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Experimental Therapeutics, Preclinical Pharmacology |
Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, Tokyo 170-8455 [S. Y., Y. Y., H. H.]; Department of Neurosurgery, School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519 [S. Y., M. A., K. O., K. H.]; and Department of Molecular Medicine, Sapporo Medical University, Sapporo 060-8556 [H. H.], Japan
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ABSTRACT |
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Experimental Design: We repeatedly transduced an adenoviral vector expressing the human p53 gene (AxCIhp53) into U373MG, a p53-susceptible cell line, and established the AxCIhp53-resistant cell line U373R. Most U373R cells survived even after AxCIhp53 treatment due to reduced transduction efficiency. Expression levels of adenovirus receptors were estimated to investigate the cause of reduced transduction efficiency. The mutant vector was used to overcome the resistance.
Results: The transduction efficiency of an adenoviral vector possessing the reporter LacZ gene (AxCAZ2-F/wt) for U373R cells was 25.4-fold less than that for parent cells. The expression levels of integrins 
vß3 and vß5 were found to be decreased in U373R cells without affecting the expression levels of Coxsackievirus and adenovirus receptor. The mutant vector AxCAZ2-F/K20, with a linker and a stretch of 20 lysine residues at the COOH-terminal of the fiber protein, improved the transduction efficiency of U373R cells to 12.6-fold of that of AxCAZ2-F/wt. A mutant vector carrying the p53 gene, AxCAhp53-F/K20, dramatically induced apoptosis in U373R cells.
Conclusions: Glioma cells expressing low levels of adenovirus receptors might survive and proliferate to recur after repeated adenoviral transduction, even if the adenoviral transduction is effective at first. Changing the tropism of vectors is a potent method to overcome resistance.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are great hopes that gene therapy can manage malignant tumors, including gliomas, and various approaches to gene therapy are being tested (for review, see Ref. 3 ). High-grade gliomas show very malignant biological characteristics. The 5-year actuarial survival rate for glioblastomas is only 7.6%, despite adjuvant radiation and chemotherapy in addition to surgical removal (4) . Adenoviral vectors carrying apoptosis-inducing genes are very attractive for the treatment of malignant gliomas. Tumor suppressor protein p53 has been reported to be a promising tool for the induction of apoptosis in tumor cells (5 , 6) . We have investigated the effects of gene transfer of wild-type p53 on malignant glioma cells. During this investigation, we have found that although U373MG cells are initially susceptible to p53 gene transduction, they become resistant after repeated gene transductions. This finding prompted us to examine the hypothesis that U373MG cells might become resistant due to the reduced transduction efficiency of adenoviral vectors. A fiber mutant vector that improves transduction efficiency on glioma cells was generated and used to examine whether it could overcome the resistance.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenoviral Vectors and Their Transduction Efficiency.
The XbaI-XbaI fragment of human wild-type p53 cDNA from pRC/CMV (Invitrogen), provided by Dr. T. Takahashi (9)
, was inserted into the NheI-SmaI site of pCIcc (Promega, Madison, WI; Ref. 10
) to generate pCIhp53. The human p53 expression cassette from pCIhp53 was then ligated to the ClaI site of cosmid pAxcw, resulting in the pAxCI-hp53 cosmid.
The XbaI-XbaI fragment of human wild-type p53 cDNA and EcoRI- and AvaI-digested pCAcc were treated with T4 polymerase and ligated by T4 ligase to establish pCAhp53. The human p53 expression cassette from pCAhp53 was then ligated to the ClaI site of cosmid pAxcw, yielding the pAxCA-hp53 cosmid.
The cosmid constructs for AxCAZ2-F/wt (an adenoviral vector possessing the reporter LacZ gene) and AxCAZ2-F/K20 (a mutant vector with a linker and a stretch of 20 lysine residues at the COOH-terminal of the fiber protein) were described in detail by Yoshida et al. (11) .
Recombinant adenoviruses were generated by cotransfection of cosmids by the method described by Miyake et al. (12) . Titers of infective adenovirus particles were evaluated by the standard plaque-forming assay with 293 cells after the vectors were propagated.
