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Department of Urology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas [Y. L., R-C. P., M. C. H., A. I. S., C-P. T., Z. W., J-T. H.], and Division of Immunologic and Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania [J. M. B.]
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Introduction |
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In our laboratory, we are evaluating the efficacy of gene therapy for urogenital cancer using replication-deficient adenovirus. Recently, we observed several human bladder cancer cell lines that appeared to be resistant to viral infection. Therefore, we decided to determine the levels of CAR in those cell lines. We found that the level of CAR correlated with viral infectivity. Increased viral sensitivity could be restored in a resistant line after transfecting a functional CAR cDNA vector. We believe that these findings have significant biological and therapeutic implications.
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Materials and Methods |
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Primary Culture of Human Bladder Cancer Cells.
The primary human bladder cancer cell (SWBC1) was derived from a cancer patient diagnosed with invasive transitional carcinoma (T2N0M0) after radical cystoprostatectomy. The tumor specimen was dissected into 3–5-mm3 pieces and planted on a 60-mm dish with T medium containing 5% FBS. After cells grew out from the explants, we carefully removed contaminated fibroblasts by trypsinization and continued to pass the cells. Biochemical analysis using both cytokeratin and vimentin antibodies confirmed that this cell is of epithelial origin. Currently, these cells have been cultivated in vitro for 8 months with more than 40 passages.
Recombinant Virus Construction and Purification.
The replication-deficient recombinant virus, AdCMV-ß-gal, was generated as described previously (11)
. Another replication-deficient recombinant virus, dl312, obtained from Dr. Shenk (12)
, was labeled with [3H]thymidine as described previously (13)
. To produce a large amount of viral stock, the recombinant viruses were harvested from the cell pellet after 36 h of infection and subjected to two cycles of CsCl gradient ultracentrifugation (1)
. After dialysis overnight, the stock of viruses was aliquoted and stored at -80°C until use. The titers of viral stocks were determined using the plaque assay in triplicate and viral concentrations were measured by A260 nm.
Detection of Virus-mediated Gene Delivery, Virus Binding, and Immunostaining.
To determine the viral sensitivity of human bladder cancer cells, 5 x 105 cells were infected with different concentrations of viruses at 37°C in a 5% CO2-humidified incubator. We determined the virus-mediated gene delivery with two different approaches, ß-gal staining and activity assay. At the indicated time, the infected cells were washed with PBS, fixed, and stained for ß-gal activity (14)
. The adenovirus-infected cells were counted microscopically by the number of the positive ß-gal-positive cells. In the second approach, the ß-gal activity was determined as follows: infected cells were trypsinized and washed once with PBS, and then the protein concentration of each sample was determined by the Bradford dye-binding procedure (Bio-Rad, Hercules, CA). ß-Gal activity (15)
was measured in a 200-µl cell lysate and normalized to the protein concentration of each sample.
For virus binding assays, [3H]thymidine-labeled dl312 (106 plaque forming units) was incubated with 1 ml of cells ranging from 5 x 105 to 1 x 107 at 4°C for 1 h, and then the cell suspension was loaded onto 3 ml of PBS containing 2% BSA and 10% sucrose and centrifuged in a swinging bucket rotor at 2000 rpm for 5 min (13) . The cell pellet was lysed in 100 µl of 0.3 N NaOH solution and subjected to liquid scintillation counting to determine the amount of virus bound.
Cytometric analysis of viral receptor by monoclonal antibody (RmcB [16]) membrane fluorescence staining was performed on a single-cell suspension and FITC-conjugated secondary antibodies as described previously (17) . Fluorescence-activated cell scanning was performed with a dual-laser Vantage flow cytometer (Becton Dickinson, Mountain View, CA) delivering 50 mW at 488 nm with an Enterprise air-cooled laser. Analysis was performed using LYSYS II software (Becton Dickinson, Mountain View, CA).
Cloning of CAR cDNA by RT-PCR and Construction of an Expression Vector.
