicals, Frankfurt, Germany) complemented with 10% FCS (Gibco, Eggenstein, Germany), 2mM l-glutamine (ICN Biomedicals), 50μg/ml streptomycine and 50μg/ml penicilline (both Gibco), all other cell lines in Dulbecco´s MEM (Gibco) complemented as above.

P53-status of the cell lines 

For the co-incubation experiments of cytostatic drugs and anti-Fas-antibodies and for the blocking experiments of Fas-signalling by anti-FasL-antibodies five cell lines were chosen for their Fas-sensitivity, platinum-resistance (see below), and their p53-status. p53-sequence analysis was performed as described earlier.23, 24 We selected the UD-SCC-2 cell line as a p53 wild type cell line, the UT-SCC-9 line as an example for a p53-negative cell line, 8029DDP as a platinum-resistant subline of 8029NA, and the UD-SCC-7B, 8029NA and 8029DDP as Fas-sensitive cell lines (determined by MTT-Assay, see below). Table 1 gives an overview of the mutation status of the exons 5–8 of all five cell lines used.

Table 1. Overview of the p53-mutation status of the exons 5–8 of all five cell lines used

	Cell line	Mutation	Effect on the protein	wt-Allele present	wt-Transcript present
	UD-SCC-2	No mutation	Wild type-protein (but inactivated)	+	+
	UD-SCC-7B	Nt 957, CGG→CTG	AA 248, Arg→Leu	+	−
	UT-SCC-9	Exons 2–9 deleted	No protein	−	−
	8029 NA und 8029 DDP	Exchange of exons 2–4 by 370 Nt from 7q11.23-q21	Eventually fusion-protein	+	+

For details see. 24

Detection of Fas and FasL mRNA expression by RT-PCR 

Total RNA was prepared from cells grown to 80% confluence as a monolayer using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 300ng of total RNA were retrotranscribed with oligo-dT primers using a SuperScript reverse transcriptase (Gibco). The following primer pairs were used for amplification: Fas sense, 5′-CAG AAC TTG GAA GGC CTG CAT C-3′; Fas antisense, 5′-TCT GTT CTG CTG TCT GTT GGA C-3′; FasL sense: 5′-GGA TTG GGC CTG GGG ATG TTT CA-3′; FASLG antisense: 5′-TTG TGG CTC AGG GGC AGG TTG TTG-3′. A premix consisting of 0.2μM dNTP, 0.4μM of each primer, and 1U of Taq-polymerase (Qiagen) was prepared in a total volume of 48μl and 2μl cDNA was added. PCR amplification comprised 35 cycles of denaturing for 45s at 95°C, annealing for 45s at 60°C, and extension for 60s at 72°C for FAS, and 40 cycles of denaturing for 30s at 95°C, and annealing and extension for 60s at 67°C for FASLG. PCR was preceded by 3min at 95°C and followed by 7min at 72°C or 67°C for FAS and FASLG, respectively. Quality of RNA was controlled by RT-PCR of GAPDH. Amplified products were checked on an agarose-gels, stained with ethidium bromide.

For Southern analysis the amplified FasL-products were separated on agarose-gels, transferred to a nylon transfer membrane, and hybridized with labelled cDNA probes for FasL over night at 55°C. The membrane was developed with CDP-star (Perkin Elmer) in the dark for 5min at RT.

