Fas Antibody Research Articles

Catalase inhibition can modulate the ability of peripheral blood T cells to undergo apoptosis in Crohn's disease.

Crohn's disease (CD) is a chronic relapsing inflammatory disorder in which defective apoptosis of mucosal T cells is postulated to produce sustained inflammation and reactive oxygen species accumulation. Whether CD T cells are intrinsically resistant to apoptosis or whether this resistance is acquired at the intestinal site needs to be clarified, as the cellular mechanisms modulate the impaired apoptosis in these cells. Here, we analysed peripheral blood T cells from patients naïve to specific CD treatment at the onset and from healthy controls. Non-activated freshly purified lymphocytes were cultured and submitted to in vitro protocols for activation (CD3/CD28 antibodies) and apoptosis (Fas antibody). Cells were analysed by flow cytometry. Caspases (3, 8, and 9) and catalase activity were measured; protein levels of bax, Bcl-2, and NF-kB were detected by western blotting, and cytokines by Luminex-based assays. The results showed that CD4 T cells from CD patients are less prone to apoptosis before they can migrate to the intestinal mucosa. Caspase-9, FasR, sIL-2Rα, IL-17A, IFNγ, IL-6, TNF-α, and IL-10 were shown to be significantly different in CD but not for the rest of the analysed biological elements. Catalase activity was significantly reduced in CD T cells, which was confirmed in ex vivo experiments in which catalase inhibition in T cells from healthy controls triggered apoptosis inhibition in a dose-dependent manner. In conclusion, apoptosis inhibition of CD T cells is a feature of these cells before they can migrate to the intestinal mucosa. Noteworthy, the impaired apoptosis of T cells can be directly influenced by catalase inhibition.

Evaluation of FAS and eNOS expression in COVID-19 placenta: histopathological and immunohistochemical study.

In this study, the effects of cell adhesion, inflammation and apoptotic changes on fetal development in cases of COVID-19 placenta were investigated. Placenta tissue samples from 15 COVID-19 and 15 healthy pregnant women were taken after delivery. Tissue samples were fixed in formaldehyde, then blocked with paraffin wax and 4-6 µm thick sections were cut and stained with Harris Hematoxylene-Eosin. Sections were stained with FAS antibody and endothelial nitric oxide synthase (eNOS) antibody. In COVID-19 placenta section, deterioration of the root villus basement membrane structure in the maternal region, decidua cells and syncytial cell degeneration, significant increase in fibrinoid tissue, endothelial dysfunction in free villi and intense congestion in blood vessels, increase in syncytial nodes and bridges were observed. In terms of inflammation, eNOS expression was increased in Hoffbauer cells, dilated blood vessels endothelial cells in chorionic villi, and surrounding inflammatory cells. Positive FAS expression was also increased in the basement membranes of root and free villi, syncytial bridge and nodes, and endothelial cells. The effect of COVID-19 caused an increase in eNOS activity and acceleration of the proapoptotic process and the deterioration of cell-membrane adhesion.

Impaired Death Receptor Signaling Mediates Cross-Resistance to Immunotherapy in MM