CAR cDNA was isolated by RT-PCR with total cellular RNA isolated from both 253J and RT4 cell lines. Two sets of primer were synthesized: CAR1, 5'-AATTCCCAGGAGCGAGAG-3'; CAR2, 5'-TCCCAGAGTACTCAGAAGAG-3'; CAR3, 5'-GCCTTCAGGTGCGAGATGTTAC-3'; and CAR4, 5'-GAACACGGAGAGCACAGATGAGAC-3'. For amplifying the 5' end of CAR cDNA (nucleotides 2–684), first strand cDNA was synthesized using Superscript II reverse transcriptase (Life Technologies, Inc.) with random primers, and then one-fifth of the cDNA was subjected to PCR (40 cycles of 92°C [15 s], 55°C [30 s], and 65°C [3 min]) using primers CAR1 and CAR2. The final PCR products were cloned into pcDNA3.1/V5/His-TOPO vector and, to avoid any PCR-induced mutations, sequenced using the Thermosequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Chicago, IL). Among these clones, we identified clone pCAR12s with the sense orientation to the CMV promoter of pcDNA3.1/V5/His-TOPO. For amplifying the 3' end of CAR cDNA (nt 479-1192), first strand cDNA was synthesized using Superscript II reverse transcriptase with primer CAR4, and then one-fifth of the cDNA was subjected to PCR (40 cycles of 92°C [15 s], 55°C [30 s], and 72°C [2 min]) using primers CAR3 and CAR4. The final PCR products were cloned into PCR 2.1 vector (Invitrogen, Carlsbad CA) and sequenced. Full-length CAR cDNA was assembled in an expression vector (pTOPOCAR) by ligating a 573-bp fragment from the 3' end of CAR cDNA (digested with pflMI and NotI) with pCAR12s digested with pflMI and NotI .
Measurement of CAR mRNA and Protein Using Quantitative RT-PCR and Northern Analysis.
For quantitative RT-PCR, 2 µg of total cellular RNA from each cell line was reverse transcribed into first strand cDNA as described previously. One-fifth of the cDNA was subjected to a 100-µl PCR (30 cycles of 92°C [15 s], 55°C [30 s], and 72°C [2 min]) using both the CAR primer set (i.e., CAR3 and CAR4; 1 ng/µl each) and the GAPDH primer set (5'-TCGTGGAAGGACTCATGACC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; 0.5 ng/µl). The final PCR products (10 µl) were electrophoresed in a 2% NuSieve agarose gel (3:1, FMC Bioproducts, Rockland, ME) and quantified with BioMax 1D image analysis software (Eastman Kodak, Rochester, NY). The relative level of CAR mRNA from each sample was normalized to GAPDH transcript from the same reaction. To determine the levels of CAR expression among human bladder cell lines, we performed Northern blot analysis as described previously (17)
.
Southern Blot Analysis of CAR Gene in Human Bladder Cancer Cells.
For Southern analysis, high molecular weight DNA was purified by the procedure of Davis et al. (18)
. Twenty µg of DNA were digested with restriction endonucleases overnight at 37°C and then subjected to Southern blot analysis as described previously (17)
with a full-length CAR cDNA probe.
DNA Transfection into Human Bladder Cancer Cells.
