From the Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery and the ¶ Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and the § Introgen Therapeutics, Inc., Houston, Texas 77054
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ABSTRACT |
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A synthetic GAL4-responsive promoter consisting of five GAL4-binding sites and a TATA box (GAL4/TATA) was evaluated for its transcriptional activity in an adenoviral backbone using luciferase as the reporter. Basal luciferase activities in vitro were the same for cells infected with either adenovirus-containing luciferase cDNA driven by GAL4/TATA (Ad/GT-Luc) or adenovirus-containing luciferase cDNA not driven by a promoter (Ad/PO-Luc). In vitro induction of GAL4/TATA by coinfection of cells with adenovirus expressing the GAL4/VP16 fusion protein (Ad/3-phosphoglycerate kinase (PGK)-GV16) was dose-dependent and reached as high as 4 × 104- to 9 × 104-fold above basal levels when GAL4/TATA and GAL4/VP16 were delivered at a ratio of 10:1. In vivo studies in Balb/c mice showed no detectable luciferase activities in liver or other tissues examined in mice infused with either Ad/GT-Luc or Ad/PO-Luc. High levels of luciferase activity were, however, elicited when animals were infused with Ad/GT-Luc and Ad/PGK-GV16. Together, these results suggest that combination of the GAL4 gene regulatory system with adenovirally mediated in vivo gene delivery may be applicable to the in vivo evaluation of promoter activities and in vivo targeting of gene expression.
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INTRODUCTION |
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The yeast GAL4 gene expression system is one of the most thoroughly studied eukaryotic transcriptional regulatory systems and has been used as a research tool in a variety of fields (1). Yeast regulatory protein GAL4 binds specifically to the GAL upstream activating sequence (UASG) to activate transcription of the adjacent GAL1 and GAL10 genes. It was consequently found that four related 17-base pair dyad symmetrical sequences within the UASG are responsible for GAL4-specific binding and transcriptional activation (2). In other research, a synthetic GAL4-responsive promoter containing GAL4-binding sites and a TATA box with minimal transcriptional activity in mammalian cells was found to be efficiently transactivated in the presence of a GAL4 transactivator (3-5). Moreover, fusing the DNA binding domain of GAL4 protein to a highly acidic portion of the herpes simplex virus protein VP16 was shown to greatly increase the ability of GAL4 to activate transcription while preserving its DNA-binding specificity (6). This system was successfully used to target gene expression in Drosophila (7, 8) and transgenic mice (9) and to control gene expression in mammalian cells (5, 10). More recently, the technology has been used to induce the expression of transgenes delivered via the herpesvirus (11) and to shut off the expression of viral genes (12). Yet even though the synthetic GAL4/TATA promoter has been well characterized in cultured cells in vitro (3-5), little is known about its basal activity and GAL4-mediated inducibility in various organs in vivo. Therefore, to characterize the basal transcriptional level and inducibility of the GAL4/TATA promoter, we constructed adenoviral vectors containing reporter luciferase cDNA driven by GAL4/TATA or expressing GAL4/VP16 fusion protein. Then we tested the induction of the GAL4/TATA promoter in vivo by adenovirally mediated gene codelivery. As a result, we found that the transcriptional activities of GAL4/TATA promoter were undetectable in a variety of organs in the absence of GAL4/VP16 and induced to several orders of magnitude in its presence. Our results therefore indicate that combining the GAL4 regulatory gene expression system with adenovirally mediated gene delivery might be a powerful tool for evaluating promoter activities and specificities in vivo.
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MATERIALS AND METHODS |
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Construction of Recombinant Adenovirus Vectors-- Construction of the Ad/RSV-Luc vector has been reported elsewhere (13). Vectors Ad/GT-Luc, Ad/GT-LacZ, Ad/PO-Luc, and Ad/PGK-GV161 (Table I) were constructed as described previously (14). In brief, Ad/GT-Luc was constructed by placing luciferase cDNA from pGL3-Basic Vector (Promega, Madison, WI) at the downstream of the GAL4/TATA promoter (GT) derived from pG5EC (a gift from Dr. I. Sadowski, University of British Columbia, Vancouver, BC, Canada). A vector containing only the luciferase gene and a poly(A) signal sequence but no promoter (Ad/PO-Luc) was also constructed and used as a negative control. To induce GAL4/TATA activities in vitro and in vivo, an adenoviral vector (Ad/PGK-GV16) expressing a GAL4/VP16 fusion protein (GV16) driven by a housekeeping promoter (PGK) was constructed. This is accomplished by placing the cDNA for the GAL4/VP16 fusion protein excised from plasmid pM2/VP16 (also a gift from Dr. Sadowski) at the downstream of the mouse 3-phosphoglycerate kinase (PGK) gene promoter (15). Recombinant virus from a single plaque was identified by DNA analysis, then expanded in 293 cells, and twice purified by ultracentrifugation on a cesium chloride gradient. Virus titers were determined by both optical absorbance at A260 (one A260 unit = 1012 particles/ml) and by median tissue culture infective dose (TCID50) assays as described previously (16). Titers determined by TCID50 assay were used in subsequent experiments. All viral preparations were tested for E1+ adenovirus contamination by polymerase chain reaction (17) and for cross-contamination by Ad/RSV-Luc by polymerase chain reaction using primers located in RSV long terminal repeat and luciferase cDNA. No contamination was detected in viral preparations used in our experiments.
