This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Douglas, J. T. Articles by Curiel, D. T. Search for Related Content PubMed PubMed Citation Articles by Douglas, J. T. Articles by Curiel, D. T.

Efficient Oncolysis by a Replicating Adenovirus (Ad) in Vivo Is Critically Dependent on Tumor Expression of Primary Ad Receptors¹

Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, and the Gene Therapy Center [J. T. D., M. K., L. A. S., D. T. C.] and Medical Statistics Section, Division of Hematology and Oncology, Department of Medicine [D. E. C.], University of Alabama at Birmingham, Birmingham, Alabama 35294

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Replicating adenoviruses (Ads) are designed to replicate in and destroy cancer cells, generating viral progeny that spread within the tumor. To address the importance of the primary cellular receptor for Ads, the coxsackievirus and Ad receptor (CAR), in permitting intratumoral spread of a replicating Ad, we have used a pair of tumor cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the first direct evaluation of the relationship between the efficacy of a replicating Ad and the primary receptor levels of the host cell without the confounding influence of other variable cellular factors. We demonstrate that the absence of the primary cellular receptor on the tumor cells restricts the oncolytic potency of a replicating Ad both in vitro and in vivo. Based on these findings, it is apparent that the potential therapeutic advantages afforded by viral replication would be negated by poor intratumoral spread of the viral progeny due to the failure to infect neighboring tumor cells. Because a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect Ads to achieve CAR-independent infection will be necessary to realize the full potential of replicating Ads in the clinical setting.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The utility of replication-defective Ad vectors for cancer gene therapy is restricted by their inability to infect every cell within a solid tumor mass (1) . The realization of this limitation has led to the development of a novel class of anticancer agents, conditionally replicating Ads.³ These agents are designed to selectively replicate in and destroy cancer cells, followed by the release of the viral progeny by the lysed cells (2) . The relative specificity of viral replication in tumor versus normal cells will therefore play a major role in dictating the safety and efficacy of replicating Ads. To this end, strategies to restrict the replication of Ads to tumor cells have either involved placing the expression of viral genes, most commonly the E1A gene, under the control of tumor- or tissue-specific promoters, or have been based on the complete or partial deletion of viral genes required for replication in normal cells, but not in tumor cells (3 , 4) . However, the efficacy of replicating Ads as oncolytic agents will also be dependent on the ability of the viral progeny to achieve lateral infection and thereby spread within the tumor (3 , 4) .

The first step in Ad infection is the high-affinity binding of the COOH-terminal knob domain of the fiber capsid protein (5 , 6) to the primary cellular receptor, CAR (7 , 8) . Subsequent internalization of the virion by receptor-mediated endocytosis is potentiated by the interaction of Arg-Gly-Asp (RGD) peptide sequences in the penton base with secondary host cell receptors, integrins _vß₃ and _vß₅ (9 , 10) . A number of studies have reported that primary cancer cells express only low levels of CAR and are therefore poorly infected by Ads (11, 12, 13) . In accordance with this observation, the efficacy of Ad-mediated cancer gene therapy has been limited in preclinical and clinical studies by the resistance of the CAR-deficient tumor cells to Ad infection (14 , 15) . Consequently, considerable attention is being focused on strategies to modify Ad vectors to achieve efficient, CAR-independent gene transfer (16) .

Based on these findings with replication-defective Ad vectors, we hypothesized that a low level of CAR expression on tumor cells would also restrict the efficacy of replicating Ads. In this regard, not only would a deficiency of CAR limit the efficiency of infection by the initial viral inoculum, but, more importantly, the potential therapeutic advantages afforded by viral replication would be negated by poor intratumoral spread of the viral progeny due to the failure to infect neighboring tumor cells.

In this study, we have investigated the hypothesis that the oncolytic potency of replicating Ads could be restricted by poor dissemination of the viral progeny due to the inability to infect tumor cells expressing low levels of CAR. To address this issue, we have used a pair of tumor cell lines that differ only in the expression of a primary receptor for Ad5. This novel system thus allowed the first direct evaluation of the relationship between the efficacy of a replicating Ad and the primary receptor levels of the host cell without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary Ad receptor on the tumor cells restricts the oncolytic potency of a replicating Ad, both in vitro and in vivo. This suggests that the efficacy of replicating Ads could be improved by modifications that allow CAR-independent infection of target cancer cells.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Viruses.
Ad300wt, a wild-type human Ad serotype 5, was obtained from the American Type Culture Collection (Manassas, VA). AdGFP, an E1-/E3-deleted replication-deficient Ad5 vector that expresses GFP under the control of the cytomegalovirus promoter, has been described previously (17) . The wild-type Ad and the vector were propagated in the permissive 293 cell line and purified by two rounds of cesium chloride density centrifugation. To determine the viral particle concentration, the virus was diluted in 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.1% SDS and incubated at 56°C for 10 min, and the absorbance at 260 nm was measured. Under these conditions, an absorbance of 1 corresponds to 1.1 x 10¹² particles/ml (18) .

