CLMP

Gene Summary

Gene:CLMP; CXADR-like membrane protein
Aliases: ACAM, ASAM, CSBM, CSBS
Location:11q24.1
Summary:The CTX (see VSIG2, MIM 606011) family of proteins, including ASAM, are type I transmembrane proteins within the Ig superfamily that localize to junctional complexes between endothelial and epithelial cells and may play a role in cell-cell adhesion (Raschperger et al., 2004 [PubMed 14573622]).[supplied by OMIM, Mar 2008]
Databases:OMIM, HGNC, GeneCard, Gene
Protein:CXADR-like membrane protein
HPRD
Source:NCBIAccessed: 27 February, 2015

Ontology:

What does this gene/protein do?
CLMP is implicated in:
- integral to membrane
- plasma membrane
- tight junction
Data from Gene Ontology via CGAP

Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CLMP (cancer-related)

Zhao H, Jin G, Cui K, et al.
Novel modeling of cancer cell signaling pathways enables systematic drug repositioning for distinct breast cancer metastases.
Cancer Res. 2013; 73(20):6149-63 [PubMed] Free Access to Full Article Related Publications
A new type of signaling network element, called cancer signaling bridges (CSB), has been shown to have the potential for systematic and fast-tracked drug repositioning. On the basis of CSBs, we developed a computational model to derive specific downstream signaling pathways that reveal previously unknown target-disease connections and new mechanisms for specific cancer subtypes. The model enables us to reposition drugs based on available patient gene expression data. We applied this model to repurpose known or shelved drugs for brain, lung, and bone metastases of breast cancer with the hypothesis that cancer subtypes have their own specific signaling mechanisms. To test the hypothesis, we addressed specific CSBs for each metastasis that satisfy (i) CSB proteins are activated by the maximal number of enriched signaling pathways specific to a given metastasis, and (ii) CSB proteins are involved in the most differential expressed coding genes specific to each breast cancer metastasis. The identified signaling networks for the three types of breast cancer metastases contain 31, 15, and 18 proteins and are used to reposition 15, 9, and 2 drug candidates for the brain, lung, and bone metastases. We conducted both in vitro and in vivo preclinical experiments as well as analysis on patient tumor specimens to evaluate the targets and repositioned drugs. Of special note, we found that the Food and Drug Administration-approved drugs, sunitinib and dasatinib, prohibit brain metastases derived from breast cancer, addressing one particularly challenging aspect of this disease.

Gotoh A, Kanno T, Nagaya H, et al.
Gene therapy using adenovirus against malignant mesothelioma.
Anticancer Res. 2012; 32(9):3743-7 [PubMed] Related Publications
BACKGROUND: Adenovirus vectors have been utilized for cancer gene therapies. The present study examined the oncolytic effects of adenovirus type 5 (Ad5) and fiber-substituted conditionally replicating adenovirus (CRAD) Ad5/F35 vectors on the human malignant mesothelioma cells MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells.
MATERIALS AND METHOD: For the adenovirus, the first mRNA/protein to be made (~1 h after infection) is E1A. Ad5F35 and Ad5 CRAD vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Western blotting and cell viability assays were carried out in cells transfected with Ad5/MKp-E1 and Ad5F35/MKp-E1.
RESULTS: Coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, and CD46, a cell surface target of Ad35, were expressed in all the malignant mesothelioma cell lines examined here, as much as in HEK293 cells, with no significant differences in the expression levels among cells. Both Ad5/MKp-E1 and Ad5F35/MKp-E1 induced oncolysis of malignant mesothelioma cells in a viral particle-dependent manner, with similar efficacy.
CONCLUSION: The results of the present study suggest that both Ad5/MKp-E1 and Ad5F35/MKp-E1 are useful for the gene therapy of human malignant mesothelioma.

Ma J, Zhao J, Lu J, et al.
Coxsackievirus and adenovirus receptor promotes antitumor activity of oncolytic adenovirus H101 in esophageal cancer.
Int J Mol Med. 2012; 30(6):1403-9 [PubMed] Related Publications
Esophageal cancer is an intractable disease due to late diagnosis, high incidence of post-surgical locoregional recurrence and frequent distant metastasis. Oncolytic adenovirus (Ad) vectors are a promising method for cancer treatment. The H101 virus is a recombinant Ad which has replication-selective properties and replicates only in tumor cells. The coxsackievirus and adenovirus receptor (CAR) is considered a surrogate marker that monitors the outcome of Ad-mediated gene therapy. Accumulating evidence indicates that CAR expression levels are lower in various types of tumors such as ovarian, lung, breast and bladder when compared to their normal counterparts. In this study, we reported that trichostatin A (TSA) induced the expression of CAR in esophageal squamous cell carcinoma (ESCC) cell lines through the MAPK/ERK1/2 signaling pathway. The expression levels of CAR were positively related with the antitumor activity of H101. Our results suggest that TSA increases the antitumor activity of the oncolytic adenovirus H101 through the MAPK/ERK pathway.