The treatment landscape in various cancers is currently shifting from genotoxic drugs to immunotherapy, such as monoclonal antibodies, immunoconjugates, T-cell engaging antibodies (TCE) or CAR T-cells (CARTs). This is in particular true for multiple myeloma (MM), in which unprecedented treatment efficiencies are induced by these novel therapies; however, patients continue to relapse and a subset even faces primary resistance, with the underlying mechanisms poorly understood. The role of the death receptor signaling proteins FADD and BID in MM resistance towards immunotherapy has not yet been explored. Therefore, we first generated FADD and BID CRISPR-Cas9 knock out (KO) models in two independent MM cell lines, AMO1 and L363. We confirmed the functional impact of the KO genes on the apoptotic activity using apoptosis inducing, CD95 activating antibody (anti-FAS Ab). As hypothesized, no apoptosis was observed in our FADD KO clones following 24 hours treatment with 15µg/ml Fas antibody, compared to a 50% increase of apoptosis in the WT cells via Annexin-V PI staining. Activity of Caspase 3/7 and 8 increased 3-4 folds in the WT cells and no increase was seen in our KO models by using commercially available fluorometric assays. Detection of cleaved PARP in WT and its absence in KO clones via Western blotting further validated the inactivation of the apoptotic pathways in our KO models. Next, we treated our KO cells with 0,1µg/ml Daratumumab (Dara) along with 10:1 ratio of PBMCs from healthy donors and 5:1 and 10:1 effector to target ratio of BCMA-CARTs: Of note, killing efficacy by Daratumumab was significantly lower in our KO models compared to the WT cells (22% vs 47% cell death) after 24 hours of treatment. Similarly, BCMA-CARTs efficiency was dramatically reduced with 58% survival in our KO models compared to around 10% in WT cells. Strikingly, we did not observe such a difference in IC50 between the KO and the WT cells when we treated for 3-5 days with Bortezomib, Lenalidomide, Doxorubicin, Melphalan, or the approved ADC Belantamab mafodotin. To determine the functional profile of the CART cells we performed a commercially available cytokine quantification assay (Human IL-2, Human IFN- γ) in response to 20 hours of antigen stimulation with WT and KO cells and no difference in IL-2 and IFN-γ production was observed. In addition, we excluded CD38 or BCMA antigen loss in our KO model by Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and confirmed no significant differences in receptor density /µm2 or antigen surface distribution between the WT and the KO cells. To determine the clinical relevance of our findings we screened for decreased FADD and BID expression levels in a pilot cohort of 24 newly diagnosed (NDMM) and 54 relapsed refractory MM (RRMM) patients and normalized expression levels with 26 CD138+ bone marrow plasma cell samples from healthy donors. We did not find significant expression differences between the cohorts (that did not include patients post TCE or CARTs). However, when we ranked the patients according to their gene expression of FADD and BID the ten patients with the lowest values were predominantly Daratumumab resistant (7/10 and 9/10, respectively), whereas in the patients with highest FADD BID expression Daratumumab resistance was less pronounced (3/10 and 4/10 respectively). In conclusion, our study highlights induction of apoptosis as the main mode of action of T-cell based immunotherapies in MM. Furthermore, we provide first evidence, that gene expression of FADD and BID is essential for the action of Daratumumab and BCMA CARTs but may be dispensable for the anti-tumor effects in MM of ADC, IMiD, PI or chemotherapeutic agents. In our in vivo screen, we identified patients with low expression of FADD and BID being resistant to Daratumumab, suggesting a potential resistance mechanism.

Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis.

Simple SummarySolid tumor cells can lose or heterogeneously express antigens to become resistant to chimeric antigen receptor (CAR) T cell therapy. Here, we explore whether epigenetic manipulation to unleash antigen-independent killing mechanisms can overcome this hurdle. KDM1A is overexpressed in many cancers and removes lysine methylation on histones that keeps the DNA firmly packed to selectively activate or repress gene activity, depending on the specific lysine target. KDM1A also regulates the expression of nonhistone proteins. We inhibited KDM1A in the childhood tumor, neuroblastoma, to increase FAS expression on tumor cells. The FAS receptor can be triggered to induce cell death when bound by the FAS ligand on CAR and other activated T cells present in the tumor environment, even if the tumor cells lack the target antigen. FAS upregulation via KDM1A inhibition sensitized neuroblastoma cells to FAS-FASL-mediated killing and augmented CAR T cell therapy against antigen-poor or even antigen-negative neuroblastoma. Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.

P53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

Background & AimsA coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury.MethodsNitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2.ResultsIn a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response.ConclusionsCharacteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

The revascular and transplanted effects of vascular endothelial cells on islets grafts

Objective To investigate the revascular and transplanted effects of islet grafts after co-transplantation with vascular endothelial cells (ECs) in diabetic rats. Methods The rat ECs and islet cells were isolated and purified, then subcutaneously co-transplanted to the inbred SD male rats with diabetes mellitus, the group of islets transplanted alone, group of phosphate buffered solution, and group of normal rats served as control. Islet grafts revascularization degree, islet cells activity and apoptosis were observed by immunohistochemical double staining of CD31, insulin and factor associated suicide (Fas) antibody. The results of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT), the blood glucose and insulin levels of the rats and the survival time of the islet grafts were compared. Results The blood glucose concentrations of IPGTT and IPITT, the blood glucose and insulin levels of rats analyzed by multivariate analysis in the ECs and islets transplantation group were better than those in the islets transplantation alone group (P<0.05). The islet grafts mean survival time of the ECs and islets transplantation group was longer than that of the islets transplantation alone group (P<0.05). On the 7th day after transplantation, mean microvascular density of islet grafts per square millimeter in the ECs and islets transplantation group was 26.4±6.1, significantly greater than that in the islets transplantation alone group 18.3±5.7 (P<0.05). In the ECs and islets transplantation group, insulin staining intensity was higher than that in the islets transplantation alone group (P<0.05), while factor associated suicide(Fas) staining intensity was lower than that in the islets transplantation alone group (P<0.05). Conclusion Co-transplantation with ECs could promote the revascularization of islet grafts and improve the effect of islet transplantation. (Chin J Endocrinol Metab, 2018, 34: 516-521) Key words: Islet transplantation; Endothelial cells; Revascularization; Diabetes mellitus

Corrigendum.