Cell Lines.
Human U118 MG glioma cells were obtained from the American Type Culture Collection. U118 MG-AR cells (previously designated U118 MG-Ad5KsFv.rec cells), which express an artificial primary receptor for Ad5, have been described previously (19) . The cells were propagated at 37°C in a 5% CO₂ atmosphere in DMEM/Ham’s F-12 supplemented with 10% FCS, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. U118 MG-AR cells were maintained in 400 µg/ml G418. FCS was purchased from Life Technologies, Inc. (Grand Island, NY), and media and supplements were from Mediatech (Herndon, VA).

Ad DNA Replication.
U118 MG and U118 MG-AR cells cultured in 6-well plates were infected with Ad300wt or AdGFP at an MOI of 0.1 viral particle/cell. The culture medium was supplemented with 1 µCi/ml BrdUrd (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Attached and detached cells were harvested 4, 6, and 8 days after infection, and encapsidated DNA was purified after precipitating unencapsidated DNA with spermine (20) . The viral DNA was digested with XhoI and resolved on a 1% agarose gel. The incorporation of BrdUrd into the DNA as a result of viral replication was determined by a Southwestern blot using a mouse anti-BrdUrd primary antibody (DAKO Corp., Carpinteria, CA) followed by a HRP-conjugated rabbit antimouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and detection with a Western blot chemiluminescence reagent (New England Nuclear Life Science Products, Boston, MA).

Ad Yield Assay.
U118 MG and U118 MG-AR cells cultured in 6-well plates were infected with Ad300wt or AdGFP at an MOI of 0.1 viral particles/cell. Eight days after infection, the cells and media were harvested, and the viral titer was determined by a plaque assay on 293 cells.

CPE Assay.
U118 MG and U118 MG-AR cells cultured in 24-well plates were infected with Ad300wt or AdGFP at MOIs of 0.1 and 1 viral particle/cell. Eight days after infection, the cells were fixed and stained with crystal violet.

In Vitro Cytotoxicity Assay.
U118 MG and U118 MG-AR cells cultured in 24-well plates were infected with Ad300wt or AdGFP at an MOI of 1 viral particle/cell. Eight days after infection, a commercial cell proliferation assay (Promega, Madison, WI) was used to measure cell survival according to the manufacturer’s instructions.

Animal Experiments.
U118 MG and U118 MG-AR tumor xenografts were established by s.c. injection of 5 x 10⁶ cells into the flank of 8–10-week-old female athymic nude mice (nu/nu; National Cancer Center, Frederick, MD). On reaching 60–100 mm³ , the tumor nodules were injected with 50 µl of PBS or with a single dose of 10⁸ particles of AdGFP or 10⁶ particles of Ad300wt in 50 µl of PBS (five mice/group). Bidimensional tumor measurements were taken twice a week with calipers, and the tumor volume was calculated using the simplified formula for a rotational ellipsoid: 0.5 x length x width² (21) . Animals were followed for 38 days, until the tumor burden in some of the control groups became excessive, and the mice were sacrificed. Experiments were performed in accordance with federal and institutional guidelines for animal care.

Statistical Methods.
Descriptive statistics (mean and SD) on tumor volume (mm³ ) were calculated per day for each treatment group. The percentage change in volume was calculated for U118 MG tumors infected with Ad300wt compared with the control groups treated with AdGFP or PBS. A similar procedure was used to calculate the percentage change in the volume of U118 MG-AR tumors infected with Ad300wt compared to the two controls, AdGFP and PBS. The mean percentage change in tumor volumes was compared between U118 MG and U118 MG-AR cells infected with Ad300wt using one-way ANOVA and tests of repeated measures using SAS software (version 6.12; SAS Institute, Inc. Cary, NC). The mean tumor volumes were compared between U118 MG tumors treated with PBS and U118 MG tumors treated with AdGFP using the ANOVA (t test). Likewise, the mean tumor volumes were compared between U118 MG-AR tumors treated with PBS and U118 MG-AR tumors treated with AdGFP. Similar tests were performed to compare mean tumor volumes between controls and Ad300wt-treated tumors. P < 0.05 was considered statistically significant in all of the analyses.

Immunohistochemistry.
The presence of Ad5 capsid proteins in U118 MG and U118 MG-AR tumor sections was determined by immunohistochemical analysis using polyclonal rabbit anti-Ad5 antiserum (Cocalico, Reamstown, PA) as the primary antibody with an HRP-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Laboratories). Diaminobenzidine (Sigma, St. Louis, MO) was used as the chromogenic substrate.

	Results

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
To evaluate the hypothesis that the oncolytic potency of replicating Ads correlates with their ability to achieve intratumoral spread based on lateral infection of tumor cells by the viral progeny, we used a pair of cell lines that differ only in the expression of a primary receptor for Ad5. Human U118 MG glioma cells are refractory to Ad5 infection due to a paucity of CAR, although they express the _v integrins necessary for virus internalization (12) . We have previously generated a derivative cell line, designated U118 MG-AR, which is sensitive to Ad5 infection due to the expression on the cell surface of an artificial primary Ad5 receptor, in which the extracellular domain is derived from a single-chain antibody with specificity for the Ad5 knob (19) . In preliminary experiments, we confirmed that a replication-defective Ad5 vector expressing GFP, AdGFP, was able to infect U118 MG-AR cells, although it failed to infect the parental U118 MG cells, which lack a primary receptor for Ad (data not shown).

Monolayers of U118 MG and U118 MG-AR cells maintained in medium supplemented with BrdUrd were infected at an MOI of 0.1 viral particle/cell with a wild-type, replicating Ad5, Ad300wt, or with a replication-defective Ad5 vector, AdGFP, as a control. Encapsidated Ad DNA was isolated from equivalent numbers of cells at various times after infection, digested with XhoI, and subjected to Southwestern blot analysis using an anti-BrdUrd antibody. As shown in Fig. 1 , Ad DNA could not be detected 6 or 8 days after infection of U118 MG or U118 MG-AR cells with the replication-defective Ad5 vector AdGFP, although a very small amount of newly synthesized DNA was present at 4 days. In contrast, newly synthesized Ad DNA could be purified from cells infected with the wild-type Ad, with a significantly greater amount of encapsidated DNA present at all time points in U118 MG-AR cells compared with U118-MG cells. Thus, more Ad DNA was synthesized in the cells that express a primary Ad receptor and can therefore be infected by the replicating Ad.

View larger version (55K): [in this window] [in a new window]   Fig. 1. Southwestern blot analysis of Ad DNA synthesis. Monolayers of U118 MG and U118 MG-AR cells maintained in medium supplemented with BrdUrd were infected with AdGFP or Ad300wt at an MOI of 0.1 viral particle/cell. Encapsidated Ad DNA was isolated from equivalent numbers of cells at the indicated times after infection, digested with XhoI, and subjected to Southwestern blot analysis using an anti-BrdUrd antibody. A significantly greater amount of DNA was synthesized by the replicating Ad in U118 MG-AR cells compared to U118-MG cells. Representative results are shown.

We then examined whether the increased yield of replicating viruses in cells expressing a primary cellular receptor for Ad5 was the result of the enhanced spread of the viral progeny through the monolayer during the several viral life cycles of the 8-day experimental period. Monolayers of U118 MG and U118 MG-AR cells were infected with Ad300wt or AdGFP at an MOI of 0.1 or 1 viral particles/cell. Eight days after infection, the CPE was monitored by staining the viable cells with crystal violet. As shown in Fig. 2A , the replication-defective AdGFP failed to lyse either cell line. Although the replicating virus did not kill the parental U118 MG cells, it caused extensive CPE in the U118 MG-AR cells, which were almost completely lysed at an MOI of 1 viral particle/cell. This indicates that the primary Ad receptor on the U118 MG-AR cells permitted lateral infection by the replicating Ad, allowing the viral progeny to spread efficiently throughout the monolayer. This finding was confirmed by a quantitative assay in which viable cells were counted (Fig. 2B) . Hence, the absence of the primary Ad receptor on the U118 MG cancer cells significantly reduced the oncolytic potency of the replicating Ad in vitro.

View larger version (46K): [in this window] [in a new window]   Fig. 2. A, CPE assay. Monolayers of U118 MG and U118 MG-AR cells were infected with AdGFP or Ad300wt at the indicated MOI. Eight days after infection, the CPE was monitored by staining the viable cells with crystal violet. The replicating Ad did not kill the parental U118 MG cells but caused extensive CPE in the U118 MG-AR cells. Representative results are shown. B, cell viability assay. Monolayers of U118 MG and U118 MG-AR cells were infected with AdGFP or Ad300wt at a MOI of 1 viral particle/cell. Eight days after infection, a cell proliferation assay was performed to count viable cells. The replicating Ad did not kill the parental U118 MG cells but caused a significant reduction in survival of the U118 MG-AR cells. Results are the mean of triplicate experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Growth kinetics of s.c. U118 MG (A) and U118 MG-AR (B) tumors in athymic nude mice. Tumors were injected with a single dose of 108 particles AdGFP (green squares), 106 particles of Ad300wt (blue triangles), or with PBS (red circles). Data points represent the mean ± SE of the tumor size in each group at the indicated time points (n = 5). The oncolytic potency of the replicating Ad was significantly greater in the U118 MG-AR tumors than in the U118 MG tumors.

View larger version (134K): [in this window] [in a new window]   Fig. 4. Immunohistochemical staining of Ad5 capsid proteins in U118 MG and U118 MG-AR tumor xenografts treated with AdGFP or Ad300wt. Tumors were excised from nude mice 35 days after treatment. Tumor sections were subjected to immunohistochemistry using rabbit anti-Ad5 antiserum as the primary antibody with an HRP-conjugated goat antirabbit secondary antibody. Diaminobenzidine was used as the chromogenic substrate. Cells containing Ad5 capsid proteins are stained brown. A: cell, U118 MG; virus, AdGFP. B: cell, U118 MG; virus, Ad300wt. C: cell, U118 MG-AR; virus, AdGFP. D: cell, U118 MG-AR; virus, Ad300wt, with numerous adjacent cells stained positively for Ad5 capsid proteins, indicating the intratumoral spread of the replicating Ad.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In this study, we have investigated the hypothesis that the efficacy of replicating Ads could be restricted by poor dissemination of the viral progeny due to the inability to infect tumor cells expressing low levels of the primary Ad receptor. To address this issue, we have used a pair of tumor cell lines that differ only in the expression of a primary receptor for Ad5. We demonstrated that the oncolytic potency of a replicating Ad was significantly greater in the receptor-positive cell line, both in monolayers of cells in vitro and in solid tumors in vivo. Moreover, the greater efficacy of the replicating Ad in the receptor-positive tumors was due to increased spread of the virus. Therefore, we have shown that it is necessary for a replicating Ad to achieve efficient lateral infection of the tumor cells to realize its full potential as an anticancer agent. This novel model system thus allowed the first direct evaluation of the relationship between the efficacy of a replicating Ad and the primary receptor levels of the host cell without the confounding influence of other variable cellular factors.

A number of studies have reported that primary cancer cells from human patients express only low levels of the primary Ad receptor, CAR, and are therefore poorly infected by Ads (11, 12, 13) . Based on our results, this suggests that the efficacy of replicating viruses dependent on CAR-mediated infection pathways will be restricted in the clinical setting. In accordance with this, Phase I and II clinical trials in which patients with recurrent squamous cell carcinoma of the head and neck received direct intratumoral injection of a replicating Ad, ONYX-015, resulted in clinical benefit in less than 15% of cases (22 , 23) . Only when combined with standard chemotherapy did this oncolytic Ad cause an objective response (at least a 50% reduction in tumor size) in 19 of 30 cases, with 8 complete responses (24) .

The CAR deficiency of primary human cancer cells suggests that the efficacy of replicating Ads could be improved by modifying the viruses to allow efficient infection via a CAR-independent pathway. In this regard, Shinoura et al. (25) have reported that the potency of a replicating Ad in glioma cell lines in vitro and in vivo could be improved by the addition of a stretch of 20 lysine residues to the COOH-terminal of the fiber protein, allowing the virus to bind to cellular heparan sulfate receptors. Similarly, Suzuki et al. (26) have shown that the efficacy of a replicating Ad can be enhanced by incorporating an RGD peptide motif into the fiber protein, permitting the virus to bind to _v integrins. Each of these strategies to enhance the infectivity, and hence the potency, of replicating Ads resulted in expanded viral tropism: the viruses retained the ability to recognize the native primary Ad receptor, CAR, which is expressed by normal cells. Therefore, modifications to the fiber protein that both introduce a tumor cell-specific targeting motif and ablate recognition of CAR would simultaneously improve both the efficacy and safety of replicating viruses by permitting efficient, CAR-independent infection of tumor cells while preventing infection of normal cells (16) . This would complement other strategies to restrict the replication of Ads to tumor cells, either by placing the expression of viral genes, most commonly the E1A gene, under the control of tumor- or tissue-specific promoters or by the complete or partial deletion of viral genes required for replication in normal cells, but not in tumor cells.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a grant from the Muscular Dystrophy Association and Grant DOUGLA00I0 from the Cystic Fibrosis Foundation (both to J. T. D.) and by United States Army Medical Research and Materiel Command Prostate Cancer Research Program Grants DAMD17-98-1-8571 and DAMD17-00-1-0002, NIH Grant R01CA83821, Grant 9707 from the Susan G. Komen Breast Cancer Foundation, and a grant from CaPCURE (all to D. T. C.).

