Báo cáo khoa học: Interferon-c bolsters CD95/Fas-mediated apoptosis of astroglioma cells doc

Interferon-c bolsters CD95/Fas-mediated apoptosis of astroglioma cells Anna M. Giammarioli 1 , Rosa Vona 1 , Lucrezia Gambardella 1 , Barbara Ascione 1 , Angela Maselli 1 , Cristiana Barbati 2 , Antonella Tinari 3 and Walter Malorni 1 1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita’, Rome, Italy 2 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita’, Rome, Italy 3 Department of Technology and Health, Istituto Superiore di Sanita’, Rome, Italy Introduction Astroglioma represents the most common primary brain tumor in humans. The prognosis of patients with the most severe form of astrocytoma is poor and only marginally improved by chemotherapy. Astrogliomas are generally resistant to drug and radiation therapy and this greatly limits the available treatment options. The induction of apoptosis, the program for cell sui- cide, is the main goal of anti-tumor treatments. This is normally induced by chemotherapic drugs or by spe- cific stimuli, such as the interaction of death receptors with their ligands. Well-defined signal transduction pathways and effector mechanisms have been described that finally lead to cell demise [1,2]. Among these, a key role is played by Bcl-2 family molecules, by the mitochondria, which can release apoptogenic factors, and by a family of cysteine proteases (caspases) that act in cascade, initiating and executing programmed cell death [3,4]. Apoptosis induced by CD95 ⁄ Fas receptor was origi- nally identified as the main mechanism for inducing apoptosis in lymphoid cells, but it has also been shown to trigger programmed cell death in other cell types, including nervous system cells [5–8]. In particular, astroglioma cells constitutively express CD95 ⁄ Fas but are usually resistant to cell death as a result of the ligation of this receptor [9]. Interferons (IFNs) constitute a large family of multi- functional proteins that share antiviral, antitumor and Keywords apoptosis; astrocytoma; CD95 ⁄ Fas; interferon-c; proteasome Correspondence A. M. Giammarioli, Department of Therapeutic Research and Medicines Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanita’, viale Regina Elena 299, 00161 Rome, Italy Fax: +39 06 49903691 Tel: +39 06 49902443 E-mail: anna.giammarioli@iss.it (Received 31 March 2009, revised 21 July 2009, accepted 11 August 2009) doi:10.1111/j.1742-4658.2009.07271.x In the present study, we investigated the mechanisms of the resistance to CD95-mediated cell death and the effects of interferon-c in modulating the susceptibility to CD95-induced apoptosis of human astroglioma cells. We found that interferon-c administration sensitized cancer cells to CD95-med- iated apoptosis. The mechanism underlying this sensitization appeared to be associated with a framework of cell changes, including up-regulation of death receptor at the cell surface, pro-apoptotic molecule Bax and Bak over-expression and mitochondria hyperpolarization, as is known to be associated with cell sensitization to apoptosis. An involvement of the pro- teasome activity in the mechanism of sensitization by interferon-c was also detected, probably as a result of the differing expression of catalytic protea- some subunits. Taken together, these findings suggest that interferon-c could represent a promising candidate for modulating astroglioma cell apoptotic susceptibility. Abbreviations BrdUrd, bromodeoxyuridine; CD95R, CD95 receptor; CisPt, cisplatin; cyt c, cytochrome c; FasL, Fas ligand; FITC, fluorescein isothiocyanate; IFN, interferon; I-jB, NF-jB inhibitor; LMP, low molecular weight protein; MMP, mitochondrial membrane potential; NF-jB, nuclear factor kappa B; PKR, RNA-dependent protein kinase; STAT, signal transducer and activator of transcription. 