Adenovirus-mediated gene transduction was performed by a method described previously (10) , and the transduction efficiency of the adenoviral vectors was evaluated by X-Gal3 staining using either AxCAZ2-F/wt or AxCAZ2-F/K20 (11 , 13) , both of which possess the LacZ gene as a reporter. Briefly, glioma cells were seeded in 24-well plates on day 0. The cells were infected with AxCAZ2-F/wt for 2 h at various MOIs and washed twice with PBS, and culture medium was added on day 1. X-Gal staining was performed on day 3, and the number of positive cells per 300 cells in three fields (100 cells/field; magnification, x400) for each well was counted.
Selection of U373MG Cells Resistant to AxCIp53.
A 10-cm-diameter dish of confluent U373MG cells was infected at a MOI of 1000. The adherent cells were fed fresh, complete medium at 4-day intervals. The cells were further infected two times with AxCIhp53 (an adenoviral vector expressing the human p53 gene) at a MOI of 1000 after the apparently healthy cell population had grown out. The resistant cell line selected by this method was named U373R.
Flow Cytometry.
The expression of integrins vß3, vß5, and ß3 was analyzed with CELLQuest software using a FACScan (Becton Dickinson, San Jose, CA). Cells in exponential growth were washed three times with PBS and harvested in a single cell suspension using PBS containing 0.5 mM EDTA alone. The harvested cells were centrifuged and washed twice with PBS containing 5% FBS. Then, 5 x 105 cells were incubated with primary antibodies for 30 min at room temperature, further incubated with FITC-labeled secondary antibody for 30 min at room temperature, and washed twice with PBS containing 5% FBS. The cells were then analyzed by flow cytometry. Ten thousand cells were counted in each analysis in this experiment.
Chromatographically purified mouse IgG1 and mouse antihuman integrin vß3 and vß5 monoclonal antibodies were purchased from Chemicon International, Inc. (Temecula, CA). Mouse antihuman integrin v (CD51) and ß3 (CD61) monoclonal antibodies were obtained from Immunotech (Marseille, France). FITC-labeled antimouse IgG was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Western Blotting.
The protein expression level of integrin ß5 was evaluated by Western blotting as described previously (14)
. Briefly, cultured cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA, 0.5% NP40, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride] for 30 min on ice and centrifuged at 15,000 rpm for 30 min. Protein concentration was analyzed by a DC protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. A total of 50–200 µg of protein was extracted from 2 x 106 cells. The cell lysates were then frozen quickly and stored at -80°C until use for further analysis. An equal amount of protein (15 µg) from each extract was resolved by electrophoresis on 10% polyacrylamide gels and electroblotted to Immobilon transfer membranes (Millipore, Bedford, MA) at a constant current of 400 mA for 90 min. The membranes were blocked with 5% nonfat dried milk in PBS for 2 h and then incubated with the primary antibodies at a 1:1000 dilution at 4°C overnight. The membranes were washed in PBS, alkaline phosphatase-conjugated secondary antibodies were added at a 1:250 dilution, and the membranes were incubated for 2 h at room temperature. The immune signal was detected using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium membrane phosphatase substrate (KPL, Gaithersburg, MD).
Rabbit anti-integrin ß5 subunit polyclonal antibody was purchased from Chemicon International, Inc.
Detection of CAR mRNA by RT-PCR.
Total cellular RNA was isolated by Isogen (Nippon Gene) according to the manufacturer’s protocol. Two µg of total cellular RNA were reverse transcribed using oligo(dT)12–18 as primer (Boehringer Mannheim, Indianapolis, IN) and Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim) in a 20-µl reaction. One µl of reverse transcript was used directly for each reaction. In a 50-µl reaction, 25 nmol of forward and reverse primers; 200 µM each of dATP, dCTP, dGTP, and dTTP (Pharmacia); and 47 µl of super Taq premix kit (Sawady Technology, Tokyo, Japan) were used for PCR. The PCR primers used were as follows: (a) CAR sense primer, 5'-CCG-GAA-TTC-ACC-A100TG-GAT-TTC-GCC-AGA-AGT-TTG-AGT-ATC-ACT128-3'; (b) CAR antisense primer, 5'-CGC-GGA-770TCC-AGC-TTT-ATT-TGA-AGG-AGG-GAC-AAC-GTT741-3'; (c) ß-actin sense primer, 5'-886TGG-AAT-CCT-GTG-GCA-TCC-ATG-AAA-C910-3'; and (d) ß-actin antisense primer, 5'-1234TAA-AAC-GCA-GCT-CAG-TAA-CAG-TCC-G1210-3' (15)
. EcoRI and NcoI sites were added to the 5' end of the CAR sense primer, and a BamHI site was added to the 5' end of the CAR antisense primer. Reactions were incubated in a thermal cycler for 20, 30, and 35 cycles (denaturation, 60 s, 95°C; annealing, 60 s, 60°C; and extension, 90 s, 72°C). The same samples were amplified with ß-actin-specific primers to obtain a ratio of CAR:housekeeping gene signal for semiquantitative analysis. The signal intensity was measured by NIH Images version 1.55. Total RNA in 293 cells was used as a positive control for CAR expression.
Cell Adhesion and Growth.
The ability of cells to adhere to culture plates was estimated by counting the number of cells at 1 and 4 h after seeding 2 x 105 cells in each well of 6-well culture plates (Iwaki Glass). Cells in exponential growth were washed three times with PBS, treated with PBS containing 0.5 mM EDTA alone, harvested in a single cell suspension with culture media containing 10% fetal bovine serum, and deposited in the plates for adhesion. After the cells were cultured for an appropriate time at 37°C, they were trypsinized, harvested, and counted using a cell counter (CDA500; Sysmex, Kobe, Japan). For the growth assay, 1 x 104 cells were seeded on day 0. The numbers of cells were counted on days 2, 4, and 6. The culture medium was changed on days 2 and 4.
Statistical Analysis.
Data are shown as the mean ± SD. The ED50 was calculated using a logarithmic equation (CA-Cricket Graph III, version 1.5J; Computer Associates International). Data were analyzed by two-sample t test, one-way factorial ANOVA, or two-way factorial ANOVA using StatViewJ, version 4.5 (Abacus Concepts, Berkeley, CA). P < 0.05 was considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the effects of AxCIhp53 on the growth of U373MG cells. AxCIhp53 strongly inhibited the growth of U373MG cells even at a MOI of 100 (P < 0.005 compared with AxCAZ2-F/wt transduced cells). Only 3.6% of the seeded cells survived on day 4 at a MOI of 500. FACScan analysis using the terminal deoxynucleotidyl transferase-mediated nick end labeling method and transmission electron microscopy revealed the induction of apoptosis in U373MG cells by transduction with AxCIhp53 but not AxCAZ2-F/wt on day 2 (data not shown).
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Transduction Efficiency of AxCAZ2-F/wt for U373R Cells.
Fig. 2
shows the effects of AxCIhp53 on the growth of U373R cells at a MOI of 1000. The growth of AxCIhp53-transduced U373R cells was similar to that of AxCAZ2-F/wt-transduced cells. The number of AxCIhp53-transduced U373R cells grew 17.5-fold from the level of seeded cells by day 6, demonstrating the ineffectiveness of AxCIhp53 on U373R cells. To clarify the mechanism by which U373R cells acquired resistance, we first evaluated the transduction efficiency of adenoviral vectors for U373R cells. Fig. 3
shows the results of transduction efficiency of AxCAZ2-F/wt for U373R cells and U373MG cells, as estimated by X-Gal staining. The average ED50 of U373MG cells was 31.24, and 99.42% of cells were positive for X-Gal staining at a MOI of 250. In contrast, the average ED50 of U373R cells was 794, significantly greater than that for U373MG cells (P < 0.05). The transduction efficiency of U373R cells was 92.8% even at a MOI of 5000.
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vß3 and integrin vß5. The expression levels of integrins vß3 and vß5 were evaluated by flow cytometry using FACScan (Fig. 4A)
. U373MG cells abundantly expressed both integrins vß3 and vß5, whereas the expression levels of both integrins vß3 and vß5 were much lower in U373R cells. We further studied the expression levels of each subunit of the integrins, v, ß3, and ß5. Fig. 4A
shows the expression levels of the v and ß3 subunits estimated by flow cytometry, both of which were lower in U373R cells than in U373MG cells. Western blot analysis revealed that the expression level of integrin subunit ß5 was much lower in U373R cells than in U373MG cells (Fig. 4B)
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. Human embryonic kidney 293 cells, adenoviral vector-producing cells, were used as a positive control in this experiment. No signal was detected after 20 cycles of amplification with primers specific for CAR as well as primers specific for ß-actin in any of the three cell lines. After 30 cycles of amplification, U373R cells showed a weak PCR signal with primers specific for CAR, with a signal intensity similar to that of U373MG cells. The U373R cell line gave a much weaker PCR signal with primers specific for CAR than did the human embryonic kidney 293 cell line (P < 0.005), although the PCR signals with primers specific for ß-actin were similar in both. The PCR signals became similar among the three cell lines after 35 cycles of amplification (P = 0.08).
