Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

Mast cells derived from Bruton’s tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon FcRI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.     

Herpes simplex virus thymidine kinase and ganciclovir suicide gene therapy for human pancreatic cancer

AIM: To investigate the in vitro effects of suicide gene therapy system of herpes simplex virus thymidine kinase gene (HSV-TK) in combination with the treatment of nucleotide analog-ganciclovir (GCV) on human pancreatic cancer, and to provide a novel clinical therapeutic method for human pancreatic cancer. METHODS: We used a replication defective recombinant retrovirus vector GINaTK (bearing HSV-TK gene) to make packaging cell PA317 produce progeny virions. We then transferred the HSV-TK gene to target cells SW1990 using these progeny virions, and treated these gene-modified tumor cells with GCV to study the sensitivity of the cells to GCV and their bystander effects by routine MTT-method. RESULTS: Packaging cell PA317/TK was successfully constructed, and we acquired SW1990/TK through virus progeny infection. These gene-modified pancreatic cancer cells were sensitive to the treatment of GCV compared with unmodified tumor cells (t=4.15, n=10, P<0.0025). We also observed a remarkable bystander effect by mixing two kinds of cells at different ratio. CONCLUSION: Our data demonstrate that HSV-TK/GCV suicide gene therapy system is effective for treating experimental human pancreatic cancer, which is largely resistant to the common therapies, so the suicide gene therapy system may be a potential treatment approach for pancreatic cancer.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.     

GFRα3 is an orphan member of the GDNF/neurturin/persephin receptor family

GDNF, neurturin, and persephin are transforming growth factor β-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor α components 1 and 2, GFRα1 or GFRα2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFRα3 that is encoded by a gene located on human chromosome 5q31.2–32. GFRα3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFRα3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFRα3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFRα1 and GFRα2. Fibroblasts expressing Ret and GFRα3 do not respond to any of the known members of the GDNF family, suggesting that GFRα3 interacts with an unknown ligand or requires a different or additional signaling protein to function.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G₀-G₁ phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G₁→S transition thereby resulting in an arrest of cells in G₀-G₁ phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.     

Herpes simplex virus infection of dendritic cells: balance among activation,inhibition, and immunity

Several lines of evidence suggest that dendritic cells (DCs), the most potent antigen-presenting cells known, play a role in the immunological control of herpes simplex virus (HSV) infections. HSV infection of DCs induced submaximal maturation, but DCs failed to mature further in response to lipopolysaccharide (LPS). LPS induced interleukin (IL)-12 secretion, and the induction of primary and secondary T cell responses were impaired by infection. Ultimately, DC infection resulted in delayed, asynchronous apoptotic cell death. However, infected DCs induced HSV recall responses in some individuals. Furthermore, soluble factors secreted by DCs after infection induced DC maturation and primed for IL-12 secretion after LPS stimulation. These data support a pathogenetic model of HSV infection, in which initial delay in the generation of immune responses to HSV at peripheral sites is mediated by disruption of DC function but is overcome by bystander DC maturation and cross-presentation of HSV antigens.     

A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.     

Herpes simplex virus protein kinase US3 activates and functionally overlaps protein kinase A to block apoptosis

Herpes simplex virus 1 encodes at least four genes whose functions include blocking apoptosis induced by exogenous agents (e.g., sorbitol, Fas ligand, and BAD protein) or replication-incompetent mutants (e.g., the d120 mutant lacking both copies of the alpha 4 gene). U(S)3, one of these four genes, encodes a serine-threonine kinase that has been demonstrated to block apoptosis induced by proapoptotic cellular proteins or by the d120 mutant. The amino acid context of serine-threonine phosphorylated by U(S)3 is similar to that of the cAMP-dependent protein kinase PKA. We report that (i) the pattern of proteins phosphorylated by U(S)3 in transduced cells or in cells infected with WT virus overlaps that of phosphoproteins targeted by PKA, (ii) activation of PKA blocks apoptosis induced by d120 mutant or by BAD protein independently of U(S)3, (iii) U(S)3 protein kinase phosphorylates peptides containing the serine or threonine targeted by PKA including that present in the regulatory type II alpha subunit of PKA, and (iv) in WT virus-infected cells the regulatory type II alpha subunit is phosphorylated in a U(S)3-dependent manner. We conclude that a major determinant of the antiapoptotic activity of the U(S)3 protein kinase is the phosphorylation of PKA substrates by either or both enzymes.     

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways

Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-alpha, and whether the production of IFN-alpha in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-alpha in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-alpha was produced by other cell types. Moreover, IFN-alpha production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-alpha in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-alpha production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-alpha in response to HSV-1 via both TLR9-independent and -dependent pathways.     

Radiation and stress-induced apoptosis: A role for Fas/Fas ligand interactions

The lpr gene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of the lpr Fas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas/Fas ligand (FasL) interactions, we quantitated in vitro apoptosis by flow cytometry measurement of DNA content in splenic T and B cells from irradiated 5- to 8-month-old B6/lpr mice. Total apoptosis of both lpr and control cells was substantial after treatment; however there was a significant difference between B6 (73%) and lpr (25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells from lpr showed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired in lpr. The finding that γ-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas–Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas/FasL interactions may thus play an important role in identifying and eliminating damaged cells after γ-irradiation and other forms of injury.     

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid(-/-) neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid(-/-) mice (-67% and -41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.     

Expression of the Na+/Ca2+ exchanger emerges in hepatic stellate cells after activation in association with liver fibrosis

Activation of hepatic stellate (Ito) cells is a final common pathway of liver fibrosis. The findings presented in this paper indicate that expression of Na⁺/Ca²⁺ exchanger (NCX) emerges in rat hepatic stellate cells after activation in vitro during primary culture or in vivo in response to intoxication with CCl₄. NCX mRNA became detectable by Northern blot analysis in cultured stellate cells on day 3, as was α-smooth muscle actin, an indicator not only of smooth muscle differentiation but also of stellate cell activation. Western blot analysis showed expression of the exchanger protein in the activated stellate cells. Functional expression of the exchanger, monitored by Ni²⁺-sensitive, verapamil-insensitive intracellular free Ca²⁺ increases in response to reduction of extracellular Na⁺ concentration, became sizable by using Fura-2 in stellate cells by 7 days in culture. Furthermore, increased expression of the exchanger mRNA was found predominantly in stellate cells freshly isolated from the CCl₄ model rat of hepatic fibrosis. Thus, it is concluded that NCX expression is closely associated with activation of hepatic stellate cells in vitro and in vivo. Because, even at the whole liver level, increased expression of NCX mRNA became observable after induction of liver fibrosis, it is suggested that NCX expression serves a useful diagnostic marker of liver fibrosis or cirrhosis.