Fas-mediated Apoptosis Research Articles

Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome

SUMMARYGoldmann-Favre syndrome, also known as enhanced S-cone syndrome, is an inherited retinal degeneration disease in which a gain of photoreceptor cell types results in retinal dysplasia and degeneration. Although microglia have been implicated in the pathogenesis of many neurodegenerative diseases, the fundamental role of these cells in this disease is unknown. In the current study, sequential analyses suggest that microglia are recruited and appear after outer nuclear layer folding. By crossing rd7 mice (a model for hereditary retinal degeneration owing to Nr2e3 mutation) with mice carrying the macrophage Fas-induced apoptosis (Mafia) transgene, we generated double-mutant mice and studied the role of the resident retinal microglia. Microglial cells in these double-mutant mice express enhanced green fluorescent protein (EGFP) and a suicide gene that can trigger Fas-mediated apoptosis via systemic treatment with AP20187 (FK506 dimerizer). We demonstrated that more than 80% of the EGFP+ cells in retinas from rd7/rd7;Tg/Tg mice express Iba-1 (a microglial marker), and resident microglia are still present in the retina because AP20187 does not cross the blood-brain barrier. Hence, only circulating bone marrow (BM)-derived microglia are depleted. Depletion of circulating BM-derived microglia accelerates retinal degeneration in rd7 mice. An increased number of autofluorescent (AF) spots is a consequence of resident microglia proliferation, which in turn establishes an inflammatory cytokine milieu via the upregulation of IL-1β, IL-6 and TNFα expression. This inflammation is likely to accelerate retinal degeneration. This study not only identifies inflammation as a crucial step in the pathogenesis of retinal degeneration, but also highlights the involvement of specific cytokine genes that could serve as future treatment targets in retinal degenerations.

Suppressors of Cytokine Signaling Promote Fas-Induced Apoptosis through Downregulation of NF-κB and Mitochondrial Bfl-1 in Leukemic T Cells

Suppressors of cytokine signaling (SOCS) are known as negative regulators of cytokine- and growth factor-induced signal transduction. Recently they have emerged as multifunctional proteins with regulatory roles in inflammation, autoimmunity, and cancer. We have recently reported that SOCS1 has antiapoptotic functions against the TNF-α- and the hydrogen peroxide-induced T cell apoptosis through the induction of thioredoxin, which protects protein tyrosine phosphatases and attenuates Jaks. In this study, we report that SOCS, on the contrary, promote death receptor Fas-mediated T cell apoptosis. The proapoptotic effect of SOCS1 was manifested with increases in Fas-induced caspase-8 activation, truncated Bid production, and mitochondrial dysfunctions. Both caspase-8 inhibitor c-Flip and mitochondrial antiapoptotic factor Bfl-1 were significantly reduced by SOCS1. These proapoptotic responses were not associated with changes in Jak or p38/Jnk activities but were accompanied with downregulation of NF-κB and NF-κB-dependent reporter gene expression. Indeed, p65 degradation via ubiquitination was accelerated in SOCS1 overexpressing cells, whereas it was attenuated in SOCS1 knockdown cells. With high NF-κB levels, the SOCS1-ablated cells displayed resistance against Fas-induced apoptosis, which was abrogated upon siBfl-1 transfection. The results indicate that the suppression of NF-κB-dependent induction of prosurvival factors, such as Bfl-1 and c-Flip, may serve as a mechanism for SOCS action to promote Fas-mediated T cell apoptosis. SOCS3 exhibited a similar proapoptotic function. Because both SOCS1 and SOCS3 are induced upon TCR stimulation, SOCS would play a role in activation-induced cell death by sensitizing activated T cells toward Fas-mediated apoptosis to maintain T cell homeostasis.

Apoptotic Volume Decrease (AVD) Is Independent of Mitochondrial Dysfunction and Initiator Caspase Activation

Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation.

