anti-Fas Antibody Research Articles

Human Herpesvirus 8 Protein K1 Interferes with Fas-Mediated Apoptosis Induces Clonal Growth and Lymphoid Hyperplasia

Background: Expression of Human herpesvirus 8 (HHV-8) K1 causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 ± 0.6% vs. 50 ± 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis. We mapped the region that K1 uses to bind to Fas as an immunoglobulin (Ig) chainlike domain by expressing deletion mutants of K1. Overexpression of an Ig domaincontaining protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. Ig-deleted K1 (K1dIg)-transfected mice were not protected (0/6), and K1- transfected mice were protected (7/10, P < 0.01) against the lethal effects of agonistic anti-Fas (Jo2) antibody. K1dIg expressed in mice did not form complexes with Fas, suggesting that the Ig domain is essential for K1-Fas binding and suppression of apoptosis.Conclusion: These results confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation. K1 blocks Fas-mediated apoptosis and competing peptides can reinstate apoptosis.

HTLV-1 Triggers HIAP-1 and cFLIP Expression in CD8+ T-Cells While Impairing DNA Repair in CD4+ T-Cells.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL), a CD4+ mature lymphoproliferation that is preceded by the persistent clonal expansion of CD4+ and CD8+ infected lymphocytes. It depends on the expression of the viral oncogene tax that is restricted to the preleukemic phase of the infection. The virus prevents cell death in CD8+ lymphocytes whereas recruiting CD4+ lymphocytes into the cell cycle (J Clin Invest. 2006; 116:974–983). In vitro, Tax+ cell lines overexpress the antiapoptotic genes cFLIP (Genes to Cells 2006; 11:177–191; Blood 2006; 107:4491–4499), HIAP-1 (Blood 2006; 107:3933–3939), and TRAIL (J Virol. 2005; 79:1367–1378). Furthermore HTLV-1 causes defects on DNA repair in cell lines while cloned HTLV- 1 positive tax-expressing CD4+ lymphocytes cumulate morphologic cellular defects characteristic of genetic instability. Here we explored certain mechanisms underlying apoptosis inhibition and genetic instability in CD4+ and CD8+ cells. 56 clones harboring distinct and unique TCRs, including 40 uninfected (27 CD4+, 13 CD8+) and 16 infected clones (10 CD4+, 6 CD8+), were generated by limiting dilution cloning of PBMCs deriving from an infected individual without malignancy. To investigate cell death inhibition, we first analyzed the expression of the genes involved in apoptosis. Microarrays confirmed by real-time RT-PCR revealed that upon infection, CD8+ cells specifically underexpressed FasL while overexpressing cFLIP-l (p=0.012) and HIAP-1 (p=0.0063). cFLIP-s expression was not influenced by HTLV-1 infection in CD8+ and CD4+ cells. In contrast to CD8+ cells, infected and uninfected CD4+ lymphocytes displayed the same amount of cFLIP-l and HIAP-1 transcripts. HIAP-1 and tax expression correlated in both CD4+ (p=0.002) and CD8+ cells (p=0.001) whereas a negative correlation between tax and cFLIP-s characterized CD8+ cells (p=0.043). Anti-Fas antibody and recombinant Fas ligand triggered cell death in all CD4+ clones, in uninfected CD8+ clones and in 4/6 infected CD8+ clones. Thus the defect in FasL expression, that is restricted to infected CD8+ clones, seems accounting for preventing cell death in ~67% of HTLV-1+-CD8+ clones whereas an additional defect downstream the Fas pathway is involved for the remaining HTLV-1+-CD8+ clones. We investigated DNA repair mechanisms through comet assays in the same cloned lymphocytes. We analyzed 4945 cells before and after 10 Gy irradiation. Infected and uninfected CD8+ clones displayed the same proportion of cells with repaired DNA. CD4+ clones displayed 2 significantly distinct patterns of time-dependent DNA repair. Clones with no or moderate tax expression displayed no significant difference in time-dependent DNA repair when compared to uninfected CD4+ clones. Before irradiation, the pattern of DNA repair of tax-expressing CD4+ clones was indistinguishable from that of control cells but at 2, 6, and 25 hours after irradiation, the mean proportion of infected CD4+ cells with repaired DNA was 9, 2, and 1.2 fold lower than that of uninfected cells. A significant correlation was observed between tax expression and the tail moment for the 1454 cells deriving from HTLV-1+CD4+ clones (p<10–4); in contrast, no significant correlation could be observed for the 1000 cells deriving from CD8+HTLV-1+ clones. These data indicate that tax expression impairs DNA repair in naturally infected CD4+Tax+ cells whereas HTLV-1 infection does not influence DNA repair in CD8+ cells. We next investigated the expression of the genes involved in DNA repair in cloned lymphoid cells from infected patients. Microarray analysis revealed that upon infection, there was no significant transcriptional deregulation of these genes in CD8+ cells; in contrast, the expression of 17 genes directly or indirectly involved in DNA repair was found to be significantly deregulated in CD4+HTLV-1+Tax+ clones. The deregulation of some of these genes was consistent with present comet findings. In conclusion, present results suggest that 1°) the CD8+-restricted anti-apoptotic effect of HTLV-1 relies on a tax-dependent overexpression of HIAP-1, a tax-independent overexpression cFLIP-l, together with a defect in the Fas pathway with evidence for clonal heterogeneity; 2°) upon tax expression, CD4+ but not CD8+ HTLV-1 naturally infected cells are phenotypically and genotypically impaired for DNA repair.

