Death of solid tumor cells induced by Fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressing muscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Levels of soluble Fas ligand in myocarditis

Removable dies used in fixed prosthodontics typically exhibit movement. A solid working cast permits the technician to perfect the interproximal contacts of fixed prostheses. This saves the clinician time at the insertion appointment. This article describes a quick and easy procedure for making a solid working cast with easily visualized margins.     

Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand

Anchietia salutaris tea is traditionally used in Brazil to treat allergies, suggesting it contains compounds with antagonistic activity on the allergic mediators. We have evaluated extracts and semi-purified fractions of Anchietia salutaris as a source of compounds having this type of antagonism on the contraction induced in guinea-pig lung parenchymal strips and on platelet aggregation and shape change. After 10 min pre-incubation dichloromethane extracts containing 30 or 100 microg mL(-1) inhibited the contraction induced by prostaglandin D2 (PGD2) in guinea-pig lung parenchymal strips with dose ratios (DR) of 0.76+/-0.14 and 0.93+/-0.19, respectively; the amount of inhibition depended both on the concentration and on the time of pre-incubation (DR after 30 min pre-incubation was 1.21+/-0.51). The dichloromethane extract and its semi-purified fractions also inhibited the contractions induced by U46619, a more potent, stable, synthetic agonist of thromboxane A2 (TxA2) prostanoid (TP) receptors, the receptors acted upon by PGD2 to produce lung contractions. The dichloromethane extract did not inhibit the lung parenchymal contractions induced by histamine, leukotriene D4 (LTD4) or platelet-activating factor (PAF). Platelet aggregation induced by U46619, adenosine 5'-diphosphate (ADP) or PAF was not inhibited by the dichloromethane extract. Indeed, the extract potentiated platelet aggregation induced by low concentrations of these agonists and also potentiated the shape change induced by U46619. These results imply that the dichloromethane extract of Anchietia salutaris and its semi-purified fractions contain an active principle that competitively inhibits TxA2 TP receptors, the stimulation of which causes lung parenchymal contraction. The inhibition seems to be selective for this receptor subtype, because the extract fails to inhibit platelet aggregation or shape change. This provides additional support of earlier reports suggesting the occurrence of TP receptor subtypes.     

Fibrogenic effect of oxidative stress on rat hepatic stellate cells

Members of the dynamin-related proteins family have been identified in a wide range of organisms, however their precise functions remain elusive. We have identified a new member of that GTPase family in the fission yeast Schizosaccharomyces pombe. We show that Msp1+ is an essential nuclear gene encoding a 101 kDa protein whose closest homologue is the S. cerevisiae MGM1 gene product. We also report that msp1 conditional loss of function affects the maintenance of mitochondrial DNA and leads to growth arrest associated with respiratory deficiency.     

An aggressive nasal lymphoma accompanied by high levels of soluble Fas ligand

10 adults' performances on visual training through recognition of speech spectrograms were examined. All subjects completed the training within eight 1-hr. sessions. Success and retention of training were also evident in the subjects' performances on two posttests.     

Inhibition of integrin signaling with Arg-Gly-Asp motifs in rat hepatic stellate cells

BACKGROUND/AIMS: Activation of hepatic stellate cells plays a key role in liver fibrogenesis. Disruption of normal hepatic stellate cell-matrix interactions may contribute to this process. However, little is known about the molecular events leading from integrin-extracellular matrix interaction to hepatic stellate cell function. Therefore, we investigated the role of integrin signaling in tyrosine phosphorylation of focal adhesion kinase and cytoskeletal assembly in rat hepatic stellate cells using soluble Arg-Gly-Asp containing peptides. METHODS: Hepatic stellate cells were isolated from normal rat livers. Integrin alpha5beta1 expression in hepatic stellate cells was analyzed by immunoprecipitation and immunocytochemistry. The cytoskeletal assembly and tyrosine phosphorylation of focal adhesion kinase were determined by immunocytochemistry and immunoblotting. We also analyzed the effect of Arg-Gly-Asp containing peptides on the expression of smooth muscle alpha actin by immunocytochemistry and immunoblotting. RESULTS: We identified integrin alpha5beta1 in rat hepatic stellate cells. Stress fiber formation and cell shape were different when hepatic stellate cells were plated on various extracellular matrix components. Treatment of hepatic stellate cells with soluble Arg-Gly-Asp peptides diminished the adhesion-induced tyrosine phosphorylation of focal adhesion kinase and inhibited the formation of stress fibers. The peptides also reduced the expression of smooth muscle alpha actin. CONCLUSIONS: Our results demonstrate that adhesion to extracellular matrix induces tyrosine phosphorylation of focal adhesion kinase and promotes actin stress fiber formation and focal adhesion assembly in rat hepatic stellate cells, and that these events are disturbed by soluble Arg-Gly-Asp peptides.     