² To whom requests for reprints should be addressed, at Gene Therapy Center, University of Alabama at Birmingham, 1824 Sixth Avenue South, WTI 620, Birmingham, AL 35294. Phone: (205) 975-2897; Fax: (205) 975-7476; E-mail: Joanne.Douglas{at}ccc.uab.edu

³ The abbreviations used are: Ad, adenovirus; BrdUrd, bromodeoxyuridine; CAR, coxsackievirus and adenovirus receptor; CPE, cytopathic effect; GFP, green fluorescent protein; HRP, horseradish peroxidase; MOI, multiplicity of infection.

Received 10/ 2/00. Accepted 12/ 4/00.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Gomez-Navarro J., Curiel D. T., Douglas J. T. Gene therapy for cancer.. Eur. J. Cancer, 35: 867-885, 1999.
2. Heise C., Kirn D. H. Replication-selective adenoviruses as oncolytic agents.. J. Clin. Investig., 105: 847-851, 2000.[Medline]
3. Alemany R., Balague C., Curiel D. T. Replicative adenoviruses for cancer therapy.. Nat. Biotechnol., 18: 723-727, 2000.[Medline]
4. Curiel D. T. The development of conditionally replicative adenoviruses for cancer therapy.. Clin. Cancer Res., 6: 3395-3399, 2000.[Abstract/Free Full Text]
5. Henry L. J., Xia D., Wilke M. E., Deisenhofer J., Gerard R. D. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.. J. Virol., 68: 5239-5246, 1994.[Abstract/Free Full Text]
6. Louis N., Fender P., Barge A., Kitts P., Chroboczek J. Cell-binding domain of adenovirus serotype 2 fiber.. J. Virol., 68: 4104-4106, 1994.[Abstract/Free Full Text]
7. Bergelson J. M., Cunningham J. A., Droguett G., Kurt-Jones E. A., Krithivas A., Hong J. S., Horwitz M. S., Crowell R. L., Finberg R. W. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5.. Science (Washington DC), 275: 1320-1323, 1997.[Abstract/Free Full Text]
8. Tomko R. P., Xu R., Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.. Proc. Natl. Acad. Sci. USA, 94: 3352-3356, 1997.[Abstract/Free Full Text]
9. Bai M., Harfe B., Freimuth P. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.. J. Virol., 67: 5198-5205, 1993.[Abstract/Free Full Text]
10. Wickham T. J., Mathias P., Cheresh D. A., Nemerow G. R. Integrins _vß₃ and _vß₅ promote adenovirus internalization but not virus attachment.. Cell, 73: 309-319, 1993.[Medline]
11. Dmitriev I., Krasnykh V., Miller C. R., Wang M., Kashentseva E., Mikheeva G., Belousova N., Curiel D. T. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism.. J. Virol., 72: 9706-9713, 1998.[Abstract/Free Full Text]
12. Miller C. R., Buchsbaum D. J., Reynolds P. N., Douglas J. T., Gillespie G. Y., Mayo M. S., Raben D., Curiel D. T. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.. Cancer Res., 58: 5738-5748, 1998.[Abstract/Free Full Text]
13. Li Y., Pong R-C., Bergelson J. M., Hall M. C., Sagalowsky A. I., Tseng C-P., Wang Z., Hsieh J-T. Loss of adenoviral receptor expression in human bladder cells: a potential impact on the efficacy of gene therapy.. Cancer Res., 59: 325-330, 1999.[Abstract/Free Full Text]
14. Rancourt C., Rogers B. E., Sosnowski B. A., Wang M., Piche A., Pierce G. F., Alvarez R. D., Siegal G. P., Douglas J. T., Curiel D. T. Basic fibroblast growth factor enhancement of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer.. Clin. Cancer Res., 4: 2455-2461, 1998.[Abstract/Free Full Text]
15. Sterman D. H., Treat J., Litzky L. A., Amin K. M., Coonrod L., Molnar-Kimber K., Recio A., Knox L., Wilson J. M., Albelda S. M., Kaiser L. R. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a Phase I clinical trial in malignant mesothelioma.. Hum. Gene Ther., 9: 1083-1092, 1998.[Medline]
16. Krasnykh V. N., Douglas J. T., van Beuschem V. W. Genetic targeting of adenoviral vectors.. Mol. Ther., 1: 391-405, 2000.[Medline]
17. Tillman B. W., de Gruijl T. D., Luykx-de Bakker S. A., Scheper R. J., Pinedo H. M., Curiel T. J., Gerritsen W. R., Curiel D. T. Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.. J. Immunol., 162: 6378-6383, 1999.[Abstract/Free Full Text]
18. Mittereder N., March K. L., Trapnell B. C. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.. J. Virol., 70: 7498-7509, 1996.[Abstract]
19. Douglas J. T., Miller C. R., Kim M., Dmitriev I., Mikheeva G., Krasnykh V., Curiel D. T. A system for the propagation of adenoviral vectors with genetically modified receptor specificities.. Nat. Biotechnol., 17: 470-475, 1999.[Medline]
20. Hardy S., Kitamura M., Harris-Stansil T., Dai Y., Phipps M. L. Construction of adenovirus vectors through Cre-lox recombination.. J. Virol., 71: 1842-1849, 1997.[Abstract]
21. Dethlefsen L. A., Prewitt J. M., Mendelsohn M. L. Analysis of tumor growth curves.. J. Natl. Cancer Inst. (Bethesda), 40: 389-405, 1968.
22. Kirn D., Hermiston T., McCormick F. ONYX-015: clinical data are encouraging.. Nat. Med., 4: 1341-1342, 1998.[Medline]
23. Ganly I., Kirn D., Eckhardt S. G., Rodriguez G. I., Soutar D. S., Otto R., Robertson A. G., Park O., Gulley M. L., Heise C., Von Hoff D. D., Kaye S. B. A Phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer.. Clin. Cancer Res., 6: 798-806, 2000.[Abstract/Free Full Text]
24. Khuri F. R., Nemunaitis J., Ganly I., Arseneau J., Tannock I. F., Romel L., Gore M., Ironside J., MacDougall R. H., Heise C., Randlev B., Gillenwater A. M., Bruso P., Kaye S. B., Hong W. K., Kirn D. H. A controlled trial of intratumoral ONYX-015, a selectively replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.. Nat. Med., 6: 879-885, 2000.[Medline]
25. Shinoura N., Yoshida Y., Tsunoda R., Ohashi M., Zhang W., Asai A., Kirino T., Hamada H. Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of gliomas.. Cancer Res., 59: 3411-3416, 1999.[Abstract/Free Full Text]
26. Suzuki K., Fueyo J., Krasnykh V., Reynolds P. N., Curiel D. T., Alemany R. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency.. Clin. Cancer Res., 7: 120-126, 2001.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. Gaballah, A. Hills, D. Curiel, G. Hallden, P. Harrison, and M. Partridge Lysis of Dysplastic but not Normal Oral Keratinocytes and Tissue-Engineered Epithelia with Conditionally Replicating Adenoviruses Cancer Res., August 1, 2007; 67(15): 7284 - 7294. [Abstract] [Full Text] [PDF]