Hiwasa K, Nagaya H, Terao S, et al.
Improved gene transfer into bladder cancer cells using adenovirus vector containing RGD motif.
Anticancer Res. 2012; 32(8):3137-40 [PubMed] Related Publications
BACKGROUND: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in high-grade human bladder cancer cells is generally extremely low due to the non-expression of coxsackie and adenoviral receptor (CAR). We investigated whether fiber-modified adenovirus vector containing an RGD motif in the HI loop of the adenovirus fiber knob could increase the transduction efficiency of Ad5 into human bladder cancer cells in vitro.
MATERIALS AND METHODS: We examined the expressions of CAR, and of α(v), β(3) and β(5) integrin, and the transduction efficacy of fiber-modified adenovirus vector in four human bladder cancer cell lines (TCC-SUP, 253J, T24 and KK47).
RESULTS: The expression of CAR was lower and those of α(v) and β(3) integrin were higher in four human cancer cell lines compared with the control cell line, KK47. The transduction efficacy of fiber-modified adenovirus vector increased by 20- to 470-fold compared with Ad5.
CONCLUSION: Fiber-modified adenovirus vector may be useful in order to establish new effective gene therapy strategies for the treatment of high-grade human bladder cancer.

Nagaya H, Tagawa M, Hiwasa K, et al.
Fiber-substituted conditionally replicating adenovirus for oncolysis of human renal carcinoma cells.
Anticancer Res. 2012; 32(7):2985-9 [PubMed] Related Publications
BACKGROUND: Adenovirus vectors have lately been highlighted in gene therapies. We investigated the oncolytic effects of a chimeric adenovirus type 5 (Ad5) with replacement of Ad5 fiber knob with adenovirus type 35 (Ad35) fiber knob (Ad5F35) on human renal cell carcinoma (RCC).
MATERIALS AND METHODS: The conditionally replicating Ad5F35 vector was constructed and infected into RCC cell lines 786-O, ACHN, and RCC4-VHL. For these cells, reverse transcription-polymerase chain reaction and western blotting were carried out and the cell viability was assayed.
RESULTS: In all RCC cell lines, it was found that CD46, a cell surface target of Ad35, was well-expressed, while coxsackie and adenovirus receptor (CAR), a cell surface target of Ad5, was considerably less expressed. The Ad5F35 vector induced oncolysis of RCC cells, with significantly higher efficacy as compared with that for the Ad5 vector.
CONCLUSION: Ad5F35 vector could be a candidate for promising gene therapy of human RCC.

Coughlan L, Vallath S, Gros A, et al.
Combined fiber modifications both to target α(v)β(6) and detarget the coxsackievirus-adenovirus receptor improve virus toxicity profiles in vivo but fail to improve antitumoral efficacy relative to adenovirus serotype 5.
Hum Gene Ther. 2012; 23(9):960-79 [PubMed] Related Publications
Achieving high-efficiency tumor targeting after systemic delivery is a considerable challenge facing oncolytic gene therapists. Efficient retargeting should be combined with efforts to improve in vivo safety, reduce hepatotoxicity, minimize off-target interactions, and improve antitumoral potency and efficacy. We previously described the successful retargeting of adenovirus serotype 5 (Ad5) to α(v)β(6), an integrin that is highly overexpressed in numerous human carcinomas. In this study, we have further modified this construct by introducing mutations that ablate coxsackievirus-adenovirus receptor (CAR) binding and putative interactions with factor IX (FIX)/C4b-binding protein (C4BP). We have found that the resulting vector, Ad5-477dlTAYT(A20), displays a desirable in vivo safety profile. This vector does not agglutinate human erythrocytes, fails to cause thrombocytopenia after intravenous delivery, has limited induction of proinflammatory cytokines, and results in low-level toxicity (aspartate aminotransferase/alanine aminotransferase) when compared with Ad5-EGFP(WT). Furthermore, it has reduced accumulation in Kupffer cells (1 hr) and limited hepatocyte transduction at later time points (24 and 96 hr). The parental vector, Ad5-EGFP(A20), also displayed many of these desirable properties. As a result of the improved safety profile of both A20-modified vectors, we escalated the dose from 2×10(10) to 4×10(10) viral particles in an antitumoral efficacy study. We observed improvements in reducing percent tumor growth at early time points (96 hr) when compared with Ad5-EGFP(WT), although increasing the dose did not affect the therapeutic outcome beneficially. On completion of the experiment, we detected increased E1A staining in the tumors of all A20-treated groups and we determined that E1A expression was localized largely within α(v)β(6)(+) tumor cells. However, in spite of apparently efficient tumor transduction, this did not result in enhanced antitumoral efficacy as the virus failed to disseminate effectively throughout the tumor mass, presumably due to physical intratumoral restrictions. This highlights a remaining challenge that needs to be overcome before such vectors can be developed for future cancer gene therapy applications.

Chung SK, Kim JY, Lim JY, et al.
Transcription factor Sp1 is involved in expressional regulation of coxsackie and adenovirus receptor in cancer cells.
J Biomed Biotechnol. 2011; 2011:636497 [PubMed] Free Access to Full Article Related Publications
Coxsackie and adenovirus receptor (CAR) was first known as a virus receptor. Recently, it is also known to have tumor suppressive activity such as inhibition of cell proliferation, migration, and invasion. It is important to understand how CAR expression can be regulated in cancers. Based on an existence of putative Sp1 binding site within CAR promoter, we investigated whether indeed Sp1 is involved in the regulation of CAR expression. We observed that deletion or mutation of Sp1 binding motif (-503/-498) prominently impaired the Sp1 binding affinity and activity of CAR promoter. Histone deacetylase inhibitor (TSA) treatment enhanced recruitment of Sp1 to the CAR promoter in ChIP assay. Meanwhile, Sp1 binding inhibitor suppressed the recruitment. Exogenous expression of wild-type Sp1 increased CAR expression in CAR-negative cells; meanwhile, dominant negative Sp1 decreased the CAR expression in CAR-positive cells. These results indicate that Sp1 is involved in regulation of CAR expression.

