Caspase-9 pathway activation by inhibiting endogenous fibroblast growth factor signaling in human glioma cells.
The cell survival activity of human glioma cells is largely dependent on autocrine fibroblast growth factor (FGF) signaling. Caspases, a family of cysteine proteases, play an integral part in the execution phase of apoptosis. To better understand the mechanism of resistance to apoptosis in human glioma cells, we investigated the effect of a blockade of endogenous FGF signaling through the expression of the dominant negative type I FGF receptor (DNFGFR) in U251MG cells. The cells were infected with adenovirus vector expressing DNFGFR (AdDNFGFR) and apoptosis was semi-quantified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method and flow cytometric annexin V assay. The activation of caspase-3, -8, and -9, the activation of Akt, a serine/threonine protein kinase, and the cleavage of poly(ADP-ribose) polymerase (PARP) were analyzed by immunoblotting. The infection with AdDNFGFR (multiplicity of infection of 200) induced marked apoptosis, along with a down-regulation of akt phosphorylation, and activation of caspase-9 and -3, but not -8. By contrast, LacZ virus (a control) had minimal effects. The level of the cleaved form of PARP was increased in a time-dependent fashion, and this increase was inhibited by adding Z-DEVD-FMK, a caspase-3 inhibitor, and Z-LEHD-FMK, a caspase-9 inhibitor. Moreover, ultraviolet exposure (100 J/m(2)) induced apoptosis and caspase-8, but not caspase-9, activation. Our data suggested that the induction of apoptosis through the inhibition of endogenous FGF signaling is caspase-9 pathway- dependent. The suppression of this or other specific anti-apoptotic pathways may lead to genetic or pharmacological manipulations that favorably modulate the malignant behavior of human gliomas.
Fukushima
Shintaro
S
Department of Neurosurgery, School of Medicine, Kurume University, Kurume, Japan.
Kato
Seiya
S
Maeda
Mitsuhide
M
Shigemori
Minoru
M
D017209:Apoptosis; D001932:Brain Neoplasms / Q000201:enzymology*; D053453:Caspase 9 / Q000096:biosynthesis*; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000201:enzymology*; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D015398:Signal Transduction
D061945:Caspase Inhibitors; D009842:Oligopeptides; D017468:Receptors, Fibroblast Growth Factor; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D005346:Fibroblast Growth Factors; D011065:Poly(ADP-ribose) Polymerases; D053453:Caspase 9