| | Is there a role for the Fas-/Fas-Ligand pathway in chemoresistance of human squamous cell carcinomas of the head and neck (SCCHN)?Received 4 February 2008; received in revised form 31 March 2008; accepted 1 April 2008. published online 14 July 2008. Summary The aim of the present investigation was to determine the expression of the Fas-receptor/ligand system in established cell lines of squamous cell carcinomas of the head and neck (SCCHN), and to study it’s functional impact on chemotherapy-induced apoptosis in these SCCHN cell lines. We observed constitutive expression of Fas and FasL in 13 SCCHN cell lines by RT-PCR, Southern-blotting and immunocytochemistry, respectively. Administration of the agonistic Fas-antibody CH-11 led to a significant reduction of viable cells in the colorimetric MTT-assay in 5 out of 13 (38%) cell lines tested and preincubation with Interferon-γ (IFN-γ) rendered 3 (23%) primarily resistant cell lines sensitive. Cisplatin (cDDP) and bleomycin (BLM) caused dose-dependent cytotoxicity in all cell lines as determined by the 50% inhibitory concentration (IC50) and induction of apoptosis. Furthermore, both antineoplastic agents led to an enhanced surface expression of Fas and FasL in all cell lines, and this effect was independent of the respective p53-status. This upregulation of Fas/FasL surface expression increased preexisting Fas-sensitivity only, but failed to make primarily resistant cell lines undergo Fas-mediated growth reduction or apoptosis. Vice versa, blockade of Fas-receptor–ligand-interactions by monoclonal antibodies directed against FasL was able to attenuate the cytotoxic effect of cDDP and BLM in 2 out of 5 (40%) cell lines tested only. In conclusion, in contrast to many other solid tumors, the Fas/FasL-system does not seem to play an exclusive role in anticancer drug mediated apoptosis in SCCHN. Introduction  Human head and neck cancer is known to be widely chemoresistant to commonly administered antineoplastic drugs resulting in very poor prognosis once the tumor has left the stage in which it is amenable to surgical interventions or radiotherapy. Despite extended basic research for new substances and a huge amount of clinical trials investigating multimodality treatment regimens over the past 30 years the outcome of head and neck cancer of stages III and IV remains uncertain.1 The surprising regression of even large tumors upon administration of chemotherapy is often followed by the outgrowth of some small populations of resistant cells giving rise to recurrence of the disease. Apoptosis or programmed cell death is a distinct, multi-step intrinsic cellular death program that can be triggered by a wide range of factors and circumstances including signaling by death receptors, growth factor withdrawal, ultra violet or γ-irradiation or by chemotherapeutic drugs.2 It is characterized by typical morphological and biochemical changes including cell shrinkage, nuclear DNA fragmentation and membrane blebbing.3 Recent evidence indicates that the Fas (CD95/APO-1)-system, a central mediator of receptor–ligand induced apoptosis, might play a pivotal role in the development of apoptotic resistance at least in some tumors.4 The type I transmembrane receptor Fas of the TNF/nerve growth factor family, together with his corresponding ligand or upon ligation by agonistic anti-Fas-antibodies like CH-11 or APO-1, effectively induces apoptosis in sensitive cells in vivo and in vitro. The cytotoxic signals are integrated and processed by a number of cysteinyl aspartate proteinases (caspases) and converge in the cleavage of endogenous proteins.5 Though expressed on a variety of untransformed and neoplastic cells, the role of the Fas system in tissue homeostasis has mainly been investigated in the immune system.6 Since then, the expression of the Fas-receptor was studied in several tissues of healthy and diseased state, but so far not on head and neck cancer.7, 8 The natural ligand to the Fas-receptor, FasL (CD95L, CD177, APO-1-L), is a type II transmembrane molecule and as well part of the TNF/nerve growth factor family, which can – cleaved by metalloproteinases – also occur in a soluble form.9 FasL is predominantly expressed on activated T-cells, but also on a wide range of tissues which became known as “immune privileged sites”.10 Moreover, several tumor cells were recently shown to express functional FasL, which may contribute to a phenomenon called the counterattack of the tumor, since activated antitumoral T-cells are widely sensitive to Fas-mediated killing.11, 12 It has been shown that death of tumor cells by anticancer treatment is not only the result of direct cytotoxic effects but also of an active cellular program which also involves the