Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis Jeanette Blomberg, Kristina Ruuth, Maria Jacobsson, Andreas Ho ¨ glund, Jonas A. Nilsson and Erik Lundgren Department of Molecular Biology, Umea ˚ University, Sweden Fas (CD95 ⁄ Apo-1) is a cell surface receptor that is important for the mediation of cell death, and is one of eight different death receptors that have been char- acterized to date [1]. The role of the Fas ligand (FasL) and receptor interaction has been emphasized in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis [2,3]. Oligomerization of Fas via binding of its cognate ligand (FasL) induces a signalling cascade that culminates in the controlled degradation of cellular components [4]. The apical caspases-8 ⁄ 9, together with the downstream effector caspase-3, have been documented to be crucial players in the mediation of death receptor-induced apop- tosis [5]. Keywords CpG methylation; ERK activation by PD98059; Fas; Fas expression; TNF-a resistance Correspondence J. Blomberg, Department of Molecular Biology, Umea ˚ University, S-90187 Umea ˚ , Sweden Fax: +46 90 771420 Tel: +46 90 7852535 E-mail: jeanette.blomberg@molbiol.umu.se (Received 16 July 2008, revised 6 November 2008, accepted 12 November 2008) doi:10.1111/j.1742-4658.2008.06790.x Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeo- stasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas- induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-a stimulation, but the mRNA expression of tumour necro- sis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-a stimulation, sug- gesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experi- ments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation. Abbreviations AKT, protein kinase B; CpG, cytosine-phosphate-guanine; ERK, extracellular signal-regulated kinase; FasL, Fas ligand; FLIP, Flice-like inhibitory protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP-kinase, mitogen-activated protein kinase; MEK, mitogen- activated ERK-activating kinase; P.U. cells, parental U937 cells; PARP, poly (ADP-ribose) polymerase; PI3-kinase, phosphoinositide 3-kinase; qRT-PCR, quantitative RT-PCR; SOCS-1, suppressor of cytokine signalling 1; TNFR, tumour necrosis factor receptor; TNF-a, tumour necrosis factor-a; TRAIL, TNF-related apoptosis-inducing ligand. FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS 497 Expression of Fas and susceptibility to FasL activa- tion are common traits in most tissues [6]. In contrast, tumour cells frequently display impaired death recep- tor functions [7]. Nonfunctional Fas signalling has been implicated in the resistance to apoptosis induced by chemical stimuli. Moreover, a lack of Fas function enables tumour cells to evade surveillance by the immune system and facilitates metastatic progression [8,9]. Several different mechanisms that contribute to impaired Fas signalling have been described in a range of tumours, such as suppressed expression of Fas at both the mRNA and protein levels [10,11]. Epigenetic silencing of the FAS promoter has recently been shown to be regulated by oncogenic Ras [12,13]. Furthermore, mutations or deletions in FAS have been found to cause an autoimmune lymphoproliferative syndrome [14]. The Fas signalling pathway can be modified by an array of proteins, e.g. Flice-like inhibitory proteins (FLIPs), Bcl-2 family members, Fas-associated protein tyrosine phosphatase 1 and inhibitors of apoptosis proteins [15]. To establish which molecular alterations occur dur- ing the acquisition of Fas resistance, we established Fas-resistant U937 cells by prolonged growth in pro- gressively increasing concentrations of apoptosis-induc- ing Fas antibody. We have demonstrated previously that the resistant phenotype is associated with multiple molecular changes, such as reduced Fas expression, increased cFLIP expression and altered activities of both protein tyrosine kinases and protein tyrosine phosphatases. Moreover, selection for Fas resistance results in coselection for resistance to other death receptor ligands [16]. In this study, individual clones derived from the Fas-resistant population were used to dissect the resistance mechanisms in the heterogeneous resistant population described earlier. The results showed that a decrease in Fas expression was the most prominent reason for resistance, and that the reintro- duction of Fas expression abolished Fas resistance. Notably, the reduced amount of Fas in the resistant clones was a result of impaired FAS transcription. Results Downregulation of Fas as a means to develop Fas resistance Previously, we have developed Fas-resistant U937 cells by prolonged growth in progressively increasing con- centrations of stimulating Fas antibody [16]. To inves- tigate the molecular basis that underlies the resistance