Coxsackievirus And Adenovirus Receptor Expression Research Articles

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C′- or N′-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.

Coxsackievirus and adenovirus receptor is essential for cardiomyocyte development

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that is known to be a site of viral attachment and entry, but its physiologic functions are undefined. CAR expression is maximal in neonates and wanes rapidly after birth in organs such as heart, muscle, and brain, suggesting that CAR plays a role in the development of these tissues. Here, we show that CAR deficiency resulted in an embryonic lethal condition associated with cardiac defects. Specifically, commencing approximately 10.5 days postconception (dpc), CAR-/- cardiomyocytes exhibited regional apoptosis evidenced by both histopathologic features of cell death and positive staining for the apoptotic marker cleaved caspase 3. CAR-/- fetuses invariably suffered from degeneration of the myocardial wall and thoracic hemorrhaging, leading to death by 11.5 dpc. These findings are consistent with the view that CAR provides positive survival signals to cardiomyocytes that are essential for normal heart development.

Coxsackievirus and Adenovirus Receptor Expression on Primary Osteosarcoma Specimens and Implications for Gene Therapy with Recombinant Adenoviruses

To the Editor: We have read with great interest the article by Gu and coworkers reporting the coxsackievirus and adenovirus receptor (CAR) status on primary musculoskeletal tumors [(1)][1]. We are particularly interested in their findings on osteosarcomas because we are developing gene therapy

Enhanced oncolysis by a tropism-modified telomerase-specific replication-selective adenoviral agent OBP-405 (‘Telomelysin-RGD’)

Replication-competent oncolytic viruses are being developed for human cancer therapy. We previously reported that an attenuated adenovirus (OBP-301, 'Telomelysin'), in which the hTERT promoter element drives expression of E1A and E1B genes linked with an IRES, could replicate in cancer cells, and causes selective lysis of cancer cells. We further constructed OBP-405 ('Telomelysin-RGD') that contains an RGD motif in the HI loop of the fiber knob. We examined whether OBP-405 could be effective in overcoming the limitations of OBP-301, specifically their inefficient infection into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). By flow cytometric analysis, H1299 (lung) and SW620 (colorectal) tumor cells showed high levels of CAR expression, whereas LN444 (glioblastoma), LNZ308 (glioblastoma), and H1299-R5 (lung) tumor cells were negative for CAR expression. A quantitative real-time PCR analysis demonstrated that fiber-modified OBP-405 infected more efficiently than OBP-301, although the intracellular replication rate of both viruses was consistent. The comparative antitumor effect of fiber-modified OBP-405 and unmodified OBP-301 for human cancer cells was evaluated in vitro by XTT assay as well as in vivo by using athymic mice carrying xenografts. OBP-405 had a profound oncolytic effect on human cancer cell lines compared to OBP-301, in particular on cells with low CAR expression. Intratumoral injection of 10(7) plaque-forming units of OBP-405 into CAR-negative H1299-R5 lung tumor xenografts in nu/nu mice resulted in a significant inhibition of tumor growth and long-term survival in all treated mice. Moreover, selective replication of OBP-405 in the distant, uninjected H1299-R5 tumors was demonstrated. Our results suggest that fiber-modified replication-competent adenovirus OBP-405 exhibits a broad target range by increasing infection efficiency, an outcome that has important implications for the treatment of human cancers.

Targeted Adenovirus Vectors

Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy and the study of gene function because they are relatively easy to construct, can be produced at high titer, and have high transduction efficiency. However, in some applications gene transfer with Ad vectors is less efficient because the target cells lack expression of the primary receptor, coxsackievirus and adenovirus receptor (CAR). Another problem is the wide biodistribution of vector in tissue following in vivo gene transfer because of the relatively broad tissue expression of CAR. To overcome these limitations, various approaches have been developed to modify Ad tropism. In one approach, the capsid proteins of Ad are modified, such as with the addition of foreign ligands or the substitution of the fiber with other types of Ad fiber, in combination with the ablation of native tropism. In other approaches, Ad vectors are conjugated with adaptor molecules, such as antibody and fusion protein containing an anti-Ad single-chain antibody (scFv) or the extracellular domain of CAR with the targeting ligands, or chemically modified with polymers containing the targeting ligands. In this paper, we review advances in the development of targeted Ad vectors.