Characteristics of U373R Cells.
The morphology of U373R cells is quite different from that of the parental U373MG cells. The U373MG cells appear fan-shaped and show distinct ruffling around the cytoplasm, whereas U373R cells form short processes and show little ruffling (data not shown).
Fig. 5A
shows the abilities of U373MG and U373R cells to adhere to the culture plate. U373MG cells adhere readily to the culture plate. After 1 h of incubation, 86.5% of seeded U373MG cells adhered to the culture plate, and by 4 h, all of the seeded cells had attached themselves to the culture plate. In contrast, U373R cells were less capable of attaching themselves to the culture plate. Only 56.5% and 62.0% of seeded U373R cells adhered to the culture plate after 1 and 4 h of incubation, respectively (P < 0.005 and P < 0.0001, respectively).
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. The number of U373R cells counted on days 2, 4, and 6 was 80.4%, 73.7%, and 83.4% that of U373MG cells, respectively (P < 0.01, 0.005, and 0.05 on days 2, 4, and 6, respectively).
Transduction Efficiency of AxCAZ2-F/K20 and the Effect of AxCAhp53-F/K20, a Mutant Vector Carrying the p53 Gene.
As reported previously, we generated a fiber mutant adenoviral vector that has a linker and a stretch of 20 lysine residues added to the COOH terminus of the fiber. The vector with a fiber mutant, AxCAZ2-F/K20, showed remarkably enhanced efficiency of gene transduction in human glioma cells (11)
. We transduced AxCAZ2-F/K20 into U373R cells to determine whether the vector could improve the transduction efficiency for the U373R cell line as for other glioma cell lines. The ED50 in U373MG and U373R cells was 31.24 and 794, respectively, when the cells were transduced with AxCAZ2-F/wt. AxCAZ2-F/K20 significantly improved the transduction efficiency for both U373MG and U373R cell lines. The ED50 of U373MG and U373R was 7.03 and 63.2, respectively (Fig. 6A)
. AxCAZ2-F/K20 decreased the ED50 of U373MG and U373R cells to 4.44- and 12.56-fold of AxCAZ2-F/wt, respectively. Fig. 6B
shows the results of X-Gal staining of U373R cells transduced with either AxCAZ2-F/wt or AxCAZ2-F/K20 at a MOI of 250. Ninety-three percent of U373R cells treated with AxCAZ2-F/K20 were positive. In contrast, only 22.7% of U373R cells treated with AxCAZ2-F/wt were positive.
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. AxCAhp53-F/K20 successfully induces apoptosis in U373R cells and inhibits their growth. The number of cells adherent to the culture plates was 1.47, 0.90, and 0.46 of the seeded cells at MOIs of 100, 250, and 500, respectively, whereas the number of untransduced cells increased to 3.06-fold of seeded cells on day 2. The ratios were 1.17, 0.06, and 0.00 on day 6, and the number of untransduced cells increased to 16.6-fold.
AxCIhp53 possesses a CMV promoter (CI), whereas AxCAhp53-F/K20 possesses a chicken ß-actin promoter (CA). To confirm that the apoptosis induced by AxCAhp53-F/K20 is due to improved transduction efficiency rather than to the effect of the CA promoter, we compared the effects of AxCAhp53-F/wt and AxCIhp53 on U373R cells (Fig. 6D)
. As shown in the figure, the effects of AxCAhp53-F/wt were similar to those of AxCIhp53, demonstrating that the dramatic induction of apoptosis is due to an improvement in transduction efficiency rather than an effect of the CA promoter.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v and ß3, and integrin vß5 than normal brain tissue (22, 23, 24)
, it would be advantageous to use adenoviruses as a vector for gene therapy for gliomas. However, here we provide the first evidence that human tumor cells expressing low levels of adenovirus receptors will survive and proliferate to recur after repeated gene transduction, although the gene transduction is effective at first.