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In Vitro Studies--
H1299 and A549 cells were plated at
densities of 2 × 106/100-mm plate. The cells were
then infected with recombinant adenoviral vectors at a multiplicity of
infection (MOI)1 of 10 and grown at 37 °C. Cells were
harvested 48 h after infection and frozen at 20 °C until used
for protein and luciferase assays.
Animal Studies-- All animals were cared for according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 85-23) and the institutional guidelines of The University of Texas, M. D. Anderson Cancer Center. In vivo infusion of adenoviral vectors into and subsequent tissue removal from Balb/c mice were done as described previously (14).
Biochemical Analysis-- Cultured cells were lysed or tissues from Balb/c mice were homogenized in luciferase assay buffer. Cell or tissue debris was then removed by microcentrifugation. Protein concentrations were determined using a kit from Life Technologies, Inc. according to the manufacturer's instructions. Luciferase activities were determined using a luminometer and a luciferase assay system according to the manufacturer's instructions (Promega, Madison, WI).
DNA Assays-- DNA was isolated from tissues of Balb/c mice infused with vectors or buffer alone. A semiquantitative assay of viral DNA in the tissues was performed via the polymerase chain reaction as described previously (14). The following polymerase chain reaction primers were used: 5'-TGCCTAGGCAAAATAG-3' and 5'-CATCATCAATAATATAC-3', located at the right end of the adenovirus genome.
Statistical Analyses-- Differences between treatment groups were assessed by analysis of variance using SPSS software (SPSS, Inc., Chicago, IL). P values less than 0.05 were considered significant.
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RESULTS AND DISCUSSION |
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GAL4/TATA Activities in Cultured Human Cell Lines-- In our studies, luciferase was used as a reporter to evaluate GAL4/TATA promoter activity in vivo because firefly luciferase assays are sensitive enough to detect even trace levels of promoter activity. Established human lung carcinoma cell lines H1299 and A549 were used to determine basal activity levels of GAL4/TATA in the adenoviral backbone. In brief, cells were infected with Ad/GT-Luc, Ad/PO-Luc, Ad/RSV-Luc, and Ad/GT-LacZ at MOI 10. Preliminary experiments showed that over 80% of the H1299 cells and over 20% of the A549 cells were transduced at MOI 10. Cells were then harvested 48 h after infection. Mock-infected cells were used as background controls. Luciferase activity was measured and expressed as relative light units/µg of cellular protein (Fig. 1). In both H1299 and A549 cells, the luciferase activity was significantly higher in cells infected with Ad/GT-Luc or Ad/PO-Luc than in cells infected with Ad/GT-LacZ (p < 0.05). These results are consistent with earlier reports of low basal activity of GAL4/TATA-driven reporters in HeLa and Chinese hamster ovary cells (3). Nevertheless, the same basal level of luciferase activity was observed in constructs without promoters (Ad/PO-Luc), thus suggesting that the basal activity may be unrelated to the GAL4/TATA promoter.
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In Vitro Induction of GAL4/TATA Activities-- It has been reported previously by others that expression of the GAL4-VP16 fusion protein via plasmid cotransfection increases the in vivo induction of GAL4/TATA activity at least 1200-fold (3). To test whether the induction of GAL4/TATA activities might be similarly induced through adenovirally mediated gene codelivery, H1299 and A549 cells were infected at MOI 10 with a mixture of Ad/GT-Luc and Ad/PGK-GV16 in ratios of 1000:1, 100:1, and 10:1. Luciferase activities were then determined 48 h postinfection. As the ratio moved from 1000:1 to 10:1, the luciferase activities increased about 1 × 104- to 4.1 × 104-fold in H1299 cells and 75- to 9 × 104-fold in A549 cells. In contrast, no change in luciferase activity was observed in cells infected with mixtures of Ad/PO-Luc and Ad/PGK-GV16 at the same ratios. Moreover, mixing Ad/GT-LacZ or Ad/RSV-Luc with Ad/PGK-GV16 at a ratio of 10:1 had no significant effect on luciferase activities either. Thus, induction by adenovirally mediated gene codelivery appears to be dose-dependent and highly effective. In addition, the difference between the two cell lines in their response to the induction may be reflected by their sensitivity to adenoviral infections (12).
