Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the Coxsackievirus and adenovirus receptor

2000 ◽  
Vol 92 (6) ◽  
pp. 1002-1008 ◽  
Author(s):  
Katsuyuki Asaoka ◽  
Mitsuhiro Tada ◽  
Yutaka Sawamura ◽  
Jun Ikeda ◽  
Hiroshi Abe
Keyword(s):  
Gene Therapy ◽  
Cell Lines ◽  
Malignant Gliomas ◽  
Transduction Efficiency ◽  
Wild Type ◽  
Content Type ◽  
Expression Levels ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor ◽  
Fine Print

Object. Recombinant adenovirus is used as a competent vector in a wide spectrum of cancer gene therapies because of its high efficiency in gene delivery. To study the feasibility of gene therapy in malignant gliomas, the authors examined the antiproliferative effect of the adenovirally transduced wild-type p53 tumor suppressor gene by using 15 different high-grade glioma cell lines.Methods. Although growth suppression in association with a high adenoviral p53 transduction efficiency was seen in five of 15 cell lines, it was not observed in the remaining 10 cell lines. To clarify the underlying mechanism, we examined the expression levels of the Coxsackievirus and adenovirus receptor (CAR), which is the primary receptor for adenovirus, and of the integrins αvβ3 and αvβ5, which promote adenoviral internalization. The expression level of the CAR gene showed a close correlation to adenoviral gene transduction efficiency in the tested cell lines, whereas the expression levels of the integrins did not. The CAR expression was decreased by wild-type p53 transduction in U251MG cells harboring mutant p53 and increased by antisense inhibition of p53 in LN443 cells with endogenous wild-type p53.Conclusions. The results of this study indicate that CAR expression is a critical determinant of transduction efficiencies in adenovirus-based gene therapy for human malignant gliomas.

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy

2005 ◽  
Vol 103 (6) ◽  
pp. 1058-1066 ◽  
Author(s):  
Weijun Wang ◽  
Nian-Ling Zhu ◽  
Jason Chua ◽  
Steve Swenson ◽  
Fritz K. Costa ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Cell Lines ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Transduction Efficiency ◽  
Fibroblast Growth ◽  
Content Type ◽  
Fine Print

Object. Adenovirus vector (AdV)—mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (αvβ3 and αvβ5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, αvβ3, and αvβ5 expression. Methods. A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. Conclusions. Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

Interaction between p53 and p16 expressed by adenoviral vectors in human malignant glioma cell lines

2002 ◽  
Vol 97 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Seung-Ki Kim ◽  
Kyu-Chang Wang ◽  
Byung-Kyu Cho ◽  
Hyun-Tai Chung ◽  
Young-Yim Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Western Blotting ◽  
Glioma Cell ◽  
Malignant Gliomas ◽  
G1 Arrest ◽  
Moderate Degree ◽  
Wild Type ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Fine Print

Object. Multiple gene replacements have been examined as a potential treatment modality for malignant gliomas. Nevertheless, no reports are available that detail the synergy, additivity, or antagonism of multiple genes. The aim of this study was to assess the interaction between p53 and p16 genes in the growth of glioma cell lines. Methods. The human glioma cell lines U87MG and U373MG were transduced using an adenoviral vector with Ad-p53, Ad-p16, or both. Western blotting was performed to determine the expression of the protein products of the transduced p53 and p16 genes. To establish whether the combination of Ad-p53 and Ad-p16 would be beneficial, the effects of gene combinations at the median inhibitory concentration level were analyzed using the isobologram method. Annexin assays and cell cycle analyses were performed on the transduced cells. Western blotting demonstrated the expression of p53 and p16 in transduced cells. Simultaneous exposure to Ad-p53 and Ad-p16 produced additive effects in both glioma cell lines. Experimental data points in U373MG lay near the Mode I line, indicating that the vectors had a different mode of action. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis, whereas in the wild-type p53 cell lines, the overexpression of wild-type p53 resulted in a moderate degree of apoptosis and G1 arrest. Furthermore, Ad-p16 induced more marked G1 arrest than Ad-p53 in cells with wild-type p53. Conclusions. The results show that interaction between Ad-p53 and Ad-p16 is additive, regardless of p53 gene status.