XenotransplantationVolume 23, Issue 2 p. 171-172 CorrigendumFree Access Corrigendum This article corrects the following: Expression of Fas but not Fas ligand on fetal pig β cells Lijun Bai, Kathrin Maedler, Marc Donath, Bernard E. Tuch, Volume 11Issue 5Xenotransplantation pages: 426-435 First Published online: August 10, 2004 First published: 22 April 2016 https://doi.org/10.1111/xen.12200AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Bai et al. 1, Figures 1 and 5 were published incorrectly, and the correct figures and their legends are illustrated below: The authors regret the error. Figure 1Open in figure viewerPowerPoint Fas expression on fetal pig pancreatic cells. Fetal pig ICCs stained with (A) anti-Fas polyclonal antibody. Positively stained cells are red. Bar = 20 μm. (B) Anti-insulin antibody. Positively stained cells are green. (C) The enriched single β-cells stained with Fas antibody. Positively stained cells are red. Bar = 20 μm. (D) The enriched single β-cells stained with Fas antibody and analyzed by flow cytometry. M1 is the gate for positive cells. The percentage of positive cells in this Figure is 73%. (E) Negative control for flow cytometry. Figure 5Open in figure viewerPowerPoint Expression of FLIP on fetal pig pancreas. (A) Double immunostaining for FLIP in red and insulin in green in a fetal pig pancreatic tissue section. (B) Immunoblotting of FLIPL, actin, caspase 8 and 3. Fetal pig ICCs cultured with or without IL-1β or anti-Fas antibody were analyzed after 24-, 72-, and 96-h incubation. Samples were loaded in parallel and anti-FLIP and anti-caspase 3 as well as anti-caspase 8 and anti-actin were blotted on the same membrane, respectively, after stripping. One representative out of four experiments is shown. Reference 1Bai LJ, Maedler K, Donath M, Tuch BE. Expression of Fas but not Fas ligand on fetal pig β cells. Xenotransplantation 2004; 11: 426– 435. Volume23, Issue2March/April 2016Pages 171-172 FiguresReferencesRelatedInformation

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

Apolipoprotein A1 as a potential biomarker in the ascitic fluid for the differentiation of advanced ovarian cancers

Context: Primary ovarian cancer and ovarian metastasis from non-ovarian cancers in advanced stage are closely mimicking conditions whose therapeutics and prognosis are different.Objective: To identify biomarkers that can differentiate the two variants of advanced ovarian cancers.Methods: Gel-based proteomics and antibody-based assays were used to study the differentially expressed proteins in the ascitic fluid of fourteen patients with advanced ovarian cancers.Results: Programmed Cell Death 1-Ligand 2, apolipoprotein A1, apolipoprotein A4 andanti-human fas antibody are differentially expressed proteins.Conclusions: Apolipoprotein A1 with a 61.8 ng/ml cut-off is a potential biomarker with the best differentiating statistical parameters.

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 expression promotes proteasomal activity in toxin-induced parkinsonism.

Fas (CD95), commonly categorised as a death receptor due to its well-defined role in apoptosis, can paradoxically also promote neuroprotection. We have previously found that defects in Fas signalling render mice highly susceptible to neural degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD). Decreased activity of the ubiquitin proteasome system and accumulation of protein aggregates are implicated in PD pathogenesis. Here, we investigate the relationship between Fas and ubiquitin proteasomal activity in neuronal cells. We performed proteasome assays in neuroblastoma cells and in midbrain cultures of wild-type and Fas-deficient mice. Neuroblastoma cells upregulated proteasomal activity in response to an activating Fas antibody in vitro. Furthermore, neural tissue from Fas-deficient mice showed decreased proteasomal activity compared with the tissue from wild-type mice when exposed to a PD-inducing toxin in vivo. These findings suggest that mechanisms for Fas-mediated neuroprotection may include Fas-induced upregulation of proteasomal activity, and consequently less accumulation of toxic protein aggregates.

Proteomics of the ascitic fluid for the differentiation of advanced ovarian cancer.

e15510 Background: Ovarian cancers are classified as primary, if it arises in the ovary and secondary or metastatic, if the origin is from other parts of the body. The clinical manifestations of these cancers in the advanced stage are very similar making it difficult to distinguish clinically, hiostopathologically and radiologicaly. The therapeutics and management of the primary and secondary malignancies are completely different. While, the advanced primary malignancies are treated by cytoreduction followed by chemotherapy, the metastatic tumors are treated mainly with palliative chemotherapy. The prognosis is better for primary than secondary ovarian cancer making their diagnosis very crucial for patient care. Methods: 1D and 2D-gel based proteomic approaches were used to study the differentially expressed proteins in the ascitic fluid of patients (10 primary and 4 secondary) with advanced ovarian cancer. The relative ratios of the protein expression were estimated by densitometric analysis. The bands/spots with more than three fold difference were subjected to in-gel trypsin digestion and identified by mass spectrometric analysis. The differential expression of one of the proteins was further validated by western blot experiments and ELISA. Results: Programmed Cell Death 1-Ligand 2 and apolipoprotein A1 were seen to be up regulated in the advanced primary ovarian cancer while apolipoprotein A4, and chain L, humanized version of the anti-human fas antibody Hfe7a were seen to be up regulated in the metastatic variant. Validation for the expression of apolipoprotein A1 shows that a 61.8ng/ml cut off is ideal to differentiate the primary and secondary advanced ovarian cancers. The assay has 100% sensitivity, 75% specificity, positive predictive value of 90.9%, negative predictive value of 100%, accuracy of 92.85% and a pre-test odds positive of 2.5. Conclusions: Proteomics of ascitic fluid is useful for the differentiation of advanced ovarian cancers. There are proteins which are differentially expressed in the ascitic fluid of patients with primary and secondary ovarian cancer. Apolipoprotein A1 is a potential biomarker that can be used to differentiate the closely mimicking clinical scenarios of advanced ovarian cancer.

Abstract 5385: Effect of lenalidomide on antileukemia activity in vitro against adult T-cell leukemia (ATL)

Abstract Background: Lenalidomide is an FDA approved agent for del 5q myelodysplastic syndromes and in combination with dexamethasone for previously treated multiple myeloma. Recently, phase II clinical trial has shown its anti-tumor activity against recurrent and refractory T-cell lymphomas. However, the mechanism underlining its effect is poorly understood. Adult T-cell leukemia (ATL) is a mature T cell malignancy caused by Human T-cell leukemia virus type I (HTLV-I) that is endemic in southwestern Japan, the Caribbean islands and Central Africa, and it still remains as one of the most difficult malignancies to be cured. We have evaluated the anti-ATL activity of lenalidomide in vitro using HTLV-I positive cell lines. Methods: The effect of lenalidomide (10-3-102µM) and Fas antibody (25-400 ng/mL) was assessed on the proliferation of HTLV-I positive cell lines by CellTiter-Glo Luminescent Cell Viability Assay. The cytotoxic effect against HTLV-I positive cell lines of lenalidomide on IL-2 primed peripheral blood mononuclear cells (PBMNCs) as well as sorted CD56 positive and CD3 negative cells from IL-2 primed PBMNCs from healthy volunteers was studied by 51Cr releasing assay. Results: Lenalidomide alone did not inhibit cellular proliferation. Even 6 day incubation did not inhibit or stimulate proliferation of HTLV-1 positive cell lines. Cytotoxic activity of IL-2 primed PBMNCs from healthy volunteers against HTLV-I positive cell lines was enhanced by lenalidomide. Sorted NK cells have also shown enhanced cytotoxic activities on HTLV-1 positive cells. That effect was observed regardless of Tax or cellular FLICE-inhibitory protein (cFLIP) expression. Therefore, even Fas resistant HTLV-1 positive cell lines were succumbed to death by enhanced cytotoxicity by lenalidomide, whereas lenalidomide did not interact Fas inducing apoptotic pathways. Conclusions: These results have shown that lenalidomide enhances cytotoxic effects against HTLV-I positive cells, without direct anti-proliferative effect on those cell lines. That effect was mediated through CD56 positive and CD3 negative cells stimulated by lenalidomide. Further study is now on-going to elucidate the mechanism of those actions in an in vivo setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5385. doi:1538-7445.AM2012-5385