CD40 ligand inhibits Fas/CD95-mediated apoptosis of human blood-derived dendritic cells

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) in the immune system. In this study, we analyzed the regulation of apoptosis of human peripheral blood-derived DC. DC were generated from adherent peripheral blood mononuclear cells that had been cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. These cells displayed phenotypic properties of DC, including dendritic processes, expression of CD1a and lack of expression of CD14, and were very potent at presenting soluble antigens to T cells. Blood-derived DC were demonstrated to express the Fas/CD95 antigen and an agonist antibody to CD95 strongly induced apoptotic cell death in these cells. Soluble trimeric CD40 ligand potently inhibited both CD95-mediated and spontaneous apoptosis in DC. The data suggest that interactions between members of the tumor necrosis factor family of ligands expressed by T cells with their receptors on DC play an important role in the regulation of apoptosis in DC during antigen presentation and may, therefore, regulate the duration of T cell expansion and cytokine production.     

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.     

Involvement of Fas/Fas ligand system-mediated apoptosis in the development of concanavalin A-induced hepatitis

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.     

Fas ligand is constitutively secreted by prostate cancer cells in vitro

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.     

Induction of Fas ligand in murine bone marrow NK cells by bacterial polysaccharides

Bacterial polysaccharides have a wide range of activities in mammals. We have studied the effect of LPS and poly-beta-(1-->4)-D-mannuronate (mannuronan, poly-M), an exopolysaccharide from Pseudomonas aeruginosa, on the cytotoxicity mediated by murine bone marrow cells (BMC). Addition of LPS or mannuronan to BMC induced a time- and dose-dependent cytotoxicity against Jurkat cells. The LPS- or mannuronan-induced cytotoxicity was due to increased Fas ligand (FasL) expression by BMC, since 1) Fas-transfected L1210-Fas target cells were more susceptible to lysis than the Fas(low)-expressing parent L1210 cells, 2) stimulated BMC from FasL-defective gld/gld mice were not cytolytic and, 3) the cytolytic activity of normal BMC was inhibited by a Fas-Fc fusion protein. Flow cytometry showed an increase in surface FasL in LPS-stimulated BMC. RT-PCR analysis of BMC revealed constitutive expression of FasL mRNA, which was increased after LPS stimulation. Immunomagnetic depletion of NK1.1-, CD2-, or CD32/16-expressing cells from BMC abrogated the LPS-induced BMC cytotoxicity against L1210-Fas cells, suggesting that NK cells were the cytotoxic effector cells. Depletion of CD45R/B220-, Gr-1-, or CD11b/Mac-1-expressing cells only partially decreased BMC-mediated cytotoxicity, and depletion of CD4- or CD8a-expressing cells had no effect. The results support the conclusion that LPS and mannuronan induce expression of cytotoxic FasL on bone marrow NK cells.     

Fas-mediated apoptosis of hepatic cells

While the fas/fas ligand system has been extensively investigated in immuno-competent cells, the place of this system in the physiology and pathophysiology of liver cells remains to be clarified. Although we know that fas is present at the surface of hepatocytes--the main hepatic cells--the role of this membranous protein in physiological conditions is not yet elucidated. However it is the localization of fas on the plasma membrane of hepatocytes which explains why these cells are mainly destroyed by apoptosis--in a picture resembling human fulminant hepatitis--when mice are administered with anti-fas antibodies or fas ligand. It is also established that fas is surexpressed in some human chronic liver diseases, such as those induced by hepatitis B or C virus, a situation which could explain the pathogenesis of some liver lesions occurring during these diseases, such as the apoptosis of hepatocytes in piecemeal necrosis. Finally the fact that caspases, a group of cysteine proteases activated in fas-induced apoptosis, opens the way to inhibition of these enzymes by synthetic peptides and to prevent and treat hepatocyte apoptosis. Demonstration of this possibility has been recently reported in animals presenting fulminant hepatitis induced by anti-fas antibodies.