Iguchi K, Sakurai F, Tomita K, et al.
Efficient antitumor effects of carrier cells loaded with a fiber-substituted conditionally replicating adenovirus on CAR-negative tumor cells.
Cancer Gene Ther. 2012; 19(2):118-25 [PubMed] Related Publications
Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.

Kim DR, Park MY, Lim HJ, et al.
Combination therapy of conditionally replicating adenovirus and histone deacetylase inhibitors.
Int J Mol Med. 2012; 29(2):218-24 [PubMed] Related Publications
Combination therapy of adenoviral gene therapy and a histone deacetylase (HDAC) inhibitor is important due to the enhancing effect of HDAC inhibitors on adenoviral transduction and transcription. However, contradictory results have been reported on the effect of combination of CRAd (conditionally replicating adenovirus) and HDAC inhibitors. This study was designed to investigate the interaction of CRAd and HDAC inhibitors and determine the ideal way to combine the two agents. Combination of HDAC inhibitors (SK7041, SBHA and vorinostat) at pre- and post-transductional periods with CRAd enhanced the transduction of CRAd and expression of luciferase expression from Δ24-luc in vitro. However, suppression of luciferase expression from Δ24-luc injected tumor mass was observed by in vivo tumor bioluminescence imaging and drug interaction analysis also showed an antagonistic interaction that was probably related with the inhibitory effect of the HDAC inhibitor on adenoviral replication. Suppression of p21 induction by p21 siRNA reversed the suppressive effect of vorinostat on the replication of CRAd, but still failed to reverse the antagonistic interaction. Addition of vorinostat at the pre-transductional period revealed an improvement in the transduction efficiency of CRAd and also induced a synergistic interaction between CRAd and vorinostat, which was possibly related with prevention of the suppressive effect of vorinostat on adenoviral replication. In conclusion, the addition of HDAC inhibitor before CRAd injection showed synergistic antitumor effects, which warrants further investigation on the sequence of HDAC inhibitor and CRAd treatment in an animal tumor model.

Azab B, Dash R, Das SK, et al.
Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells.
J Cell Physiol. 2012; 227(5):2145-53 [PubMed] Free Access to Full Article Related Publications
Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.

Majhen D, Stojanović N, Špeljko T, et al.
Increased expression of the coxsackie and adenovirus receptor downregulates αvβ3 and αvβ5 integrin expression and reduces cell adhesion and migration.
Life Sci. 2011; 89(7-8):241-9 [PubMed] Related Publications
AIMS: Coxsackie and adenovirus receptor (CAR) is a tumor suppressor and a primary receptor for adenovirus type 5 (Ad5). Our study aims to examine the influence of forced expression of CAR in rhabdomyosarcoma cells (RD) on expression levels of integrins implicated in Ad5 entry, and the effect of CAR on cell-extracellular matrix adhesion and migration.
MAIN METHODS: CAR expressing clones were established from RD cells by stable transfection. Flow cytometry was used to evaluate the expression of CAR and integrins. Adhesion was measured in plates previously coated with vitronectin or fibronectin. Boyden chambers were used to investigate migration. Transfection of cells with siRNA was used to achieve integrin silencing. Ad5-mediated transgene expression was measured by β-gal staining.
KEY FINDINGS: Increased expression of CAR in RD cells reduces the expression of αvβ3 and αvβ5 integrins. Cells overexpressing CAR exhibit significantly reduced adhesion to vitronectin and fibronectin, and reduced cell migration. Specifically silencing αvβ3 integrin in RD cells reduced cell migration indicating that reduced migration could be the consequence of αvβ3 integrin downregulation. This study also demonstrates the negative effect of reduced levels of αvβ3 and αvβ5 integrins on Ad5-mediated transgene expression with Ad5 retargeted to αv integrins.
SIGNIFICANCE: The pharmacological upregulation of CAR aimed to increase Ad5-mediated transgene expression may actually downregulate αvβ3 and αvβ5 integrins and thus alter Ad5-mediated gene transfer. The mechanism of decreased cell migration, a prerequisite for metastasis and invasion, due to increased CAR expression may be explained by reduced αvβ3 integrin expression.

Wang L, Yao B, Li Q, et al.
Gene therapy with recombinant adenovirus encoding endostatin encapsulated in cationic liposome in coxsackievirus and adenovirus receptor-deficient colon carcinoma murine models.
Hum Gene Ther. 2011; 22(9):1061-9 [PubMed] Related Publications
Adenovirus (Ad)-based antiangiogenesis gene therapy is a promising approach for cancer treatment. Downregulation or loss of coxsackievirus and adenovirus receptor (CAR) is often detected in various human cancers, which hampers adenoviral gene therapy approaches. Cationic liposome-complexed adenoviral vectors have been proven useful in CAR-deficient cells to enhance therapeutic gene transfer in vivo. Here, we investigated the antitumor effects of recombinant adenovirus encoding endostatin (Ad-hE) encapsulated in cationic liposome (Ad-hE/Lipo) on CAR-deficient CT26 colon carcinoma murine models. In vitro, Ad-hE/Lipo enhanced adenovirus transfection in CAR-deficient cells (CT26), and endostatin gene expression was measured by both qualitative and quantitative detection. In addition, an antibody neutralizing assay indicated that neutralizing serum inhibited naked adenovirus 5 (Ad5) at rather higher dilution than the complexes of Ad5 and cationic liposomes (Ad5-CL), which demonstrated that Ad5-CL was more capable of protecting Ad5 from neutralization. In vivo, Ad-hE/Lipo treatment in the murine CT26 tumor model by intratumoral injection resulted in marked suppression of tumor growth and prolonged survival time, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells. In conclusion, recombinant endostatin adenovirus encapsulated with cationic liposome effectively inhibited CAR-deficient tumor growth through an antiangiogenic mechanism in murine models without marked toxicity, thus showing a feasible strategy for clinical applications.