Fas-system by causing upregulation of cell surface expression of the receptor and the ligand and thus activating an autocrine suicide and a paracrine fratricide death machinery.13, 14, 15 It has been suggested that functional wild type p53 is able to transactivate Fas gene transcription via a p53-responsive element in this gene.16 The antineoplastic agents cisplatin (cDDP) and bleomycin (BLM) are components of many standard protocols in the treatment of metastatic or recurrent SCCHN. In this report we provide the results of experiments designed to elucidate the role of the Fas-system and the caspases in cisplatin- and bleomycin-induced death of SCCHN cell lines. Failure to undergo programmed cell death in response to anticancer drugs may explain resistance, deregulation of the expression of proapoptotic molecules may contribute to tumor progression and tumor escape. A better understanding of the molecular events that regulate anticancer drug mediated apoptosis and that allow cancer cells to abscond from the apoptotic machinery might open up new opportunities for molecular based cancer therapy and drug design. Materials and methods 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), 2 mM 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. 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 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: 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. Results 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). 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). 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). 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. 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. Discussion Primary or acquired resistance to cytotoxic agents is a major problem in the treatment of many solid tumors that clinicians have to face in their work nearly every day. It is meanwhile commonly accepted, that tumors and neoplasms of different origin, if at all, respond to cytotoxic agents used in therapy by the activation of an intrinsic cellular program called apoptosis.13, 38 The Fas-system as a cornerstone of apoptosis has been shown to be an essential mediator of the cytotoxic effects of chemotherapeutic agents in many leukaemic and solid tumors.14, 16, 39 Vice versa, resistance of tumors to chemotherapy might be explained by a defective activation of the apoptotic cascade. These findings also promised to provide new insights into the molecular basis of the clinically apparent multidrug-resistance in SCCHN and directed the development of new strategies to overcome this chemo- and apoptotic crossresistance. In our study we demonstrate a low constitutive surface expression of Fas and FasL on SCCHN cell lines using different detection methods. In contrast to other investigators, we did not find a gain in FasL expression during tumor progression or an asymmetry of FasL-expression over the tumor tissue specimens (data not shown).7, 8, 40 Looking at the function we saw that the majority of cell lines was primarily not susceptible towards Fas-mediated cytotoxicity. This is in congruence with the results of many other groups testing different solid tumors like bladder carcinoma41, renal cell cancer42, or prostate cancer.43 In order to sensitize tumor cells towards Fas-mediated cytotoxicity, we used IFN-γ, which is known to elevate cell surface expression of Fas and FasL on HT-29 cells by enhancing Fas gene transcription and mRNA-stabilisation.44 Additionally, it promotes a p53-independent apoptotic pathway by directly and indirectly inducing selected apoptosis-related genes.37, 45 Using this pretreatment, only 40% of the cell lines remained Fas-resistant, suggesting that function of Fas or part(s) of the subsequent cascade might have been altered during tumor progression but can be restored by a mere cytokine preincubation. Therefore, down-regulation of Fas-sensitivity under a critical level might contribute to tumor progression in our SCCHN cell lines tested. Cytotoxic drugs can activate Fas-signaling pathways by upregulation and binding of the receptor and the ligand, but are also able to trigger the downstream signaling cascade by multimerization of the overexpressed receptor independent of the ligand.46 We focused on the action of antineoplastic drugs on five SCCHN cell lines selected for Fas-sensitivity, p53-status and chemosensitivity. cDDP as well as BLM led to cell death in a dose-dependent manner. This was abolished by incubation with the broad spectrum caspase inhibitors Ac-YVAD-CMK (predominantly inhibits Caspase-1) and Ac-DEVD-CMK (mainly blocks Caspase-3), at least when administered in concert, confirming that the major mechanism of action of cDDP and BLM in our system involves