CD74 Is a Regulator of Fas-Mediated Apoptotic Signaling

Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood 2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res. 2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol. 1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.

A Promising Therapeutic Implication of a Novel Bcl-2 Family Inhibitor ABT-737 for Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-cell lymphotrophic virus type I. Conventional chemotherapeutic regimens used against other malignant lymphoma have been administered to ATLL patients, but the treatment outcome of acute and lymphoma type ATLL remains very poor. Therefore, new therapeutic strategies are needed to improve patient outcome. Anti-apoptotic proteins, Bcl-2 and Bcl-XL have been reported to be highly expressed in ATLL cells, and confer resistance to chemotherapy. The aim of this study is to elucidate the therapeutic implication of ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a potent small-molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w for ATLL. We first examined the direct toxicity of ABT-737 against tumor cell lines in vitro. ABT-737 significantly inhibited growth of ATLL cell lines (MT-1, MT-2 and HuT102) and an acute T-cell leukemia cell line, Jurkat with IC50 values at 72h of 2.5μM, 0.4μM, 0.01μM and 0.75μM, respectively. In contrast, Burkitt lymphoma cell lines, Raji and Ramos were resistant to ABT-737 with IC50 values at 72h of 24μM and 7.0μM, respectively. Importantly, Bcl-2 protein was highly expressed in MT-1, MT-2, HuT102 and Jurkat cells, but not in Raji and Ramos cells. We next examined the mechanism of ABT-737 induced growth inhibition using MT-1 and MT-2 cells. ABT-737 induced apoptosis with cleavage of caspase 9 and caspase 3 without modulating cell cycle distribution. Furthermore, ABT-737 significantly induced apoptosis in fresh tumor cells obtained from ATLL patients. We finally elucidated the potential of ABT-737 to enhance the apoptosis induced by anti-Fas monoclonal antibody (mAb), and cytotoxicity induced by conventional chemotherapeutics. ABT-737 enhanced apoptosis induced by anti-Fas mAb in MT-1 and MT-2 cells. Most importantly, ABT-737 synergistically enhanced the cytotoxicity induced by either of doxorubicin, vincristine or etoposide. These results suggest that ABT-737, used either alone or in combination with other conventional cytotoxic drugs, represents a promising novel targeted approach to overcome drug resistance and improve patient outcome in ATLL.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi s sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF -B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer s patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 200 mg vs. 94 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with antikappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 1% of splenocytes from K1 mice versus 17 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 0.6% vs. 50 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Reactive Oxygen Species Regulate Spontaneous and Fas-Mediated Apoptosis in SBDS-Deficient Cells.