Hamdan S, Verbeke CS, Fox N, et al.
The roles of cell surface attachment molecules and coagulation Factor X in adenovirus 5-mediated gene transfer in pancreatic cancer cells.
Cancer Gene Ther. 2011; 18(7):478-88 [PubMed] Related Publications
Transduction of 11 pancreatic cancer cell lines with a replication-deficient adenovirus 5 expressing enhanced green fluorescent protein (Ad5EGFP) was analyzed and variable EGFP levels were observed, ranging from <1% to ∼40% of cells transduced, depending on the cell line. Efficient Ad5EGFP transduction was associated mainly with higher levels of cell surface Coxsackie and adenovirus receptor (CAR) but not with expression of α(v)β(3) and α(v)β(5) integrins and was fiber dependent. Reduction of CAR by RNA interference resulted in a corresponding decrease in Ad5EGFP transduction. Pre-treatment of Ad5EGFP with blood coagulation Factor X increased virus entry even in the presence of low CAR levels generated by RNA interference, suggesting a potential alternative route of Ad5 entry into pancreatic cancer cells. Immunohistochemistry carried out on 188 pancreatic ductal adenocarcinomas and 68 matched controls showed that CAR was absent in 102 (54%) of adenocarcinomas, whereas moderate and strong staining was observed in 58 (31%) and 28 (15%) cases, respectively. Weak or absent CAR immunolabeling correlated with poor histological differentiation of pancreatic cancer. In normal tissue, strong immunolabeling was detected in islet cells and in the majority of inter- and intralobular pancreatic ducts.

Kim J, Nam HY, Kim TI, et al.
Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA.
Biomaterials. 2011; 32(22):5158-66 [PubMed] Free Access to Full Article Related Publications
Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG)(n). The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG(500)-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG(500)-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.

Stecker K, Vieth M, Koschel A, et al.
Impact of the coxsackievirus and adenovirus receptor on the adenoma-carcinoma sequence of colon cancer.
Br J Cancer. 2011; 104(9):1426-33 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Coxsackie and adenovirus receptor (CAR) has been suggested to function as a tumour suppressor. Its impact on the adenoma-carcinoma sequence of the colon, however, is unclear.
METHODS: Coxsackie and adenovirus receptor was analysed in non-cancerous and neoplastic colon samples using immunohistochemistry and quantitative RT-PCR. The function of CAR in colon cancer cell lines was determined following application of CAR siRNA or ectopic expression of a human full-length CAR cDNA.
RESULTS: Compared with healthy mucosa, increased CAR-mRNA expression was found in adenomas, whereas primary cancers and metastases displayed a marked decline. At the plasma membrane, CAR was present in normal mucosa samples (93%), adenomas, and metastases (100% ea.), whereas in colon cancers, it was found less frequently (49%, P<0.0001). Cytoplasmic CAR immunopositivity increased from normal mucosa (22%), to adenomas (73%, P=0.0006), primary cancers (83%, P<0.0001), and metastases (67%, P=0.0019). In cancer cell lines, CAR inhibition resulted in increased proliferation, whereas enforced ectopic CAR expression led to opposite results. Blocking the extracellular portion of CAR increased cell invasion in vitro. In mice, xenotransplants of colon cancer cells with enforced CAR expression formed significantly smaller tumours, whereas CAR inhibition increased the formation of liver metastases.
CONCLUSION: We conclude that CAR facilitates complex effects during colon carcinogenesis, potentially mediated by its stage-dependent subcellular distribution; high CAR expression potentially prevents apoptosis in adenomas, loss of CAR at the plasma membrane promotes growth, and dissemination of primary cancers, and high membranous CAR presence may support the establishment of distant metastases.

Zhao Y, Li Y, Wang Q, et al.
Increased antitumor capability of fiber-modified adenoviral vector armed with TRAIL against bladder cancers.
Mol Cell Biochem. 2011; 353(1-2):93-9 [PubMed] Related Publications
Adenoviral vectors are widely used for cancer therapy and show a tumor-suppressing effect. However, bladder cancers are found to be resistant against infection of Ad5-derived adenoviral vector, limiting the application of the existing strategy of gene therapy. Therefore, efforts to develop novel types of adenoviral vector aimed for improving the viral infection and enhancing expression level of tumor-inhibiting transgene is urgently required. We constructed a 5/35 fiber-modified E1A-deleted adenoviral vector armed with TRAIL gene. Its ability to express this gene for inhibition of bladder cancer cell growth was investigated in our work. The results showed that this modification in fiber region facilitates adenoviral infection to bladder cancer, perhaps due to high expression of CD46 on target cell surface. Subsequently, we found an enhanced expression level of TRAIL mediated by 5/35 fiber-modified adenoviral vectors in bladder cancer cells, leading to an increased tumor-inhibiting capability of 5/35 adenoviral vector against bladder cancer cells. Consistently, growth of xenograft tumors in mice was also effectively inhibited by 5/35 fiber-modified vector-mediated gene therapy strategy. The 5/35 fiber-modified adenoviral vector-based gene transfer shows an improved efficacy against bladder cancers. The application of this novel gene therapy vector may benefit the patients in clinical bladder cancer treatment.