5920 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS immunomodulatory activities [10]. They have also been implicated in the modulation of apoptosis [11–14]. Interferons are included in therapeutic protocols against human malignancies either as single agents or, more frequently, in combination with chemotherapeutic drugs [15,16]. For example, IFN-c, a type II inter- feron, has been suggested to increase apoptotic suscep- tibility of some tumor cell types (e.g. melanoma cells) [17–19]. In the present study, we show that IFN-c sensitizes astroglioma cells to CD95-mediated apopto- sis, facilitating the induction of biochemical events of relevance in cell death, such as an increased expression of CD95 receptor at the cell surface, the modulation of Bcl-2 family proteins and mitochondrial hyperpolariza- tion, as is known to be associated with cell sensiti- zation to apoptosis [20,21]. Finally, we also suggest that IFN-c could sensitize astroglioma cells to CD95- induced apoptosis by modulating catalytic proteasome activity. Results Sensitization to apoptosis by IFN-c To determine the susceptibility of astroglioma cells to apoptosis, ADF, LN229 and U373 cell lines were treated with monoclonal CD95 serum (clone CH11) and IFN-c, or a combination of these. Astroglioma cell lines were also treated with a chemotherapic com- pound of widespread use in the clinical practice (cis- platin; CisPt), a drug that provided some beneficial effect in the treatment of different astrocytic tumors [22]. Because it was previously reported that sensitiza- tion by IFN-c in other tumor cell lines occurred after at least 48–72 h of treatment [23], astroglioma cells were pre-treated with this cytokine for 72 h before the addition of monoclonal anti-CD95 serum or CisPt. The optimal dose of IFN-c (500 IU) was determined after a dose–response curve assay (see Experimental procedures). As shown in Fig. 1, cell treatment with monoclonal anti-CD95 serum (CH11) for 24 h or IFN-c given alone did not exert any significant pro- apoptotic effect in ADF, LN229 and U373 cell lines, whereas the combined treatment of IFN-c with mono- clonal anti-CD95 serum (CH11) cooperated to trigger apoptosis (Fig. 1A). When we analyzed the effect of CisPt (i.e. cells were treated as indicated in Experimen- tal procedures), we observed a significant increase of apoptotic cells in combined treatment with IFN-c in ADF cells, but not in LN229 and U373 astroglioma cell lines (Fig. 1B). In brief, sensitization to apoptosis by IFN-c occurred in anti-CD95-treated cells in all astroglioma cell lines (although by different degrees: ADF > LN229 > U373) (Fig. 1A), whereas increased apoptotic rates as a result of CisPt were observed in ADF astroglioma cells only (Fig. 1B). On the basis of these results, we decided to investigate the mechanisms underlying IFN-c-mediated sensitization to CD95- mediated apoptosis in the ADF astroglioma cell line. Caspase activity To identify the CD95-mediated apoptotic pathway in IFN-c pre-treated cells, caspase activity was analyzed in ADF cells by both flow cytometry and western blot- ting. In particular, caspase 8 and 10 (mainly involved in receptor-mediated apoptosis) and caspase 9 (mainly involved in mitochondria-mediated apoptosis) were considered. After 72 h of IFN-c treatment followed by monoclonal anti-CD95 serum for a further 24 h, we demonstrated the powerful activation of apical caspas- es (caspase 8 and 10) (Fig. 2A,B). An increase of caspase 9 activity was also evident (Fig. 2C). Western blotting confirmed the results obtained by flow cytom- etry (Fig. 2, right). Accordingly, specific caspase inhibitors significantly impaired apoptotic execution (Fig. 3). In detail, ADF cells pre-treated with IFN-c were pre-incubated with the pan-caspase inhibitor Z-VADfmk and with selec- tive inhibitors of caspase 8 (Z-IETDfmk), caspase 10 (Z-AEVDfmk) or caspase 9 (Z-LEHDfmk) for 2 h before the administration of monoclonal anti-CD95 serum. Notably, inhibition of caspase 10 and 9 was highly effective in impairing the apoptotic process (Fig. 3C,D). The role of mitochondria To further characterize the apoptotic pathway trig- gered by IFN-c ⁄ anti-CD95 in ADF cells, we also focused our attention on the possible role of mito- chondria, which are well known regulators of cell death [24]. In particular, previous studies suggested that a significant decrease of mitochondrial mem- brane potential (MMP) and cytochrome c (cyt c) release into the cytosol can be considered as a hall- mark of apoptosis execution [21,25]. According to the results reported above (Fig. 1), we observed a MMP loss in IFN-c ⁄ anti-CD95-treated cells, whereas no changes were detected with IFN-c and ⁄ or monoclonal anti-CD95 serum given alone. In Fig. 4, graphs of MMP loss from a representative experiment are reported (Fig. 4A). Parallel analyses were carried out on cytosolic cyt c released from