The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure.
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The coxsackie and adenovirus receptor (CAR) is identified as a high-affinity receptor for adenovirus type 5. We observed that invasive bladder cancer specimens had significantly reduced CAR mRNA levels compared with superficial bladder cancer specimens, which suggests that CAR may play a role in the progression of bladder cancer. Elevated CAR expression in the T24 cell line (CAR-negative cells) increased its sensitivity to adenovirus infection and significantly inhibited its in vitro growth, accompanied by p21 and hypophosphorylated retinoblastoma accumulation. Conversely, decreased CAR levels in both RT4 and 253J cell lines (CAR-positive cells) promoted their in vitro growth. To unveil the mechanism of action of CAR, we showed that the extracellular domain of CAR facilitated intercellular adhesion. Furthermore, interrupting intercellular adhesion of CAR by a specific antibody alleviates the growth-inhibitory effect of CAR. We also demonstrated that both the transmembrane and intracellular domains of CAR were critical for its growth-inhibitory activity. These data indicate that the cell-cell contact initiated by membrane-bound CAR can elicit a negative signal cascade to modulate cell cycle regulators inside the nucleus of bladder cancer cells. Therefore, the presence of CAR cannot only facilitate viral uptake of adenovirus but also inhibit cell growth. These results can be integrated to formulate a new strategy for bladder cancer therapy. (+info)
An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression.
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The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to alpha(v)beta(3) and alpha(v)beta(5) integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials. (+info)
CAR-binding ablation does not change biodistribution and toxicity of adenoviral vectors.
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Intravenous administration of adenoviral vectors results mostly in hepatocyte transduction and subsequent hepatotoxicity. Because hepatocytes express high levels of the primary adenovirus receptor CAR, untargeting hepatocytes requires CAR-binding ablation. The amino acid residues of the viral fiber responsible for CAR-binding are known. We have constructed a mutant adenoviral vector unable to bind CAR and studied vector biodistribution and hepatotoxicity after intravenous administration. In contrast to a vector with wild-type fiber, the infectivity of the CAR-ablated vector is greatly reduced and not susceptible to inhibition with wild-type knob. Biodistribution and hepatotoxicity are, however, not affected by CAR-binding ablation. A possible explanation could be related to an increased blood persistence detected for the CAR-ablated vectors combined with their residual infectivity through other receptors. (+info)
Efficient c-kit receptor-targeted gene transfer to primary human CD34-selected hematopoietic stem cells.
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We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit(+) human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer. (+info)
Forced myofiber regeneration promotes dystrophin gene transfer and improved muscle function despite advanced disease in old dystrophic mice.
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Duchenne muscular dystrophy (DMD) is caused by defects in the dystrophin gene. In young dystrophic mdx mice, immature regenerating myofibers represent the principal substrate for adenovirus vector (AdV)-mediated dystrophin gene transfer. However, in DMD patients immature regenerating myofibers are generally sparse. Such a situation also exists in old mdx mice, which may represent a more realistic model. Therefore, here we have used old mdx mice (of 14- to 17 months of age) to test the hypothesis that one-time administration of a myonecrotic agent can transiently re-establish a population of immature myofibers susceptible to AdV-mediated dystrophin gene transfer. This strategy led to upregulation of the coxsackie/adenovirus attachment receptor by means of induction of regenerating myofibers, significantly augmented AdV-mediated dystrophin gene expression, and enhanced force-generating capacity. In addition, it led to an increased resistance to contraction-induced injury compared with untreated controls. The latter protective effect was positively correlated with the number of dystrophin-expressing myofibers (r=0.83, P<0.05). Accordingly, the risk:benefit ratio associated with the sequential use of forced myofiber regeneration and AdV-mediated dystrophin gene transfer was favorable in old mdx mice despite advanced disease. These findings have implications for the potential applicability of AdV-mediated gene therapy to DMD and other muscle diseases in which immature regenerating myofibers are lacking. (+info)
The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction.
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The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces. (+info)
Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus.
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Adenovirus type 5 (Ad) based vectors transduce vascular endothelial cells (EC) and have been widely used for vascular gene transfer. However, many cell types express the Ad receptor (cox-sackievirus adenovirus receptor; CAR), preventing selective EC infection and precluding clinical use. We previously isolated the human EC-binding peptides SIGYPLP and LSNFHSS by phage display and demonstrated by means of a bispecific antibody that SIGYPLP directs efficient, high-level, EC-selective Ad-mediated gene transfer. We now generate genetically modified Ad fiber proteins with selective EC tropism by engineering these peptides into the HI loop of the Ad fiber. SIGYPLP, but not LSNFHSS, enhanced EC selectivity, demonstrating maintenance of peptide-cell binding fidelity upon incorporation into virions. Combining fiber mutations that block CAR binding (detargeting) with SIGYPLP insertion (retargeting) generated a novel Ad vector, AdKO1SIG, in a single component system. AdKO1SIG demonstrated efficient and selective tropism for EC compared with control Ad vectors. This is the first demonstration of genetic incorporation of a novel, mammalian, cell-selective ligand that retains its targeting fidelity in the Ad fiber HI loop, in combination with point mutations that abolish fiber-CAR interaction. This study demonstrates the potential for improving the cell-selectivity and safety of adenoviral vectors. (+info)
Absence of germline infection in male mice following intraventricular injection of adenovirus.
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The possibility of inadvertent exposure of gonadal tissue to gene therapy vectors has raised safety concerns about germline infection. We show here that the receptor for coxsackie B viruses and adenoviruses 2 and 5 (CXADR) is expressed in mouse germ cells, suggesting the possibility that these viruses could infect germ cells. To directly assess the risk of germline infection in vivo, we injected an adenovirus carrying the germ-cell-specific protamine promoter fused to the bacterial lacZ reporter gene into the left ventricular cavity of mice and then monitored expression of the reporter gene in germ cells. To differentiate between infection of stem cells and differentiating spermatogenic cells, we analyzed expression of the reporter cassette at different times after viral delivery. Under all conditions tested, mice did not express the Escherichia coli beta-galactosidase protein in developing spermatids or in mature epididymal spermatozoa. Primary germ cells cultured in vitro were also refractory to adenoviral infection. Our data suggest that the chance of vertical germline transmission and insertional mutagenesis is highly unlikely following intracoronary adenoviral delivery. (+info)