Dietel M, Häfner N, Jansen L, et al.
Novel splice variant CAR 4/6 of the coxsackie adenovirus receptor is differentially expressed in cervical carcinogenesis.
J Mol Med (Berl). 2011; 89(6):621-30 [PubMed] Related Publications
The coxsackie adenovirus receptor (CAR) is a component of the tight junction complex and involved in cell adhesion. Loss of CAR expression can affect cell adhesion which in the context of carcinogenesis may influence both invasion and metastatic spread. Functional inactivation of CAR may also result from the interaction with its soluble isoforms. To relate alterations of CAR expression to tumor progression, we aimed to establish a highly specific real-time PCR protocol for quantification of all splice variants. In the process of cloning, we identified a novel splice variant termed CAR4/6 that lacked exon 5 but retained exon 6 encoding the transmembrane domain. Localization of CAR4/6 in the cell membrane was confirmed by ectopic expression in HT1080 cells. Expression analyses using cDNA arrays revealed that most normal tissues, including those of the female genital tract, express full-length CAR (CAR6/7) but not CAR4/6. Differential expression of both CAR splice variants was validated in microdissected epithelia (n = 66) derived from normal cervical ectodermal tissue, high-grade cervical intraepithelial neoplasia (CIN2/3) and invasive squamous cervical carcinoma. CAR4/6 was not expressed in normal cervical tissue but in 42% of CIN2/3 and in most cervical carcinomas (p < 0.001). In contrast, CAR6/7 was detected in all of the microdissected samples. As for CAR4/6 expression levels of CAR6/7 were significantly lower in normal tissue as compared with CIN2/3 and cancer (p < 0.01). Ectopic expression of CAR4/6 in different cell lines enhanced the proliferative and invasive properties indicating a possible role in cancer progression.

Vindrieux D, Le Corre L, Hsieh JT, et al.
Coxsackie and adenovirus receptor is a target and a mediator of estrogen action in breast cancer.
Endocr Relat Cancer. 2011; 18(3):311-21 [PubMed] Related Publications
The involvement of the coxsackie and adenovirus receptor (CAR), an adhesion molecule known to be the main determinant of adenovirus transduction of the cells, in cancer is currently under investigation. Recent reports suggest that CAR levels are elevated in breast cancer, and this may have an impact on its use as means of delivery for gene therapy. In this study, we show that estradiol (E(2)) treatment of the estrogen receptor (ER)-positive breast cancer cell MCF-7 increases CAR levels and, in turn, enhances adenoviral transduction. Employing the transfection of CAR promoters in breast cancer cells, we show that this regulation of CAR expression occurs at the transcriptional level. In addition, and by chromatin immunoprecipitation, we have identified a crucial region of CAR promoter that controls E(2) responsiveness of CAR gene through the recruitment of ER. Moreover, utilizing CAR antibodies or CAR silencing by RNA interference repressed the estrogen-dependent growth of breast cancer cells, whereas the stable expression of CAR in MCF-7 or MDA-MB-231 cells led to an increased proliferation. Altogether, our data suggest that CAR is a novel estrogen-responsive gene, which is involved in the E(2)-dependent proliferation of breast cancer cells.

Sakakibara A, Tsukuda M, Kondo N, et al.
Examination of the optimal condition on the in vitro sensitivity to telomelysin in head and neck cancer cell lines.
Auris Nasus Larynx. 2011; 38(5):589-99 [PubMed] Related Publications
OBJECTIVE: Telomelysin (OBP-301) is a telomerase-specific replication-competent adenovirus with a human telomerase reverse transcriptase (hTERT) promoter. Telomelysin has a strong antitumor effect on a variety of cancers, including head and neck squamous cell carcinoma (HNSCC), and combining telomelysin treatment with paclitaxel or cisplatin enhances the antitumor effect on HNSCC. In the present study, we investigated the relationship between the antitumor activity of telomelysin and tumor cell doubling time(DT), S-phase fraction, and E1A expression. We also investigated whether the antitumor effects of OBP-301-resistant tumor cells are enhanced by cisplatin, paclitaxel, or streptolysin O.
METHODS: The tumor cell DT of 17 human HNSCC cell lines was examined. Antitumor activities of telomelysin (OBP-301) for each HNSCC cell line were examined by MTT assay. Cell cycle analysis was conducted by flowcytometry. E1A gene expressions after infection with telomelysin, hTERT, CAR (Cocksackie Adenovirus Receptor), and c-Myc were examined by quantitative PCR, and E1A expressions were examined again after pretreatment with cisplatin, paclitaxel, or streptolysin O. Correlations were analyzed by Spearman's correlation coefficient.
RESULTS: There was a significant relationship between telomelysin sensitivity and DT, S-phase fraction and early E1A expression, and pretreatment with cisplatin, paclitaxel, and streptolysin O increased infectivity of telomelysin-resistant HNSCC cell lines.
CONCLUSION: These findings are useful for advancing clinical trials, and suggest that adjuvant telomelysin treatment would be effective even in telomelysin-resistant HNSCC cell lines.