Immunocytochemistry for Fas and FasL 

Cells were plated on polystyrol-slides (Nunc, Kamstrup, Denmark), washed three times with TBS (20mM Tris/HCl, pH 7.4, 150mM NaCl), fixed for 5min with 70% acetone at −20°C, and air-dried. Slides were blocked for 1h in 5% skim milk powder (ICN Biomedicals) in TBS and for 1h in normal goat serum (Dako, Hamburg, Germany) diluted 1:4 in TBS supplemented with 10% BSA and 5% skim milk powder. Slides were washed with TBS and incubated at 4°C overnight in 5% skim milk/TBS with specific primary antibodies APO-1 for Fas and N-20 for FasL (Dako and Santa Cruz Biotechnology, Santa Cruz, USA, respectively). The detection was done by the APAAP-FastRed-method (Dako) according to manufacturer’s instructions. Slides were counterstained with Mayer´s hemalaun. In all experiments, goat non-immune-serum (Dako) was used as control. The classification of expression levels was done by multiplication of the scores for the percentage of staining cells (0: no positive cells, 1: ⩽25% positive cells, 2: 26–50% positive cells, 3: 51–75% positive cells to 4: ⩾76% positive cells) with the score of the staining intensity (0: no staining reaction, 1=weak, 2=medium and 3=strong staining reaction). While the N-20-antibody of Santa Cruz™ is numbered among a group of polyclonal anti-Fas-L-antibodies with at least questionable specifity,25, 26, 27 the APO-1-antibody was used in a study of Schmitz et al. on the specificity of anti-human Fas-antibodies in Western Blotting as a reference antibody for surface staining of Fas in FACS-analysis.28, 29

Treatment with cytostatic drugs 

The cell lines were treated with cDDP (Bristol-Myer´s Squibb, Bristol, UK) at a dose range of 0.25–25μg/ml or with BLM (Heinrich Mack Nachf., Illertissen, Germany) ranging from 0.25 to 25,000μg/ml for 72h. The serum concentrations obtained under clinical standard dosages are 0.4–1.6μg/ml for cDDP30 and 1.5–3μg/ml for BLM.31

In vitro growth inhibition/cytotoxicity assay 

Antitumor effects were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT, Sigma, Munich, Germany). This colorimetric assay is based on the reduction of a non-toxic water soluble yellow tetrazolium salt to a purple-colored water-insoluble formazan precipitate by the reductive capacity of cytoplasmic and mitochondrial dehydrogenases present only in living metabolically active cells.32, 33 Exponentially growing tumor cells were harvested from culture flasks, plated on 96-well-microtiter plates (6000 cells/200μl culture medium/well; exceptions: UD-SCC-3, UM-SCC-17A and UT-SCC-9: 12,000 cells/well and UD-SCC-2: 18,000 cells/well). After 72h (day 3) at 37°C and 5% CO2 the medium was discarded and 200μl of fresh MEM containing the cytostatic drugs or agonistic antibodies (CH11, Beckman-Coulter) were added, and controls received medium alone. After another 72h (day 6) of incubation at the same conditions the medium was discarded, 50μl of MTT was added, and cells were incubated for another 4h. Finally, the precipitated dye was solubilized by the addition of 150μl of DMSO (Sigma) and the OD was measured on a spectrophotometric plate reader (Tecan, Männedorf, Switzerland) at λ=570nm. Wells with all components of the mixtures except cells served as blanks. In previous investigations, this experimental procedure has been identified to produce a good linear correlation between absorbance and the number of viable tumor cells.34 The rate of viable cells was then calculated using the following formula:

Combination effect of simultaneous administered CH11-antibodies and cytostatic drugs 

Cells were trypsinized, resuspended and plated as described for 72h. Cells were then incubated with the respective IC50 of cDDP or BLM and after 72h (day 3) the CH11-antibody (Beckman-Coulter) was administered at final concentrations of 10, 100 and 1000ng/ml for another 48h before viability was assessed using the MTT-assay as described above (day 5). Controls received CH11-antibodies or cytostatic drugs or culture medium alone.

Statistical analysis 

All MTT-assays were performed as 6-fold determinations and the coefficient of variation (Vc=standard deviation×1/mean value×100) of repeats never exceeded 10%. The 50% inhibitory drug concentration (IC50) for cDDP and BLM were determined in at least three independent experiments employing a non-linear estimation procedure (SAS-PC 6.08; SAS-Institute, Carey, USA) resulting in sigmoid dose–response curves.35 To distinguish additive, subadditive and synergistic effects of the simultaneous administration of the agonistic anti-Fas-antibody CH-11 and cDDP or BLM, we established theoretical, additive dose–response curves according to the logit transformation method of Pöch and Holzmann.36