Basal GAL4/TATA Promoter Activity in Adenoviral Vectors in Vivo in the Balb/c Animal Model-- To determine the basal level of GAL4/TATA promoter activity in vivo, 6-8-week-old female Balb/c mice were infused through the tail vein with Ad/RSV-Luc, Ad/PO-Luc, Ad/GT-Luc, or Ad/GT-LacZ at a dose of 1 × 109/mouse. Mice infused with phosphate-buffered saline alone served as negative controls. All mice were killed 2 days after infusion, after which liver, spleen, lung, kidney, intestine, ovary, and brain were collected and homogenized for luciferase and protein assays. Luciferase activities were readily detected in liver, lung, spleen, heart, ovary, and kidney of mice infused with Ad/RSV-Luc. Levels of luciferase activities were absent or very low in all organs tested in animals infused with other viral constructs, including those infused with Ad/GT-Luc (Fig. 2). Together, these results demonstrated that the GAL4/TATA promoter was not active in vivo in the adenoviral backbone.
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In Vivo Induction of GAL4/TATA Promoter Activity-- To test whether GAL4/TATA promoter activity could be induced in vivo by adenovirally mediated gene codelivery as we demonstrated in vitro, a group of Balb/c mice infused with 1 × 109 plaque-forming unit of vector mixtures containing Ad/GT-Luc and Ad/PGK-GV16 (10:1) was included in the animal experiment mentioned above. Mice infused with Ad/RSV-Luc plus Ad/PGK-GV16 or Ad/PO-Luc plus Ad/PGK-GV16 at the same dose and ratio were used as controls. Although no luciferase activity was detected in mice infused with Ad/GT-Luc alone, luciferase activities were dramatically induced in all organs of mice treated with Ad/GT-Luc plus Ad/PGK-GV16. The induction ranged from 1.4 × 104-fold in brain to 9.3 × 106-fold in liver. No induction was found in animals treated with Ad/PO-Luc plus Ad/PGK-GV16, and no significant differences in luciferase activities were observed between animals infused with Ad/RSV-Luc alone or Ad/RSV-Luc plus Ad/PGK-Luc. Thus, these results demonstrated that in vivo induction of GAL4/TATA by GAL4/VP16 through adenovirally mediated gene codelivery is highly specific and efficient. These results also ruled out loss of vector infectivity as the reason why luciferase activity was undetectable in animals treated with Ad/GT-Luc alone.
In conclusion, the undetectable transcriptional activity of GAL4/TATA in vivo supports our hypothesis that viral genes can be inactivated by promoter replacement (12), a concept that may be used for the development of viral vectors or vaccines. Our results also suggest that combination of the GAL4 gene regulatory system and adenovirally mediated gene delivery will be sensitive tools for evaluating promoter activities and for inducing or amplifying gene expression in vivo. Thus, this technology may be applied in two ways: 1) in vivo evaluation of promoter activity and 2) targeted expression of transgenes. In the first case, tissue- or cell type-specific promoters can be easily evaluated by substituting a testing promoter for the PGK promoter to drive GAL4/VP16 cDNA. The sensitivity of in vivo measurement for a testing promoter could be markedly increased through the magnification of gene expression via the GAL4 regulatory system. As for the targeted expression of transgenes, replacing the PGK promoter with a tissue- or cell type-specific promoter may result in high levels of tissue- or cell type-specific expression of the transgenes driven by the GAL4/TATA promoter, thus overcoming the fact that most tissue-specific promoters have relatively low activities. Moreover, because current vector technology allows several expression cassettes to be incorporated into one vector, the efficiency of gene codelivery might be even further increased. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. I. Sadowski for providing us the plasmids used in this study, Jude Richard for editorial review, Patricia Koch for technical assistance, and Monica Contreras for assistance in preparing this manuscript.
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FOOTNOTES |
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* This work was funded by a grant from The University of Texas Physicians Referral Service, National Institutes of Health Grant P50-CA70907 for a Specialized Program of Research Excellence in Lung Cancer, and a sponsored research agreement with Introgen Therapeutics, Inc. (Austin, TX).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Texas
M. D. Anderson Cancer Center, Dept. of Thoracic and Cardiovascular Surgery, Box 109, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-6932; Fax: 713-794-4901.
1 The abbreviations used are: PGK, 3-phosphoglycerate kinase; MOI, multiplicity of infection; RSV, Rous sarcoma virus.
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REFERENCES |
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