Application of p27 gene therapy for human malignant glioma potentiated by using mutant p27

2004 ◽  
Vol 101 (3) ◽  
pp. 505-510 ◽  
Author(s):  
Kyung-Ho Park ◽  
Jaeho Lee ◽  
Chul-Gyu Yoo ◽  
Young Whan Kim ◽  
Sung Koo Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Wild Type ◽  
Antitumor Effects ◽  
Poor Prognostic Factor ◽  
Content Type ◽  
Fine Print

Object. Malignant glioma could be an ideal candidate for local gene therapy because its invasion is local and it has little metastatic potential. A low expression level and high degradation activity of p27 are known to constitute an independent poor prognostic factor in patients with malignant glioma. In this study, the authors investigated the roles of wild-type p27 and mutant p27 on the treatment of malignant glioma. Methods. The authors used two adenoviruses: one expressed wild-type p27 (ad-p27wt) and the other, containing a mutation at the major metabolic site, expressed mutant p27 (ad-p27mt). The antitumor effects of the two adenoviruses were compared with respect to cell growth arrest, cell cycle alteration, apoptosis induction, and in vitro tumorigenicity in three glioblastoma mutiforme (GBM) cell lines and in a primary GBM cell line. Transduction with ad-p27wt or ad-p27mt induced the production of p27 and the dephosphorylation of pRB. The protein level of mutant p27 was significantly higher than that of wild-type p27. The ad-p27wt induced cell cycle arrest at the G1—S transition point, whereas the ad-p27mt induced arrest at the G2—M point. Both ad-p27wt and ad-p27mt induced a growth-inhibiting effect, apoptosis, and suppression of in vitro tumorigenicity; however, ad-p27mt displayed a stronger antitumor effect than ad-p27wt in brain tumor cell lines. Conclusions. Gene therapy involving p27, especially mutant p27, has the potential to become a novel and powerful therapy for malignant glioma.

Thymidine kinase-mediated killing of rat brain tumors

1993 ◽  
Vol 79 (5) ◽  
pp. 729-735 ◽  
Author(s):  
David Barba ◽  
Joseph Hardin ◽  
Jasodhara Ray ◽  
Fred H. Gage
Keyword(s):  
Gene Therapy ◽  
Brain Tumors ◽  
Tumor Cells ◽  
Wild Type ◽  
Cns Disorders ◽  
Content Type ◽  
Potential Applications ◽  
Ganciclovir Treatment ◽  
The Brain ◽  
Fine Print

✓ Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 µg ganciclovir/ml; C6, 5.0 µg ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

Molecular determinants of glioma cell migration and invasion

2001 ◽  
Vol 94 (6) ◽  
pp. 978-984 ◽  
Author(s):  
Christine Wild-Bode ◽  
Michael Weller ◽  
Wolfgang Wick
Keyword(s):  
Cell Lines ◽  
Cell Invasion ◽  
Glioma Cell ◽  
Growth Patterns ◽  
Migration And Invasion ◽  
Integrin Expression ◽  
Content Type ◽  
Expression Levels ◽  
Glioma Cell Lines ◽  
Fine Print

Object. Migration and invasion are important prerequisites for the infiltrative and destructive growth patterns of malignant gliomas. Infiltrative growth prevents complete tumor resection and causes significant neurological morbidity and mortality. Methods. The authors assessed the expression of matrix metalloproteinases (MMPs) at messenger RNA and protein levels, MMP-2 and MMP-9 activities, and expression levels of a panel of anti- and proapoptotic proteins of the BCL-2 family. They then correlated their findings with αVβ3 integrin expression and the migratory and invasive potentials in 12 human malignant glioma cell lines. Multiple MMPs were expressed by most cell lines. The levels of MMP-2 and MMP-3 and the activities of MMP-2 and MMP-9 correlated with tumor cell invasion. Migration and invasion were also correlated. Although the expression levels of αVβ3 integrin did not predict migration or invasion, a neutralizing αVβ3 integrin antibody inhibited migration and invasion selectively in cell lines that contained a high level of αVβ3 integrin expression, thus indicating the important role of αVβ3 integrin for migration and invasion in this subset of cell lines. An expression pattern of BCL-2 family proteins that favor resistance to apoptosis was associated with enhanced migration, invasion, and MMP activity. Wild-type p53 cell lines migrated farther than mutant p53 cell lines. Conclusions. Activities of MMP-2 and MMP-9 are the best predictors of glioma cell invasion. The αVβ3 integrin mediates migration and invasion in a subset of glioma cell lines, but these processes do not depend on αVβ3 integrin expression. Antiapoptotic BCL-2 family protein expression is a predictor of efficient migration and invasion.