Flow cytometric analysis of Fas and FasL membrane expression 

Tumor cells were harvested, resuspended and plated at 180,000 cells per well of a 6-well plate (exceptions: UD-SCC-2 and UT-SCC-9: 360,000 cells). After an incubation period of 72h at 37°C and 5% CO2 the medium was discarded and 6ml of fresh medium containing the respective IC50 of the cytostatic drugs cDDP or BLM were added for another 72h. Cells were washed two times with PBS (Gibco), resuspended and incubated for 2h at 37°C and 5% CO2 under gentle repetitive shaking to avoid adhesion during reconstitution of the surface receptors. Fas expression was determined by flow cytometry upon incubating the cells for 1h at 4°C with the monoclonal mouse-IgG1-FITC-antibody UB2 (Coulter, Marseille, France; 1:10 dilution) or a mouse-isotype matched control-FITC-antibody (Coulter, 1:10 dilution) in FACS-medium (0.5% sodium acid, 1.5% HEPES, 2% FCS in HBSS, PAA-Laboratories, Linz, Austria). FasL expression was measured by incubating the cells for 1h with the mouse IgG-antibody NOK-1 (BD Biosciences Pharmingen, San Diego, USA). After two washes with FACS-medium, cells were incubated for 30min with a FITC-labelled rabbit–anti-mouse IgG (DAKO, 1:20 dilution), washed again and fixed with 0.2% Formalin-Hank´s (Merck). For each experiment 10,000 cells were analysed on a Cytoron Absolute Flow Cytometer (Ortho Clinical Diagnostics, Raritan, USA). Experiments were repeated in triplicate.

Apoptosis assay 

Cells were trypsinized, washed and plated as described above. After 72h (day 3) caspase-inhibitors (Ac-YVAD-CMK and Ac-DEVD-CMK, both 10μM, Sigma) and blocking anti-FasL-antibodies (NOK-1, 10μg/ml, BD Biosciences Pharmingen, and 4H9, 1μg/ml, Beckman–Coulter) were added. After 6h the medium was supplemented with apoptosis-inducing agents (cytostatic drugs cDDP, BLM, and/ or the agonistic antibodies CH11, 1μg/ml, Beckman-Coulter). Early apoptotic cells were determined by staining with fluorescein–isothiocyanate labelled annexin V according to the manufacturer’s recommendations (Beckman–Coulter). Annexin V binds to phosphatidylserine exposed on the outer leaflet of apoptotic cell membranes. Floating cells were collected by centrifugation at 800rpm. Adherent cells were trypsinized, washed twice with PBS (Gibco-Invitrogen Corp.) and re-plated for 2h to allow recovering of the cell surface structures. Cells were gently trypsinized, centrifuged at 800rpm at 4°C, and resuspended in MEM. Annexin V-FITC (10μl) were added to 980μl of the cell suspension and incubated for 15min on ice. To discriminate late apoptotic and necrotic cells, 10μl of propidium-iodide were added just before the analysis on the flow cytometer.

Detection of Fas and FasL mRNA expression in HNSCC-cell lines by RT-PCR 

The majority of studies to date have found constitutive expression of mRNA for the Fas-receptor in a wide range of SCCHN lines tested. In agreement with this, Fas mRNA was expressed in all SCCHN cell lines as well as the control cell lines Jurkat, HaCaT and HT-29. In two cases (UD-SCC-4 and -5), a smaller RT-PCR product was obtained additional to the full length Fas amplificate, suggesting the presence of an alternative spliced variant. (Fig. 1, upper panel). For Fas-Ligand, southern hybridisation to the PCR-products left only two cell lines (UD-SCC-1 and UM-SCC-11B) negative for FasL mRNA (Fig. 1, lower panel).

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Figure 1. Constitutive Fas- and FasL-mRNA-Expression on 16 human head and neck cancer cell lines (SCCHN). The cell lines HT-29 and HaCaT were used as controls for Fas. The Jurkat cell line was used untreated and upon stimulation with 400ng/ml Ionomycin and 10ng/ml PMA. Marker at 600bp; the second plot of the cell lines UD-SCC-4 und UD-SCC-5 at 572bp was interpreted as a splice variant of the receptor (upper panel). For Southern analysis the amplified FasL-products were separated on agarose-gels, transferred to a nylon transfer membrane, and hybridized with labelled cDNA probes for FasL over night at 55°C. The membrane was developed in the dark for 5min at RT (lower panel).