Mechanism of Regulating Fas Signaling by Promyelocytic Leukemia Protein (PML) and Its Dominant-Negative Mutant PMLRARα

Abstract 1327 Objective:Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods:Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results:The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIPL/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P<.001). Livers from PMLRARα -transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions:PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

Regulation of Fas-mediated Apoptosis in B-Cell Lymphomas by Nucleolin.

Abstract 2406The death receptor Fas has a key role in mediating homeostasis, elimination of defective cells and more recently promotion of cancer. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas- apoptotic signaling is a basis for primary as well as acquired resistance to chemotherapy. We hypothesized that Fas is subjected to direct regulation and inhibition of Fas attained by cancer cells and may explain the emergence of chemoresistance. To screen for potential binding modulators of Fas, we analyzed lymphoma cells for Fas binding proteins.We first purified Fas associated proteins by using activating CH-11 antibody bound to intact BJAB cells. After immunoprecipitation, any remaining Fas, considered activation–resistant, was subjected to the second immunoprecipitation with Fas antibody B-10 followed by liquid chromatography and tandem mass spectroscopy. This purification scheme identified high scoring peptides derived from nucleolin, a nuclear protein known to be overexpressed in cancer. Nucleolin is selectively expressed on the surfaces of cancer cells and blood vessels undergoing angiogenesis. In a cell culture system, we confirmed binding of nucleolin to Fas and the presence of nucleolin-Fas complexes on the surface of lymphoma cells by surface biotin labeling. Using deletion mutants of nucleolin, we identified RNA binding domain 4 and glycine/arginine rich region to be required for the binding to Fas. BJAB cells with partially knockdown (KD) nucleolin showed significantly higher rates of apoptosis in response to stimulation with CH-11 and FasL when compared to nontarget KD controls. Importantly, the lower levels of nucleolin in knockdown cells did not affect total and surface Fas expression. Nucleolin present on the cell surface prevented binding of FasL and CH-11 to the receptor and thus provides a mechanism for blocking activation of Fas apoptosis. To examine the role of nucleon in vivo, we transfected mice with nucleolin-expressing plasmids using the hydrodynamic transfection method. The mice overexpressing nucleolin showed significantly higher survival rates than vector control transfected mice (P=.01) after a challenge with a lethal dose of agonistic anti-Fas antibody.We next examined the expression of nucleolin in human lymphomas. Cell lines derived from lymphomas of different histological types consistently expressed nucleolin protein. We found nucleolin expressed on the surface of cells in over 20 primary lymphoma isolates, whereas peripheral blood lymphocytes showed low or undetectable levels. Lymphoma tissue microarray staining showed a correlation between nucleolin and Ki-67 expression. Whether nucleolin expression also correlates with adverse clinical features in lymphoma is currently under evaluation.Taken together, we show here that the known cancer associated protein nucleolin is overexpressed on surface of lymphoma cells where it binds to Fas receptor and blocks Fas signaling and apoptosis. We expect that further analysis of nucleolin properties will reveal how Fas-nucleolin interaction can be targeted to enhance killing of cancer cells leading eventually to cell surface nucleolin targeting therapy. Disclosures:Fayad:Roche: Research Funding.