				W. J. Van Houdt, H. Wu, J. N. Glasgow, M. L. Lamfers, C. M. Dirven, G. Y. Gillespie, D. T. Curiel, and Y. S. Haviv Gene delivery into malignant glioma by infectivity-enhanced adenovirus: In vivo versus in vitro models Neuro-oncol, July 1, 2007; 9(3): 280 - 290. [Abstract] [Full Text] [PDF]

				M. Wakayama, M. Abei, R. Kawashima, E. Seo, K. Fukuda, H. Ugai, T. Murata, N. Tanaka, I. Hyodo, H. Hamada, et al. E1A, E1B Double-Restricted Adenovirus with RGD-Fiber Modification Exhibits Enhanced Oncolysis for CAR-Deficient Biliary Cancers Clin. Cancer Res., May 15, 2007; 13(10): 3043 - 3050. [Abstract] [Full Text] [PDF]

				Y. Tsuruta, L. Pereboeva, J. N. Glasgow, D. T. Rein, Y. Kawakami, R. D. Alvarez, R. P. Rocconi, G. P. Siegal, P. Dent, P. B. Fisher, et al. A Mosaic Fiber Adenovirus Serotype 5 Vector Containing Reovirus {sigma}1 and Adenovirus Serotype 3 Knob Fibers Increases Transduction in an Ovarian Cancer Ex vivo System via a Coxsackie and Adenovirus Receptor Independent Pathway Clin. Cancer Res., May 1, 2007; 13(9): 2777 - 2783. [Abstract] [Full Text] [PDF]