to Fas-induced apoptosis, the resistant cells were seeded out as single cells in microtitre wells and 39 different clones were established. These clones showed various degrees of Fas sensitivity, ranging from 50% sensitivity to complete resistance compared with paren- tal U937 (P.U.) cells (Fig. 1A). (Apoptosis was detected as illustrated in Fig. S1.) When the surface expression of Fas was determined by flow cytometry in the 39 resistant clones, it was evident that 82% of the clones contained a reduced amount of Fas (Fig. 1B). As no correlation between Fas expression and Fas sen- sitivity was detected in this experiment (R = 0.1446), four clones with low Fas expression (F7, F23, F30 and F35) and two with high Fas expression (F1 and F33) within the more resistant area (< 10% apoptosis) were randomly selected. All six clones were resistant for up to 48 h of Fas stimulation, which demonstrated that a persistent resistance had been obtained (data not shown). Four of the six clones contained reduced expression of Fas protein when assayed both with flow cytometry and immunoblot (Fig. 1C,D). The decrease in Fas expression at the cell surface was approximately 60%. When activation of the Fas signalling cascade was investigated with immunoblot on cleaved caspase-8 and its downstream caspase target, poly(ADP-ribose) polymerase (PARP), no cleavage was detected in any of the resistant clones compared with parental cells (Fig. 1E). In summary, the data illustrate that four of the six resistant clones (F7, F23, F30 and F35) exhibit reduced Fas expression, whereas clones F1 and F33 have acquired alternative abnormalities that abolish caspase-8 activation. A decreased expression of Fas could be the result of alterations at many different levels, including transcrip- tion, mRNA half-life, translation and protein turnover. As an initial attempt to elucidate the mechanism behind Fas resistance, conventional RT-PCR and quantitative RT-PCR (qRT-PCR) were performed to explore whether FAS transcription was reduced in the resistant clones. Three different primer pair sets (Fig. 2A) were used: one control set designed against the mRNA encoding the ubiquitous enzyme gly- ceraldehyde-3-phosphate dehydrogenase (GAPDH), another set encompassing exon–exon boundaries of the Fas mRNA, with which steady-state mRNA levels could be measured, and a third set encompassing the exon–intron boundary of the Fas pre-mRNA, with which newly synthesized transcripts could be mea- sured. As shown in Fig. 2B, clones F7, F23, F30 and F35 exhibited a reduced steady-state level of Fas tran- script compared with clone F1, clone F33 and P.U. cells. The reduction in clones F7, F23, F30 and F35 measured 40% or less by qRT-PCR (Fig. 2C). Inter- estingly, the same four clones also showed an 80% decrease in the amount of Fas pre-mRNA (Fig. 2D,E), Fas resistance caused by reduced transcription J. Blomberg et al. 498 FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS suggesting that the reduced amount of Fas protein was a result of events regulating FAS transcription. qPCR on genomic FAS did not reveal any differences between parental and resistant cells (data not shown). Thus, the decrease in FAS transcription did not depend on a reduced amount of FAS gene copies in the resistant clones. Mutations in both the promoter and coding sequences of FAS have been identified in a vast range of tumours (reviewed in [8]). However, no acquired mutations in the Fas cDNA or FAS promoter, spanning the region from )1781 to )22 bp, were detected in our resistant clones when compared with P.U. cells (data not shown). Fas-resistant clones with reduced Fas expression are partially cross-resistant to tumour necrosis factor-a (TNF-a) independent of restored Fas transcription Cross-resistance to the activation of other members of the death receptor family was investigated in the clones with reduced Fas expression. Rather surprisingly, the Fig. 1. Reduced Fas expression in resistant clones. Thirty-nine individual clones were isolated from the Fas-resistant variant of U937 described earlier. (A) Fas sensitivity was monitored by propidium iodide staining and flow cytometry after 15 h of stimulation with 20 ngÆmL )1 a-Fas in P.U. cells and resistant clones (numbered). Surface expression of Fas was investigated by flow cytometry in 39 Fas- resistant clones (B) and six resistant clones that were randomly selected for further studies (C). Fas was stained with a nonapoptosis-induc- ing antibody, as described in Experimental procedures, and the secondary antibody alone served as a negative control. (D) Immunoblotting was performed to determine the total amount of Fas protein in the six Fas-resistant clones. (E) Cells were treated with or without 15 ngÆmL )1 stimulating Fas antibody for 15 h before the processing