FAS Mutations Are Associated with Therapeutic Resistance in Follicular Lymphoma

AbstractAbstract 1549 Introduction:We recently reported on recurrent genomic alterations present in genome, transcriptome or exome data from 127 cases of NHL (Morin et al. Nature. 2011 Jul 27;476:298–303). Our data suggests FAS acts as a tumor suppressor in B cells based on a selection bias towards mutations that would result in truncated or altered protein, most of which were in the functional death domain. FAS is the prototypical death receptor involved in the extrinsic apoptotic pathway. Germline mutations in FAS have a dominant-negative phenotype, leading to dysfunctional FAS-mediated apoptosis, accumulation of lymphocytes and autoimmune lymphoproliferative syndrome. The role of FAS in lymphomagenesis is unclear. The FAS mutations in our study were limited to 6 cases of germinal center B lymphomas, including 3 cases of aggressive follicular lymphoma (FL). Herein we investigate the role of FAS in therapeutic resistance in FL. Methods:One of the FL cases had treatment-refractory disease. Biopsies taken at diagnosis and after progression following 2 cycles of cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) were available for transcriptome sequencing. Owing to our discovery of FAS mutations in this and other FL patients, we sequenced exon 7–9 of FAS on an extended cohort of 214 clinically-annotated FL samples to determine its potential for association with clinical outcome. To functionally validate our findings we transfected two lymphoma cell lines that are sensitive to FAS-induced apoptosis with either FAS wild-type, FAS mutant (Y232*) or empty vector control. Single clones (wild type and Y232*) with similar expression of FAS protein by flow cytometry were compared for their ability to undergo apoptosis after exposure to the FAS agonist antibody CH-11 and therapeutic levels of cyclophosphamide, adriamycin, vincristine and etoposide. We defined apoptotic cells as those expressing annexin V+/propidium iodine-negative and activated caspase 3 by flow cytometry and cells demonstrating cleaved caspase 8 by western blotting. We further determined if etoposide, chosen because it has no background fluorescence, could change the expression of FAS or FAS ligand in primary lymphocytes using multi-color flow cytometry. Results:We demonstrate that FAS mutations are associated with clinically-aggressive FL. In a patient with treatment-refractory FL, only 3 mutations were confirmed to be somatic and significantly enriched in the post-treatment biopsy: CTSS, RPS24 and FAS Y232* (p values <0.05), where CTSS and RPS24 have no known function in lymphocytes. In an extended cohort of FL samples, 6/214 (3%) had non-synonymous mutations in FAS predicting for a severely altered or truncated protein. Coding FAS mutations were associated with a trend towards an earlier time to progression (1 year versus 2.8 years, p=0.08) and an increased risk of histological transformation to DLBCL (n=4/6, p=0.036). Indeed, 5 of the 6 developed early resistance to primary rituximab-based chemotherapy. In vitro studies demonstrated that the FAS Y292* impairs extrinsic apoptosis induced by CH-11 (decrease in cleaved caspase 8, activated caspase 3 and Annexin V+/PI- cells). However, there was no difference in apoptosis between FAS Y232* mutants and FAS wild type cells after exposure to chemotherapy in vitro. These results led us to speculate that other factors are involved in resistance to chemotherapy. Using primary tonsilar lymphocytes in culture, we observed a significant increase in both FAS expression on primary B cells and the cognate ligand FASL expression in primary T cells following a 24-hour exposure to etoposide. Conclusion:Mutations in FAS are associated with clinical therapeutic resistance in FL. FAS mutations can impair FAS-mediated apoptosis. Our results suggest that chemotherapy may potentiate an immune response in part by increasing FAS expression in B cells and FASL expression in T cells. This may enhance killing of FAS wild type but not FAS mutant expressing tumor cells. Thus the role the tumor microenvironment in chemotherapy-induced cell death may be under appreciated in FL. Disclosures:No relevant conflicts of interest to declare.

Immunogenetic Characterization of Primary Immune Thrombocytopenia (ITP) in Adults: Preliminary Results of the Unit Study.

