Helper virus-free gutless adenovirus (HF-GLAd): a new platform for gene therapy.

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades.

No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants.

Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing [BCL2L12] and [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA.

Enhancement of TRAIL-induced apoptosis by 5-fluorouracil requires activating Bax and p53 pathways in TRAIL-resistant lung cancers.

Lung cancer, especially lung adenocarcinoma, is one of the main causes of death worldwide. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a primary anticancer agent and a member of the tumor necrosis factor family that selectively induces apoptosis in various tumor cells, but not in normal cells. Combination chemotherapy can be used for treating specific cancer types even at progressive stages.

DUSP6 is a novel transcriptional target of p53 and regulates p53-mediated apoptosis by modulating expression levels of Bcl-2 family proteins.

p53 regulates various cellular responses through transcriptional regulation of distinct sets of target genes. Dual specificity phosphatase 6 (DUSP6) is a cytosolic phosphatase that inactivates the extracellular-signal-regulated kinase 1/2 (ERK1/2). This study demonstrates that p53 transactivates DUSP6 in human colorectal HCT116 cells to regulate ERK1/2 in p53-mediated cell death.

p53-Independent up-regulation of a TRAIL receptor DR5 by proteasome inhibitors: a mechanism for proteasome inhibitor-enhanced TRAIL-induced apoptosis.

Gliomas are the most common brain tumors in adults and account for more than half of all brain tumors. Despite intensive clinical investigations, average survival for the patients harboring the malignancy has not been significantly improved. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), shown to have potent and cancer-selective killing activity, has drawn considerable attention as a promising anti-cancer therapy.

Preclinical studies for pharmacokinetics and biodistribution of Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy.

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed.

Suppression of IRF4 by IRF1, 3, and 7 in Noxa expression is a necessary event for IFN-γ-mediated tumor elimination.

IFN-γ plays a critical role in tumor immunosurveillance by affecting either immune cells or tumor cells; however, IFN-mediated effects on tumor elimination are largely unknown. In this study, we showed that IFN regulatory factors (IRF) modulated by IFNs up- and downregulated Noxa expression, a prodeath BH3 protein, in various cancer cells. Inhibition of Noxa expression using short hairpin RNA in tumor cells leads to resistance against lipopolysaccharide (LPS)-induced tumor elimination, in which IFN-γ is known as a critical effecter in mice.

Effective knockdown of multiple target genes by expressing the single transcript harbouring multi-cistronic shRNAs.

Gene silencing by RNA interference (RNAi) using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a valuable tool for evaluating the target gene function. Here, we report an approach for silencing multiple target genes simultaneously by expressing one single transcript encoding different target shRNAs. We first constructed the cytomegalovirus (CMV) promoter-driven expression vectors, each of which expresses microRNA mir-30-mimicked shRNA specifically targeting X-chromosome-linked inhibitor of apoptosis protein (XIAP), Akt, or Bcl-2.

The cell death-inducing activity of the peptide containing Noxa mitochondrial-targeting domain is associated with calcium release.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain.

DOBI is cleaved by caspases during TRAIL-induced apoptotic cell death.

Downstream of Bid (DOBI) known as Pus10, has been identified as a modulator of TRAIL-induced cell death using RNAi library screening. The crystal structure of DOBI has revealed that it is a crescent-shaped protein containing the pseudouridine synthase catalytic domain and a THUMP-containing domain. Here, we demonstrated that DOBI is expressed in various tissues such as heart and lung, and is also expressed in various tumor cells such as HeLa and A549.

Homer1 regulates the susceptibility to TRAIL.

TRAIL is an apoptotic cell death-inducing ligand that belongs to a TNF superfamily. To identify the regulators that govern the susceptibility to TRAIL, TRAIL-resistant HeLa (TR) cells were established by repeatedly treating HeLa cells with TRAIL. Here we showed that scaffolding protein Homer1 plays a decisive role in regulating the apoptotic susceptibility to TRAIL.

Hypoxia protects articular chondrocytes from thapsigargin-induced apoptosis.

The present study investigates the effect of low oxygen concentrations on thapsigargin-induced apoptosis and reactive oxygen species (ROS)-related signaling in articular chondrocytes. Chondrocytes were obtained from normal canine knee cartilage and were treated with different concentrations of thapsigargin for 24h under normoxic (21% oxygen tension) or hypoxic (1% oxygen tension) conditions. The cells treated with thapsigargin under normoxic conditions showed a dose-dependent induction of apoptosis.

Possible novel therapy for malignant gliomas with secretable trimeric TRAIL.

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress.

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography.

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn(2+) ions as affinity ligands.

Up-regulation of IAPs by PI-3K: a cell survival signal-mediated anti-apoptotic mechanism.

Under normal cell physiology, a balance between cell survival and apoptosis is crucial for homeostasis. Many studies have demonstrated that apoptosis is modulated by cell survival stimuli. Active Akt, a common mediator of cell survival signals, has been shown to inhibit apoptosis by attenuating activity of pro-apoptotic factors Bad and caspase-9.

Blockade of processing/activation of caspase-3 by hypoxia.

Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells.

A novel protein, MUDENG, induces cell death in cytotoxic T cells.

A screening system comprised of a randomized hybrid-ribozyme library has previously been used to identify pro-death genes in Fas-mediated apoptosis, and short sequence information of candidate genes from this system was previously reported by Kawasaki and Taira [H. Kawasaki, K. Taira, A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries, Nucleic Acids Res.

The role of mitochondria in apoptosis.

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways.

Stress-governed expression and purification of human type II hexokinase in Escherichia coli.

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL21 (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E.

Regulation of DR-5 protein and mitochondrial transmembrane potential by gemcitabine, a possible mechanism of gemcitabine-enhanced TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. Gemcitabine, chemotherapeutic agent is well known to be involved in synergistic cytotoxic effect with TRAIL. But the mechanism of this synergistic effect is still unclear.

Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay.

Conformational changes in hexokinase are induced by its binding to glucose, thus providing an excellent example of an 'induced fit' model. To observe glucose-induced fluorescence restoration in hexokinase II using split-enhanced, green fluorescent protein (EGFP) in a process involving the reconstitution of split EGFP, E. coli cells expressing the chimeric NEGFP:HXK:CEGFP recombinant protein were treated with glucose and visualized via fluorescence read-outs.

A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element.

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases.

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage.

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis.

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1beta. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer.