Signaling In Glioma Cells Research Articles

Down-regulation of Integrin αvβ3 Expression and Integrin-mediated Signaling in Glioma Cells by Adenovirus-mediated Transfer of Antisense Urokinase-type Plasminogen Activator Receptor (uPAR) and Sense p16 Genes

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

C2-ceramide signaling in glioma cells: synergistic enhancement of CD95-mediated, caspase-dependent apoptosis.

Most human malignant glioma cell lines are susceptible to CD95 ligand (CD95L)-induced apoptosis. Here, we report that glioma cells are also susceptible to the cytotoxic effects of exogenous C2-ceramide. This form of cell death exhibits some morphological features of apoptosis as assessed by electron microscopy, but is unaffected by the broad spectrum caspase inhibitor, zVAD-fmk. Further, CD95L-induced apoptosis is synergistically enhanced by coexposure of the glioma cells to CD95L and C2-ceramide. CD95L-induced caspase 3-like activity, cytochrome c release and cleavage of caspases 3, 8, 9 and poly(ADP-ribose)polymerase (PARP) increase substantially after cotreatment with CD95L and C2-ceramide compared with CD95L treatment alone. None of these events occur in response to cytotoxic concentrations of C2-ceramide alone. C2-ceramide does not alter CD95 expression. Gene transfer-mediated enhancement of CD95 expression results not only in increased susceptibility to CD95L, but also in increased sensitivity to C2-ceramide. We conclude that (i) synergistic induction of apoptosis by C2-ceramide and CD95L depend on a cross-talk between the two signal transduction pathways and that (ii) C2-ceramide, independently of its sensitizing effects on CD95-dependent caspase activation, is also capable of triggering an apoptotic signaling cascade that is unaffected by zVAD-fmk-mediated caspase inhibition, but promoted by high levels of CD95 expression.

Analysis of intra- and intercellular calcium signaling in a mouse malignant glioma cell line.

Intra- and intercellular calcium signaling in glioma cells was examined by mechanical stimulation of a monolayer cell line of methylcholanthrene-induced mouse ependymoblastoma, 203-glioma, with a fine round-tip glass needle. A fura-2 fluorescence image of the glioma revealed a four- to eightfold increase in the cytosolic calcium ion concentration in directly stimulated signal cells. The increased calcium spread to surrounding cells at a speed of 20 microns/sec for a distance of up to 200 microns. Calcium was transmitted between adjacent cells and even in cells up to 200 microns distant from the initially stimulated cell. Microinjection of Lucifer yellow dye showed no gap junctional communication between cells. Depletion of extracellular calcium ion inhibited both cytosolic calcium elevation and propagation to neighboring cells by mechanical stimulus. An intracellular calcium blocker, TMB-8, eliminated the cytosolic calcium mobilization in a mechanically stimulated cell, but had no effect on calcium diffusion to surrounding cells. Nifedipine and verapamil, antagonists of voltage-dependent calcium channels, did not act on the mechanically induced calcium response. This suggests that some stimulating factor may trigger transmission of calcium, which may be ejected directly from single stimulated cells and mediated via a membrane receptor but not through a gap junction. The calcium signaling in a mechanically stimulated cell may be related to both an influx and a redistribution of intracellular calcium from internal stores, while calcium propagation to neighboring cells may involve calcium influx alone.