mitochondria. Cyt c localization was analyzed by immunofluorescence microscopy after double-labeling A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5921 of cyt c and mitochondria (Fig. 4B). In particular, to visualize mitochondria, ADF cells were loaded with Mitotracker Green, a fluorescent dye that loads into mitochondria (see Experimental procedures). As shown in Fig. 4B, treatments that did not induce apoptosis display a yellow punctuate pattern result- ing from the overlap of green and red fluorescence and consistent with cyt c (red fluorescence) localiza- tion into the mitochondrial compartment (green fluorescence). By contrast, after IFN-c ⁄ anti-CD95 treatment, cyt c exhibited a more diffuse red fluores- cent staining that corresponded to the release of cyt c from the mitochondria into the cytosol (Fig. 4B). As shown in Fig. 4C, quantitative analysis of cyt c release was performed by using a commercial ELISA Kit (note the significant cyt c release in IFN-c ⁄ anti- CD95-treated cells with respect to cells treated with anti-CD95 only). In Fig. 4D, the results of morpho- metric analyses carried out in samples shown in Fig. 4B and obtained by counting at least 300 stained cells for each condition are reported (mean values from three different experiments performed in triplicate are shown). Note the high percentage of cells showing the diffuse red fluorescence. Effects of IFN-c Because the combined treatment of IFN-c and mono- clonal anti-CD95 serum strongly enhanced the induc- tion of apoptosis in ADF cells, and because this 80 A B Control C * * * 30 40 50 60 70 Anti-CD95 IFN-γ IFN-γ/anti-CD95 Apoptotic cells (%) ADF LN229 U373 0 10 20 IFN-γ IFN-γ/CisPt Apoptotic cells (%) 40 50 60 70 80 * CisPt Control 0 10 20 30 ADF LN229 U373 Control 4.1% 2.1% 6.5% 2.5% Control 4.5%3.2% 4.3% PI 45% 7.3% 21% 7.5% 4.0% 53% 9.8% 10.5% 3.6% Ann e xin V -FIT C IFN-γ IFN-γ IFN-γ/anti-CD95 IFN-γ/CisPt CisPt Anti-CD95 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Fig. 1. Evaluation of apoptosis in astrogli- oma cell lines. Histograms show the per- centage of AnnexinV positive cells after anti-CD95 (A) or CisPt (B) administration in the absence or presence of IFN-c in ADF, LN229 and U373 cells. Data were obtained from four different experiments. Note the significant increase of apoptosis in cells pre- treated with IFN-c (*P < 0.01). (C) A repre- sentative flow cytometry experiment with ADF cells is shown. Numbers reported in the bottom and top right quadrants represent the percentage of annexin V single-positive cells and annexin V ⁄ PI double-positive cells, respectively. IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al. 5922 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS increase was the result of a simple additive effect of single agents, we further evaluated the effects of IFN-c administration per se. The aim was to assess the mech- anism involved in the sensitization to apoptosis described above. Up-regulation of CD95 receptor and the release of Fas ligand (FasL) IFN-c has previously been shown to mediate up-regula- tion of CD95 receptor (CD95R) in various types of Caspase 8 activity Caspase 10 activity Caspase 9 activity 50 A B C Positive cells (%) 0 10 20 30 40 * Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 Pro-caspase 8 Caspase 8 α-actin Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 50 0 Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 10 20 30 40 * Positive cells (%) Positive cells (%) Pro-caspase 10 Caspase 10 α-actin Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 20 30 40 50 * 0 10 Pro-caspase 9 Caspase 9 α-actin Control IFN-γ IFN-γ /anti-CD95 Anti-CD95 Control 4.9% 5.6% Counts Fluorescence intensity 7.2% 38.7% IFN-γ Anti-CD95 IFN-γ/anti-CD95 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 300 0 300 0 0 300 300 4% Control 5.2% Fluorescence intensity Counts 5.8% 33.8% IFN-γ Anti-CD95 IFN-γ/anti-CD95 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 300 0 300 0 0 300 300 IFN-γ Control Counts 28.7% 8.7% 6.7% Anti-CD95 4.5% IFN-γ/anti-CD95 Fluorescence intensity 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 300 0 300 0 300 0 300 Fig. 2. Caspase activity. Left: caspase 8 (A), caspase 10 (B) and caspase 9 (C) activity were measured by a colorimetric assay. Data are expressed as the mean ± SD of four independent experiments (*P < 0.01 