				D. Bourbeau, C. J. Lau, J. Jaime, Z. Koty, S. P. Zehntner, G. Lavoie, A.-M. Mes-Masson, J. Nalbantoglu, and B. Massie Improvement of Antitumor Activity by Gene Amplification with a Replicating but Nondisseminating Adenovirus Cancer Res., April 1, 2007; 67(7): 3387 - 3395. [Abstract] [Full Text] [PDF]

				M. Rajecki, A. Kanerva, U.-H. Stenman, M. Tenhunen, L. Kangasniemi, M. Sarkioja, M. Y. Ala-Opas, H. Alfthan, A. Sankila, E. Rintala, et al. Treatment of prostate cancer with Ad5/3{Delta}24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo Mol. Cancer Ther., February 1, 2007; 6(2): 742 - 751. [Abstract] [Full Text] [PDF]

				R.-C. Pong, R. Roark, J.-Y. Ou, J. Fan, J. Stanfield, E. Frenkel, A. Sagalowsky, and J.-T. Hsieh Mechanism of Increased Coxsackie and Adenovirus Receptor Gene Expression and Adenovirus Uptake by Phytoestrogen and Histone Deacetylase Inhibitor in Human Bladder Cancer Cells and the Potential Clinical Application. Cancer Res., September 1, 2006; 66(17): 8822 - 8828. [Abstract] [Full Text] [PDF]

				L. Kangasniemi, T. Kiviluoto, A. Kanerva, M. Raki, T. Ranki, M. Sarkioja, H. Wu, F. Marini, K. Hockerstedt, H. Isoniemi, et al. Infectivity-Enhanced Adenoviruses Deliver Efficacy in Clinical Samples and Orthotopic Models of Disseminated Gastric Cancer. Clin. Cancer Res., May 15, 2006; 12(10): 3137 - 3144. [Abstract] [Full Text] [PDF]

				J. M. Mathis, P. L. Stewart, Z. B. Zhu, and D. T. Curiel Advanced Generation Adenoviral Virotherapy Agents Embody Enhanced Potency Based upon CAR-Independent Tropism. Clin. Cancer Res., May 1, 2006; 12(9): 2651 - 2656. [Full Text] [PDF]

				M. D. Lacher, M. I. Tiirikainen, E. F. Saunier, C. Christian, M. Anders, M. Oft, A. Balmain, R. J. Akhurst, and W. M. Korn Transforming Growth Factor-{beta} Receptor Inhibition Enhances Adenoviral Infectability of Carcinoma Cells via Up-Regulation of Coxsackie and Adenovirus Receptor in Conjunction with Reversal of Epithelial-Mesenchymal Transition Cancer Res., February 1, 2006; 66(3): 1648 - 1657. [Abstract] [Full Text] [PDF]

				S. Kesmodel, I. Prabakaran, R. Canter, C. Menon, K. Molnar-Kimber, and D. Fraker Virus-Mediated Oncolysis of Thyroid Cancer by a Replication-Selective Adenovirus Driven by a Thyroglobulin Promoter-Enhancer Region J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3440 - 3448. [Abstract] [Full Text] [PDF]

				D. T. Rein, M. Breidenbach, T. O. Kirby, T. Han, G. P. Siegal, G. J. Bauerschmitz, M. Wang, D. M. Nettelbeck, Y. Tsuruta, M. Yamamoto, et al. A Fiber-Modified, Secretory Leukoprotease Inhibitor Promoter-Based Conditionally Replicating Adenovirus for Treatment of Ovarian Cancer Clin. Cancer Res., February 1, 2005; 11(3): 1327 - 1335. [Abstract] [Full Text] [PDF]

				T. O. Kirby, A. Rivera, D. Rein, M. Wang, I. Ulasov, M. Breidenbach, M. Kataram, J. L. Contreras, C. Krumdieck, M. Yamamoto, et al. A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity Clin. Cancer Res., December 15, 2004; 10(24): 8697 - 8703. [Abstract] [Full Text] [PDF]

				M. K. Merrill, G. Bernhardt, J. H. Sampson, C. J. Wikstrand, D. D. Bigner, and M. Gromeier Poliovirus receptor CD155-targeted oncolysis of glioma Neuro-oncol, July 1, 2004; 6(3): 208 - 217. [Abstract] [PDF]