T24 cells (2 x 105 per p-35 plate) were transfected with 2 µg of pcDNA3.1/V5/His-TOPO or pTOPOCAR using LipofectAMINE transfection reagent. Forty-eight h after transfection, cells were split and were selected for neomycin resistant clones with 600 µg/ml G-418. Resistant colonies were either pooled or cloned by ring isolation after 2 weeks of selection.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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To test this hypothesis, we examined the infectivity of human bladder cancer cells by a recombinant adenovirus, AdCMV-ß-gal. As shown in Fig. 1A
, the positive blue cells were very visible in the RT4 cells infected with virus at m.o.i. 1, whereas no blue cells were seen in cells treated with the buffer control. The infectivity also increased with longer viral incubation (Table 1)
. In contrast to both 253J and RT4 cells, WH, TCC, and T24 cells showed either few or no blue cells even 48 h after infection; these results correlated with the previous results obtained using different kinds of adenoviruses (20)
. Similarly, in the presence of the same amount of AdCMV-ß-gal, the ß-gal activity per cell in RT4 cells was at least 50-fold higher than that in T24 cells 48 h after infection (data not shown). Both RT4 and 253J cells bound significantly more radiolabeled virus than T24 cells did (Fig. 1B)
. As summarized in Fig. 1C
and Table 1
, human bladder cancer lines exhibited a wide spectrum of sensitivity to virus attachment and virus-mediated gene delivery. RT4 and 253J bound the most virus and were the most sensitive to the ß-gal viral infection. TCC and T24 bound the least virus and were resistant to viral infection. It is known that the entry of adenovirus is mediated by the presence of a specific receptor on the target cells (1, 2, 3, 4, 5)
. Therefore, these data suggested that human bladder cancer cells may possess different levels of receptor for adenovirus and that receptor expression may correlate with sensitivity to adenoviral infection.
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using RNA extracted from these human cancer cells, and we observed a significant difference (approximately 20-fold) in the intensity of CAR PCR product (i.e., 714 bp) between T24 and RT4 cells with no difference in the intensity of the GAPDH (i.e., 452 bp) control. CAR mRNA levels paralleled the viral sensitivity in human bladder cancer lines tested (Fig. 2A)
. The CAR PCR product was cloned into a PCR vector and sequenced. Sequences of this PCR product were completely identical to those of CAR cDNA previously reported (6, 7, 8)
. Using CAR cDNA as a probe (Fig. 3C)
, we detected at least four different sizes (i.e., 2.4, 4.8, 6.4, and 8.0 kb) of RNA transcript in human cancer cell lines as well as other cells (7
, 8)
, suggesting that there may be a gene family or different splicing variants associated with the adenoviral receptor gene. CAR mRNA (Fig. 3C)
levels were significantly higher in RT4 cells than in T24 cell line.
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, the data indicated that the overall hybridization intensity of CAR gene in both RT4 and T24 cells is similar to each other, indicating that gene amplification does not account for the elevated levels of CAR mRNA in RT4 cells. In addition, the restriction enzyme patterns of the CAR gene in both RT4 and T24 cells were identical revealed by five different enzymes (such as BamHI, EcoRI, HindIII, PstI, and MspI) suggested that no large gene alteration in the CAR gene can account for the low levels of CAR mRNA in T24 cells. In four other human bladder cancer cell lines tested, their restriction enzyme patterns of the CAR gene were identical. Taken together, these data suggested that neither DNA amplification nor DNA arrangement of the CAR gene can be accounted for such dramatic difference in CAR mRNA levels in these human bladder cancer cells. Therefore, transcriptional regulation of the CAR gene may be critical for modulating CAR levels in each cell line. More detailed analyses of the CAR gene in T24 cells are warranted.
Increased Viral Infectivity after CAR cDNA Transfection.
To test whether the CAR protein is responsible for adenoviral infection in human bladder cancer cells, we constructed a mammalian CAR expression vector and transfected it into T24 cells. After G-418 selection, three independent clones (N2, N3, and N4) and vector-transfected clones (T6, T8, and T10) were chosen based on their different DNA integration pattern (Fig. 3A)
. Data from the quantitative RT-PCR (Fig. 3B)
indicated that the levels of CAR mRNA expression among those transfected sublines were N2 > N4 > N3. The control sublines were completely negative. Northern blot analysis indicated that these three sublines expressed a single CAR mRNA band with a predicted size of 1.1 kb (only transcribed from the open reading frame of CAR cDNA) that was identical to the 2.4 kb of CAR mRNA detected in RT4 cells (Fig. 3C)
. Furthermore, results obtained from virus binding and ß-gal activity showed that the N2 subline, consistent with its higher levels of CAR mRNA expression, was the most sensitive cell to adenoviral infection (Table 2)
. We also noticed that the ß-gal activity 24 h after viral infection in N4 cells is relatively high, but the receptor binding, determined by 1 h binding assay, to this subline as similar to that in parental T24 cells. This may be due to the fact that the kinetics for virus binding in N4 cells is much slower than the other two sublines.