of caspase-8 and PARP was investigated by immunoblot. Actin was used as a loading control in all immunoblot experiments. J. Blomberg et al. Fas resistance caused by reduced transcription FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS 499 sensitivity to TNF-a was reduced in all four clones when compared with P.U. cells (Fig. 3A, P < 0.02). In contrast, susceptibility to TNF-related apoptosis- inducing ligand (TRAIL) was similar to that of P.U. cells in all clones, except clone 23, which showed a 70% decrease in the apoptotic response (P = 0.03) (Fig. 3B). This cross-resistance prompted us to investi- gate whether a general defect in apoptosis had been acquired. We therefore treated U937 cells and the Fas- resistant clones with the anticancer drug etoposide, which is a known activator of caspases and inducer of apoptosis, independent of Fas expression [17,18]. As the sensitivity to etoposide was similar in all clones and P.U. cells (Fig. 3C), it was concluded that there was no general apoptosis defect in the resistant clones. In addition, because only one clone displayed cross- resistance to TRAIL, we hypothesized that the decreased response to TRAIL represented a second independent event, whereas the reduced sensitivity to TNF-a was associated with acquired Fas resistance. U937 cells have been reported to express both tumour necrosis factor receptor 1 (TNFR1) and TNFR2 [19]. To investigate whether the reduced sensi- tivity to TNF-a in the Fas-resistant clones could be explained by a concomitant decreased expression of TNFRs, qRT-PCRs were performed with isoform- specific primers. No drastic decrease in either TNFR1 or TNFR2 mRNAs was detected in the Fas-resistant clones compared with P.U. cells which correlated with the reduced sensitivity to TNF-a stimulation (Fig. 3D,E). Thus, the partial cross-resistance to TNF-a-induced apoptosis is not caused by a general suppression of death receptor transcription. The significance of the reduced Fas expression in the resistant clones was studied by the reintroduction of Fas by transfection into two of the clones. The success of Fas expression was confirmed by immunoblot anal- ysis (Fig. 4A) of the total lysate and flow cytometry of Fas expressed on the cell surface (Fig. 4B). Stimulation of Fas confirmed that the re-established Fas expression mediated susceptibility to Fas-induced apoptosis in the resistant cells (Fig. 4C). The restored apoptotic response to Fas stimulation also correlated with a normal activation of caspase-8 (Fig. 4D). Despite the fact that the Fas-resistant clones were cross-resistant to TNF-a, the reintroduction of Fas did not mediate renewed sensitivity to TNF-a. This suggests that either the FAS gene and components of TNF signalling are downstream of a common regulator mutated in these clones, or these two resistance mechanisms have arisen independently of each other (Fig. 4E). Potential regulators of Fas expression It has been shown that oncogenic factors, such as Ras and p53, are important regulators of Fas expression. Ras inhibition of Fas expression is associated with hypermethylation of cytosine-phosphate-guanine (CpG)-rich regions in the FAS promoter [12,13], and demethylation of CpG sites in the first intron of FAS Fig. 2. Decreased amount of Fas pre-mRNA in the resistant clones. (A) Schematic presentation of the location of the primers used for RT-PCR and qRT-PCR studies. GAPDH was used as a control and the primers were designed to allow the determination of contami- nating genomic DNA. RT-PCR (B) and qRT-PCR (C) were performed with primer pair I to analyse Fas mRNA expression in the different clones. Primer pair II was used for the study of Fas pre-mRNA expression with RT-PCR (D) and qRT-PCR (E). RT-PCR products were visualized with ethidium bromide staining after separation on a 1% agarose gel. Fas resistance caused by reduced transcription J. Blomberg et al. 500 FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS has been illustrated to enhance p53-induced Fas expression [11]. As the Fas-resistant cells contained a reduced amount of Fas pre-mRNA (Fig. 2D,E), a potential repression of FAS expression through epige- netic silencing was investigated. The FAS gene con- tains a 650 bp CpG island in the 5¢-flanking region of the transcriptional start site (as illustrated in Fig. 5A). However, none of the unmethylated CpGs (black boxes) in the CpG island were altered in any of the resistant clones when assayed with genomic sequencing of bisulfite-modified DNA (Fig. 5B). In addition, methylation-specific PCR of a CpG region in the first intron of FAS revealed no methylations in any of the clones (Fig. 5C). Methylation-specific PCR of suppres- sor of cytokine signalling 1 (SOCS-1) was used as a positive control, as it has been reported to be methy- lated