Adenovirus infection starts with the attachment of the viral fiber protein to its cellular receptor, CAR (for review, see Refs. 16
and 17
). After fiber-mediated attachment to the cell, the penton base binds via an RGD motif to the vß3 and vß5 integrin receptors. The cell surface-bound viruses are internalized via receptor-mediated endocytosis. The viruses are then released into the cytosol and bind to the nuclear pore complexes. Next, they release their DNA into the nucleus, and DNA transcription starts (for review, see Ref. 17
). The inhibition of any of these steps will cause resistance to infection. In the present study, the ED50 of AxCAZ2-F/wt for U373R was 25.4 times higher than that for U373MG cells, suggesting that reduced transduction efficiency is the major cause of the acquired resistance. FACScan analysis, Western blotting, and RT-PCR revealed that U373R cells express reduced levels of penton base receptors and integrins vß3 and vß5, without changing the expression levels of the fiber knob receptor CAR. Integrins vß3 and vß5 are known to be receptors for vitronectin, the main adhesive protein in routine cell culture media (25)
. Cell interaction with vitronectin may induce cell spreading and migration and have positive effects on cell growth (26)
. U373R cells showed little ruffling, grew more slowly, and were less capable of adherence to the culture plate than parental U373MG cells. All of these characteristics are compatible with the reduced expression levels of integrins, further supporting the notion that resistance is caused by low expression levels of integrins. The decrease in the expression levels of integrin subunits v and ß3 in U373R cells was not so prominent as that of integrins vß3 and vß5. Integrin subunit v forms heterodimers not only with integrin subunits ß3 and ß5 but also with integrin subunits ß1, ß6, and ß8. Similarly, integrin subunit ß3 forms a heterodimer with IIb (27)
. The total amounts of integrin subunits v and ß3 on the cell surface of U373R cells may not decrease as expected. Recently, other integrin subunits have been suggested to modulate adenovirus-mediated gene transfer (28)
. We have not examined the expression levels of integrins other than integrins vß3 and vß5, and other integrins might also contribute to the transduction efficiency of adenoviral vectors. CAR is indispensable for adenovirus adsorption (18
, 19)
, and the expression level of CAR is reported to be more critical for adenoviral infection than those of integrins (29, 30, 31)
. These reports focused mainly on the expression levels of CAR. None or few of the integrins were subjected to evaluation of their expression levels, although multiple integrins have the potential to mediate adenoviral endocytosis into cells (32)
. In this report, we estimated the expression levels of both CAR and integrins and found that the expression levels of integrins in U373R cells were lower than those in U373MG cells, whereas both cell lines showed similar expression levels of CAR. Our results clearly demonstrate the significance of the integrins on adenoviral infection under similar CAR levels. Pearson et al. (28)
also indicated the significance of integrin expression. According to Asaoka et al. (33)
, wild-type p53 may repress the expression levels of CAR. In our experiments, no significant difference in the expression levels of CAR was detected between U373R and U373MG cells, despite the fact that U373R cells were selected by wild-type p53. First of all, U373MG cells are very susceptible to wild-type p53, and few cells survive after transduction of the p53 gene. Secondly, adenovirally transduced gene expression is transient, and it is unlikely that the transduced gene affects exponentially growing cells. Therefore, adenovirally transduced p53 seems to have little effect on the expression levels of CAR in U373R cells. In our laboratory, the efficacy of adenoviral-delivered wild-type p53 tends to correlate at least with the expression level of CAR,4
as other laboratories have indicated (30
, 31)
, which would corroborate de novo sensitivity to restored wild-type p53 as a therapy for gliomas. However, there might also be other mechanisms that regulate susceptibility of glioma cells to wild-type p53.
