Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O2) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O2 concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the ␣-subunit of hypoxia-inducible factor 1. At higher O2 concentrations (1-3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O2 or O2-derived species as signaling molecules. Diversion of O2 away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells.AMP-activated protein kinase ͉ hypoxia-inducible factor 1␣ ͉ hypoxia ͉ nitric oxide

We use lentiviral-delivered RNA interference (RNAi) to inhibit the growth of a model of primary effusion lymphoma (PEL) in vitro and in vivo. RNAi is a phenomenon allowing the sequencespecific targeting and silencing of exogenous and endogenous gene expression and is being applied to inhibit viral replication both in vitro and in vivo. We show that silencing of genes believed to be essential for the Kaposi sarcomaassociated herpesvirus (KSHV) latent life cycle (the oncogenic cluster) has a varied effect in PEL cell lines cultured in vitro, however, concomitant silencing of the viral cyclin (vcyclin) and viral FLICE (Fasassociating protein with death domainlike interleukin-1␤-converting enzyme) inhibitory protein (vFLIP) caused efficient apoptosis in all PEL lines tested. We demonstrate that in a murine model of PEL, lentiviral-mediated RNA interference both inhibits development of ascites and can act as a treatment for established ascites. We also show that the administered lentiviral vectors are essentially limited to the peritoneal cavity, which has advantages for safety and dosage in a therapeutic setting. This shows the use of lentiviral-mediated RNA interference in vivo as a potential therapeutic against a virally driven human cancer. IntroductionRNA interference (RNAi) is being exploited to treat or prevent infection and as a therapeutic against cancer. 1,2 Attractive targets include foreign (eg, viral) and mutated, fused, or overexpressed genes (ie, cancer). [3][4][5] In vitro, RNAi has been shown to block infection and replication of various pathogens including human immunodeficiency virus (HIV-1), 6,7 influenza, 8 and viruses implicated in oncogenesis such as Epstein Barr virus (EBV), 9,10 hepatitis B, 11,12 hepatitis C, 13 and human papilloma virus (HPV). 14,15 In experimental in vivo models, RNAi has been shown to prevent chemical-and viral-induced hepatitis. [16][17][18] The efficient delivery of therapies that knock-down specific RNA remains one obstacle to translate RNAi into a realistic treatment option for human disease. 19 The direct delivery of antisense RNA to treat or prevent CMV retinitis is one of the few successful clinical applications thus far of RNA-targeted treatment. 20,21 Although antisense provides significantly less robust inhibition of gene expression compared to RNAi, the major problem with both therapies remains effective delivery to the site of disease. One use of RNAi is based around injecting large quantities of synthetic double-stranded RNA (dsRNA) or DNA encoding short hairpin RNA (shRNA) intravenously or using hydrodynamic transfection. 16,18 This approach is not realistic for treating human disease, except for diseases involving sites where delivery of synthetic dsRNA is more straightforward. The development of shRNA to knock-down gene expression 22,23 and the incorporation of shRNA into lentiviral- 24,25 or adenoviral-based 26,27 vectors offers the opportunity to use these vectors to target RNA efficiently in vivo. Vesicular stomatitis virus-g envelope (VSV-g)-pseudoty...

Background and Aim The early diagnosis of biliary tract cancer (BTC) remains challenging and there are few effective therapies. We investigated whether the M2 isotype of pyruvate kinase (M2-PK), which serves as the key regulator of cellular energy metabolism in proliferating cells, could play a role in the diagnosis and therapy of BTC. Methods Plasma and bile M2-PK concentrations were measured by ELISA in 88 patients with BTC, 79 with benign biliary diseases and 17 healthy controls. M2-PK expression was sought in BTC tissue array by immunohistochemistry. The role of M2-PK in tumour growth, invasion and angiogenesis was evaluated in BTC cell lines by retrovirus-mediated M2-PK transfection and shRNA silencing techniques. Results Sensitivity (90.3%) and specificity (84.3%) of bile M2-PK for malignancy were significantly higher than those for plasma M2-PK and serum CA19-9. M2-PK expression was specific for cancer cells and correlated with microvessel density. M2-PK positivity was a significant independent prognostic factor by multivariable analysis. Transfection of M2-PK in a negatively expressed cell line (HuCCT-1cells) increased cell invasion whereas silencing in a M2-PK positive cell line (TFK cells) decreased tumour nodule formation and cellular invasion. A significant increase in endothelial tube formation was noted when supernatants from M2-PK transfected cells were added to an in vitro angiogenesis assay whereas supernatants from silenced cells negated tube formation. Conclusions Bile M2-PK is a novel tumour marker for BTC and correlates with tumour aggressiveness and poor outcome. shRNA mediated inhibition of M2-PK indicates the potential of M2-PK as a therapeutic target.

Human stem cells could revolutionize the field of medicine by providing a diverse range of cell types for tissue replacement therapies and drug discovery. To achieve this goal, genetic tools need to be optimized and developed for controlling and manipulating stem cells ex vivo. Here we describe a lentiviral delivery system capable of high infection rates in human mesenchymal and embryonic stem cells. The lentiviral backbone was modified to express mono-and bi-cistronic transgenes and was also used to deliver short hairpin ribonucleic acid for specific silencing of gene expression in human stem cells. We show that lentiviral transduction can be used to alter gene expression without altering the genes' ability to differentiate in vitro. These vectors will enable rapid analysis of gene function in stem cells and permit the generation of knock-in / knock-out models of human disease in the rapidly developing field of gene therapy.