in U937 (Fig. 5C) [20]. Treatment with a deme- thylating agent, 5-aza-2¢-deoxycytidine, did not restore either Fas surface expression or Fas sensitivity (data not shown). Furthermore, no activating mutations of codons 12, 13 and 61 in the genes encoding H-, K- and N-Ras [21] could be detected in any of the clones (data not shown), which would have provided an explanation for the altered FAS transcription. In summary, this clearly shows that epigenetic silencing through CpG methylations does not account for the resistance to Fas-induced apoptosis in our system. Survival signalling pathways downstream of Ras, such as the phosphoinositide 3-kinase (PI3-kinase) and the mitogen-activated protein kinase (MAP-kinase) sig- nalling cascades, have been suggested to regulate death receptor-induced apoptosis [22,23]. Before we analysed the RAS genes for mutations, we performed immuno- blot analyses and used specific inhibitors to determine whether the PI3-kinase and MAP-kinase signalling cas- cades contributed to Fas resistance. The Fas-resistant clones with decreased Fas expression (F7, F23, F30 and F35) contained elevated levels of phosphorylated protein kinase B (pAKT), whereas resistant clones with normal Fas expression (F1 and F33) contained less pAKT as well as total AKT protein (Fig. 6A). Quanti- fication showed that the increase in pAKT was slight, as it reached statistical significance only for clones F7 and F23 (Fig. S2). Inhibition of the PI3-kinase path- way with wortmannin for 1.5 h completely abolished AKT phosphorylation (Fig. 6B). However, wortman- nin pretreatment for 1 h before Fas stimulation did not restore the sensitivity to Fas stimulation in the Fig. 3. Partial cross-resistance to TRAIL and TNF-a, but not etoposide, treatment in the resistant clones. Cells were stimulated with 2ngÆmL )1 TNF-a (A) and 2 ngÆmL )1 TRAIL (B) for 20 h. (C) Cells were treated with dif- ferent concentrations of etoposide for 16 h. Apoptosis was assayed with propidium iodide staining and flow cytometry in all experiments. Normal expression of TNFR1 and TNFR2 was detected in the resistant clones when mRNA expression of TNFR1 (D) and TNFR2 (E) was measured with qRT-PCR. J. Blomberg et al. Fas resistance caused by reduced transcription FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS 501 Fig. 5. Methylation of the FAS promoter does not account for the reduced expression in the resistant clones. (A) Schematic illustration of CpGs in the 650 bp 5¢-flanking region of the FAS promoter. Black boxes are individual CpGs and the numbers in the white boxes represent the number of nucleotides between each site. (B) Schematic illustration of the sequenced CpGs in the FAS promoter in parental and resis- tant clones, where the boxes represent unmethylated CpG sites (black), methylated CpG sites (grey, none present) and other nucleotides (white). (C) Primers that recognized either unmethylated or methylated sequences in the first intron of FAS were used to determine the methylation status with methyl-specific PCR. Methylated primers for suppressor of cytokine signalling 1 (SOCS-1) were used as a positive control. The PCR products were visualized with ethidium bromide staining after separation on a 2% agarose gel. Fig. 4. Reintroduction of Fas abolishes the insensitivity to Fas, but not TNF-a-induced apoptosis. The introduced Fas expression was con- firmed by immunoblot (A), and the increased surface Fas expression was investigated by flow cytometry of surface-expressed Fas (B). (C) Vector control and Fas-expressing cells were stimulated with 15 ngÆmL )1 of anti-Fas for 15 h, and apoptosis was assayed with propidium iodide staining and flow cytometry. (D) Restored processing of caspase-8 on Fas activation was studied by immunoblot. (E) TNF-a sensitivity was monitored in vector control and Fas-expressing cells by stimulation with 2 ngÆmL )1 of TNF-a for 15 h, followed by propidium iodide staining and flow cytometry. Fas resistance caused by reduced transcription J. Blomberg et al. 502 FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS resistant clones and did not reduce the apoptotic response in parental cells (Fig. 6C). The same results were obtained with another PI3-kinase inhibitor, LY294002 (data not shown). These experiments demonstrate that the altered level of pAKT is not responsible for the Fas resistance. By performing immunoblots, we identified resistant clones containing increased levels of phosphorylated extracellular signal-regulated kinase 1 (pERK1), possi- bly as a consequence of increased expression of the ERK1 protein (Fig. 6D). As these clones were not amongst those with lower Fas expression, we ruled out ERK1 as a mediator of FAS silencing. ERK2 exhib- ited variable expression between experiments, and there was no correlation between