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, RT4 cells exhibited a dramatic shift in the fluorescence intensity with RmcB antibody (thick line) staining compared to control antibody (thin line) staining, indicating that a high levels of CAR protein expression was detected in RT4 cells. In contrast to RT4 cells, parental T24 cells did not have any shift between RmcB and control antibodies, indicating that T24 cells did not have any detectable CAR protein. However, the fluorescence intensity in the N2 cells with RmcB antibody staining had a moderate increase compared to control antibody staining (Fig. 3D)
, indicating that N2 cells did express the CAR protein on their cell membrane. In addition, the profile of CAR staining exhibited a wide spectrum of intensity on their cell membrane, suggesting that the levels of CAR protein expressed in N2 cells were very heterogeneous (Fig. 3D)
. Similar results were seen with ß-gal staining (data not shown). Although, the total CAR mRNA levels in N2 cells were equivalent to that in RT4 cells (Fig. 3C)
, the majority of N2 positive cells expressed low levels of CAR protein on their cell membrane (Fig. 3D)
. With further cloning of N2 cells, we did not obtain any single clone with the enriched positive cells. We have found, on the basis of selecting more than 30 clones from two separate transfection studies, that most clones had only 10–20% positive cells (data not shown). Therefore, it is possible that the CAR protein levels in T24 cells may be modulated posttranscriptionally.
The Biological Significance of Viral Receptor in Human Bladder Cancer Lines.
Gene therapy is an innovative approach to the treatment of malignant and benign disorders characterized by genetic alterations that may serve as therapeutic targets. Several practical and theoretical considerations (21)
make recombinant adenovirus an attractive vector for cancer gene therapy. One advantage is that the adenoviral receptor CAR is ubiquitous and is detected in a variety of organs (7)
. In this study, we observed a wide spectrum of adenoviral sensitivity in several human bladder cancer lines (Table 1)
. Although those cell lines originate from the transitional epithelium, each line exhibits a diversity of phenotypes and growth potential. For example, the RT4 cell line is derived from well-differentiated transitional cell papilloma, and the T24 cell line is a rapidly growing transitional carcinoma with a mutated ras oncogene. Obviously, more detailed studies will be required to understand whether CAR levels correlate with the grade of cancer cells. If similar results can be observed clinically, these data will certainly have an impact on the efficacy of adenovirus-based gene therapy.
Adenoviruses are nonenveloped DNA viruses that can infect target cells by binding to cellular receptors. More than 40 human adenovirus serotypes have been identified based on nucleic acid homology, oncogenic potential, and their protein component. The receptor for adenovirus type 5, which is frequently used as a viral vector for gene therapy, has been cloned (6 , 7) . Sequence analysis indicates that this adenoviral receptor CAR cDNA encodes a typical immunoglobulin-like membrane protein with two immunoglobulin domains that may interact with adenovirus fiber protein. In addition to the extracellular domain, CAR cDNA contains a 22-amino acid transmembrane domain and a 107-amino acid intracellular domain that has a putative tyrosine phosphorylation site. Based on the CAR protein structure, it is likely that CAR not only may function as the receptor for adenovirus but also may have other physiological functions, such as a cell adhesion molecule.