AbstractAbstract 2192 Background.In adults, ITP displays variable clinical presentation and different response to steroid, Rituximab, and other immune suppressive agents. The pathophysiological differences underlying these different behaviours are mostly unknown and a better knowledge of this biological heterogeneity might help identifying more targeted and rationale treatments for this disease. Purpose.To identify immunogenetic features distinguishing ITP patients from control and different subsets of ITP patients based on clinical presentation and responsiveness to steroid or Rituximab. Several biological analyses were based on the model of autoimmune lymphoprolipherative syndrome (ALPS), a pediatric disease due to genetic defects decreasing function of the Fas death receptor involved in shutting off the immune response and cytotoxic cell function. ALPS displays polyclonal lymphoproliferation, peripheral blood (PB) expansion of TCRαβ+ T-cells double negative for CD4 and CD8 (DN T-cells) and autoimmune manifestations frequently including thrombocytopenia. ALPS is mostly due to deleterious mutations of the Fas gene, but the +1239A>C single nucleotide polymorphism (SNP) of the osteopontin gene (OPN) and several variations of the perforin gene (PRF1) can act as disease modifier. Moreover, defective Fas function and these OPN and PRF1 variations are also displayed by subsets of patients with multiple sclerosis, type-I diabetes mellitus, systemic lupus erythematosus, progressive sclerosis, and chronic inflammatory demyelinating polyneuropathy. Patients and Methods.Adult patients with ITP and matched controls were selected for the analyses and stratified in different clinical subsets according to the disease severity and responsiveness to therapy (asymptomatic, steroid or Rituximab sensitive vs. steroid or Rituximab refractory). Analyses included evaluation of Fas-mediated apoptosis in T cell cultures; proportions of DN-T cells in PB; typing of the +1239A>C SNP of OPN and sequencing of PRF1. All patients were also investigated for TCR monoclonality, whereas BCR monoclonality was analysed in Rituximab-untreated patients only. Results.Analysis of Fas function and DN T-cell expansion was assessed in 100 ITP patients and showed that they displayed higher frequency of defective Fas function than the controls (17/100 vs. 5/100; P<0.05). Expansion of DN T-cells was detected in 2/17 (12%) patients displaying defective Fas function and 3/83 (4%) of those with normal Fas function. Analysis of PRF1 and OPN was performed in 64 patients. Sequencing of PRF1 detected three patients carrying two rare variations; two carried the N252S amino acid substitution (previously described in ALPS) and one the novel R385W amino acid substitution. The overall frequency of these rare variations was higher in the patients than in the controls (4.7% vs. 0.8%, P<0.05). By contrast, the OPN +1239A>C SNP displayed a similar distribution in the patients and the controls. TCR monoclonality was assessed in 76 patients and was detected in 4 of them (5%). BCR monoclonality was assessed in 17 patients in PB and bone marrow and it was always absent. No statistical differences of these parameters were detected comparing patients refractory vs. sensitive to either Rituximab or steroid treatments. However, a trend was found for DN T-cell expansion that tended to be more frequent in Rituximab resistant vs. sensitive patients (0/13 vs. 4/20, P=0.13). Conclusions.These preliminary analyses detected some differences between ITP patients and controls suggesting that defects involved in ALPS development may play a role in adult ITP too. Increasing the patient number is needed to confirm these data and, possibly, to detect differences between clinical subgroups. Disclosures:Off Label Use: Rituximab in ITP.

Tyrosine Kinase Inhibitor-Mediated Apoptosis Is Potentiated by FasL and Increased with IFN-α in Chronic Myeloid Leukemia

AbstractAbstract 4428The Fas death receptor (CD95/TNFSFR6) conveys death and non-death signals through binding to its cognate ligand, FasL (CD95L)1,2. Fas is expressed constitutively in CD34+ cells of patients with chronic myeloid leukemia3. In order to explore the implication of Fas in CML patient's response, we aimed at analyzing expression and function of Fas in the imatinib-sensitive human Bcr-abl+ Fas+ AR 230S cell line, and in its imatinib-resistant counterpart AR 230R.We analysed the Fas-mediated apoptosis of AR230S and showed that not only imatinib (Figure 1) but also nilotinib and dasatinib (data not shown) potentiate Fas-L mediated apoptosis. This potentiation is dose dependent (Figure 2). Interestingly, there is no potentiation of TKI-mediated apoptosis by FasL in the imatinib-resistant counterpart AR 230R which is Fas negative. The Fas knockdown with a lentivirus-expressing a FAS-shRNA in AR 230S was associated with a disappearance of potentiation effect, whereas the up regulation of Fas expression in AR230 S cell line using lentivirus-expressing human Fas (hFas) induced an emphasized potentiation effect confirming the pivotal role of Fas in the potentiation of TKI-mediated apoptosis.Notably, IFN-α is able to enhance expression of Fas on CD34+ cells from CML patients3. We thus explored the action of IFN-α on the Fas potentiation of TKI-mediated apoptosis. We observed that preincubation with IFN-α increased potentiation in a dose-dependant manner (Data not shown).In conclusion, our preliminary results demonstrate the pivotal role of Fas in the potentiation of TKI-mediated apoptosis and highlight the role of IFN-α in the design of CML treatments. [Display omitted] [Display omitted] Disclosures:Legros:Novartis: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Celgene: Speakers Bureau.