the presence of ele- vated phosphorylation and increased Fas resistance. In addition, the upstream ERK kinases, mitogen-acti- vated ERK-activating kinase 1 (MEK1) and MEK2, were not excessively phosphorylated in any of the clones, suggesting that whatever causes the irregular hyperphosphorylation of ERK1 ⁄ 2 represents a nonca- nonical pathway (Fig. 6E). (For the quantification of total and phosphorylated levels of ERK1 ⁄ 2 and MEK1 ⁄ 2, see Fig. S2.) To lend further support to this notion, the inhibition of MEK1⁄ 2 for 1.5 h with PD98059 resulted in increased ERK1 ⁄ 2 phosphoryla- tion in both Fas-resistant clones and P.U. cells (Fig. 6F), which is opposite to what is expected with this inhibitor. The PD98059-induced phosphorylation of ERK1 ⁄ 2 was stable for at least 16 h after adminis- tration (Fig. S3A). These obscure results were not caused by dysfunction of PD98059, as it blocked the fetal bovine serum-induced activation of ERK1 ⁄ 2in starved HeLa cells and P.U. cells (Fig. S3B). Neverthe- less, PD98059 did not confer Fas resistance to P.U. cells (Fig. 6G), indicating that increased phosphoryla- tion of ERK1 ⁄ 2 is not sufficient to mediate the resis- tance to Fas-induced apoptosis. In conclusion, this suggests that the canonical MAP-kinase signalling cascade does not have a direct regulatory role in the resistant cells. Discussion Tumour development is a multistep process that evolves with time. It is driven by a progressive increase in the acquisition of mutations and genetic aberrations. The acquisition of genetic lesions that abrogate sensitivity to cell death signals is an important part of tumour devel- opment and progression, as it is needed to support increased proliferation [24]. In addition, resistance to cell death enables tumour cells to avoid elimination triggered by both cytotoxic immune cells and thera- peutic agents [25]. Through an increased understanding of the mechanisms that mediate resistance to apoptosis, important improvements in therapeutic interventions can be made. In this article, we have described a model system in which acquired Fas resistance is shown to be dependent on more than one mechanism; reduced Fas receptor expression was studied in detail. Fig. 6. Altered activities of the PI3-kinase and MEK ⁄ ERK signalling pathways in resistant clones do not contribute to the reduced susceptibil- ity to Fas. (A) Lysates were subjected to immunoblot of phosphorylated and total levels of AKT. (B) Reduced phosphorylation of AKT with 1 l M wortmannin after 1.5 h of treatment was confirmed by immunoblot. (C) Cells were pretreated with or without 1 lM wortmannin for 1 h before 10 ngÆmL )1 of anti-Fas was added for 15 h. Apoptosis was detected by propidium iodide staining and flow cytometry. Phosphory- lated and total levels of ERK1 ⁄ 2 (D) and MEK1 ⁄ 2 (E) were investigated by immunoblot. (F) Cells were treated with or without 50 l M PD98059 for 1.5 h before the phosphorylated levels of ERK1 and ERK2 were investigated by immunoblot. (G) Parental cells were pretreated with or without 50 l M PD98059 for 1 h before 10 ngÆmL )1 of a-Fas was added. Cells were harvested after 15 h of Fas stimulation, and apoptosis was determined by propidium iodide staining and flow cytometry. J. Blomberg et al. Fas resistance caused by reduced transcription FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS 503 The reduced expression of death receptors has been detected in a considerable number of different tumours, but the mechanism contributing to this phe- notype is still not well defined and needs to be deter- mined [8]. In this article, we have shown that a decrease in Fas expression is the major phenotype in U937 cells selected for resistance to Fas, making these cells a tractable model for the identification of the pathways involved in the regulation of Fas expression and resistance. Interestingly, many of the clones exhi- bit cross-resistance to other death receptor signals. Indeed, communication between different death recep- tors has been postulated as they share certain intracel- lular signalling molecules [26,27]. In addition, the presence of TNFRs is believed to be important for appropriate susceptibility to Fas stimulation in mouse T cells and macrophages [28–30]. However, a depen- dence of Fas expression on TNFR signalling has not been reported, which would have provided an explana- tion for our data if it had not been for the results showing that the re-expression of Fas did not reverse TNF-a resistance. The partial cross-resistance to TNF-a is puzzling and highlights the complexity of cell death signalling. Death receptor activation has been illustrated to mediate nonapoptotic signalling, which is most promi- nent in TNF signalling [31]. The balance between pro- and anti-apoptotic signalling is tightly regulated