				J. Davydova, L. P. Le, T. Gavrikova, M. Wang, V. Krasnykh, and M. Yamamoto Infectivity-Enhanced Cyclooxygenase-2-Based Conditionally Replicative Adenoviruses for Esophageal Adenocarcinoma Treatment Cancer Res., June 15, 2004; 64(12): 4319 - 4327. [Abstract] [Full Text] [PDF]

				W. Gu, A. Ogose, H. Kawashima, M. Ito, T. Ito, A. Matsuba, H. Kitahara, T. Hotta, K. Tokunaga, H. Hatano, et al. High-Level Expression of the Coxsackievirus and Adenovirus Receptor Messenger RNA in Osteosarcoma, Ewing's Sarcoma, and Benign Neurogenic Tumors among Musculoskeletal Tumors Clin. Cancer Res., June 1, 2004; 10(11): 3831 - 3838. [Abstract] [Full Text] [PDF]

				K. Toth, H. Djeha, B. Ying, A. E. Tollefson, M. Kuppuswamy, K. Doronin, P. Krajcsi, K. Lipinski, C. J. Wrighton, and W. S. M. Wold An Oncolytic Adenovirus Vector Combining Enhanced Cell-to-Cell Spreading, Mediated by the ADP Cytolytic Protein, with Selective Replication in Cancer Cells with Deregulated Wnt Signaling Cancer Res., May 15, 2004; 64(10): 3638 - 3644. [Abstract] [Full Text] [PDF]

				A. M. Witlox, V. W. van Beusechem, B. Molenaar, H. Bras, G. R. Schaap, R. Alemany, D. T. Curiel, H. M. Pinedo, P. I. J. M. Wuisman, and W. R. Gerritsen Conditionally Replicative Adenovirus with Tropism Expanded towards Integrins Inhibits Osteosarcoma Tumor Growth in Vitro and in Vivo Clin. Cancer Res., January 1, 2004; 10(1): 61 - 67. [Abstract] [Full Text] [PDF]

				R.-C. Pong, Y.-J. Lai, H. Chen, T. Okegawa, E. Frenkel, A. Sagalowsky, and J.-T. Hsieh Epigenetic Regulation of Coxsackie and Adenovirus Receptor (CAR) Gene Promoter in Urogenital Cancer Cells Cancer Res., December 15, 2003; 63(24): 8680 - 8686. [Abstract] [Full Text] [PDF]

				K. Fukuda, M. Abei, H. Ugai, E. Seo, M. Wakayama, T. Murata, T. Todoroki, N. Tanaka, H. Hamada, and K. K. Yokoyama E1A, E1B Double-restricted Adenovirus for Oncolytic Gene Therapy of Gallbladder Cancer Cancer Res., August 1, 2003; 63(15): 4434 - 4440. [Abstract] [Full Text] [PDF]

				Y. Kawakami, H. Li, J. T. Lam, V. Krasnykh, D. T. Curiel, and J. L. Blackwell Substitution of the Adenovirus Serotype 5 Knob with a Serotype 3 Knob Enhances Multiple Steps in Virus Replication Cancer Res., March 15, 2003; 63(6): 1262 - 1269. [Abstract] [Full Text] [PDF]

				Y. S. Haviv, J. L. Blackwell, A. Kanerva, P. Nagi, V. Krasnykh, I. Dmitriev, M. Wang, S. Naito, X. Lei, A. Hemminki, et al. Adenoviral Gene Therapy for Renal Cancer Requires Retargeting to Alternative Cellular Receptors Cancer Res., August 1, 2002; 62(15): 4273 - 4281. [Abstract] [Full Text] [PDF]

				G. J. Bauerschmitz, J. T. Lam, A. Kanerva, K. Suzuki, D. M. Nettelbeck, I. Dmitriev, V. Krasnykh, G. V. Mikheeva, M. N. Barnes, R. D. Alvarez, et al. Treatment of Ovarian Cancer with a Tropism Modified Oncolytic Adenovirus Cancer Res., March 1, 2002; 62(5): 1266 - 1270. [Abstract] [Full Text] [PDF]

				S. A. Vorburger and K. K. Hunt Adenoviral Gene Therapy Oncologist, February 1, 2002; 7(1): 46 - 59. [Abstract] [Full Text] [PDF]

				A. Hemminki, I. Dmitriev, B. Liu, R. A. Desmond, R. Alemany, and D. T. Curiel Targeting Oncolytic Adenoviral Agents to the Epidermal Growth Factor Pathway with a Secretory Fusion Molecule Cancer Res., September 1, 2001; 61(17): 6377 - 6381. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Douglas, J. T.