The Coxsackievirus and Adenovirus Receptor Interacts with the Multi-PDZ Domain Protein-1 (MUPP-1) within the Tight Junction

The coxsackievirus and adenovirus receptor (CAR) is a component of the epithelial cell tight junction. In a yeast two-hybrid screen we identified the multi-PDZ domain protein MUPP1 as an interaction partner for the CAR cytoplasmic domain. CAR and MUPP1 were found to colocalize at the tight junction, to coprecipitate from epithelial cells, and to interact in vitro. The interaction was found to specifically involve the PDZ-binding motif within the CAR C terminus and MUPP1 PDZ domain 13. In transfected cells, CAR recruited MUPP1 to cell-cell contacts. The inhibition of CAR expression with small interfering RNA inhibited MUPP1 localization to the tight junction. The results indicated that CAR interacts with MUPP1 and is involved in MUPP1 recruitment to the tight junction.

Targeted Adenovirus Vectors

Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy and the study of gene function because they are relatively easy to construct, can be produced at high titer, and have high transduction efficiency. However, in some applications gene transfer with Ad vectors is less efficient because the target cells lack expression of the primary receptor, coxsackievirus and adenovirus receptor (CAR). Another problem is the wide biodistribution of vector in tissue following in vivo gene transfer because of the relatively broad tissue expression of CAR. To overcome these limitations, various approaches have been developed to modify Ad tropism. In one approach, the capsid proteins of Ad are modified, such as with the addition of foreign ligands or the substitution of the fiber with other types of Ad fiber, in combination with the ablation of native tropism. In other approaches, Ad vectors are conjugated with adaptor molecules, such as antibody and fusion protein containing an anti-Ad single-chain antibody (scFv) or the extracellular domain of CAR with the targeting ligands, or chemically modified with polymers containing the targeting ligands. In this paper, we review advances in the development of targeted Ad vectors.

Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro.

Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells. Bispecific antibodies, constructed as conjugates between an anti-adenovirus fiber knob Fab' fragment and anti-EpCAM or anti-PSMA monoclonal antibodies, were incubated with an eGFP-expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non-prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined. Receptor-specific transduction was obtained for both EpCAM and PSMA. PSMA-retargeting was shown to be selective for the prostate cancer cell lines. PSMA serves as a tissue-specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer.

A cox-2 promoter-based replication-selective adenoviral vector to target the cox-2-expressing human bladder cancer cells.

Cyclooxygenase-2 (Cox-2), an enzyme that catalyzes the synthesis of prostaglandins, is overexpressed in a variety of premalignant and malignant conditions, including urinary bladder cancer. In the present study, we examined the feasibility of using Cox-2 promoter-based replication-selective adenovirus for targeting bladder cancer cells that express Cox-2 transcriptional activity. A series of human cancer cell lines, including three bladder cancer cell lines (KK47, T24, and 5637), were evaluated for their Cox-2 and CAR (the Coxsackievirus and adenovirus receptor) mRNA expression levels by quantitative real-time PCR. AdE3-cox2-327, a replication-selective adenovirus in which the expression of E1a is controlled by the Cox-2 promoter, was generated, and its tissue-specific activity was tested in vitro and in vivo. Three bladder cancer cell lines express higher levels of Cox-2 mRNA than does the human prostate cancer cell line PC3, the primary cultured human benign prostatic fibroblast, PF cells, and the human colon cancer cell line Colo320. Relatively higher expression of CAR mRNA was detected in the KK47, 5637, respectively, and Colo320 than in the T24, PC-3, and PF cells. In vitro assays revealed significant growth suppression of both Cox-2- and CAR-expressing bladder cancer cells KK47 and 5637 in comparison with the other cells that lack Cox-2 expression and/or CAR expression. The present study demonstrated both specificity and efficacy of AdE3-cox2-327, a selectively replicated adenovirus, toward the Cox-2-expressing bladder cancer cells in vitro and in vivo. We also found that CAR expression in the target cancer cells is an important factor for the efficacy of selectively replicated adenovirus-based gene therapy.