by important transcription factors, such as nuclear factor- jB and c-Jun, which have also been demonstrated to regulate FAS transcription [32–34]. The interplay and regulation of different transcription factors are highly complex, and the cellular context is critical for deter- mining the contribution of different factors for FAS transcription [35]. Preliminary data have illustrated that there is reduced protein expression of c-Jun in our resistant clones with low Fas expression when com- pared with P.U. cells (data not shown). The precise function of c-Jun, however, is still controversial, as it has different effects depending on the type of cell and expression levels [36]. Thus, the binding pattern of several different transcription factors to the FAS promoter is an interesting topic for further study. Apoptosis is only one of several cell death pathways, e.g. necrosis and autophagy [37]. Reports by others have shown that, if the apoptosis signalling pathway via Fas is defective, an alternative route leading to necrosis can be activated [38,39]. Fas has also been shown recently to stimulate autophagy when activated by autoantibodies in neuroblastoma [40]. However, the importance and interplay between the different cell death pathways are not fully understood, and the involvement of death receptor expression has not been investigated thoroughly. Thus, the cooperation of death receptors in cell death is an interesting topic to explore further. Studies have implied that, even if tumour cells con- tain multiple genetic and epigenetic alterations, they can be completely dependent on only one or a few important gene alterations for survival and prolifera- tion [41]. It has been shown that oncogenic factors exist which deregulate Fas expression [12,13,42–44]. However, the sequencing of RAS did not reveal any alterations in our resistant cells (data not shown), and it is unlikely that dysfunction of the tumour suppressor p53 can account for the resistant phenotype in our sys- tem, as U937 cells are TP53-null cells (data not shown [45]). Somatic mutation of FAS itself was first detected in lymphoid tumours, and it has been reported to be present in considerable proportions of non-Hodgkin’s lymphoma [8]. Sequencing of the coding region and the promoter, containing the core, enhancer and silen- cer region, of FAS did not reveal any mutations in the resistant cells. Because tumour progression is tightly coupled to genetic changes, complete genomic sequenc- ing, comparative genomic hybridization or single nucleotide polymorphism analysis of resistant clones would be the most straightforward way to elucidate the genetic aberrations that occur during the acquisi- tion of Fas resistance. The genomic sequencing of tumours has been shown to be a powerful method of identifying reoccurring alterations in the complex heterogenicity of different tumours [46]. Epigenetic changes, through promoter methylations or other global chromatin modifications, have recently received increasing attention in cancer research, as it has become evident that alterations in methylation status are one of several important tumour character- istics [47]. We could not detect methylation of any CpG sites in a 650-bp 5¢-flanking region of the FAS promoter by genomic sequencing of bisulfite-modified DNA. This region has been studied by others, because it is recognized as a CpG island that contains many CpG sites [11]. In addition, regions upstream of the FAS promoter and in intron 1 have also been shown to be methylated [11,12]. However, as the inhibition of DNA methylation did not result in an elevated level of Fas expressed on the surface, and did not restore sensitivity to Fas stimulation in resis- tant cells (data not shown), we conclude that epige- netic changes through methylation cannot account for the reduced expression of Fas. Survival pathways, such as the PI3-kinase and MAP-kinase pathways, regulate a multitude of cellular responses and have a major impact on cell viability. Importantly, signalling molecules in these pathways Fas resistance caused by reduced transcription J. Blomberg et al. 504 FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS can interfere, either directly or indirectly, with several of the components in the apoptosis signalling cascade [22,23]. In our resistant cells, we detected increased phosphorylation of signalling proteins in both the PI3- kinase and MEK ⁄ ERK pathways, even though the three RAS genes were devoid of activating mutations. Specific inhibitors of PI3-kinase did not restore Fas sensitivity in resistant clones, demonstrating that this pathway is not essential for Fas resistance. Moreover, it is unlikely that the MEK ⁄ ERK pathway is impor- tant for Fas resistance because the MEK inhibitor PD98059 did not cause inhibition