statistically significant compared to control cells). Middle panel: a representative experiment is shown with respect to the activity of caspase 8, caspase 10 and caspase 9, respectively. Right: caspase 8, caspase 10 and caspase 9 activity by western blotting. Note cleaved caspase 8 (18 kDa), caspase 10 (30 kDa) and caspases 9 (35 kDa) in IFN-c ⁄ anti-CD95-treated cells. Blots were reprobed for a-actin to establish equivalent loading. A representative blot is shown. A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5923 tumors. Up-regulation of CD95R was demonstrated to be related to an increased susceptibility to CD95-depen- dent apoptosis in neuroblastoma, leukemia, melanoma, colon adenocarcinoma and cervical cancer cells [6,26–28]. To evaluate the possible role of up-regulation of CD95R in CD95-mediated cell death, we investigated the constitutive and IFN-c induced expression of CD95R in the ADF cell line. Under our experimental conditions, ADF cells showed a constitutively low expression of CD95R, whereas IFN-c treatment leads to a time-dependent increase of surface receptor expression (Fig. 5A). More- over, time-dependent analysis of FasL released into the medium from IFN-c-treated cells showed only a slight increase of FasL release at all time points (Fig. 5B). Bcl-2 family proteins Sensitization activity of IFN-c has also been corre- lated with Bcl-2 family protein modulation [11]. To investigate the possibility that IFN-c treatment could sensitize ADF cells to CD95 apoptosis through the regulation of Bcl-2 family proteins, we also performed flow cytometry and western blotting analyses to evalu- ate the expression of some important Bcl-2 proteins. Both pro-apoptotic and anti-apoptotic Bcl-2-family proteins were considered. We found that IFN-c admin- istration did not result in any significant change of the anti-apoptotic molecules Bcl- XL and Bcl-2 (Fig. 6A). By contrast, the expression of pro-apoptotic molecules Bax and Bak was found to be significantly increased (Fig. 6B,C). Mitochondria analysis In consideration of the key role of mitochondria in apoptosis execution and on the basis of the above results (Figs 2 and 4), we investigated the effects of IFN-c administration on this organelle. We found A 45 60 75 90 * 0 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Anti-CD95 IFN-γ/anti-CD95 IFN-γ + z-VAD z-VAD + z-VAD + z-VAD 15 30 Apoptotic cells (%) B 45 60 75 90 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Anti-CD95 IFN-γ/anti-CD95 IFN-γ + z-IETD z-IETD + z-IETD + z-IETD Apoptotic cells (%) 0 15 30 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Anti-CD95 IFN-γ/anti-CD95 IFN-γ + z-AEVD z-AEVD + z-AEVD + z-AEVD Apoptotic cells (%) C 45 60 75 90 * 0 15 30 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Anti-CD95 IFN-γ/anti-CD95 IFN-γ + z-LEHD z-LEHD + z-LEHD + z-LEHD Apoptotic cells (%) 45 60 75 90 * D 0 15 30 Fig. 3. Inhibition of CD95-mediated apoptosis by different caspase inhibitors. ADF cells pre-treated with IFN-c were preincubated with selec- tive caspase inhibitors for 2 h before the administration of monoclonal anti-CD95 serum. Histograms show the percentage of Annexin V posi- tive cells treated in the absence or presence of caspase inhibitors: (A) z-VAD-fmk, pan-caspase inhibitor; (B) z-IETD-fmk, caspase 8 inhibitor; (C) z-EAVD-fmk, caspase 10 inhibitor; (D) z-LEHD-fmk, caspase-9 inhibitor. Data are expressed as the mean ± SD of four independent experi- ments using flow cytometry. Note that inhibitors significantly impair the apoptosis of IFN-c ⁄ CD95 treated cells (*P < 0.01). IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al. 5924 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS that cell treatment with IFN-c induced a profound mitochondrial remodeling that was characterized by ultrastructural modifications (Fig. 7A) and re-distribu- tion (Fig. 7B). Moreover, it was recently demon- strated in lymphoid cells that an elevation of MMP is associated with an increased sensitivity to apoptosis and that this hyperpolarization of mitochondria occurs before any activation of caspases and before phosphatidylserine externalization [20,21,29]. Interest- ingly, a marked increase of MMP was detected in IFN-c-treated astroglioma cells (mean values from four different experiments: 24 ± 6% of cells with hyperpolarized mitochondria versus 67 ± 7% detected