Targeting adenovirus to CD80 and CD86 receptors increases gene transfer efficiency to malignant glioma cells

2007 ◽  
Vol 107 (3) ◽  
pp. 617-627 ◽  
Author(s):  
Ilya V. Ulasov ◽  
Angel A. Rivera ◽  
Yu Han ◽  
David T. Curiel ◽  
Zeng B. Zhu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Brain Tumors ◽  
Malignant Glioma ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Transduction Efficiency ◽  
Wild Type ◽  
Malignant Glioma Cells

Object Gene therapy protocols for malignant gliomas utilize adenoviral vectors that rely almost exclusively on the adenovirus serotype 5 (Ad5) backbone. The authors have previously shown that chimeric vectors that bind to the Ad3 receptor, or CD46, increase the transduction efficiency of malignant brain tumors. In light of the debate regarding the efficacy of CD46 compared with CD80/CD86 in binding Ad3 virions, the authors now examine the expression and transduction efficiency of Ad5/3 chimeras that bind via CD80/CD86. Methods The authors first analyzed CD80/CD86 expression in glioma cell lines. They then used three replication-defective vectors containing a luciferase reporter gene: Ad5/3 (containing the tail and shaft domain of Ad5 and the knob domain of Ad3); Ad3/5 (containing the tail of Ad5, shaft of Ad3, and knob of Ad5); and Ad3/3 (containing the tail of Ad5, shaft of Ad3, and knob of Ad3). These vectors were analyzed both in vitro and in vivo against malignant glioma cells. To examine further the effect of Ad5/3 fiber modification, the authors created an oncolytic vector, conditionally replicative Ad5/3 (CRAd5/3). Results The Ad5/3 vector showed a 10- to 100-fold enhanced transduction efficiency of malignant glioma compared with replication-defective wild-type adenovirus (reAd5) (p < 0.05). Moreover the use of Ad5/3 reduced transgene expression by more than 90% in normal human brain cells compared with reAd5. Finally, the use of CRAd5/3 inhibited tumor cell proliferation by 43% more than replication-competent wild-type virus in vitro (p < 0.05). Conclusions The results of this study demonstrate that the Ad5/3 vector offers superior transduction efficiency and low toxicity in the setting of brain tumors, and therefore represents a potential new approach to gene therapy for malignant gliomas.

scholarly journals Spectrum-Wide Exploration of Human Adenoviruses for Breast Cancer Therapy

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1403 ◽  
Author(s):  
Nicolas Mach ◽  
Jian Gao ◽  
Lukas Schaffarczyk ◽  
Sebastian Janz ◽  
Eric Ehrke-Schulz ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cancer Therapeutics ◽  
Expression Levels ◽  
Oncolytic Adenoviruses ◽  
Reporter Assays ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor ◽  
Low Expression

Oncolytic adenoviruses (Ads) are promising tools for cancer therapeutics. However, most Ad-based therapies utilize Ad type 5 (Ad5), which displays unsatisfying efficiency in clinical trials, partly due to the low expression levels of its primary coxsackievirus and adenovirus receptor (CAR) on tumor cells. Since the efficacy of virotherapy strongly relies on efficient transduction of targeted tumor cells, initial screening of a broad range of viral agents to identify the most effective vehicles is essential. Using a novel Ad library consisting of numerous human Ads representing known Ad species, we evaluated the transduction efficiencies in four breast cancer (BC) cell lines. For each cell line over 20 Ad types were screened in a high-throughput manner based on reporter assays. Ad types featuring high transduction efficiencies were further investigated with respect to the percentage of transgene-positive cells and efficiencies of cellular entry in individual cell lines. Additionally, oncolytic assay was performed to test tumor cell lysis efficacy of selected Ad types. We found that all analyzed BC cell lines show low expression levels of CAR, while alternative receptors such as CD46, DSG-2, and integrins were also detected. We identified Ad3, Ad35, Ad37, and Ad52 as potential candidates for BC virotherapy.

Camptothecin analogs in malignant gliomas: comparative analysis and characterization

2003 ◽  
Vol 98 (3) ◽  
pp. 570-577 ◽  
Author(s):  
Prakash Sampath ◽  
Eric Amundson ◽  
Monroe E. Wall ◽  
Betty M. Tyler ◽  
Mansukh C. Wani ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Cell Lines ◽  
Topoisomerase I ◽  
Glioma Cell ◽  
Malignant Gliomas ◽  
Sodium Dodecyl ◽  
Local Therapy ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Fine Print