118. Efficient Infection of Head and Neck Cancer Cells by Ad5/35 Hybrid Virus

Top of pageAbstract Most adenoviral vectors that are currently being employed as gene delivery vehicles for cancer gene therapy are derivatives of adenovirus serotype 5 (Ad5). These vectors enter target cells via two specific interactions: between the viral fiber knob and the coxsackievirus and adenovirus receptor (CAR) and the penton base and integrins αVβ3 and αVβ5. However, adenovirus-mediated gene transfer to many important gene therapy targets is limited due to the low expression levels of CAR. Redirecting Ad5-based vectors to other, preferentially target specific cellular receptors and simultaneously abrogating the interaction between the viral capsid proteins and their cellular counterparts, has thus become a major goal in gene therapy. Group B adenovirus-based vectors have recently gained interest as attractive gene delivery vehicles. Subgroup B2 adenoviruses, including Ad35, infect cells in a CAR-independent manner and may thus transduce cells that are resistant to Ad5 infection. Notably, human CD46 was recently identified as the cellular attachment receptor for these viruses. Squamous cell carcinoma of the head and neck (HNSCC) has several characteristics that make it a suitable target for gene therapy. Most HNSCC are localized and thus easily accessible by direct intratumoral injection. In addition, metastases of the tumor are a late-stage occurrence and therefore early loco-regional control of the disease is paramount. On the basis of these favorable characteristics, gene delivery strategies that utilize adenoviruses have been developed for HNSCC. However, effective in vivo transduction has been a limiting factor in the initial clinical trials using standard Ad5-based vectors for this disease. In order to determine the feasibility of adenoviral retargeting to head and neck cancer, primary HNSCC cells collected from cancer patients were used. Full clinical as well as histological data is available on these cells. Our retargeting scheme was based on the Ad5/35 hybrid system, where the serotype 5 fiber has been replaced with the fiber of serotype 35. Flow cytometric analyses demonstrated that the level of CD46 expression was uniformly high in all primary cells studied (85–99%, average positive ratio 96%). In contrast, CAR expression was clearly lower and also highly variable (1.6–62%, average positive ratio 30%). αV integrin expression was between 39–98% with an average expression of 82%. In situ stainings for b-galactosidase gene expression demonstrated that Ad5/35.lacZ was clearly more effective than Ad5.lacZ in transducing primary HNSCC cells, and quantification of b-galactosidase expression revealed 3 to 58-fold differences in transduction efficacy between the two viruses. As expected, the infection efficacies of the viruses correlated with the receptor expression levels. When the viral transduction and receptor expression data was compared against the clinical data, the difference between the efficacies of the two viruses was found to be clearest in tumors with a high degree of malignancy, although the trend was not statistically significant. These data suggest that the group B2 adenovirus cell entry pathway is highly efficient in transducing HNSCC cells. These results warrant further development of the Ad5/35 hybrid system for head and neck cancer.

Monoclonal antibody against mouse CAR following genetic immunization.

To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR. Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.

Expression of coxsackievirus and adenovirus receptor in neointima of the rat carotid artery

Our previous study revealed that the coxsackievirus and adenovirus receptor (CAR) is a homophilic cell adhesion molecule and may function as a sensor of cell–cell interactions in the brain and damaged heart. In this study, we investigated if CAR expression is involved in the formation of neointimal hyperplasia using a balloon injury model of rat carotid artery. Cultured vascular smooth muscle cells (SMCs) from rat aorta were also studied. CAR antigen was constitutively detected in the endothelial cells (ECs) but not in SMCs before injury. On Day 5 after balloon injury, CAR was expressed strongly in the first layer of medial SMCs. Neointimal hyperplasia was observed on Day 7, and strong expressions of CAR concomitantly with proliferating cell nuclear antigen (PCNA) were obvious in the neointimal SMCs, while CAR in medial SMCs disappeared. The expression of CAR mRNA reached a peak on Day 7 and declined gradually to the basal levels. When the ECs regenerated on Day 14, CAR antigen was observed in the ECs but disappeared in the neointima. CAR together with PCNA was expressed abundantly in the proliferating SMCs in vitro and diminished in cells grown to a confluent state. The abundant expression of CAR in the neointima may facilitate an adenoviral gene therapy.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing.

Adenovirus vectors are expected to be a powerful tool for gene therapy to treat severe fractures. Adenovirus invades cells through binding to the coxsackievirus and adenovirus receptor (CAR) on the cell membrane. CAR expression is low in normal adult animals, but it is induced on regenerating cells in some experimental models. We made a rib fracture model in mice and evaluated the histological changes and CAR mRNA expression by RT-PCR 1, 5, 10, 14, and 21 days after the fracture. CAR mRNA was expressed exclusively in the fractured ribs at each time point, but not in the normal ribs. We detected the CAR protein immunohistochemically in fibroblast-like cells in the fracture callus on days 10 and 14 after fracture. In situ hybridization showed that these fibroblast-like cells expressed mRNA of type I collagen and osteopontin, but not osteocalcin, defining the cells as immature osteoblasts. We then transferred small doses (10(4)-10(8) PFU) of lacZ-expressing adenovirus vector into immature osteoblasts on day 14. beta-galactosidase was detected only on the immature osteoblasts at every dose. Immature osteoblasts play an important role in the matrix replacement step in fracture healing. CAR-mediated gene transfer into immature osteoblasts can be reasonable for adenovirus-mediated treatment of fracture healing.