in IFN- c-treated cells). The results obtained from a representative flow cytometry experiment are shown in Fig. 7C. 6% 8.5% 43% 8.9% J-monomers J-aggregates IFN-γ IFN-γ/anti-CD95 Anti-CD95 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Control IFN-γ IFN-γ/anti-CD95 Anti-CD95 Control Cyt C Mitochondria Merged * Release cyt c (ng·mL –1 ) 40 30 20 10 0 50 40 30 20 10 0 * % of cells with diffuse red staining 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 A B CD Fig. 4. Analysis of MMP and cyt c release. (A) Cytofluorometric analysis of the MMP performed using the JC-1 probe. The num- bers in the bottom quadrants represent the percentages of cells with depolarized mito- chondria (MMP). Note that, after 72 h of IFN-c plus monoclonal anti-CD95 serum for a further 24 h, the mitochondrial depolariza- tion becomes evident as a drop in the MMP. The results obtained from a repre- sentative flow cytometry experiment are shown. (B) Fluorescence microscopy analy- sis of cyt c and mitochondria. The release of cyt c from mitochondria is evident in cells treated with IFN-c and monoclonal anti- CD95 serum. Note that yellow staining in the merged images is lost in IFN-c ⁄ anti- CD95 treated cells where green (mitochon- dria) and red (cyt c) fluorescence are obser- vable. The results obtained from a representative experiment are reported. (C) The release of cyt c is evaluated by a sensitive and specific immunoassay using a commercial ELISA Kit. Data are expressed as the mean ± SD of three independent experiments. (D) Morphometric analyses. The percentage of cells with diffuse red staining have been calculated. Data are expressed as the mean ± SD of four independent florescence microscopy experiments. Note that the treatment with IFN-c ⁄ anti-CD95 significantly increased the release of cyt c. All the analyses were performed 72 h after treatment with IFN-c (*P < 0.01). A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5925 IFN-c and cell signaling Several molecules with important signaling functions are known to be induced by IFN-c. Among these is the double-stranded RNA-dependent protein kinase (PKR), which has also been implicated in the apop- totic process. PKR can provoke apoptosis through its ability to control protein translation in part by activating nuclear factor kappa B (NF-jB) by phos- phorylating its inhibitor, IjB, (23). Moreover, PKR inhibits protein synthesis by phosphorylating eukary- otic translational initiation factor 2 [30]. To deter- mine whether sensitization by IFN-c could be mediated by these pathways, further analyses were carried out. However, flow cytometry analysis of IFN-c-treated cells showed a slight but not signifi- cant increase of phosphorylated eukaryotic transla- tional initiation factor 2 (Fig. 8A). Strikingly, the total protein content, evaluated as described previ- ously [31], was found to be significantly modified (mean values from three different experiments: 310 ± 25 lgÆlL )1 in control cells versus 585 ± 30 lgÆlL )1 in IFN-c-treated cells; P < 0.01). With respect to NF-jB, IFN-c was shown to stimulate a specific signaling that requires components of the NF-jB pathway [32–34]. However, the relationship between IFN-c and NF-jB activation concerning the modulation of tumor cell apoptosis remains contro- versial [35–37]. We therefore decided to evaluate the role of NF-jB in ADF cell death and its possible regulation by IFN-c. We studied the nuclear translo- cation of NF-jB by immunofluorescence analysis in ADF cells treated with IFN-c for 72 h. Control and IFN-c-treated cells were negative for nuclear translo- cation of NF-jB, with both exhibiting a clear cyto- plasmic staining (data not shown). Because the activation ⁄ translocation of NF-jB to the nucleus is regulated by a ubiquitin–proteasome pathway thro- ugh the cytosolic degradation of I-jB, we analyzed the level of the I-jB in IFN-c-treated ADF cells. A significant up regulation of I-jB was detected in IFN-c-treated cells compared to control ADF cells (Fig. 8B, left). This suggests a probable inhibition of proteasome by IFN-c. A recent study has also shown that constitutive proteasome catalytic subun its (b1, b5, b2) can be replaced by their IFN-c inducible counterparts, namely low molecular weight proteins (LMP2 and LMP7) and multicatalytic endopeptidase complex-like 1, resulting in alternatively