Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response.

Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin ß1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFß1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFß1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression.

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.

An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression.

The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.

Long-circulating vectors for the systemic delivery of genes.

The development of vectors capable of treating systemic diseases is an important goal for gene therapy protocols. In order for a carrier system to preferentially accumulate at sites of systemic disease, such as tumors, sites of inflammation and sites of infection, the carrier must exhibit long circulation lifetimes following intravenous injection. Unfortunately, most gene delivery systems, including viral vectors as well as non-viral vectors, e.g., lipoplexes, polyplexes and lipopolyplexes, are rapidly cleared from the circulation and are preferentially taken up by the 'first-pass' organs such as liver, lung and spleen. Here we review recent literature concerning the ability of non-viral vectors to act as systemic gene therapy agents. The most promising systemic vectors are liposomal systems in which plasmid DNA is encapsulated within a lipid bilayer. The stabilized plasmid-lipid particle (SPLP) system, for example, exhibits circulation half-lives of the order of 6 h following intravenous injection, and preferentially accumulates in distal tumors with gene expression primarily localized to the tumor site.

Cationic lipid-mediated transfection of cells in culture requires mitotic activity.

Cationic lipid-based delivery systems such as lipoplexes or stabilized plasmid-lipid particles (SPLP) represent a safer alternative to viral systems for gene therapy applications. We studied the impact of cell cycle status on the efficiency of transfection of human ovarian carcinoma tumor cells using two cationic-lipid based delivery systems. Cells arrested in the G1 phase of the cell cycle by treatment with aphidicolin were compared with an asynchronous dividing population of cells. Treatment of the cells with aphidicolin had no effect on the rate of internalization of the lipid formulated DNA or on the level of gene expression observable in stably transfected cells. However, cells treated with aphidicolin exhibited 20-fold lower reporter gene activity than asynchronous control cells upon incubation with lipoplexes. When cells arrested in the G1 phase were allowed to proceed through the cell cycle in the presence of the lipoplex or SPLP, transgene expression was found to coincide with the transition of cells from the G2/M phase into the G1 phase of the subsequent cell cycle. In addition, higher levels of reporter gene expression were observed when the cells were incubated with lipoplexes or SPLP during, or just before, mitosis. These results suggest that it may be possible to augment cationic lipid-mediated transfection by manipulating the cell cycle status of the target cells.

Stabilized plasmid-lipid particles: construction and characterization.

A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.

Progress towards a synthetic virus for systemic gene therapy.

Here we discuss recent progress in non-viral gene therapy with an emphasis on developments which specifically attempt to address the limitations of current systems and their inability to overcome the first barrier to systemic gene delivery, delivery to the disease site and the target cell. Other issues associated with the efficiency of transfection, such as intracellular delivery, the endosomal release of vectors internalized through endocytosis and nuclear delivery are also discussed.

Distinct domains of IkappaB-alpha inhibit human immunodeficiency virus type 1 replication through NF-kappaB and Rev.

Among the regulators of human immunodeficiency virus (HIV) replication is the cellular transcription factor NF-kappaB, whose activity is regulated through inhibition by IkappaB family members. We have shown previously that I kappaB-alpha inhibits HIV type 1 (HIV-1) replication, and unexpectedly, IkappaB-alpha was found both to suppress HIV-1 transcription and to inhibit Rev function. The relative contributions and specificities of these mechanisms to HIV replication were unknown. Here, we report that the region of IkappaB-alpha which blocks Rev function is separable from that required for inhibition of NF-kappaB. Molecular mutagenesis revealed that the N terminus of IkappaB-alpha is required for inhibition of Rev function, whereas mutants lacking the N terminus retained the ability to inhibit NF-kappaB function. Interestingly, the nuclear export sequence of IkappaB-alpha was not required for inhibition of Rev or NF-kappaB function in mammalian transfection assays. Conversely, the C terminus of IkappaB-alpha was not required for the inhibition of Rev, while deletion of this region resulted in a loss of NF-kappaB inhibition. Another IkappaB family member with a distinct amino-terminal sequence, IkappaB-beta, inhibited NF-kappaB but not Rev function. These studies indicate that the inhibition of Rev by IkappaB-alpha is independent of NF-kappaB. Mutants defective in inhibition of either Rev or NF-kappaB retained the ability to inhibit HIV-1 replication, suggesting that both functions may contribute to the inhibition of HIV replication by I kappaB-alpha.

Molecular characterization of quail apolipoprotein very-low-density lipoprotein II: disulphide-bond-mediated dimerization is not essential for inhibition of lipoprotein lipase.