Van den Bossche J, Al-Jamal WT, Yilmazer A, et al.
Intracellular trafficking and gene expression of pH-sensitive, artificially enveloped adenoviruses in vitro and in vivo.
Biomaterials. 2011; 32(11):3085-93 [PubMed] Related Publications
Recombinant adenovirus (Ad) has shown great promise in gene therapy. Artificial envelopment of adenovirus within lipid bilayers has previously been shown to decrease the immunogenicity and hepatic affinity of naked Ad in vivo. Unfortunately, this also resulted in a significant reduction of gene expression, which we attributed to poor endosomal release of the Ad from its artificial lipid envelope. In this work, we explored the artificial envelopment of Ad within pH-sensitive DOPE:CHEMS bilayers and characterized this vector by TEM, AFM, dot blot, dynamic light scattering and zeta potential measurements. The artificially enveloped viral vectors exhibited good stability at physiological pH but immediately collapsed and released naked Ad virions at pH 5.5. Intracellular trafficking using confocal laser scanning microscopy (CLSM) revealed that Cy3-labelled Ad enveloped in DOPE:CHEMS bilayers exhibited the characteristic Ad distribution within the cytoplasm that led to virion accumulation around the nuclear membrane, indicating endosomal release of Ad. We obtained equivalent levels of gene expression as those of naked Ad in a series of CAR-positive (CAR+) and CAR-negative (CAR-) cell lines. This suggested that the mechanism of infection for the artificially enveloped Ad remained dependent on the presence of CAR receptors. Finally, the pH-sensitive enveloped Ad were injected intratumorally in human cervical carcinoma xenograft-bearing nude mice, also illustrating their capacity for efficient in vivo marker gene expression. This study is a step forward toward the engineering of functional, artificially enveloped adenovirus vectors for gene transfer applications.

Majhen D, Brozovic A, Buger T, et al.
Vincristine-resistant human laryngeal carcinoma cells demonstrate increased Rous sarcoma virus promoter activity.
Life Sci. 2010; 87(15-16):468-74 [PubMed] Related Publications
AIMS: Gene therapy is a candidate approach for treating cancer patients whose tumors have developed resistance to some drugs. Our study aims to examine possible alteration in Ad5RSVβgal-mediated transgene expression in a vincristine-resistant cells (VK2) derived from the human laryngeal carcinoma cell line HEp2, and the underlying mechanism(s) thereof.
MAIN METHODS: Adenovirus-mediated transgene expression in HEp2 and VK2 cells was measured by β-gal staining. Semiquantitative PCR was used to evaluate attachment of adenovirus to the cell surface and adenovirus internalization into cells. After transfection of cells with plasmid DNA, promoter activity was measured by semiquantitative RT-PCR.
KEY FINDINGS: We show here that VK2 cells exhibited increased Ad5RSVβgal-mediated transgene expression, despite moderately decreased Ad5RSVβgal attachment and internalization, as compared with HEp2 cells. The increased transgene expression was also observed with a virus (Ad5FbΔ639RSVβgal) that does not use the coxsackie-adenovirus receptor (CAR), suggesting that increased transgene expression is independent of CAR. Upon transfection of VK2 cells with a plasmid expressing a reporter gene under the control of the RSV promoter or a plasmid containing the complete Ad5RSVβgal genome, RSV promoter activity was 33- and 4.7-fold higher, respectively, than in HEp2 cells.
SIGNIFICANCE: The increased Ad5RSVβgal-mediated transgene expression in the VK2 cells is due to the increased RSV promoter activity in VK2 cells. Our results point out that (i) drug-resistance may be accompanied with an alteration in promoter activity; (ii) the proper choice of promoter could contribute to a decrease in the vector dose required to achieve a therapeutic effect during gene therapy.

Shim SH, Lee CT, Lee JJ, et al.
A combination treatment with SAHA and ad-p63/p73 shows an enhanced anticancer effect in HNSCC.
Tumour Biol. 2010; 31(6):659-66 [PubMed] Related Publications
Suberoylanilide hydroxamic acid (SAHA) is one of the most widely used histone deacetylase inhibitors. However, the potential advantage of SAHA has not been sufficiently validated as an adjunct to gene therapy of head and neck squamous cell carcinoma (HNSCC). SAHA has been shown to boost the efficiency of gene transfer by upregulating the expression of coxsackie adenoviral receptor on treated cells. The p53 family genes, p63 and p73, have been shown to have characteristics similar to p53, and although they are not confirmed as tumor suppressors, DNA-damaging signals induce their overexpression. We previously reported that the adenovirus-mediated transfer of p63 or p73 showed an effective cancer-killing effect similar to that of p53. In this study, we combined SAHA with adenoviral delivery of p63 or p73 to enhance the efficiency of gene therapy. This combination resulted in a significantly enhanced cancer-killing effect in HNSCC cell lines but had no effect on normal human fibroblasts. SAHA treatment added to ad-p63/p73 gene delivery caused an increase in p21 expression and cleaved poly-ADP ribose polymerase. Our results indicate that adjuvant SAHA treatment could be developed as a therapeutic strategy to enhance the efficiency of adenoviral gene transfer in the treatment of cancer.