A single round of gene delivery may not be sufficient to eradicate malignant tumors in clinical use, and multiple administration of vectors will be indispensable. However, we have shown the possibility that tumors become resistant to vectors by repeated administration because the population of cells with low expression levels of receptors will survive and proliferate irrespective of the genes transduced. To overcome this resistance, a new device for improving transduction efficiency is indispensable. There are several strategies to enhance the efficiency of adenovirus gene transduction. The simplest approach is to use higher MOIs to get higher transduction. This approach, although simple, is costly and laborious in clinical gene therapy situations. Moreover, dose-limiting vector-related toxicities (e.g., immune reaction, reaction to toxic adenovirus components, and replication-competent adenovirus) may limit the overall therapeutic index achievable with adenoviral vectors. Another approach is to test chemical methods and physical conditions to enhance adenovirus infection efficiency (34) . An alternative strategy is to develop adenovirus mutants. Mutations in the fiber knob or penton base will change the tropism of target receptors. The advantage of the F/K20 adenoviral vector on glioma cells was described previously in detail by Yoshida et al. (11) . In the present study, the F/K20 adenoviral vector also improved the transduction efficiency into U373R cells, and AxCAhp53-F/K20 strongly induced apoptosis in U373R cells. The results indicate the advantages of using F/K20 adenoviral vector not only for established glioma cell lines but also for cells whose receptors for F/wt adenoviral vectors are significantly reduced. Wickham et al. have generated either seven lysine residues (35) or variable numbers (36) of lysine residues at the COOH-terminal end of the fiber. The receptor molecules for these fibers with lysine oligomers are supposed to be the negatively charged cell surface molecules such as heparin and heparan sulfate. These heparan sulfate proteoglycans are expressed in large amounts in gliomas, especially in high-grade gliomas (37 , 38) . There might be a positive correlation between the expression levels of heparan sulfate proteoglycans and the transduction efficiency of F/K20 adenovirus vectors, leading to efficient gene transduction into human glioma cells as demonstrated by Yoshida et al. (11) . The expression levels of heparan sulfate proteoglycans may be independent of those of integrins, and heparan sulfate proteoglycans may remain abundant in U373R cells as they do in U373MG cells. Consequently, F/K20 adenovirus could improve the transduction efficiency of U373R cells and thereby induce apoptosis by p53 protein. Further study is necessary to elucidate the mechanism.
This report suggests the disadvantages of repeated administration of the same vector and further advantages of F/K20 adenovirus. In general, tumor cells are heterogeneous in terms of levels of integrins. Therefore, some cells intrinsically resistant to adenoviral infection because of low expression of adenovirus receptors will survive and proliferate to recur, which is very critical in clinical use. This problem may not be limited to gliomas but could also be applicable to other malignant tumors. It may also be said that this is not only a problem with adenoviral vectors with wild-type fiber knob but also in other vectors that are integrated through specific receptors. Changing the tropism of vectors is a potent method to overcome resistance. In addition to low expression levels of vector receptors, the Achilles’ heel of this and all p53-based gene transfer approaches is that it is simply not possible to deliver the transgene into all cells. Therefore, these approaches will always have a population of tumor cells that will regrow. Multiple gene therapy strategies (e.g., suicide or prodrug strategies, immunogene therapy, and antiangiogenesis) should also be applied together with this replacement/therapeutic gene transfer.
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FOOTNOTES |
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1 Supported in part by grants from the Ministry of Education, Culture, and Science of Japan and by grants from the Ministry of Health and Welfare of Japan. 
2 To whom requests for reprints should be addressed, at Department of Neurosurgery, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81-3-5803-5266; Fax: 81-3-5803-0140; E-mail yamamoto.nsrg{at}med.tmd.ac.jp
3 The abbreviations used are: X-Gal, 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside; CAR, Coxsackievirus and adenovirus receptor; MOI, multiplicity of infection; ED50, MOI for transduction of 50% of the population; RT-PCR, reverse transcription-PCR; CMV, cytomegalovirus.
Received 9/ 5/01; revised 12/17/01; accepted 12/20/01.
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vß5 integrin. Biochem. Biophys. Res. Commun., 240: 88-92, 1997.[CrossRef][Medline]
5ß1-mediated adenovirus infection is enhanced by the integrin-activating antibody TS2/16. J. Virol., 71: 6204-6207, 1997.[Abstract]
v-integrins. Hum. Gene Ther., 9: 507-520, 1998.[Medline]
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; transduction with AxCIhp53, •; no transduction (control), x. The data shown are representative of two independent experiments. Values are the means ± SD (bars) of duplicate determinations.
x174 DNA HaeIII digest (New England Biolabs, Inc., Bevery, MA); Lane 2, U373MG cells; Lane 3, U373R cells; Lane 4, 293 cells. The data shown are representative of three independent experiments.