Postexposure Application of Fas Receptor Small-Interfering RNA to Suppress Sulfur Mustard–Induced Apoptosis in Human Airway Epithelial Cells: Implication for a Therapeutic Approach

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Requirement of Apoptotic Protease-Activating Factor-1 for Bortezomib-Induced Apoptosis but Not for Fas-Mediated Apoptosis in Human Leukemic Cells

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

Up-regulated Expression of Fas Antigen in Peripheral T cell Subsets in Patients with Myasthenia Gravis

Recent reports have linked various autoimmune diseases to defective Fas-mediated apoptosis or Fas expression. Here we aimed to determine whether Fas-mediated apoptosis is involved in the pathogenesis of myasthenia gravis (MG). The expression of Fas antigen in peripheral T cell subsets from 17 Chinese patients with MG and 13 healthy individuals was determined by flow cytometry, and its associations with clinical classification, thymus pathology, the concomitance with hyperthyroidism (HT) and corticosteroid treatment were investigated. Compared with normal controls, a significantly up-regulated expression of Fas antigen was observed in the peripheral CD4+, CD4+CD8- and CD4-CD8- T cell subsets from patients with MG. Fas expression in CD4-CD8+ T cells of MG patients with normal thymus was significantly higher than that of patients with thymoma. Fas expressions in CD4+CD8+ T cells in MG patients with HT was significantly higher than controls and the ones without HT. Enhanced Fas expressions was found in CD4-CD8+ and CD4-CD8- T cells of MG patients with corticosteroid treatment, but no significant difference of Fas expression in peripheral T cells between patients with ocular MG (OMG) and general MG (GMG) was observed. Fas antigen may play a role in the pathogenesis of MG. It may be involved in the mechanisms of corticosteroid treatment, and with the occurrence of HT. OMG may represent a systemic disease, similar to that of GMG.

ERK-Mediated Activation of Fas Apoptotic Inhibitory Molecule 2 (Faim2) Prevents Apoptosis of 661W Cells in a Model of Detachment-Induced Photoreceptor Cell Death

In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death.

Unphosphorylated STAT1 Promotes Sarcoma Development through Repressing Expression of Fas and Bad and Conferring Apoptotic Resistance

STAT1 exists in phosphorylated (pSTAT1) and unphosphorylated (uSTAT1) forms each regulated by IFN-γ. Although STAT1 is a key mediator of the IFN-γ signaling pathway, an essential component of the host cancer immunosurveillance system, STAT1 is also overexpressed in certain human cancers where the functions of pSTAT1 and uSTAT1 are ill defined. Using a murine model of soft tissue sarcoma (STS), we show that disruption of the IFN effector molecule IRF8 decreases pSTAT1 and increases uSTAT1 in STS cells, thereby increasing their metastatic potential. We determined that the IRF8 gene promoter was hypermethylated frequently in human STS. An analysis of 123 human STS specimens revealed that high uSTAT1 levels in tumor cells was correlated with a reduction in disease-specific survival (DSS), whereas high pSTAT1 levels in tumor cells were correlated with an increase in DSS. In addition, uSTAT1 levels were negatively correlated with pSTAT1 levels in these STS specimens. Mechanistic investigations revealed that IRF8 suppressed STAT1 transcription by binding the STAT1 promoter. RNAi-mediated silencing of STAT1 in STS cells was sufficient to increase expression of the apoptotic mediators Fas and Bad and to elevate the sensitivity of STS cells to Fas-mediated apoptosis. Together, our findings show how the phosphorylation status of pSTAT1 determines its function as a tumor suppressor, with uSTAT1 acting as a tumor promoter that acts by elevating resistance to Fas-mediated apoptosis to promote immune escape.