of pERK, indicating that ERK phosphorylations are regulated by an alter- native pathway in U937 cells. The unexpected increase in ERK1 ⁄ 2 phosphorylation by PD98059 suggests that this alternative pathway is negatively regulated by MEK, but nonessential, as its activation is insufficient to confer Fas resistance to P.U. cells. Future studies need to be performed to identify this potentially inter- esting mode of activation by the PD98059 ‘inhibitor’, as studies like this may provide additional knowledge on how the MEK ⁄ ERK pathway is regulated by scaf- fold proteins, kinases and phosphatases. Taken together, the observed alterations in the AKT- and MAP-kinase pathways illustrate that the selection pres- sure imposed on the Fas-resistant cells affects them in a profound way, requiring several important signalling cascades to be activated in order to support the devel- opment of resistance. This is an example of one of the major challenges in the elucidation of tumour develop- ment, namely to discriminate between secondary sup- portive alterations and important tumour-maintaining aberrations [48]. In other studies on acquired resistance to Fas- induced apoptosis, it has been illustrated that the resistant phenotype is associated with a loss of Fas function through mutations in FAS or a dysfunctional activation of the sphingomyelin–ceramide pathway [49–51]. In this article, we have reported that resistance to Fas-induced apoptosis is caused by a decrease in FAS transcription by an incompletely understood mechanism. The clones described here may therefore be utilized in unbiased screens for components involved in acquired Fas resistance, which may gener- ate new targets for anticancer treatments. Experimental procedures Cell culture and reagents The human monocytic cell line U937 was obtained from the American Type Culture Collection, Manassas, VA, USA. Cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum. All cell cultures were maintained in 5% CO 2 at 37 °C. Anti-Fas CH-11 IgM was obtained from MBL (Woburn, MA, USA), and TNF-a and TRAIL from PeproTech (London, UK). Caspase-8 (1 : 1000), PARP (1 : 1000), pAKT (1 : 1000), AKT (1 : 1000), pERK1 ⁄ 2 (1 : 1000), ERK1 ⁄ 2 (1 : 1000), pMEK1 ⁄ 2 (1 : 1000) and MEK1 ⁄ 2 (1 : 1000) antibodies were obtained from Cell Signalling Technology (Beverly, MA, USA). The Fas antibody C-20 (1 : 1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the actin anti- body (1 : 7500) from Sigma (St Louis, MO, USA). The Fas DX2 antibody was purchased from BD Pharmingen (Frank- lin Lakes, NJ, USA) and the Alexa Fluor 488 anti-mouse Ig antibody (1 : 300) from Molecular Probes Inc. (Eugene, OR, USA). Propidium iodide and wortmannin were obtained from Sigma. PD98059 was obtained from Calbiochem (EMD Biosciences Inc., Darmstadt, Germany). Generation of FasL-resistant cells and clones Fas-resistant U937 cells were established as described previ- ously [16]. In order to isolate individual resistant clones from the resistant cell population, 0.1 and 0.3 cells per well were seeded out into microtitre wells and 39 single colonies were expanded. Four clones with low Fas expression and two with high Fas expression within the more resistant area (< 10% apoptosis) were randomly selected for further studies. Apoptosis detection with flow cytometry Cells were stimulated with different concentrations of Fas, TNF-a, TRAIL and etoposide, and analysed at the time points indicated in the figures. For inhibition studies, cells were pretreated with either 1 lm wortmannin or 50 lm PD98059 for 60 min before stimulation with 15 ngÆmL )1 Fas antibody for 15 h. Cell pellets were dissolved in propi- dium iodide solution (0.1% v ⁄ v Nonidet P-40, 20 m Tris pH 7.5, 100 mm NaCl, 50 l g ÆmL )1 propidium iodide and 20 lgÆmL )1 RNAse) and incubated for 30 min at 4 °C before analysis was performed with a FACSCalibur flow cytometer equipped with cell quest pro software (BD Biosciences, San Jose, CA, USA). At least 1 · 10 4 cells were acquired per sample and the sub-G1 population was scored as apoptotic cells. Live and apoptotic cells were identified on the FL-2- and fetal bovine serum–height plots, and particles smaller than approximately 1000-fold relative to live cells were excluded. Doublets were discriminated for in the FL-2 area versus FL-2 width plots (see Fig. S1). Fas staining for flow cytometry Cells (1 · 10 6 ) were labelled with 0.4 lg of anti-Fas DX2 for 60 min, followed by Alexa Fluor 488 anti-mouse anti- body for 60 min. Fas expression was quantified with a J. Blomberg et al. Fas resistance caused by reduced transcription FEBS Journal 276 (2009) 497–508 ª 2008 The Authors Journal compilation ª 2008 FEBS 505 FACSCalibur flow cytometer. The secondary antibody alone was used as negative control. Immunoblot analysis For studies on