Our Northern blot analysis indicated that there are four CAR mRNA transcripts detected in RT4 cells using a full-length CAR cDNA. Biologically, these RNA transcripts could represent various isoforms of CAR that are likely to have different viral binding affinities or other distinct physiological function. Based on our RT-PCR results using both CAR3 and CAR4 primers, including both transmembrane and intracellular domains, we only observed one single PCR product from all of the human bladder cancer lines tested. However, with both CAR1 and CAR2 primers, including extracellular domain, we did observe several larger size transcripts from RT-PCRs. Thus far, we have sequenced at least 20 different clones, and none of them appeared to be CAR homologues (data not shown). Other studies using RmcB antibody also indicated that a single protein of 46 kDa was detected by both Western blot and immunoprecipitation (6 , 7) . Taken together, these results suggest that the higher molecular weight RNA transcripts may be splicing intermediates of CAR mRNA.
Although neither CAR mRNA nor protein levels are detectable in T24 cells, the genomic structure of the CAR gene in T24 cells and several other human bladder cancer lines is identical. This suggests that DNA rearrangement or mutation of the CAR gene does not account for the dramatic difference between RT4 and T24 cells. Therefore, transcriptional regulation of the CAR gene appears to be an important aspect of modulating CAR gene activity. Obviously, understanding the gene regulation of CAR leading to the induction of endogenous CAR gene activity could be a new strategy for increasing the efficiency of gene delivery.
Results from this study demonstrate that the expression of adenoviral receptor is heterogeneous among human bladder cancer cells. This may have significant implications concerning the design and efficacy of gene therapy trials for human bladder cancer. Our data suggest that determining the receptor status of given patient’s tumor prior to adenovirus-based gene therapy may be important.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the National Institute of Health Grant CA 73017 (to J-T. H.), AI35667, HL 54734, and an Established Investigator Award from the American Heart Association (to J. M. B.). 
2 The first two authors contributed equally in this project.
3 To whom requests for reprints should be addressed, at Department of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9110. Phone: (214) 648-3988; Fax: (214) 648-8786; E-mail: Hsieh{at}utsw.swmed.edu
4 The abbreviations used are: m.o.i., multiplicity of infection; CAR, coxsackie and adenovirus receptor; ß-gal, ß-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription PCR; CMV, cytomegalovirus; Ad, adenovirus.
Received 9/15/98. Accepted 11/25/98.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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M. Qin, S. Chen, T. Yu, B. Escuadro, S. Sharma, and R. K. Batra Coxsackievirus Adenovirus Receptor Expression Predicts the Efficiency of Adenoviral Gene Transfer into Non-Small Cell Lung Cancer Xenografts Clin. Cancer Res., October 15, 2003; 9(13): 4992 - 4999. [Abstract] [Full Text] [PDF]
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Q. Wu, R. Mahendran, and K. Esuvaranathan Nonviral Cytokine Gene Therapy on an Orthotopic Bladder Cancer Model Clin. Cancer Res., October 1, 2003; 9(12): 4522 - 4528. [Abstract] [Full Text] [PDF]
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P. P. Connell and R. R. Weichselbaum Gene Therapy: The Challenges of Translating Laboratory Research Into Clinical Practice J. Clin. Oncol., June 15, 2003; 21(12): 2230 - 2231. [Full Text] [PDF]