Immunocytochemical detection of Fas and FasL in SCCHN cell lines 

Immunocytochemically, Fas protein was detected in all 18 cell lines tested. Antibody APO-1 showed an expression in the cytoplasma and the cell membrane in over 50% of the tumor cells for all cell lines. (Fig. 2). FasL was detected by a weak–moderate staining reaction in 35–85% of the cells. Of the 18 cell lines tested, five were positive (++) and 12 weakly positive (+) for Fas, whereas five were strongly positive (++++), six clearly positive (+++), two positive (++) and four weakly positive for FasL (+) (data not shown).

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Figure 2. Immunocytochemical staining of Fas and FasL. Cells were grown on polystyrol-slides, harvested upon 80% confluence, fixed with acetone, air-dried and stained with the APO-1-antibody (DAKO) for Fas and the N-20-antibody (Santa Cruz) for FasL using the APAAP-FastRed-method according to the manufacturer’s instructions and counterstained with Mayer´s hemalaun for 25s. (a) Strong staining pattern for Fas in the cell line UD-SCC-7C, magnification: ×100. (b) Strong staining pattern for FasL in the cell line UD-SCC-1, magnification: ×166.

Resistance to Fas-mediated apoptosis 

If a tumor expresses a large quantity of death receptors but still is capable of escaping from immunosurveillance operating with these receptors, they might bear functional impairments that facilitate tumor growth. To elucidate this, we investigated the effect of the agonistic anti-Fas-antibody CH11 on human SCCHN cell lines using the colorimetric MTT-cytotoxicity assay. Because 11 of 16 cell lines (69%) turned out to be primarily resistant towards Fas-mediated cytotoxicity, we preincubated the cells with Interferon-γ (IFN-γ), which is known to sensitize tumor cells of different origin to apoptotic cell death by inducing the expression of the apoptosis-signaling receptors CD95 and TNFR1 and various interleukin-1b-converting enzyme (Ice) family members.37 IFN-γ alone conferred low toxicity to the tumor cells. Five out of the eleven Fas-resistant cell lines (45%; the control lines HT-29 and HaCaT as well as UD-SCC-5, UD-SCC-6 and UT-SCC-9) changed to the sensitive status. In addition, IFN-γ made primarily sensitive cell lines (i.e. UD-SCC-7A, UD-SCC-7B, HLaC 79, 8029NA, and 8029DDP) even more susceptible to Fas-mediated cytotoxicity (Fig. 3). Six of sixteen cell lines (38%) retained IFN-γ-refractory Fas-resistance: UD-SCC-1, UD-SCC-2, UD-SCC-3, UD-SCC-4, UM-SCC-14C, and UM-SCC-17A). Table 2 gives a gross classification of the cell lines in Fas-resistant, Fas-inducible, Fas-sensitive, IFN-γ-resistant or -sensitive and the combination of the mentioned properties (see Fig. 3).

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Figure 3. Dose dependence of cell death induced by agonistic anti-Fas-antibodies (CH11) alone or in combination with IFN-γ in several SCCHN cell lines as determined by MTT-assay. Cells were prepared as described in Section “Materials and Methods”, harvested, resuspended and seeded per 6000 (12,000 and 18,000, respectively) in 96-well-plates. After 72h, the medium was discarded, cells were incubated with IFN-γ in various concentrations for another 48h. Then, the antibodies (CH-11) or control-mouse-IgM was added. 36h later, viability was assessed by the MTT-assay. Values are the mean of at least four experiments and bars represent 1 SEM (standard error of the mean).

Table 2. Gross classification of the cell lines according to their Fas- and IFN-γ sensitivity as obtained by the MTT-assay

		Fas-resistant	Fas-inducible	Fas-sensitive
	IFN-γ-sensitive		HT-29
		UD-SCC-2	HaCaT	UD-SCC-7A
		UD-SCC-3	UD-SCC-5	UD-SCC-7B
		UD-SCC-4	UD-SCC-6	8029 NA
			UT-SCC-9
	IFN-γ-resistant	UD-SCC-1
		UD-SCC-4		HLaC 79
		UM-SCC-14C		8029 DDP

Cells were seeded at 6000 per well, preincubated on day 3 with IFN-γ, followed by administration of the agonistic antibody in the concentration of 10, 100 and 1000ng/ml, respectively, on day 5 for another 36h. Classification was done upon graphical analysis of the results shown in Figure 4.