initiator and executioner caspases (data not shown).47 Both antineoplastic agents did enhance cell surface expression of Fas and, to a lesser extent, of FasL as determined by FACS-analysis. These findings are in congruence with other groups who tested cells of solid tumors or leukaemic cells.14, 48, 49 This upregulation was seen in tumor cells containing wild type, mutant or no p53, indicating that upregulation of Fas and FasL seems to be a p53-independent event in our system. Interestingly, enhancement of Fas-receptor and -ligand surface expression after cDDP incubation was seen in the same manner in both the cDDP sensitive cell line 8029NA and it’s resistant subline 8029DDP. In order to detect the functional relevance of this upregulation we blocked the receptor–ligand communication by two antagonistic antibodies and saw that this significantly diminished chemosensitivity in the two Fas-sensitive cell lines UD-SCC-7B and 8029 NA. Drug cytotoxicity was not impaired by blocking the receptor–ligand-interactions in the other three cell lines including 8029DDP, the cDDP-resistant subline of 8029NA. Of note, in the cDDP-resistant cell line 8029DDP Fas- and chemoresistance ran parallel throughout our experiments. In our combination experiments again, neither cDDP nor BLM were able to turn Fas-resistant cell lines (UD-SCC-2, 8029DDP or UT-SCC-9) to the sensitive status but did enhance preexisting Fas-sensitivity (8029NA and UD-SCC-7B). On the other hand, cDDP-sensitivity in 8029DDP cells was not restored by agonistic Fas-antibodies as it has been described for other entities like bladder carcinomas.41 Thus, although the essential components of the Fas death machinery seem to be in place in the SCCHN cell lines and can be activated by agonistic Fas-antibodies, at least in concert with cofactors like CHX or IFN-γ, the upregulation of the surface expression of the Fas-receptor and the corresponding ligand by cDDP and BLM does result only in more effective engagement of the extrinsic Fas-mediated system if it is primarily functional active. Furthermore, our study does not support a major role of the p53-status on Fas-sensitivity, since the mutant p53 cell lines 8029NA, 8029DDP or UD-SCC-7B are Fas-sensitive while the wild type p53 cell line UD-SCC-2 is non-reactive to CH-11-antibodies even after preincubation with IFN-γ. Furthermore, the difference in chemo- and Fas-sensitivity between the parental cell line 8029NA and the platinum-resistant cell line 8029DDP is rather not due to the p53-status since they share the same transcriptional and protein p53-status. This ineffective activation of the Fas mediated-pathway by anticancer drugs puts SCCHN in contrast to many other solid tumors, where the antiproliferative effect of cytostatic drugs is mediated at least in part by the Fas-/Fas-Ligand-system via p53-dependent mechanisms.16 One explanation of our results might be, that recently characterized other members of the p53 network (i.e. p63 and p73) are able to take over functions in p53-mediated responses to DNA-damage agents.50 A cross-talk between the p53-family members has been demonstrated for other tumor entities like epithelial ovarian cancer.51 Furthermore, Müller et al. have showed newly that the functional status of TAp73/ΔNp73 is an important determinant of chemosensitivity in hepatoma cells and that TAp73β acts as a transcriptional factor of the Fas-gene.52 Gressner et al. demonstrated that the Fas-gene is a transcriptional target also of TAp63α in hepatocellular carcinoma.53 They propose an interference network in which each of the p53-family members can – upon engagement by antineoplastic drugs – induce and activate or downregulate and inhibit several death receptors, their ligands, regulatory caspases, executioner caspases, the mitochondrial way, apoptosis regulating proteins and effector caspases and thereby act as an important operational centre of chemoresistance in HCC. 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a Department of Maxillo- and Plastic Facial Surgery, Westdeutsche Kieferklinik, Heinrich-Heine-University, Düsseldorf, Germany b Department of Otorhinolaryngology, Heinrich-Heine-University, Düsseldorf, Germany c Department of Gynecology and Obstetrics, Heinrich-Heine-University, Düsseldorf, Germany d Department of Pharmacology, Heinrich-Heine-University, Düsseldorf, Germany e Department of Pathology, Heinrich-Heine-University, Düsseldorf, Germany f Department of Otorhinolaryngology, Technical University, Munich, Germany  Corresponding author. Tel.: +49 211 81 0 40 92; fax: +49 211 81 1 88 77.
PII: S1368-8375(08)00101-2 doi:10.1016/j.oraloncology.2008.04.003 © 2008 Elsevier Ltd. All rights reserved. | |
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