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L. C. Pagliaro, A. Keyhani, D. Williams, D. Woods, B. Liu, P. Perrotte, J. W. Slaton, J. A. Merritt, H. B. Grossman, and C. P. Dinney Repeated Intravesical Instillations of an Adenoviral Vector in Patients With Locally Advanced Bladder Cancer: A Phase I Study of p53 Gene Therapy J. Clin. Oncol., June 15, 2003; 21(12): 2247 - 2253. [Abstract] [Full Text] [PDF]
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M. Anders, C. Christian, M. McMahon, F. McCormick, and W. M. Korn Inhibition of the Raf/MEK/ERK Pathway Up-Regulates Expression of the Coxsackievirus and Adenovirus Receptor in Cancer Cells Cancer Res., May 1, 2003; 63(9): 2088 - 2095. [Abstract] [Full Text] [PDF]
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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]
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 L.-Q. Zhang, Y.-F. Mei, and G. Wadell Human adenovirus serotypes 4 and 11 show higher binding affinity and infectivity for endothelial and carcinoma cell lines than serotype 5 J. Gen. Virol., March 1, 2003; 84(3): 687 - 695. [Abstract] [Full Text] [PDF]
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 M. Anders, R. Hansen, R.-X. Ding, K. A. Rauen, M. J. Bissell, and W. M. Korn Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor PNAS, February 18, 2003; 100(4): 1943 - 1948. [Abstract] [Full Text] [PDF]
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A. Hemminki, A. Kanerva, B. Liu, M. Wang, R. D. Alvarez, G. P. Siegal, and D. T. Curiel Modulation of Coxsackie-Adenovirus Receptor Expression for Increased Adenoviral Transgene Expression Cancer Res., February 15, 2003; 63(4): 847 - 853. [Abstract] [Full Text] [PDF]
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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]
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N. Belousova, V. Krendelchtchikova, D. T. Curiel, and V. Krasnykh Modulation of Adenovirus Vector Tropism via Incorporation of Polypeptide Ligands into the Fiber Protein J. Virol., July 29, 2002; 76(17): 8621 - 8631. [Abstract] [Full Text] [PDF]
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K. A. Rauen, D. Sudilovsky, J. L. Le, K. L. Chew, B. Hann, V. Weinberg, L. D. Schmitt, and F. McCormick Expression of the Coxsackie Adenovirus Receptor in Normal Prostate and in Primary and Metastatic Prostate Carcinoma: Potential Relevance to Gene Therapy Cancer Res., July 1, 2002; 62(13): 3812 - 3818. [Abstract] [Full Text] [PDF]
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 W. Russell Adenovirus Gene Therapy for Ovarian Cancer J Natl Cancer Inst, May 15, 2002; 94(10): 706 - 707. [Full Text] [PDF]
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A. Hemminki, K. R. Zinn, B. Liu, T. R. Chaudhuri, R. A. Desmond, B. E. Rogers, M. N. Barnes, R. D. Alvarez, and D. T. Curiel In Vivo Molecular Chemotherapy and Noninvasive Imaging With an Infectivity-Enhanced Adenovirus J Natl Cancer Inst, May 15, 2002; 94(10): 741 - 749. [Abstract] [Full Text] [PDF]
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 A. Hasenburg, D.-C. Fischer, X.-W. Tong, A. Rojas-Martinez, R. H. Kaufman, I. Ramzy, P. Kohlberger, M. Orlowska-Volk, E. Aguilar-Cordova, and D. G. Kieback Adenovirus-Mediated Thymidine Kinase Gene Therapy for Recurrent Ovarian Cancer: Expression of Coxsackie-Adenovirus Receptor and Integrins {alpha}v{beta}3 and {alpha}v{beta}5 Reproductive Sciences, May 1, 2002; 9(3): 174 - 180. [Abstract] [PDF]
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J. I. Izawa, P. Sweeney, P. Perrotte, D. Kedar, Z. Dong, J. W. Slaton, T. Karashima, K. Inoue, W. F. Benedict, and C. P. N. Dinney Inhibition of Tumorigenicity and Metastasis of Human Bladder Cancer Growing in Athymic Mice by Interferon-{beta} Gene Therapy Results Partially from Various Antiangiogenic Effects Including Endothelial Cell Apoptosis Clin. Cancer Res., April 1, 2002; 8(4): 1258 - 1270. [Abstract] [Full Text] [PDF]