assembled immunoproteasomes [38]. To characterize the sensi- tivity to apoptosis induced by IFN-c in astrocytoma cells, the expression of proteasome catalytic subunits was then evaluated. We found an increase in LMP2 and LMP7 protease subunits (Fig. 8B, middle and right, respectively). Western blotting confirmed the results obtained by flow cytometry (Fig. 8C). These changes may favor the assembly of immunoprotea- somes, thus modifying the proteasome activity. To better elucidate this point, the effects of a specific proteasome inhibitor such as lactacystin, was also evaluated. Different concentrations of lactacystin (10, 5, 2.5 and 1.25 lm) were given alone or in combina- tion with IFN-c for 72 h and ⁄ or monoclonal anti- CD95 serum for further 24 h to trigger apoptosis. Samples treated with lactacystin alone or in combi- nation with IFN-c did not show any significant change in the apoptotic rates compared to control cells. Conversely, a significant dose-dependent increase of apoptotic cells was evident in lactacy- stin ⁄ anti-CD95-treated cells (Fig. 8D). Interestingly, the sensitizing effect was more evident on adding IFN-c to lactacystin ⁄ anti-CD95. 4 5 6 7 A B Control IFN-γ CD95 expression FasL release 0 1 2 3 0 24 h 48 h 72 h Control IFN-γ 15 20 5 10 24 h 48 h 72 h 0 Fig. 5. CD95 and FasL expression in IFN-c-treated cells. (A) Time course analysis of cell surface expression of CD95 by flow cytome- try on ADF cells treated with IFN-c. Note that surface expression of CD95 was found to be significantly increased in IFN-c-treated cells, starting from 24 h, with respect to control cells. (B) Time- dependent analysis of FasL released into the medium from IFN-c- treated cells. A slight increase of FasL release was detected at all time points studied. The release of FasL into the culture medium was evaluated by a sensitive and specific immunoassay using a commercial ELISA Kit. Data are expressed as the mean ± SD of three independent experiments. IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al. 5926 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS Cell cycle analysis On the basis of these results, and to better define the therapeutic potential of IFN-c in the treatment of as- troglioma cells, the antiproliferative activity of the cytokine was also evaluated. Cell growth was evaluated in terms of viable cell counts after 24, 48 and 72 h of culture. As shown in Fig. 9A, a time-dependent cell growth inhibition was detectable in IFN-c cultured cells. This effect was detectable after 24 h of IFN-c culture and gradually rose, reaching values of  50% and 60% of cell growth inhibition at 72 h. To further investigate the mechanism underlying cell growth inhibition, IFN-c -treated cells were analyzed for cell cycle distribution: subconfluent ADF cultures were treated with IFN-c and harvested after 72 h of culture. IFN-c induced an increased percentage of cells in the G0 ⁄ G1 and a corresponding decrease of cells in the S and G2-M phases. Representative plots of bromodeoxy- uridine (BrdU) versus propidium iodide (PI) are shown in Fig. 9B,C. Discussion In the present study, we demonstrated the ability of IFN-c to significantly increase the sensitivity to Counts Counts 30 60 90 120 150 Bax expression (a.u.) Bcl-XI expression Bcl-2 expression Bax expression Bak expression * Counts 0 90 120 150 * Control Bak α-actin IFN-γ Control IFN-γ Counts 0 30 60 Bak expression (a.u.) Control Control M = 102 Bak α-actin IFN-γ IFN-γ M = 108 Control IFN-γ Control M = 9 IFN-γ M = 8.3 Control M = 54 IFN-γ M = 126 Control M = 55 IFN-γ M = 112 10 0 0 200 10 1 10 2 10 3 10 4 0 200 0 200 0 200 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 A B C Fig. 6. IFN-c effects on pro- and anti-apoptotic proteins. (A) Flow cytometry analysis shows that IFN-c treatment (72 h) did not modify the expression of anti-apoptotic molecules Bcl-2 and Bcl- XL . Conversely, the expression of Bax (B) and Bak (C) were found to be significantly increased by both flow cytometry (left) and western blotting (right). Histograms obtained from four independent experiments (mean ± SD) are shown (*P < 0.01). A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5927 CD95-mediated apoptosis of astroglioma cells. This appears to occur via a mechanism that brings into play: (a) the over-expression of death receptor CD95R; (b) the over-expression