Ingram N, MacCormac LP, Oxley NT, et al.
Role of cell surface molecules and autologous ascitic fluid in determining efficiency of adenoviral transduction of ovarian cancer cells.
Cancer Gene Ther. 2010; 17(10):684-93 [PubMed] Related Publications
Adenovirus is the most frequently used virus in gene therapy clinical trials. There have been conflicting reports on the ability of adenovirus to transduce primary ovarian cancer samples and the expression of relevant cell surface molecules. These factors were examined using primary ovarian cancer cells cultured from ascites and solid tumor to gain insights into the clinical use of adenovirus in ovarian cancer. The level of transduction of primary cultures was much higher than uncultured cells and established cell lines, and correlated with higher levels of coxsackie-adenovirus receptor (CAR) and integrin expression. Growth of primary cultures in autologous ascitic fluid prevented an increase in CAR expression and inhibited transduction compared with cells treated in supplemented RPMI. Cells at the periphery of solid tumor samples were transduced using a replication-incompetent virus and correlated with CAR expression. However, transduction was abolished by autologous ascitic fluid, despite the expression of CAR. We conclude that the use of adenoviruses for ovarian cancer gene therapy will require testing in the presence of inhibitory factors in ascitic fluid. The clinical use of adenoviral vectors may require circumvention of such inhibitory factors and the use of replication competent adenovirus to enable efficient viral penetration of the cancer.

Botta G, Perruolo G, Libertini S, et al.
PED/PEA-15 modulates coxsackievirus-adenovirus receptor expression and adenoviral infectivity via ERK-mediated signals in glioma cells.
Hum Gene Ther. 2010; 21(9):1067-76 [PubMed] Related Publications
Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.

Kim HS, Kim CH, Park MY, et al.
Efficient co-transduction of adenoviral vectors encoding carcinoembryonic antigen and survivin into dendritic cells by the CAR-TAT adaptor molecule enhance anti-tumor immunity in a murine colorectal cancer model.
Immunol Lett. 2010; 131(1):73-80 [PubMed] Related Publications
Because multiple tumor antigens, including carcinoembryonic antigen (CEA) and survivin (SVV), have been frequently observed in human colorectal cancer, we investigated whether the expression of both CEA and SVV by co-transduction of adenovirus vectors into dendritic cells (DCs) could improve anti-tumor immunity in a murine colorectal cancer model. The adaptor fusion protein of Coxsackie and adenovirus receptor and TAT-protein transduction domain (CAR-TAT) enhanced co-transduction of adenovirus vectors encoding CEA (AdCEA) and SVV (AdSVV) into DCs, and increased anti-tumor immunity. DCs expressing both CEA and SVV in the presence of CAR-TAT (DC-AdCEA/AdSVV+CAR-TAT) induced T-cell responses specific for CEA and SVV, and enhanced cytotoxic T-cell activity on MC38/CEA2 cells expressing CEA and SVV compared with DCs expressing either CEA or SVV alone. Particularly, DC-AdCEA/AdSVV+CAR-TAT induced higher number of CEA-specific IFN-gamma secreting T cells compared with DC-AdCEA+CAR-TAT. Vaccination with DC-AdCEA/AdSVV+CAR-TAT also more efficiently inhibited tumor growth compared with DCs expressing either CEA or SVV alone in therapeutic tumor models. These results suggest that efficient co-transduction of multiple adenovirus vectors by CAR-TAT could be used to develop various strategies for therapeutic DC vaccines.

Dash R, Dmitriev I, Su ZZ, et al.
Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells.
Cancer Gene Ther. 2010; 17(7):447-56 [PubMed] Related Publications
Gene therapy is being examined as a potential strategy for treating prostate cancer. Serotype 5 adenovirus (Ad.5) is routinely used as a vector for transgene delivery. However, the infectivity of Ad.5 is dependent on Coxsackie-adenovirus receptors (CARs); many tumor types show a reduction in this receptor in vivo, thereby limiting therapeutic gene transduction. Serotype chimerism is one approach to circumvent CAR deficiency; this strategy is used to generate an Ad.5/3-recombinant Ad that infects cancer cells through Ad.3 receptors in a CAR-independent manner. In this report, the enhanced transgene delivery and efficacy of Ad.5/3-recombinant virus was evaluated using an effective wide-spectrum anticancer therapeutic melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24). Our data show that in low CAR human prostate cancer cells (PC-3), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than an Ad.5 virus encoding mda-7/IL-24 (Ad.5-mda-7) in infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor 'bystander' effect in a nude mouse xenograft model. Considering the fact that Ad.5-mda-7 has shown significant objective responses in a phase I clinical trial for solid tumors, Ad.5/3-mda-7 is predicted to exert enhanced therapeutic benefit in patients with prostate cancer.

Giaginis C, Zarros A, Alexandrou P, et al.
Evaluation of coxsackievirus and adenovirus receptor expression in human benign and malignant thyroid lesions.
APMIS. 2010; 118(3):210-21 [PubMed] Related Publications
Coxsackievirus and adenovirus receptor (CAR) expression on tumor cells is associated with sensitivity to adenoviral infection, being considered as a surrogate marker for monitoring and/or predicting adenovirus-mediated gene therapy. The aim of this study was to evaluate the clinical significance of CAR expression in human benign and malignant thyroid lesions. CAR protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 107 patients with benign and malignant lesions and was statistically analyzed in relation to histopathologic type; tumor size; lymph node metastasis; capsular, lymphatic and vessel invasion; as well as follicular cells' proliferative capacity. CAR immunoreactivity was characterized as negative/weak in 53 (49.53%), moderate in 31 (28.97%) and strong in 23 (21.50%) of 107 thyroid cases. CAR immunoreactivity was significantly increased in malignant compared with that in benign thyroid lesions (p = 0.00002). Both malignant and benign thyroid lesions with enhanced follicular cells' proliferative capacity showed significantly increased CAR immunoreactivity (p = 0.00027). In malignant thyroid lesions, enhanced CAR immunoreactivity was significantly associated with larger tumor size (p = 0.0067). The current data revealed that CAR immunoreactivity could be considered of diagnostic utility in thyroid neoplasia. Further research effort is warranted to delineate whether CAR could be considered clinically important for both diagnosis and future (gene) therapeutic applications in thyroid neoplasia.