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 Q. Yu, L. G. Que, and D. C. Rockey Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver Am J Physiol Gastrointest Liver Physiol, March 1, 2002; 282(3): G565 - G572. [Abstract] [Full Text] [PDF]
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V. W. van Beusechem, J. Grill, D. C. J. Mastenbroek, T. J. Wickham, P. W. Roelvink, H. J. Haisma, M. L. M. Lamfers, C. M. F. Dirven, H. M. Pinedo, and W. R. Gerritsen Efficient and Selective Gene Transfer into Primary Human Brain Tumors by Using Single-Chain Antibody-Targeted Adenoviral Vectors with Native Tropism Abolished J. Virol., February 22, 2002; 76(6): 2753 - 2762. [Abstract] [Full Text] [PDF]
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B. Geoerger, J. Grill, P. Opolon, J. Morizet, G. Aubert, M.-J. Terrier-Lacombe, B. Bressac de-Paillerets, M. Barrois, J. Feunteun, D. H. Kirn, et al. Oncolytic Activity of the E1B-55 kDa-deleted Adenovirus ONYX-015 Is Independent of Cellular p53 Status in Human Malignant Glioma Xenografts Cancer Res., February 1, 2002; 62(3): 764 - 772. [Abstract] [Full Text] [PDF]
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D. M. Shayakhmetov, Z.-Y. Li, S. Ni, and A. Lieber Targeting of Adenovirus Vectors to Tumor Cells Does Not Enable Efficient Transduction of Breast Cancer Metastases Cancer Res., February 1, 2002; 62(4): 1063 - 1068. [Abstract] [Full Text] [PDF]
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C. J. Cohen, Z. Q. Xiang, G.-P. Gao, H. C. J. Ertl, J. M. Wilson, and J. M. Bergelson Chimpanzee adenovirus CV-68 adapted as a gene delivery vector interacts with the coxsackievirus and adenovirus receptor J. Gen. Virol., January 1, 2002; 83(1): 151 - 155. [Abstract] [Full Text] [PDF]
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E. A. Kashentseva, T. Seki, D. T. Curiel, and I. P. Dmitriev Adenovirus Targeting to c-erbB-2 Oncoprotein by Single-Chain Antibody Fused to Trimeric Form of Adenovirus Receptor Ectodomain Cancer Res., January 1, 2002; 62(2): 609 - 616. [Abstract] [Full Text] [PDF]
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A. Kanerva, G. V. Mikheeva, V. Krasnykh, C. J. Coolidge, J. T. Lam, P. J. Mahasreshti, S. D. Barker, M. Straughn, M. N. Barnes, R. D. Alvarez, et al. Targeting Adenovirus to the Serotype 3 Receptor Increases Gene Transfer Efficiency to Ovarian Cancer Cells Clin. Cancer Res., January 1, 2002; 8(1): 275 - 280. [Abstract] [Full Text] [PDF]
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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]
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T. Okegawa, R.-C. Pong, Y. Li, J. M. Bergelson, A. I. Sagalowsky, and J.-T. Hsieh The Mechanism of the Growth-inhibitory Effect of Coxsackie and Adenovirus Receptor (CAR) on Human Bladder Cancer: A Functional Analysis of CAR Protein Structure Cancer Res., September 1, 2001; 61(17): 6592 - 6600. [Abstract] [Full Text] [PDF]
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P. K. Tan, A.-I. Michou, J. M. Bergelson, and M. Cotten Defining CAR as a cellular receptor for the avian adenovirus CELO using a genetic analysis of the two viral fibre proteins J. Gen. Virol., June 1, 2001; 82(6): 1465 - 1472. [Abstract] [Full Text]
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T. P. Cripe, E. J. Dunphy, A. D. Holub, A. Saini, N. H. Vasi, Y. Y. Mahller, M. H. Collins, J. D. Snyder, V. Krasnykh, D. T. Curiel, et al. Fiber Knob Modifications Overcome Low, Heterogeneous Expression of the Coxsackievirus-Adenovirus Receptor That Limits Adenovirus Gene Transfer and Oncolysis for Human Rhabdomyosarcoma Cells Cancer Res., April 1, 2001; 61(7): 2953 - 2960. [Abstract] [Full Text]