Object. The authors compared and characterized several new classes of camptothecin (CPT) analogs (a total of 22 drugs) directed against human and murine glioma cell lines in vitro, trying to identify CPT analogs that can be used for local therapy in future clinical trials. Camptothecin is a naturally occurring alkaloid that inhibits the DNA-replicating enzyme topoisomerase I. Moreover, CPT and its analogs have shown promising antitumor activity against both systemic and intracranial neoplasms. Because the CPTs have poor bioavailability and are unable to cross the blood—brain barrier, they may best be delivered to the central nervous system by polymers. The authors have previously shown that local delivery of Na-CPT by implantable polymers prolongs survival in a rat intracranial glioma model. In recent years, a number of newly synthesized CPT analogs have been developed that exhibit more potency and stability than Na-CPT. Methods. Cytotoxicities of the drugs were tested using modified clonogenic and monotetrazolium assays in three glioma cell lines. A potassium chloride—sodium dodecyl sulfate coprecipitation assay was used to determine the frequency of drug-stabilized cleavable complexes. Of the CPT analogs analyzed, the 10,11-methylenedioxy (MD) class consistently demonstrated the greatest cytotoxicity. Three of these analogs, 10,11-MD-20(RS)-CPT, 10,11-MD-20(S)-CPT-glycinate ester (Gly).HCl, and 9-amino-10,11-MD-20(S)-CPT-Gly, exhibit significantly greater antiproliferative activities than CPT, Na-CPT, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against all three glioma cell lines. In addition, the 10,11-MD20(RS)-CPT analog induces more cleavable complexes than Na-CPT at every concentration. Conclusions. The increased potency and greater stability of CPT analogs hold promise for more effective local antitumor treatments against malignant intracranial brain tumors. The greater cytotoxicity of 10,11-MD CPTs in comparison with other CPT analogs as well as CPT, BCNU, or Na-CPT, may present an ideal candidate drug class for development against both primary and metastatic brain tumors.

scholarly journals Artificial Extension of the Adenovirus Fiber Shaft Inhibits Infectivity in Coxsackievirus and Adenovirus Receptor-Positive Cell Lines

2002 ◽  
Vol 76 (3) ◽  
pp. 1100-1108 ◽  
Author(s):  
Toshiro Seki ◽  
Igor Dmitriev ◽  
Elena Kashentseva ◽  
Koichi Takayama ◽  
Marianne Rots ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Lines ◽  
Receptor Interaction ◽  
Cellular Receptor ◽  
Wild Type ◽  
Shaft Length ◽  
Receptor Interactions ◽  
Serotype 5 ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor

ABSTRACT Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 β-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype 5 (Ad5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the Ad5 shaft. We show that “longer-shafted” Ad vectors (up to 32 β-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.

Overexpression of bax in human glioma cell lines

1999 ◽  
Vol 91 (3) ◽  
pp. 483-489 ◽  
Author(s):  
Michael A. Vogelbaum ◽  
Jianxin X. Tong ◽  
Rajashri Perugu ◽  
David H. Gutmann ◽  
Keith M. Rich
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Human Glioma Cell ◽  
Glioma Cell Lines ◽  
Constitutive Overexpression ◽  
Increased Sensitivity ◽  
Fine Print

Object. Cells that lose their ability to undergo apoptosis may promote the development of neoplasms and result in resistance to clinical treatment with DNA-damaging modalities such as radio- and chemotherapy. Four established human glioma cell lines that are resistant to apoptosis were transfected with the proapoptotic gene bax and assessed for their sensitivity to a proapoptotic stimulus.Methods. Two cell lines had a wild-type p53 genotype (U87 and D247MG) and two had mutant p53 genotypes (U138 and U373). Constitutive overexpression of murine bax was achieved in U138 and U373 only, which resulted in an increased sensitivity of these lines to the apoptosis-inducing effect of cytosine arabinoside (ara-C). Multiple attempts to produce constitutive overexpression of bax in U87 and D247MG cells resulted in spontaneous, near-complete cell loss. Vector-only control transfections were successful in all four cell lines. Inducible overexpression of bax was achieved in the U87 cells and elevated levels of BAX were observed as early as 6 hours after gene induction. This overexpression of BAX resulted in the spontaneous induction of apoptosis in these cells.Conclusions. Overexpression of BAX in four human glioma cell lines resulted in increased sensitivity to apoptosis. In the two lines that had a wild-type p53 genotype, overexpression of BAX produced spontaneous apoptosis. In contrast, the lines that had mutant, nonfunctional P53 did not undergo spontaneous apoptosis, but they were rendered more sensitive to the apoptosis-inducing effect of ara-C. Modulation of BAX expression may be a useful therapeutic modality for gliomas, regardless of p53 genotype.

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