Upregulation of cell surface expression of Fas and FasL by engagement of anticancer agents 

FACS-analysis showed a uniform moderate basal expression of the Fas-receptor (Fig. 4). In contrast, the corresponding ligand FasL was barely detectable without any stimulation or preincubation with antineoplastic drugs (Fig. 5), therefore matching the results of the RT-PCR and immunohistochemistry analyses. Treatment of the cells with IC50 of either cDDP or BLM for 72h led to an upregulation of Fas and FasL cell surface expression for all cell lines tested, and this effect was independent of the mechanism of cytotoxic action, i.e. direct interaction with cellular proteins and DNA in the case of cDDP or inhibition of the DNA-polymerase by inducing single- or double-strand DNA breaks in the case of BLM. In addition, upregulation of both Fas and FasL was not affected by the p53-status of the cells since the wt-p53 cell line UT-SCC-9 as well as the p53-null cell line UD-SCC-2 did show upregulation of the receptor as well as the ligand upon engagement of cytotoxic drugs. Table 3 gives an overview over the basal surface presence of Fas and FasL and the multiplicity of escalation upon administration of cDDP and BLM in the respective cell lines.

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Figure 4. Upregulation of the surface expression of Fas on five SCCHN cell lines: UD-SCC-2, UD-SCC-7B, 8029 NA, 8029 DDP and UT-SCC-9-cells were incubated for 72h with DMEM, then the cytostatic drugs cDDP and BLM were added in the IC50, respectively. After that, cells were detached, resuspended and incubated with the antibodies: they were incubated for 2h at 4°C with the FITC-conjugated anti-Fas-antibody UB-2 (1:10; open histograms) or a mouse-isotype matched control-FITC-antibody (1:10; filled histograms). For analysis cells were fixed with 0.2% Formalin-Hank’s.

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Figure 5. Upregulation of the surface expression of Fas-Ligand on five SCCHN cell lines: UD-SCC-2, UD-SCC-7B, 8029 NA, 8029 DDP and UT-SCC-9. Cells were incubated for 1h with the anti-FasL-antibody NOK-1 (1:10; open histograms) followed by a FITC-labeled rabbit anti mouse-IgG for 30min. Controls were done by omission of the 1st antibody (filled histograms). Cells were fixed by 0.2% Formalin-Hank’s before flow cytometric analysis.

Table 3. Surface expression of Fas and Fas-Ligand on human HNSCC-cell lines native and upon exposure to the IC50 of cDDP and BLM, respectively

	Cell line	CD95-receptor			CD95-ligand
		Native	IC50 cDDP	IC50 BLM	Native	IC50 cDDP	IC50 BLM
	UD-SCC-2	3.91	17.41	19.79	1.14	3.14	3.95
	UD-SCC-7B	5.62	9.73	7.93	1.58	4.07	3.35
	UT-SCC-9	2.38	8.39	11.91	1.31	2.95	3.83
	8029 NA	4.36	5.35	6.82	1.47	3.11	4.04
	8029 DDP	9.98	22.25	20.09	1.2	4.06	4.58

For each experiment, 10,000 cells were analysed; values are the mean fluorescence intensity (MFI) of one representative of a minimum of three experiments.

Combination effect of simultaneous administered CH-11-antibodies and cytostatic drugs 

Cells were trypsinized, resuspended and seeded as described above. For three days they were incubated with the IC50 of cisplatin or bleomycin and after 72h the CH-11-antibody was administered in final concentrations of 10, 100 and 1000ng/ml for another 48h before viability was assessed with the MTT-assay as described above. As shown in Figure 6, only the Fas-sensitive cell lines UD-SCC-7B, 8029 NA and 8029 DDP (the latter only in the case of BLM) showed an enhancement of Fas-mediated cytotoxicity (range from 10% to 40%) by the respective drug. Interestingly, in the cDDP-resistant cell subline 8029DDP, the cytotoxic effect of the combination of CH11-antibodies and DDP did not even reach the additive level, but rather was antagonistic. The cytotoxic drugs clearly failed to confer sensitivity to the primarily Fas-resistant cell lines UD-SCC-2 and UT-SCC-9. In these cases, the effect of the agonistic antibodies and the cytostatic drugs was only additive.