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J. Grill, V. W. Van Beusechem, P. Van Der Valk, C. M. F. Dirven, A. Leonhart, D. S. Pherai, H. J. Haisma, H. M. Pinedo, D. T. Curiel, and W. R. Gerritsen Combined Targeting of Adenoviruses to Integrins and Epidermal Growth Factor Receptors Increases Gene Transfer into Primary Glioma Cells and Spheroids Clin. Cancer Res., March 1, 2001; 7(3): 641 - 650. [Abstract] [Full Text]
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J. T. Douglas, M. Kim, L. A. Sumerel, D. E. Carey, and D. T. Curiel Efficient Oncolysis by a Replicating Adenovirus (Ad) in Vivo Is Critically Dependent on Tumor Expression of Primary Ad Receptors Cancer Res., February 1, 2001; 61(3): 813 - 817. [Abstract] [Full Text]
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C. Ebbinghaus, A. Al-Jaibaji, E. Operschall, A. Schöffel, I. Peter, U. F. Greber, and S. Hemmi Functional and Selective Targeting of Adenovirus to High-Affinity Fc{gamma} Receptor I-Positive Cells by Using a Bispecific Hybrid Adapter J. Virol., January 1, 2001; 75(1): 480 - 489. [Abstract] [Full Text]
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K. Suzuki, J. Fueyo, V. Krasnykh, P. N. Reynolds, D. T. Curiel, and R. Alemany A Conditionally Replicative Adenovirus with Enhanced Infectivity Shows Improved Oncolytic Potency Clin. Cancer Res., January 1, 2001; 7(1): 120 - 126. [Abstract] [Full Text]
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C.-T. Lee, J. Y. Seol, K.-H. Park, C.-G. Yoo, Y. W. Kim, C. Ahn, Y.-W. Song, S. K. Han, J. S. Han, S. Kim, et al. Differential Effects of Adenovirus-p16 on Bladder Cancer Cell Lines Can Be Overcome by the Addition of Butyrate Clin. Cancer Res., January 1, 2001; 7(1): 210 - 214. [Abstract] [Full Text]
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W. C. Russell Update on adenovirus and its vectors J. Gen. Virol., November 1, 2000; 81(11): 2573 - 2604. [Full Text]
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F. J. Kelly, C. R. Miller, D. J. Buchsbaum, J. Gomez-Navarro, M. N. Barnes, R. D. Alvarez, and D. T. Curiel Selectivity of TAG-72-targeted Adenovirus Gene Transfer to Primary Ovarian Carcinoma Cells versus Autologous Mesothelial Cells in Vitro Clin. Cancer Res., November 1, 2000; 6(11): 4323 - 4333. [Abstract] [Full Text] [PDF]
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T. Okegawa, Y. Li, R.-C. Pong, J. M. Bergelson, J. Zhou, and J.-T. Hsieh The Dual Impact of Coxsackie and Adenovirus Receptor Expression on Human Prostate Cancer Gene Therapy Cancer Res., September 1, 2000; 60(18): 5031 - 5036. [Abstract] [Full Text]
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A. S. Pearson, P. E. Koch, N. Atkinson, M. Xiong, R. W. Finberg, J. A. Roth, and B. Fang Factors Limiting Adenovirus-mediated Gene Transfer into Human Lung and Pancreatic Cancer Cell Lines Clin. Cancer Res., December 1, 1999; 5(12): 4208 - 4213. [Abstract] [Full Text] [PDF]
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E. R. Sauter, M. Nesbit, S. Litwin, A. J. P. Klein-Szanto, S. Cheffetz, and M. Herlyn Antisense Cyclin D1 Induces Apoptosis and Tumor Shrinkage in Human Squamous Carcinomas Cancer Res., October 1, 1999; 59(19): 4876 - 4881. [Abstract] [Full Text] [PDF]
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 J. DOUKAS, D. K. HOGANSON, M. ONG, W. YING, D. L. LACEY, A. BAIRD, G. F. PIERCE, and B. A. SOSNOWSKI Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways FASEB J, August 1, 1999; 13(11): 1459 - 1466. [Abstract] [Full Text]
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