Coxsackievirus and Adenovirus Receptor, a Tight Junction Membrane Protein, Is Expressed in Glomerular Podocytes in the Kidney

In nephrosis, filtration slits of podocytes are greatly narrowed, and slit diaphragms are displaced by junctions with close contact. Freeze-fracture studies have shown that the newly formed junctions consist of tight junctions and gap junctions. Several tight-junction proteins are known as integral membrane components, including occludin and claudins; but none of them have been found in podocytes. Coxsackievirus and adenovirus receptor (CAR) has recently been identified as a virus receptor that is a 46-kDa integral membrane protein with two Ig-like domains in the extracellular region. In polarized epithelial cells, CAR is expressed at the tight junction, where it associates with ZO-1 and plays a role in the barrier to the movement of macromolecules and ions. In the present study, we investigated the expression and localization of CAR in rat kidneys treated with puromycin aminonucleoside (PAN) and in rat kidneys perfused for 15 minutes with protamine sulfate (PS). Both the experimental models have been used to induce tight junctions in podocytes. Ribonuclease protection assay and Western blot analysis revealed a distinct increase of CAR transcript and protein in glomeruli during PAN nephrosis but no increase in glomeruli by PS perfusion. Immunohistochemistry revealed a significant increase in CAR staining intensity along the glomerular capillary wall in PAN nephrosis and after PS perfusion. Immunoelectron microscopy demonstrated in both the models that the immunogold particles for CAR along the capillary wall were found predominantly at close cell-cell contact sites of podocytes but were rarely found at slit diaphragms. In cultured podocytes, CAR was localized at cell-cell contact sites. CAR distribution was identical to that of ZO-1 and different from that of a gap junction protein, connexin43. These findings indicate that CAR is an integral membrane component of tight junction in podocytes and that CAR expression in podocytes is regulated at the transcriptional level and in the redistribution of protein.

The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells.

The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy.

Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor.

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context in vitro. Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell-cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell-cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.

Peptide-Retargeted Adenovirus Encoding a Tissue Inhibitor of Metalloproteinase-1 Decreases Restenosis after Intravascular Gene Transfer

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

Fatty acid modification of the coxsackievirus and adenovirus receptor.

Membrane-proximal cysteines 259 and 260 in the cytoplasmic tail of the coxsackievirus and adenovirus receptor (CAR) are known to be essential for the tumor suppression activity of CAR. We demonstrate that these residues provide an S-acylation motif for modification of CAR with the fatty acid palmitate. Substitution of alanine for cysteines 259 and 260 results in the additional localization of CAR in perinuclear compartments with no effect on the efficiency of adenovirus infection. The results indicate that palmitylation is important for stable plasma membrane expression and biological activity of CAR but is not critical for adenovirus receptor performance.

Enhancement of the adenoviral sensitivity of human ovarian cancer cells by transient expression of coxsackievirus and adenovirus receptor (CAR).

The coxsackievirus and adenovirus receptor (CAR) is known to be a primary receptor for attachment during adenovirus infection of target cells and thus is closely related to adenoviral infection efficiency. To extend this notion to human ovarian cancer, we investigated the difference in expression levels of CAR in human ovarian cancer cell lines and whether their adenoviral sensitivities are enhanced by transient transfection of the CAR gene. Adenoviral infection efficiency was examined by flow cytometry analysis, beta-galactosidase staining, and beta-galactosidase activity assay. Expression of the CAR-specific mRNA and protein was analyzed by reverse transcription polymerase chain reaction and flow cytometry, respectively. Expression of CAR in human ovarian cancer cell lines (SKOV3, 2774, PA-1, and OVCAR3) appeared to be correlated with their susceptibilities to adenovirus-mediated gene delivery. The 2774 and PA-1 cells expressing an easily detectable level of CAR on the cell surface showed a higher susceptibility to infection with both AdCMVGFP and AdRSVbetagal than SKOV3 and OVCAR3 cells, both of which had hardly detectable levels of CAR. Ectopic expression of the CAR gene by transient transfection of these ovarian cancer cells resulted in a dramatic increase in their adenoviral sensitivities. These data show that the expression of CAR is closely related to susceptibility to adenovirus infection in human ovarian cancer cells. These results indicate that the CAR gene can be a useful tool in boosting the efficiency of adenoviral vector-mediated gene therapy against human ovarian cancer.