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Figure 6. Antineoplastic drugs enhance Fas-sensitivity in some of the cell lines but do not in others. The effect of the cytostatic drugs alone and in combination with the agonistic anti-Fas-antibody (CH11 on the proliferation of the tumor cell lines in relationship to the untreated control (=100%) is delineated by dose–effect-curves in semilogarithmic axes, the y-axis showing the proliferation, the x-axis showing the concentrations of the cytostatic drugs. Black curve: cytostatic drugs alone, the other curves delineate the real (with inserts) or the calculated theoretically-additive concentration–response curves (without inserts), red: 10ng/ml CH11, blue: 100ng/ml CH11 and green: 1000ng/ml CH11. Values are means of at least four experiments±1 standard error of the mean (SEM). The comparison of the actually measured values with the theoretically-additive curves allows us to differentiate in underadditive (e.g. for cDDP and CH-11-antibodies in the cell line 8029 DDP), additive (e.g. for BLM and CH11-antibodies in the cell line UD-SCC-2) and finally supraadditive interactions (e.g. cDDP and CH11 in the cell line 8029 NA). (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

The apoptosis-inducing effect of anticancer agents seems to be only partly mediated by engagement of the Fas-/FasL system 

Using the annexin V-binding assay we were able to demonstrate that both anticancer agents (cDDP and BLM) led to a dose-dependent induction of apoptosis in all five cell lines tested. This effect was diminished in some cell lines by co-incubation with competitive caspase-inhibitors (Ac-YVAD-CMK and Ac-DEVD-CMK). FasL-antibodies NOK-1 and 4H9 reduced cDDP- and BLM-toxicity for cell line UD-SCC-7B and – to a lesser extent – 8029DDP, only. As an example, Figure 7 shows the distribution of the different cell fractions (Annexin −/PI −; Annexin +/PI −; Annexin −/PI + and Annexin +/PI +) for UD-SCC-7B. Figure 8 summarizes the results for various incubation conditions for the five cell lines selected.

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Figure 7. Fas- and cDDP-mediated apoptosis in UD-SCC-7B cells treated with blocking anti-FasL-antibodies or Ac-YVAD-CMK and Ac-DEVD-CMK. Cells were trypsinized, washed and seeded as described in Section “Materials and Methods”. After 72h the caspase-inhibitors (Ac-YVAD-CMK and Ac-DEVD-CMK; 10μM each) and the blocking antibodies (4H9, 5μg/ml) were added, 6h later the medium was supplemented with the cytotoxic drugs (cDDP, IC75) and the CH-11-antibodies (25ng/ml and 100ng/ml, respectively). Then, cells were assayed for Fas-mediated apoptosis by the Annexin V-assay as described above and analysed by a cytoron absolute cytometer for red (PI) and green (AnnexinV-FITC) fluorescence. The percentage distribution of the different cell fractions (Annexin −/PI −; Annexin +/PI −; Annexin −/PI + and Annexin +/PI +) for the UD-SCC-7B cell line obtained by flow cytometric analysis is shown. Results are from one out of three similar experiments, 10,000 cells were counted. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

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Figure 8. Effect of blocking anti FasL-antibodies (NOK-1 and 4H9) on cDDP- and BLM-mediated apoptosis in the five cell lines. Cells were trypsinized, washed and seeded as described in Section “Materials and Methods”. After 72h the blocking antibodies (NOK-1, 10μg/ml and 4H9, 1μg/ml) were added, 6h later the medium was supplemented with the cytotoxic drugs (cDDP and BLM, IC75) or the CH-11-antibodies (1μg/ml). Then, cells were assayed for Fas-mediated apoptosis by the Annexin V-assay as described in Section “Materials and Methods”. The percentage distribution of the different cell fractions (violet: Annexin –/PI –; red: Annexin +/PI –; yellow: Annexin –/PI +) for the different cell lines obtained by flow cytometric analysis are shown. Results represent the mean of three independent experiments. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)