Reduction of chromatin assembly factor 1 p60 and C21orf2 protein, encoded on chromosome 21, in Down syndrome brain.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.

Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas.

Gene therapeutic approaches currently favor adenoviral vectors over alternatively available vector systems. Ovarian cancer represents an attractive model for an intraperitoneal adenovirus-based gene therapy, which is now under intensive clinical investigation. Adenovirus-mediated gene transfer depends on adequate virus uptake and thus on the presence of sufficient amounts of high-affinity coxsackie-adenovirus receptor (CAR) and alphavbeta3- and alphavbeta5 integrins on target cells. This fact has been ignored in most ongoing clinical trials. This investigation, therefore, determined expression of CAR by immunohistochemistry in 37 ovarian carcinomas and compared it with that of alphavbeta3 and alphavbeta5 integrins. In all samples, except one undifferentiated carcinoma, CAR was immunohistochemically demonstrable. Grade 1 tumors exhibited stronger CAR immunostaining as compared with higher-grade cancers (P < 0.03). Integrins alphavbeta3 and alphavbeta5 were detectable in 62% and 65% of carcinomas, respectively, and staining for both classes correlated positively (P < 0.005). Cancers classified as undifferentiated completely lacked alphavbeta3 expression. Furthermore, in undifferentiated and grade 3 carcinomas the three molecules studied exhibited marked distributional heterogeneity with regard to focal positivity and negativity within the same tumor. Either the absence of CAR, alphavbeta3 and alphavbeta5 or the pronounced heterogeneity in their expression might seriously compromise the efficiency of adenovirus-based gene therapy in ovarian cancer.

The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction.

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.

The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure.

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.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting.

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway.

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.

The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy.

In a recent paper, we reported a significant difference in coxsackie and adenovirus receptor (CAR) from several human bladder cancer cell lines that correlated with their sensitivities to adenoviral infection (Y. Li, R-C. Pong, J. M. Bergelson, M. C. Hall, A. I. Sagalowsky, C-P. Tseng, Z. Wang, and J. T. Hsieh, Cancer Res., 59: 325-330, 1999). In human prostate cancer, CAR protein is down-regulated in the highly tumorigenic PC3 cell line, which suggests that, in addition to its function as a viral receptor, CAR may have a pathophysiological role in prostate cancer progression. In this paper, we document that CAR does not merely enhance the viral sensitivity of prostate cancer cells but also acts as a tumor inhibitor for androgen-independent prostate cancer cells. Our data indicate that CAR is a potential therapeutic agent for increasing the efficacy of prostate cancer therapy.

Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer.

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.

The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus.

Group B coxsackieviruses are etiologically linked to many human diseases, and cell surface receptors are postulated to play an important role in mediating their pathogenesis. The coxsackievirus adenovirus receptor (CAR) has been shown to function as a receptor for selected strains of coxsackievirus group B (CVB) serotypes 3, 4, and 5 and is postulated to serve as a receptor for all six serotypes. In this study, we demonstrate that CAR can serve as a receptor for laboratory reference strains and clinical isolates of all six CVB serotypes. Infection of CHO cells expressing human CAR results in a 1000-fold increase in CVB progeny virus titer compared to mock transfected cells. CAR was shown to be a functional receptor for swine vesicular disease virus (SVDV), as CHO-CAR cells but not CHO mock transfected controls were susceptible to SVDV infection, produced progeny SVDV, and developed cytopathic effects. Moreover, SVDV infection could be specifically blocked by monoclonal antibody to CAR (RmcB). SVDV infection of HeLa cells was also inhibited by an anti-CD55 MAb, suggesting that this virus, like some CVB, may interact with CD55 (decay accelerating factor) in addition to CAR. Finally, pretreatment of CVB or SVDV with soluble CAR effectively blocks virus infection of HeLa cell monolayers.

Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells.

Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, alpha(v)beta5- and alpha(v)beta3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was 'trans-complementation' of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.

rab5 GTPase regulates adenovirus endocytosis.

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

Molecular determinants of adenovirus serotype 5 fibre binding to its cellular receptor CAR.

Adenovirus (Ad) tropism is mediated in part through the fibre protein. The common coxsackie B virus and Ad receptor (CAR) was recently identified as the major receptor for subgroup C Ad serotype 5 (Ad5) and serotype 2 (Ad2) fibres. Effects of mutations in the Ad5 fibre gene were studied to assess domains of the fibre capsomer that could alter virus tropism without altering virus assembly and replication. All mutants that accumulated as fibre monomers failed to assemble with a penton base and proved lethal for Ad5 which suggests that the absence of infectious virions resulted in part from a defect in fibre penton base assembly. Cell binding capacity of all fibre mutants was investigated in cell binding competition experiments with adenovirions using CHO-CAR cells (CHO cells that have been transfected with CAR cDNA and express functional CAR). The results suggest that the R-sheet of the Ad5 fibre knob monomer contains binding motifs for CAR and that beta-strands E and F, or a region close to them, may also be involved in receptor recognition.

Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.

Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I alpha-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency.

Coxsackievirus and adenovirus receptor cytoplasmic and transmembrane domains are not essential for coxsackievirus and adenovirus infection.

Coxsackievirus and adenovirus receptor (CAR) from which the cytoplasmic domain had been deleted and glycosylphosphatidylinositol (GPI)-anchored CAR lacking both transmembrane and cytoplasmic domains were both capable of facilitating adenovirus 5-mediated gene delivery and infection by coxsackievirus B3. These results indicate that the CAR extracellular domain is sufficient to permit virus attachment and entry and that the presence of a GPI anchor does not prevent infection.

CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts.

Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdDeltaRGDbetagal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alphaV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)betagal (bearing seven lysines on the end of fiber) (b) AdF(RGD)betagal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK betagal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.

Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy.

There is great interest in the development of gene therapeutic strategies for the treatment of benign and malignant diseases. Recombinant adenovirus has a wide spectrum of tissue specificity and is an efficient vector delivery system. Successful gene delivery, however, requires viral entry into the target cells via specific receptor-mediated uptake. Recently, a cDNA clone (the coxsackie and adenovirus receptor [CAR]) encoding a 46-kDa protein was identified as the receptor for group C adenovirus (e.g., adenovirus type 2 and 5). Currently, little is known regarding the expression of adenoviral receptor in normal tissue and cancer. In this paper, we have documented a significant difference in viral receptor levels that may be due to transcriptional regulation of the CAR gene in several human bladder cancer cell lines. The differences in viral receptor levels in these cells correlated with their sensitivity to viral infection. Transfection of receptor-negative cell line with CAR cDNA led to increased virus binding and increased susceptibility to adenovirus-mediated gene delivery. Our results demonstrate that the expression of adenoviral receptor is variable among human bladder cancer cells. This variability may have a significant impact on the outcome of adenovirus-based gene therapy.