of pro-apoptotic proteins Bax and Bak; and (c) mitochondrial changes and remodel- ing. The first is a phenomenon that was already observed in different types of tumors. Up-regulation of CD95R has been related to an increased susceptibility to CD95-dependent apoptosis in leukemia, mela- noma, colon adenocarcinoma and cervical cancer cells [6,26,27,39]. Accordingly, in the present study, apopto- sis induced by the combined treatment IFN-c ⁄ anti- CD95 correlates with cell surface expression of CD95R. By contrast, the weak release of FasL by astroglioma cells did not provoke apoptosis per se. The results obtained also indicate that, in addition to its effect on death receptor expression, IFN-c might regulate the intracellular apoptotic machinery in ADF cells. Indeed, induction of caspase expression by IFN-c resulted in activation of caspase 8 upon anti-CD95 treatment, leading to activation of downstream apop- totic pathway. In the present study, IFN-c treatment Control IFN-γ Control IFN-γ Control C 28% J-monomers J-aggregates 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 68% IFN-γ B A Fig. 7. Effects of IFN-c on mitochondria (A) Transmission electron microscopy analysis indicates that ultrastructural features of mitochondrial network appear modified by 72 h of IFN-c (exposure magnification · 20 000). (B) Fluorescence microscopy analysis clearly shows a redistribution and polarization of mitochondria in IFN-c -treated cells. (C) Quantitative cytofluorimetric analy- ses of the MMP. Note a significant mito- chondrial hyperpolarization in IFN-c-treated cells: numbers in the boxed areas represent the percentage of cells with hyperpolarized mitochondria. IFN-c sensitizes cells to apoptosis A. M. Giammarioli et al. 5928 FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS also lead to an alteration of Bcl-2 family pro-apoptotic proteins. This is in accordance with a previous study suggesting that IFN-c, by inducing signal transducer and activator of transcription (STAT)-1 binding to the c-activated sequence of the target gene, could result in an increased Bax : Bcl-2 ratio [15]. Members of the Bcl-2 family proteins play a major role in governing the mitochondria-dependent pathway of apoptosis, with proteins such as Bax functioning as inducers of apoptosis and proteins such as Bcl-2 functioning as suppressors of apoptosis. In this regard, it is interest- ing to consider the involvement of mitochondrial changes in the sensitization of astroglioma cells by IFN-c. We found a hyperpolarization of mitochondria 10 0 0 50 100 150 A B C D p-eIF2α expression I-kB expression I-kBα Control Percentage of annexin V positive cells Lactacystin Control IFN IFNγ/Lact Lact Lact/anti-CD95 Lact/IFN/anti-CD95 100 80 60 40 20 0 1.25 µ M 2.5 µM 5 µM 10 µM LMP2 LMP7 α-actin α-actin α-actin LMP2 expression LMP7 expression p-eIF2α expression (a.u.) IFN-γ M = 85 IFN-γ Control Control M = 65 IFN-γ M = 16 IFN-γ Control M = 6 Control IFN-γ Control IFN-γ Control IFN-γ M = 45 Control M = 19 IFN-γ M = 27 Control M = 10 0 200 Counts 0 0 200 Counts 200 Counts 0 200 Counts 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Fig. 8. IFN-c and cell signaling. (A, left and right) Modulation of phosphorylated eIF2a by IFN-c. Flow cytometry analyses of phosphorylated eIF2a are expressed as histograms of the mean ± SD of four independent experiments (A, left) A representative experiment of four is shown (right). Flow cytometry (B) and western blotting (C) analysis show that IFN-c treatment increased the expression of I-jB, LMP2 and LMP7 with respect to control cells. (D) Different concentrations of lactacystin (10, 5, 2.5 and 1.25 l M) were given alone or in combination with IFN-c for 72 h and ⁄ or monoclonal anti-CD95 serum for a further 24 h to trigger apoptosis. Note a significant dose-dependent increase of apoptotic cells in lactacystin ⁄ anti-CD95 treated cells and in a combination of IFN-c and lactacystin plus anti-CD95. A. M. Giammarioli et al. IFN-c sensitizes cells to apoptosis FEBS Journal 276 (2009) 5920–5935 ª 2009 The Authors Journal compilation ª 2009 FEBS 5929 [...]