Assessment of immunolocalization of Fas antigen in the ovarian follicle and the role of IGF-I in Fas-L induced granulosa cell apoptosis in water buffalo

Primordial follicle pool size in the ovary is small and rate of follicular atresia is high in water buffaloes. The objective of this experiment was to study the expression of Fas antigen in granulosa cells of water buffalo ovarian follicle, and to know the role of insulin like growth factor-1(IGF-I) on Fas-ligand (Fas-L) mediated apoptosis. The Fas expression was observed mainly in the granulosa cells of the follicles. The expression was higher in the granulosa cells, which were denuded from basement membrane, indicating apoptosis. Healthy follicles which had intact multilaminar granulosa cell layer showed negative reaction for Fas antigen. The granulosa cells from 3–6 mm diameter follicles of the buffalo ovaries collected from the slaughterhouse ovaries were cultured in vitro to assess the effect of Fas-L at 10, 20 and 50ng/ ml on granulosa cell viability. The Fas-L even at the lowest dose tested (10ng/ml) significantly decreased granulosa cells viability as compared to control; whereas addition of IGF-I to the culture significantly improved granulosa cell viability. Addition of IGF-I could significantly improve the granulosa cells viability in the Fas-L treated culture. Overall this study demonstrated the existence of Fas and Fas mediated apoptosis in the granulosa cells of the buffalo follicle, and also suggested that IGF-I could rescue granulosa cells from Fas-L mediated apoptosis.

Ethanol extract of paeonia suffruticosa Andrews (PSE) induced AGS human gastric cancer cell apoptosis via fas-dependent apoptosis and MDM2-p53 pathways

BackgroundThe root bark of Paeonia suffruticosa Andrews (PSE), also known as Moutan Cortex, has been widely used in Asia to treat various diseases. The molecular mechanisms by which PSE exerts its anti-oxidant and anti-inflammatory activities are well known, but its anti-cancer activity is not yet well understood. Here, we present evidence demonstrating that PSE can be used as a potent anti-cancer agent to treat gastric cancer.MethodsThe effects of the ethanol extract of PSE on cell proliferation were determined using an MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) assay. Cell cytotoxicity induced by the PSE extact is measured using an LDH leakage assay. Flow cytometry was used to analyze the cell cycle and to measure the subG0/G1 apoptotic cell fraction. Apoptosis induced by the PSE extact is also examined using a DNA fragmentation assay. Western blot analysis is used to measure the levels of apoptotic proteins such as Fas receptor, caspase-8, caspase-3, PARP, Bax, Bcl-2, MDM2, and p53.ResultsThis study demonstrated that treating AGS cells with the PSE extact significantly inhibited cell proliferation and induced cytotoxicity in a dose- and time-dependent manner. The PSE extract also induced apoptosis in AGS cells, as measured by flow cytometry and a DNA fragmentation assay. We found that the PSE extract induced apoptosis via the extrinsic Fas-mediated apoptosis pathway, which was concurrent with the activation of caspases, including caspase-8 and caspase-3, and cleavage of PARP. The MDM2-p53 pathway also played a role in the apoptosis of AGS cells that was induced by the PSE extract.ConclusionsThese results clearly demonstrate that the PSE extact displays growth-suppressive activity and induces apoptosis in AGS cells. Our data suggest that the PSE extact might be a potential anti-cancer agent for gastric cancer.