As part of the avian reproductive effort, large quantities of triglyceride-rich very-low-density lipoprotein (VLDL) particles are transported by receptor-mediated endocytosis into the female germ cells. Although the oocytes are surrounded by a layer of granulosa cells harbouring high levels of active lipoprotein lipase, non-lipolysed VLDL is transported into the yolk. This is because VLDL particles from laying chickens are protected from lipolysis by apolipoprotein (apo)-VLDL-II, a potent dimeric lipoprotein lipase inhibitor [Schneider, Carroll, Severson and Nimpf (1990) J. Lipid Res. 31, 507-513]. To determine whether this protection depends on dimer formation and constitutes a general mechanism to ensure high levels of yolk triglycerides for embryonic utilization in birds, we have now molecularly characterized apo-VLDL-II in the Japanese quail, a frequently used avian species. Quail apo-VLDL-II shows 72% amino acid identity with the chicken protein, with most replacements being in the C-terminal region. Importantly, quail apo-VLDL-II lacks the single cysteine residue present eight residues from the C-terminus of chicken apo-VLDL-II, which is responsible for dimerization of the chicken lipoprotein lipase inhibitor. Nevertheless, monomeric quail and dimeric chicken apo-VLDL-II display, on a molar basis, identical inhibitory effects on lipoprotein lipase, underscoring the biological importance of their function. Furthermore secondary structure prediction of the 3'-untranslated region of the quail message supports a role for loop structures in the strictly oestrogen-dependent production of the lipoprotein lipase inhibitors. Our findings shed new light on the essential role of this small, hormonally regulated, protein in avian reproduction.

Characterization of the Japanese quail oocyte receptor for very low density lipoprotein and vitellogenin.

The plasma membrane receptor for VLDL and vitellogenin from oocytes of Japanese quail (Coturnix coturnix japonica) was characterized and compared with that of another domestic fowl, the chicken (Gallus domesticus). When visualized by ligand blotting with biotinylated or 125I-labeled lipoproteins, the quail VLDL/vitellogenin receptor had an apparent M(r) of 95 kDa under nonreducing conditions, identical to that of the chicken receptor. Upon analysis by ligand blotting, binding of radiolabeled quail plasma VLDL to the quail oocyte receptor seemed to be saturable and exhibited high affinity (apparent Kd of 13.9 mg/L). Cross-reactivity, at the level of ligand recognition, was observed between quail and chicken VLDL/vitellogenin receptors, and immunological relatedness was demonstrated by Western blotting with a rabbit anti-chicken oocyte VLDL receptor antibody. In contrast, a species difference was observed in the apolipoprotein VLDL-II moiety of plasma VLDL. Chicken apolipoprotein VLDL-II, an 82-amino acid protein with a disulfide crosslink at residue 75 (the sole cysteine residue), existed as a homodimer of 9.5 kDa subunits and, to a lesser extent, as a monomer. Quail apolipoprotein VLDL-II existed only in monomeric form without reduction and lacked cysteine. The present results demonstrate that, despite a difference in an apolipoprotein moiety of VLDL, quail and chicken oocyte lipoprotein receptors share key structural and functional elements. This lends further support to the notion that receptor recognition is mediated by the common VLDL component, apolipoprotein B.

Riboflavinuria in the rd chicken. 5'-splice site mutation in the gene for riboflavin-binding protein.

Eggs of oviparous species must contain all components required for normal embryonic development. Among these, the vitamin riboflavin is deposited into egg white and yolk bound to the carrier protein, riboflavin-binding protein (ribBP). We have begun to investigate the mechanisms underlying ribBP transport pathways by molecular characterization of a relevant mutation in chicken. The autosomal recessive rd allele in Gallus gallus domesticus prevents the synthesis of functional ribBP and induces embryonic death on day 13. Polymerase chain reaction primers derived from the previously determined wild type cDNA sequence were used to amplify and clone cDNAs for ribBP from normal (Rd) and deficient (rd) animals. The rd allele was associated with a 100-nucleotide deletion in the messenger RNA for ribBP. Genomic clones were generated via polymerase chain reaction using primers flanking this 100-base pair deletion, and the resulting 2.1-kilobase pair clones were sequenced. The deletion in the rd ribBP cDNA corresponds precisely to an exon. The splice site following this exon contains a G-->A mutation at position 1 of the downstream 5'-splice donor sequence. The effect of this anomaly and the cause of the rd phenotype is the loss of the 100-base pair exon during the splicing process.

The relationship between cognitive failures, psychoneurotic symptoms and sex.

As Broadbent et al's (1) original analysis of the relationship between the Cognitive Failures Questionnaire (CFQ) and the Middlesex Hospital Questionnaire (MHQ) was conducted on an altered version of the MHQ, the present study undertook this same analysis using the full MHQ. In addition, the relationship was examined to see if it was mediated by the differences in the scoring of males and females on each questionnaire. Our results support and strengthen Broadbent et al's conclusion that high rates of cognitive failure are associated with psychoneurotic symptoms. The sex difference on the CFQ is discussed in terms of vulnerability to stress to account for the higher incidence of psychoneurotic symptoms in females.