C/EBPα down-regulation is associated with reduced hepatic cellular viability during hypoxia in vitro and in vivo

C/EBPα transcription factor is a key regulator in liver biology and was preliminarily shown to be down-regulated in hypoxic primary rat hepatocytes. The aim of this study was to explore the possible association between C/EBPα expression level and hepatocyte viability in both the in-vitro cultured hypoxic rat primary hepatocytes and two models of acute liver hypoxia induced by carbon tetrachloride or Fas antibody. C/EBPα mRNA was significantly down-regulated under hypoxic conditions both in vitro and in vivo, which was paralleled by a similar decrease in hepatocyte viability and partially reversed by 3D matrix and dexamethasone. These results suggested that C/EBPα down-regulation may be one mechanism of reduced hepatocyte viability in these settings.

S1688 Resistance to Apoptosis in Immune Cells from Crohn's Disease Patients Is Already Evident in Peripheral Non-Activated T-Lymphocytes When They Are Activated and Exposed to Agonist Fas Antibody In Vitro

S1688 Resistance to Apoptosis in Immune Cells from Crohn's Disease Patients Is Already Evident in Peripheral Non-Activated T-Lymphocytes When They Are Activated and Exposed to Agonist Fas Antibody In Vitro

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.

Maternal and neonatal lipopolysaccharide and Fas responses are altered by antenatal risk factors for sepsis

The diagnosis of neonatal sepsis is difficult, resulting in unnecessary treatment to minimize morbidity and mortality. We hypothesized that exposure to antenatal risk factors for sepsis alters the perinatal neutrophil phenotype. The study setting was a tertiary referral university-affiliated maternity and neonatal hospital. Neutrophils from adults, normal neonates, neonates with antenatal sepsis risk factors and their respective maternal samples were incubated alone, with agonistic Fas antibody or with lipopolysaccharide (LPS). Surface receptor CD11b expression and the percentage apoptosis (persistent inflammatory response) were assessed using flow cytometry. Both mothers and asymptomatic neonates exposed to maternal sepsis risk factors had increased spontaneous neutrophil apoptosis compared to their respective controls. Infants with sepsis were LPS and Fas hyporesponsive. Maternal neutrophils had a delay in apoptosis in all groups with enhanced LPS and Fas responses associated with neonatal sepsis. CD11b expression was not altered significantly between groups. Maternal neutrophil function is altered in neonatal sepsis and may have a diagnostic role. Neonatal sepsis was associated with LPS hyporesponsiveness, potentially increasing susceptibility to infection.

Glutathione Export during Apoptosis Requires Functional Multidrug Resistance-associated Proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.

Fas-Mediated Apoptotic Signal Transduction in Keloid and Hypertrophic Scar

The pathogenesis of keloid and hypertrophic scar is less well understood because of the lack of animal models. The cell-surface Fas receptor, which is widely distributed in skin components, has been shown to be an important factor that induces apoptosis in human dermal fibroblasts. In this report, the authors performed a comparative study on apoptotic signal transduction in fibroblasts derived from keloids and hypertrophic scars. Fibroblasts harvested from 10 patients with keloids and hypertrophic scars were used for this study. At first, apoptosis induction using Fas antibody and C2-ceramide were evaluated using electron microscopy and flow cytometry. Second, the expression of the apoptosis-related proteins Fas and Bcl-2 were measured by means of flow cytometry. Third, ceramide was measured by quantitative derivatization to ceramide-1-phosphate using diacylglycerol kinase and [gamma-32P] ATP. Moreover, intercellular Ca2+ was investigated using confocal microscopic analyses. In contrast to hypertrophic scar-derived fibroblasts, those derived from keloids are significantly resistant to Fas-mediated apoptosis. The intercellular ceramide and Ca were not activated. There were no significant differences in the level of expression of Bcl-2 between the two groups, but Fas expression was higher in keloid than in hypertrophic scar. Fibroblasts from the two groups were susceptible to ceramide-induced apoptosis. Blocking of the Fas-mediated apoptotic pathway in keloids occurs upstream of the second messenger. The abnormal Fas-induced apoptosis in keloids may account for the imbalance of proliferation and apoptosis. Enhancement of Fas sensitivity could be a promising therapeutic target.