Emerging role of berbamine as an anti-cancer agent in systemic malignancies besides chronic myeloid leukemia

For the Letter to the Editor “Emerging role of berbamine as an anti-cancer agent in systemic malignancies besides chronic myeloid leukemia”, we are pleased with the interest in our article (Liang et al., 2011), and appreciate the insightful comments made by Dr. Kapoor (2012). He systemically reviewed application of berbamine as an anti-cancer agent during the past few years and found that berbamine also shows anti-cancer activity for other systemic malignancies besides chronic myeloid leukemia. Extensive scrutiny of its molecular targets and mechanism of action in the past few decades has provided important insights. At the cellular level, berbamine exhibits significantly antiproliferative activity, inhibiting the proliferations of leukemia, lymphoma (Du et al., 2010), breast cancer (Liu et al., 2010), and hepatocellular carcinoma (Wang et al., 2009). At the molecular level, berbamine-mediated anti-tumor activity was shown to be involved in PI3K/Akt, nuclear factor-kappaB (NF-κB) (Du et al., 2010), Fas-mediated apoptosis (Wang et al., 2009), and P-glycoprotein (Liu et al., 2010). However, its targets and molecular mechanism of action are still largely elusive. We feel that identification of the direct molecular target of berbamine will account for the diverse biological activities.

Investigation of the Role of Cytokeratin 8/18 Intermediate Filaments in Granulosa Cell Fate.

Apoptosis of granulosa cells is an early indication of follicular atresia; but the cellular mechanisms that drive this process, especially its cell specificity, are relatively unknown. Recent evidence suggests cytoskeletal stress filaments, such as cytokeratin 8/18 (KRT8/18) intermediate filaments, intrinsically protect epithelial cells from apoptosis by influencing intracellular signaling and diminishing death receptor expression (e.g., FAS) on the cell surface. The objective of this study was to determine the relative expression of KRT8/18 filaments in a human granulosa tumor cell line (KGN cells) and evaluate their role in the context of FAS-mediated apoptosis. Flow cytometric analysis following culture revealed ~ 92% of cells stained positively for KRT8/18 expression. Caspase-Glo 3/7 and MTS assays were used to monitor cell apoptosis after 8- and 24-hour treatment of KGN cells with anti-FAS antibody, CH11 (1 μg/mL) to activate FAS. CH11 treatment caused a modest increase in caspase activity (2 fold) but had no effect on cell viability (P<0.05; n= 6 expts). However, CH11 combined with a non-toxic concentration of the protein synthesis inhibitor cycloheximide (0.25 μg/mL) provoked extensive apoptosis (~60% cells; P<0.05; n=6 expts) characterized by 10 fold increases in caspase 3/7 activity and reduced cell viability. Under these conditions, inhibition of ERK1/2 signaling with the MEK1/2 inhibitor PD98059 (30 μM) had no effect (P>0.05; n=3 expts), but inhibition of the PI3K/Akt pathway with Wortmannin (100 nM) provided modest protection from FAS-mediated apoptosis (P<0.05; n=3 expts). Initial experiments to genetically knock-down KRT8/18 expression with short interfering RNA (siRNA; 10 nM) sensitized KGN cells to FAS-induced apoptosis (P<0.05) compared to cells exposed to a scrambled form of siRNA (n=3 expts). The present study provides initial evidence that human granulosa cells express KRT8/18, and that KRT8/18 filaments are cellular determinants that prevent FAS-mediated apoptosis. Moreover, the results suggest that the synthesis of labile protein(s) and the regulation of the PI3K/Akt pathway provide granulosa cells with additional mechanisms to avoid cell death and ultimately, follicular atresia. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3+ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3+ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3+ T-cells in PBMC cultures required ‘classical’ CD14+ monocytes, which enhanced T-cell activation. CD3+ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3+ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

Toso, a Functional IgM Receptor, Is Regulated by IL-2 in T and NK Cells

We find that the cell surface receptor Toso is dramatically downregulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5-dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation-induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an antiapoptotic molecule.