Background and hypotheses: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and a marked propensity for myelodysplastic syndrome and leukemia. Approximately 90% of the patients have mutations in the SBDS gene, but the function of the gene is unknown. We previously showed that marrow cells from SDS patients and SBDS-deficient HeLa Cells are characterized by accelerated apoptosis, overexpression of Fas and hypersensitive to Fas stimulation. Involvement of reactive oxygen species (ROS; oxidative stress) have been shown to be related to Fas hypersensitivity and overexpression in a variety of cell types. Therefore, we hypothesized that functional deficiency in SBDS in cells that express Fas could lead to impaired ROS generation and a subsequent increase in spontaneous and Fas-mediated apoptosis and decrease in cell growth.Methods: We used shRNA-mediated SBDS-knockdown HeLa cells as a model. We investigated whether SBDS-deficiency increases ROS levels and if antioxidants can rescue the cell growth and apoptosis phenotype. To measure ROS formation cells were incubated with DCFH-DA and fluorescence measured in Gemini Spectra MAX microplate reader. Staining with annexin V and propidium iodide was done to determine apoptosis and necrotic cell death. MTT assay was used to measure cell viability.Results: ROS levels in SBDS knockdown cells were significantly increased compared to control. Apoptosis analysis by annexin V and propidium iodide showed a marked decrease in cell viability in the SBDS-knockdown cells. NAC treatment decreased ROS levels, enhanced ERK phosphorylation (pERK), improved cell viability, and decreased apoptotic and necrotic cell death. Stimulation of the Fas signaling pathway by CH-11 (activating anti-Fas antibody) and Fas ligand showed increased ROS production in SBDS Knockdown cells. CH-11 treatment showed a marked increase in apoptotic and necrotic cell death after 24 and 48hrs incubation. Cell viability decreased by 40% and 80% after 24 and 48hrs incubation with CH-11. Treatment with NAC lowered ROS levels, enhanced pERK expression, protected the cells from Fas-mediated early apoptosis and improved cell survival.Conclusion: We have demonstrated that stable loss of SBDS results in increased ROS levels, leading to apoptotic and necrotic cell death. Thus, increased baseline and Fas-stimulated ROS could result in increased sensitivity to apoptosis and necrotic cell death. NAC appeared to reverse the ROS-mediated decrease in cell survival and apoptotic cell death. Our data support the novel concept that SBDS may be a homeostatic regulator of oxidative stress

Aranorosin and a novel derivative inhibit the anti-apoptotic functions regulated by Bcl-2

Bcl-2 is an intracellular membrane protein that prevents cells from undergoing apoptosis in response to various cell-death signals. It negatively regulates mitochondrial outer membrane permeabilization, which is responsible for the release of apoptogenic factors and the subsequent activation of caspases. A microbial metabolite, aranorosin, was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Based on its structure, a more potent derivative, K050, was synthesized. Apoptosis could be induced in a cell line that overexpressed Bcl-2 when cells were treated with an anti-Fas antibody in addition to K050, at sub-micromolar concentrations. Furthermore, K050 inhibited anti-apoptotic functions regulated by Bcl-2, resulting in a Fas-triggered mitochondrial transmembrane potential loss, the activation of caspase-9, and a morphological change to apoptosis. Inhibition of cell-based function of Bcl-2 and its anti-apoptotic effects could serve as useful pharmacological effects. Thus, a novel aranorosin derivative, K050, could be a potent therapeutic agent against Bcl-2-overexpressing human malignancies.

Low susceptibility of NC/Nga mice to tumor necrosis factor-α-mediated lethality and hepatocellular damage with d-galactosamine sensitization