... bolster astroglioma cell apoptosis by inducing a type of cellular activation, including mitochondria hyperpolarization and the formation of the immunoproteasome When considering the importance of the induction of cell death in cancer treatment, the results obtained also indicate IFN-c as a possible new candidate for the management of an IFN-c sensitizes cells to apoptosis untreatable form of cancer... FITCconjugated Annexin V ⁄ PI apoptosis detection kit (Marine Biological Laboratory, Woods Hole, MA, USA) in accordance with the manufacturer’s instructions FITC-conjugated Annexin V-positive cells were considered as cells in the early stages of apoptosis Cells distinguished by their ability to take up both FITC-Annexin V and PI were considered as cells in the later stages of apoptosis Live cells were considered... Recently, it was also shown that the induction of immunoproteasome by IFN-c is extremely rapid, transient and self-controlled [38,42] Moreover, in some tumor cells, the induction of immunoproteasome by IFN-c causes an up-regulation of apoptosis- related genes and an increase in early apoptotic cells [11,15,16] For example, Ceruti et al [43] demonstrated that, in ADF cells normally resistant to mitochondriotropic... anti-phosphorylated form of eIF2a (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 37 °C After washing, cells were incubated for 30 min at 37 °C with FITC-labeled anti-mouse or antirabbit sera (Alexa fluor 488-coniugated IgG; Molecular Probes, Leiden, The Netherlands) Evaluation of apoptosis Quantitative evaluation of apoptosis was performed in the total population (adhering + detached cells) using... activation of several transcription factors, such as STATs and NF-jB, and it also leads to the increase of several apoptosis- related molecules, including CD95 receptor, different members of the Bcl-2 family (in particular Bax and Bak) and caspases [46] With respect to the phosphorylation of eIF2a, which provides a fundamental mechanism to couple the rate of protein synthesis with the capacity of fold... Clin Oncol 23, 630–639 45 Awasthi N & Wagner BJ (2004) Interferon-gamma induces apoptosis of lens alphaTN4-1 cells and proteasome inhibition has an antiapoptotic effect Invest Ophthalmol Vis Sci 45, 222–229 46 Gil J & Esteban M (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action Apoptosis 5, 107–114 47 Pierdominici M, Giammarioli AM, Gambardella L, De Felice... were poorly active in apoptosis sensitization, whereas higher doses (1000 and 2000 IU), although effective, were toxic to the cells (and far from possible clinical use) On the basis of these results, and in accordance with previous studies [6], a dose of 500 UI was chosen as the experimental dose for the present study Drug treatments were started 24 h after cells were plated ADF cells were exposed to:... described previously [48] In brief, cells were incubated in complete medium for 10 min at room temperature (25 °C) in the dark with 10 lgÆmL)1 JC-1 probe At the end of the incubation period, cells were washed twice in ice-cold NaCl ⁄ Pi, resuspended in a total volume of 400 lL and analyzed by flow cytometry Detection of caspase-8, -10 and -9 activity Activation state of the caspases 8 and 9 was evaluated... (2004) IFNgamma enhances TRAIL-induced apoptosis through IRF-1 Eur J Biochem 271, 4222–4228 14 Thyrell L, Erickson S, Zhivotovsky B, Pokrovskaja K, ´ Sangfelt O, Castro J, Einhorn S & Grander D (2002) Mechanisms of interferon-alpha induced apoptosis in malignant cells Oncogene 21, 1251–1262 15 Das A, Banik NL & Ray SK (2009) Molecular mechanisms of the combination of retinoid and interferongamma for inducing... induces necrosis of L929 tumor cells via a p38MAPK ⁄ NF-kappaB ⁄ NO-dependent mechanism Oncogene 27, 3780– 3788 Shultz DB, Fuller JD, Yang Y, Sizemore N, Rani MR & Stark GR (2007) Activation of a subset of genes by IFN-gamma requires IKKbeta but not interferondependent activation of NF-kappaB J Interferon Cytokine Res 27, 875–884 Moitreyee CK, Suraksha A & Swarup AS (1998) Potential role of NF-kB and RXR . mechanisms of the resistance to CD95-mediated cell death and the effects of interferon-c in modulating the susceptibility to CD95-induced apoptosis of human astroglioma cells. We found that interferon-c. considered as cells in the early stages of apoptosis. Cells distinguished by their ability to take up both FITC-Annexin V and PI were con- sidered as cells in the later stages of apoptosis. Live cells were. Interferon-c bolsters CD95/Fas-mediated apoptosis of astroglioma cells Anna M. Giammarioli 1 , Rosa Vona 1 , Lucrezia Gambardella 1 ,