protein expression of Fas, caspase-8 and PARP, cells were lysed in SDS lysis buffer (2% w ⁄ v SDS, 100 mm Tris pH 6.8) supplemented with CompleteMini Pro- tease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). For immunoblotting of the total and phosphory- lated levels of AKT, MEK1 ⁄ 2 and ERK1 ⁄ 2, cells were lysed in RIPA lysis buffer [50 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxy- cholate] containing CompleteMini Protease Inhibitor Cock- tail, 2 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 10 lgÆmL )1 p-nitrophenyl phosphate, 5 mm b-glycerophosphate and 50 lgÆmL )1 Glycine max (soybean) inhibitor. These lysates were kept on ice for 15 min and cleared by centrifugation at 20 000 g for 15 min. SDS-PAGE was performed as described previously [16]. In brief, 30 lg of protein was separated by SDS-PAGE and transferred to nitrocellulose membranes with a semidry blot (Bio-Rad Lab- oratories, Richmond, CA, USA). Primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescent substrates (Pierce Biotechnology, Rockford. IL, USA). For the quanti- fication of arbitrary units, a fluor-s multi imager and quantity one software were used (Bio-Rad Laboratories). RT-PCR and qRT-PCR Total RNA was isolated with a Nucleospin RNA II Kit (Macherey-Nagel, Du ¨ ren, Germany). First-strand cDNA was synthesized from 1 lg of total RNA with Superscript II RT (Invitrogen Life Technologies, Paisley, Renfrewshire, UK). The following primers were used: Fas mRNA, 5¢-AGATCTAACTTGGGGTGGCT-3¢ and 5¢-ATTTATT GCCACTGTTTCAGGAT-3¢; Fas pre-mRNA, 5¢-GGACC CAGAATACCAAGTG-3¢ and 5¢-GTCAGTGTTACTTC CCTAGG-3¢; TNFR1, 5¢-GTGCTGTTGCCCCTGGT CAT-3¢ and 5¢-GCTTAGTAGTAGTTCCTTCA-3¢; TNFR2, 5¢-AAACTCAAGCCTGCACTC-3¢ and 5¢-GGA TGAAGTCGTGTTGGAGA-3¢; GAPDH has been described previously [16]. For RT-PCR amplification, 25 ng of template was amp- lified with HotMaster Taq polymerase (Eppendorf, Hamburg, Germany) under the following conditions: 95 °C for 15 min; 94 °C for 45 s; 56 °C for 30 s; 72 °C for 45 s. The PCRs were run in a MastercyclerNN (Eppendorf). qRT-PCRs were performed on 25 ng of template using a QuantiMix EASY SYG KIT (Biotools ⁄ Techtum Lab AB, Umea ˚ , Sweden); the conditions applied were 95 °C for 3 min, 95 °C for 10 s and 60 °C for 45 s. Forty cycles were performed. qRT-PCRs were run in an iCycler thermal cycler and the data were analysed using icycler iq software (Bio-Rad Laboratories). The expression of each target transcript was normalized to GAPDH. Duplicates were made on three independent RNA isolations and the data represent the means ± SD. Cell transfection The expression construct was prepared in the pCEP4 expression vector (Invitrogen Life Technologies) by insert- ing Fas cDNA (IMAGE: 5202648). cDNA was extracted from the pCMV-SPORT6 vector and cloned into pCEP4 at the XhoI and KpnI restriction sites. Cells (2 · 10 7 ) were electroporated with 20 lg of plasmid at 240 V and 950 lF using a Bio-Rad Gene Pulser. Stable transfectants were selected with 300 lgÆmL )1 hygromycin B (Roche Diagnos- tics) for 3–4 weeks and subsequently analysed. Diagrams represent the triplicates of two transfections, and similar data were obtained in more than three experiments. Sequencing of bisulfite-modified genomic DNA and methylation-specific PCR Genomic DNA was purified with a DNeasy Kit (Qiagen Nordic, Solna, Sweden). DNA (1.5 lg) was denatured in 0.3 m NaOH at 42 °C for 30 min, and subsequently treated with 3.3 m sodium bisulfite plus 0.5 mm hydroquinone for 15 h at 55 °C. DNA was purified in spin columns (Pro- mega, Madison, WI, USA), denatured with 0.3 m NaOH and neutralized with 3 m ammonium acetate. Finally, DNA was precipitated with ethanol, washed and reconstituted in TE buffer (10 mm Tris ⁄ HCl, pH 7.6; 1 mm EDTA). For sequencing, PCR amplification from )575 to +8 of the Fas promoter was carried out with the primers described else- where [11]. HotStart Taq polymerase (Qiagen Nordic) was used and the conditions applied were as follows: 95 °C for 15 min; 94 °C for 45 s; 56 °C for 1 min; 72 °C for 2 min; for 35 cycles. The PCRs were run in a MastercyclerNN (Eppendorf). The PCR products were cloned into the pCR 4-TOPO vector with the TOPO TA Cloning Kit (Invitrogen Life Technologies) before sequencing. Methylation-specific PCR of CpGs in the first intron of Fas was performed on bisulfite-treated DNA, as described elsewhere [11]. Statistical analysis All data represent three independent experiments if not stated otherwise. For statistical analyses, Student’s t-test was applied and P < 0.05 was considered to be statistically significant. Acknowledgements We would like to thank Professor Staffan Bohm, Umea ˚ University, for provision of the cDNA of FAS Fas resistance caused by reduced transcription J. 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