The susceptibility of NC/Nga mice to tumor necrosis factor (TNF)-α was examined by using sensitization with d-galactosamine ( d-GalN). Administration of TNF-α and d-GalN killed none of the NC/Nga mice, whereas it killed all of the BALB/c mice. Treatment with TNF-α and d-GalN caused few hepatic lesions in NC/Nga mice but massive hepatocellular apoptosis in BALB/c mice. Unlike BALB/c mice, there was no elevation in caspase 3 and 8 activities in the livers of NC/Nga mice receiving TNF-α and d-GalN. On the other hand, administration of anti-Fas antibody definitely killed both NC/Nga and BALB/c mice via activation of caspases 3 and 8. Treatment with TNF-α and d-GalN led to translocation of nuclear factor (NF)-κB in NC/Nga and BALB/c mice. However, NF-κB translocation was sustained in NC/Nga mice, although it disappeared in BALB/c mice 7 h after the treatment. NF-κB inhibitors activated caspases 3 and 8, and enhanced TNF-α-mediated lethality in NC/Nga. Taken together, the low susceptibility of NC/Nga mice to TNF-α-mediated lethality was suggested to be responsible for the sustained NF-κB activation.

Brief cross-linking of Fas/APO-1 (CD95) triggers engulfment of pre-apoptotic target cells

Macrophage clearance of dying cells is of crucial importance to maintain tissue homeostasis. Here, we show that brief treatment (15min) of Jurkat T cells with agonistic anti-Fas antibodies or recombinant Fas ligand results in efficient phagocytosis by human monocyte-derived macrophages prior to the occurrence of common biomarkers of apoptosis. Similar findings were obtained when using primary human T cells. Macrophage engulfment of pre-apoptotic target cells was suppressed in the absence of serum. Moreover, pre-apoptotic cells secreted annexin I and administration of Boc1, a formyl peptide receptor/lipoxin receptor antagonist markedly attenuated their engulfment. Finally, pre-apoptotic Jurkat cells induced lower macrophage production of tumor necrosis factor-α and higher production of interleukin-10 in comparison to apoptotic target cells.

Rapid and significant induction of TRAIL-mediated type II cells in apoptosis of primary salivary epithelial cells in primary Sjögren’s syndrome

Expressions of the effector molecules of Fas-mediated apoptosis in primary cultured salivary gland epithelial cells (SGEC) of primary Sjögren's syndrome (pSS) remain to be clarified. We focused on Fas-mediated caspase cleavage compared to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Induction of apoptosis was performed by anti-Fas antibody coupled with PI3K inhibitor, or TRAIL. Activation of caspases, cytochrome C, and apoptotic protease activating factor-1 (Apaf-1) was determined by western blotting or immunofluorescence observed by confocal microscopy. Fas-mediated apoptosis and activation of caspase 3/8 were induced in the presence of LY294002. TRAIL-induced apoptosis in SGEC, which was stronger than that induced by anti-Fas antibody. TRAIL-induced caspase 9 cleavage accompanied by activation of cytochrome C and Apaf-1 were not mediated by anti-Fas antibody. Our results suggest that death receptor-dependent apoptosis in primary cultured SGEC is regulated by the engagement of type II cells in pSS.

Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

EDARAVONE PREVENTS FAS-INDUCED FULMINANT HEPATIC FAILURE IN MICE BY REGULATING MITOCHONDRIAL BCL-XL AND BAX

Fulminant hepatic failure is a serious disease that has a poor cure rate unless liver transplantation is performed. Edaravone, a free radical scavenger, has been approved for the treatment of acute cerebral infarction, and its mechanism of action involves scavenging free radicals generated in ischemic tissues. We assessed the ability of 3-methyl-1-phenyl-2-pyrazolim-5-one (edaravone) to prevent Fas-induced acute liver failure in mice and examined the mechanisms underlying the observed effects. BALB/c mice were administered 0.25 microg/g (i.v.) body weight of a purified hamster agonist anti-Fas monoclonal antibody (clone Jo2). The mice also received either edaravone or isotonic sodium chloride solution before or after Jo2 treatment. Edaravone improved the survival rate of the mice markedly. Histopathological findings and serum aspartate aminotransferase levels showed that edaravone reduced the degree of liver injury caused by Jo2. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining showed that edaravone reduced the number of apoptotic hepatocytes. Edaravone also prevented cytochrome c release and caspase 3 activity, recognized as markers of apoptosis after mitochondrial disruption. Therefore, we considered that the antiapoptotic activity of edaravone involved blocking signals in the mitochondria-dependent pathway of Fas-induced apoptosis. Mitochondrial Bcl-xL and Bax, which form a channel in the mitochondrial membrane and, by their balance, regulate its permeability, are involved in mitochondrial disruption. Western blotting showed that the Bcl-xL-Bax ratio of the edaravone group was much higher than that of the control group. In conclusion, edaravone might protect hepatocytes from Fas-induced mitochondria-dependent apoptosis by regulating mitochondrial Bcl-xL and Bax.

Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Death Receptors and Caspases But Not Mitochondria Are Activated in the GDNF- or BDNF-Deprived Dopaminergic Neurons

Neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote survival of midbrain dopaminergic neurons, but the death pathways activated in the dopaminergic neurons by deprivation of these factors are poorly studied. We show here that deprivation of GDNF or BDNF triggers a novel mitochondria-independent death pathway in the cultured embryonic dopaminergic neurons: cytochrome c was not released from the mitochondria to cytosol, proapoptotic protein Bax was not activated, and overexpressed Bcl-xL did not block the death. Caspases were critically required, because the death was completely blocked by caspase inhibitor BAF [boc-aspartyl(OMe)-fluoromethylketone] and overexpression of dominant-negative mutants of caspase-9, -3, and -7 significantly blocked the death. Also, the death receptor pathway was involved, because blockage of caspase-8 or FADD (Fas-associated protein with death domain), an adapter required for caspase-8 activation, inhibited death induced by GDNF or BDNF deprivation. Ligation of Fas by agonistic anti-Fas antibody induced apoptosis in the GDNF- or BDNF-maintained neurons, and inhibition of Fas by Fas-Fc chimera blocked the death of GDNF- or BDNF-deprived neurons, whereas FAIM(L) (long isoform of Fas apoptosis inhibitory molecule) could control the activity of Fas in the dopaminergic neurons.

Cytotoxic T-lymphocyte–mediated killing of human pancreatic islet cells in vitro

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Acitretin induces apoptosis through CD95 signalling pathway in human cutaneous squamous cell carcinoma cell line SCL‐1

Skin cancers are by far the most common human malignancies. Retinoids have shown promising preventive and therapeutic effects against a variety of human malignancies. The aim of this study was to investigate the apoptosis-inducing effect of acitretin on human skin squamous cell carcinoma (SCC) SCL-1 cells. We found that acitretin preferentially inhibited the growth of SCL-1 cells in a dose- and time-dependent manner, but not of non-malignant keratinocyte HaCaT cells. This inhibition appeared to be due to induction of apoptosis as revealed by enzyme-linked immunosorbent assay. AnnexinV/propidium iodide assay and morphological observation confirmed the pro-apoptotic effect of acitretin on SCL-1 cells. We further demonstrated that apoptosis was induced within 1–2 days and involved activation of caspases-8, -9, -3 and poly (ADP-ribose) polymerase (PARP). Caspase-8 inhibitor effectively suppressed acitretin-induced apoptosis whereas caspase-9 inhibitor did not. Acitretin increased the levels of CD95 (Fas), CD95-ligand and Fas-associated death domain. Neutralizing ZB4 anti-Fas antibody significantly inhibited the apoptosis in SCL-1 cells induced by acitretin. These results suggest that acitretin is able to induce apoptosis in skin cancer cells possibly via death receptor CD95 apoptosis pathway without affecting the viability of normal keratinocyte.

The natural product honokiol preferentially inhibits cellular FLICE-inhibitory protein and augments death receptor–induced apoptosis