Micklethwaite KP, Savoldo B, Hanley PJ, et al.
Derivation of human T lymphocytes from cord blood and peripheral blood with antiviral and antileukemic specificity from a single culture as protection against infection and relapse after stem cell transplantation.
Blood. 2010; 115(13):2695-703 [PubMed] Free Access to Full Article Related Publications
Viral infections and leukemic relapse account for the majority of treatment failures in patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving allogeneic hematopoietic stem cell (HSC) or cord blood (CB) transplants. Adoptive transfer of virus-specific cytotoxic T lymphocytes (CTLs) provides protection against common viruses causing serious infections after HSC transplantation without concomitant graft-versus-host disease. We have now generated CTL lines from peripheral blood (PB) or CB units that recognize multiple common viruses and provide antileukemic activity by transgenic expression of a chimeric antigen receptor (CAR) targeting CD19 expressed on B-ALL. PB-derived CAR(+) CTLs produced interferon-gamma (IFNgamma) in response to cytomegalovirus-pp65, adenovirus-hexon, and Epstein-Barr virus pepmixes (from 205 +/- 104 to 1034 +/- 304 spot-forming cells [SFCs]/10(5) T cells) and lysed primary B-ALL blasts in (51)Cr-release assays (mean, 66% +/- 5% specific lysis; effector-target [E/T] ratio, 40:1) and the CD19(+) Raji cell line (mean, 78% +/- 17%) in contrast to nontransduced controls (8% +/- 8% and 3% +/- 2%). CB-derived CAR(+) CTLs showed similar antiviral and antitumor function and both PB and CB CAR(+) CTLs completely eliminated B-ALL blasts over 5 days of coculture. This approach may prove beneficial for patients with high-risk B-ALL who have recently received an HSC or CB transplant and are at risk of infection and relapse.

Wang Y, Ma L, Wang S, et al.
Assessment of CAR- or CD46-dependent adenoviral vector-mediated TRAIL gene therapy in clinical adenocarcinoma lung cancer cells.
Oncology. 2009; 77(6):366-77 [PubMed] Related Publications
Adenoviral vector-mediated transfer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be a powerful approach to lung cancer therapy. However, the efficiency of adenoviral vector gene transfer and the sensitivity to TRAIL-induced apoptosis in the context of adenoviral vector gene transfer have yet to be characterized in primary lung cancers. In this study, we investigated the expression of adenoviral receptor CD46 expression in primary lung cancer cells. In contrast to previous reports on enhanced CD46 expression in various types of cancer cells, we show a significantly higher CD46 expression in lung adenocarcinomas compared to lung squamous cell carcinomas. Using Ad5-GFP and Ad5F35-GFP vectors, we demonstrated an improved gene transfer efficiency in primary lung cancer cells by the Ad5F35 vector. The apoptosis induction effect mediated by Ad5-TRAIL and Ad5F35-TRAIL vector gene transfer was compared in cells from 10 lung adenocarcinomas. Of 5 lung cancers in which apoptosis was induced, 2 had an enhanced effect by Ad5F35-TRAIL vector gene transfer compared to Ad5-GFP. Thus, these results indicate a method to identify TRAIL-sensitive primary lung cancers, which will also facilitate the analysis of resistance mechanisms in lung cancers.

Kim PH, Kim TI, Yockman JW, et al.
The effect of surface modification of adenovirus with an arginine-grafted bioreducible polymer on transduction efficiency and immunogenicity in cancer gene therapy.
Biomaterials. 2010; 31(7):1865-74 [PubMed] Related Publications
Adenoviral vectors offer many advantages for cancer gene therapy, including high transduction efficiency, but safety concerns related to severe immunogenicity and other side effects have led to careful reconsideration of their use in human clinical trials. To overcome these issues, a strategy of generating hybrid vectors that combine viral and non-viral elements as more intelligent gene carriers has been employed. Here, we coated adenovirus (Ad) with an arginine-grafted bioreducible polymer (ABP) via electrostatic interaction. We examined the effect of ABP-coated Ad complex at various ABP molecules/Ad particle ratios. Enhanced transduction efficiency was observed in cells treated with cationic ABP polymer-coated Ad complex compared to naked Ad. We also examined the coating of Ad with ABP polymers at the optimal polymer ratio using dynamic light scattering and transmission electron microscopy. In both high and low coxsackie virus and adenovirus receptor (CAR)-expressing cells, ABP-coated Ad complex produced higher levels of transgene expression than cationic polymer 25K PEI. Notably, high cytotoxicity was observed with 25K PEI-coated Ad complex treatment, but not with ABP-coated Ad complex treatment. In addition, ABP-coated Ad complex was not significantly inhibited by serum, in contrast to naked Ad. Moreover, ABP-coated Ad complex significantly reduced the innate immune response relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells. Overall, our studies demonstrate that Ad complex formed with ABP cationic polymer may improve the efficiency of Ad and be a promising tool for cancer gene therapy.

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