No abstract

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

According to new hypotheses astrocytomas/gliomas either arise from or attract neural stem cells. Biological markers, particularly antigenic markers, have played a significant role for the characterization of these tumour stem cells (TSCc). Because these studies have been performed with single experimental samples mostly from gliomas, we investigated the expression of the stem cell markers CD133/Prominin, Nestin, Sox-2, Musashi-1, CXCR4, Flt-4/VEGFR-3 and CD105/Endoglin in 72 astrocytomas of different WHO-grades and compared it to normal adult human brain. Expression of their mRNA was quantified by quantitative RT-PCR, of their protein by counting immunopositive cells. In contrast to normal brain, tumour samples showed a high variability for the expression of all markers. However, their mean expression was significantly increased in astrocytomas, but this depended on the WHO grade only for CD133, Nestin, Sox-2 and Musashi-1. Confocal microscopy revealed that these markers mostly could be co-stained with glial fibrillary acidic protein, a marker for astoglial cells, but less frequently with the proliferation marker Ki-67/MIB-1. These markers sometimes, but not necessarily could be co-stained with each other in complex patterns. Our results show that most astrocytomas contain considerable portions of cells expressing stem cell markers. It appears that some of these cells originate from tumour genesis (supporting the stem cell hypothesis) while others are attracted by the tumours. Further functional markers are required to differentiate these TSC-types.

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.Tumor necrosis factor (TNF), 1 originally defined by its antitumoral activity, is now recognized as a pleiotropic cytokine exerting a wide variety of immunoregulatory activities (for review, see Refs. 1-3). TNF action is mediated by two types of cell surface receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular masses, respectively. Both receptors mediate distinct TNF responses (4 -6). The majority of activities of soluble TNF appears to be mediated by TR55 (7-11). Like other cytokine receptors, the cytoplasmic domain of both TNF receptors lacks intrinsic enzymatic activities. The activation of intracellular signaling enzyme systems is initiated by a selective interplay between the cytoplasmic domain of the TNF receptors and a number of recently identified TNF receptor-associated proteins (see Ref. Results from numerous studies have revealed that TNF signaling further involves activation of downstream enzyme systems at multiple subcellular compartments such as the plasmamembrane, endosomes, mitochondria, the cytosol, and the nucleus (for review, see Refs. 11, 23, and 24). Membrane-associated enzyme systems transmitting TR55 signals include plasmamembrane-bound phospholipases such as phosphatidylcholine-specific phospholipase C (25), which generates the lipid second messenger molecule 1,2-diacylglycerol and a N-SMase (9), producing ceramide by sphingomyelin hydrolysis. Ceramide generated at the plasmamembrane triggers activation of a 97-kDa ceramide-activated protein kinase (26), recently suggested to be identical with the "kinase suppressor of ras" (27). Ceramide-activated protein kinase belongs to a family of proline-directed protein kinases (PDPK) (9), including members of the mitogen-activated protein kinases (28). TNF signaling further involves intracellular membrane compartments like caveolae and endosomes harboring an acid SMase (A-SMase) (9,29,30). In mitochondria, TNF induces reactive oxygen species that are generated at the level of the oxidative phosphorylation complex III (31). T...

P22PRG1/IEX-1 is a putative NF-kappaB target gene implicated in the regulation of cellular viability. Here, we show that in HeLa cells TNFalpha induces expression of p22PRG1/IEX-1 in an NF-kappaB dependent fashion. Blockade of NF-kappaB activation by various NF-kappaB inhibitors abolished TNFalpha-induced p22PRG1/IEX-1 expression and increased the sensitivity to apoptosis induced by TNFalpha, an activating Fas-antibody or the anti-cancer drug etoposide. Surprisingly, ectopic expression of p22PRG1/IEX-1 in HeLa cells transfected with an inducible p22PRG1/IEX-1-expression vector augments the susceptibility to apoptosis initiated by death-receptor ligands or by etoposide. In addition, p22PRG1/IEX-1 expressing HeLa cells exhibit an accelerated progression through the cell cycle. Transfection of an antisense hammerhead ribozyme targeted to p22PRG1/IEX-1 reduced the speed in cell cycle progression and decreased the apoptotic response to death ligands. Our data demonstrate that p22PRG1/IEX-1 is specifically induced during NF-kappaB activation, but this seems not to be related to the anti-apoptotic actions of NF-kappaB. Instead, NF-kappaB dependent recruitment of p22PRG1/IEX-1 might be related to a modulation in the cell cycle, and hereby, p22PRG1/IEX-1 may accelerate cell growth on the one hand, but may trigger apoptosis on the other. Oncogene (2001) 20, 69 - 76.

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligandinduced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-κB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

Lymphangiogenesis is thought to promote the progression of malignant tumors. Because the lymphangiogenic factors vascular endothelial factor (VEGF)-C and -D are expressed in endocrine cells, we investigated their expression in pancreatic endocrine tumors (PETs) and correlated these data and intratumoral lymph vessel density (iLVD) with clinicopathological features. Lymph vessels were identified with anti-podoplanin antiserum and with podoplanin/proliferating cell nuclear antigen double labeling. PETs (n ‫؍‬ 104) were investigated by immunohistochemical staining for VEGF, basic fibroblast growth factor, and VEGF-C expression. VEGF-C and VEGF-D mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. PETs showed higher iLVD than normal pancreata, but iLVD did not discriminate between benign and malignant PETs. In PETs proliferating lymph vessels were identified. High iLVD was associated with lymph vessel invasion and it was more frequent in angioinvasive/metastatic tumors than in grossly invasive tumors. The biological behavior of pancreatic endocrine tumors (PET) is difficult to predict on the basis of histological criteria. In the absence of clear signs of malignancy, such as invasion of adjacent organs, angioinvasion, or metastasis, the prognosis remains uncertain. Because most human carcinomas metastasize via lymphatic invasion, lymph node metastasis is a key prognostic factor for the clinical outcome. By which mechanism tumor cells spread through the lymph vessels is unknown. One proposed mechanism is the induction of new lymph vessels by tumor or inflammatory cells, facilitating lymphangioinvasion. Intratumoral lymph vessels have been detected in head and neck squamous cell carcinomas 1,2 and in cutaneous melanomas. 3,4 In squamous cell carcinomas intratumoral lymph vessel density (iLVD) has been shown to correlate with lymph node metastasis, whereas the results for melanomas are inconsistent. Beasley and colleagues 1 and Straume and colleagues 3 showed that intratumoral lymphatic endothelial cells were capable of proliferation, suggesting de novo lymphangiogenesis. No evidence of intratumoral lymph vessels was found in ovarian, 5 liver, 6 breast, 7,8 or cervical carcinomas. 9

The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth-and deathinducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-jB (NF-jB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-jB activity, either basal or TNFa induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-jB activation was preceded by an altered turnover of IjBa and phospho-IjBa. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-jB activity, and accelerated the degradation of IjBa. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-jB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozymetransduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1 -itself an NF-jB target gene -inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-jB.