Targeting death receptor-mediated apoptosis has emerged as an effective strategy for cancer therapy. However, certain types of cancer cells are intrinsically resistant to death receptor-mediated apoptosis. In an effort to identify agents that can sensitize cancer cells to death receptor-induced apoptosis, we have identified honokiol, a natural product with anticancer activity, as shown in various preclinical studies, as an effective sensitizer of death receptor-mediated apoptosis. Honokiol alone moderately inhibited the growth of human lung cancer cells; however, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), greater effects on decreasing cell survival and inducing apoptosis than TRAIL alone were observed, indicating that honokiol cooperates with TRAIL to enhance apoptosis. This was also true to Fas-induced apoptosis when combined with Fas ligand or an agonistic anti-Fas antibody. Among several apoptosis-associated proteins tested, cellular FLICE-inhibitory protein (c-FLIP) was the only one that was rapidly down-regulated by honokiol in all of the tested cell lines. The down-regulation of c-FLIP by honokiol could be prevented by the proteasome inhibitor MG132. Moreover, honokiol increased c-FLIP ubiquitination. These results indicate that honokiol down-regulates c-FLIP by facilitating its degradation through a ubiquitin/proteasome-mediated mechanism. Enforced expression of ectopic c-FLIP abolished the ability of honokiol to enhance TRAIL-induced apoptosis. Several honokiol derivatives, which exhibited more potent effects on down-regulation of c-FLIP than honokiol, showed better efficacy than honokiol in inhibiting the growth and enhancing TRAIL-induced apoptosis as well. Collectively, we conclude that c-FLIP down-regulation is a key event for honokiol to modulate the death receptor-induced apoptosis.

Vascular endothelial growth factor reduces Fas‐mediated acute liver injury in mice

Fulminant hepatitis is still a fatal liver disease, and no specific treatment for it has been available. Vascular endothelial growth factor (VEGF) is the focus of attention because of its various actions. We investigated the effect of vascular endothelial growth factor (VEGF) on Fas-induced fulminant hepatic failure (FHF). Male Balb/c mice were treated with an intraperitoneal injection of an anti-Fas antibody (Jo-2 Ab) with or without premedication with intraperitoneally administered human recombinant VEGF. The serum level of alanine aminotransferase (ALT) was up to 300 times higher that of normal mice following the Jo-2 Ab injection, and histological analysis revealed hepatic injury and massive hepatocyte apoptosis. The VEGF significantly suppressed an elevation in serum ALT levels and hepatocyte apoptosis. Immunohistochemically, VEGF-treated mice showed that Bcl-xL in hepatocytes was strongly expressed. Since hepatocytes do not express VEGF receptors, we speculated that VEGF acts on sinusoidal endothelial cells (SECs) and promotes production of cytokines such as hepatocyte growth factor in SECs, resulting in reducing apoptosis through an increase expression of Bcl-xL in hepatocytes. We suggest that VEGF has a potent antiapoptotic effect on hepatocytes through cell-cell interaction between SECs and hepatocytes.

P38MAPK regulates caspase-3 by binding to caspase-3 in nucleus of human hepatoma Bel-7402 cells during anti-Fas antibody- and actinomycin D-induced apoptosis

Anti-Fas antibody- and actinomycin D (FA/AD) has been shown to have anti-tumor activity in some tumor cells. However, many of the molecular mechanism of FA/AD-induced apoptosis of human hepatoma Bel-7402 cells have not been fully clarified. In the present study, therefore, the effect of FA/AD in presence or absence of p38MAPK inhibitor SB203580 on the proliferation, apoptosis, p38MAPK, caspase-3, location of p38MAPK and caspase-3, and interaction between p38MAPK and caspase-3 in Bel-7402 cell was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), annexin V-FITC/propidium iodide (PI) double staining, electron microscopy, immunoblot, immunofluorescence and immunoprecipitation/immunoblot assay, respectively. We found that FA/AD significantly resulted in the inhibition of proliferation, induction of apoptosis, activation and up-regulation of p38MAPK, activation and up-regulation of caspase-3, translocation of p38MAPK and caspase-3 from cytosol to nucleus, and formation of p38MAPK/caspase-3 complex in Bel-7402 cells. In contrast, SB203580, a p38MAPK-specific inhibitor, apparently blocked induction of apoptosis, activation and up-regulation of p38MAPK, activation and up-regulation of caspase-3, and translocation of p38MAPK and caspase-3 from cytosol to nucleus in FA/AD-treated Bel-7402 cells. Taken together, we conclude that p38MAPK regulates caspase-3 by binding to caspase-3 in nucleus of Bel-7402 cells during FA/AD-induced apoptosis.