[CANCER RESEARCH 62, 1271–1274, March 1, 2002] Advances in Brief The flt-1 Promoter for Transcriptional Targeting of Teratocarcinoma 1 Gerd J. Bauerschmitz, Dirk M. Nettelbeck, Anna Kanerva, Andrew H. Baker, Akseli Hemminki, Paul N. Reynolds, and David T. Curiel 2 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294 [G. J. B., D. M. N., A. K., A. H., P. N. R., D. T. C.], and Department of Medicine and Therapeutics, University of Glasgow, Glasgow, GI1 GNT, Scotland, United Kingdom [A. H. B.] Abstract Flt-1, a receptor for vascular endothelial growth factor, is known to display dysregulated expression in both tumor vasculature and tumor cells per se, suggesting that the flt-1 promoter might be a useful candidate to achieve tumor-specific transgene expression. In addition, adenoviral vectors containing transgenes under the control of the flt-1 promoter achieve very low levels of expression in the normal liver, the major organ responsible for blood clearance of adenoviruses and inadvertent trans- gene-related toxicity. Thus, we assessed the ability of adenoviral vectors containing the flt-1 promoter to achieve transgene expression in a range of gynecological and breast tumor lines. High transgene expression levels were detected in teratocarcinoma lines, correlating with levels of flt-1 mRNA. These results suggest that the flt-1 promoter could be useful for transcriptionally targeted gene expression to teratocarcinoma, and that evaluation in other flt-1-positive tumors is warranted. Introduction A variety of gene therapy approaches for cancer have been under- taken based on in situ molecular chemotherapy, where systemically administered prodrugs are locally converted to their toxic counter- parts. Critical to the achievement of an acceptable therapeutic index is the restriction of toxic gene expression in tumor cells (1, 2). In this regard, ectopic vector localization, with consequent expression of the delivered genes at non-tumor sites, can induce treatment-limiting toxicities (3–5). These considerations are especially relevant for Ad 3 vectors, which exhibit a marked tropism for the liver when adminis- tered i.v. (3, 5). Thus, strategies to target transgene expression have been explored for Ad vector-based gene therapy approaches for can- cer (1, 4, 6). This strategy of transcriptional targeting is based upon the use of promoters that display preferential activity in tumor cells (7). Ideally, these promoters should be capable of substantially lim- iting the expression of transgenes in the liver as a means to mitigate the potential toxicity of Ad-delivered toxic genes at this site. Thus, an ideal promoter for transcriptional targeting applications exhibits a “tumor on/liver off” phenotype when incorporated into an Ad vector (1). Tissue- and tumor-selective promoters have been defined that exhibit this desirable phenotype. In this respect, the gene for the vascular endothelial growth factor receptor type I (flt-1) has been shown recently to have dysregulated expression in tumors (8 –10). Furthermore, our recent studies have demonstrated that the flt-1 promoter exhibits a “liver off” phenotype when used in Ad vectors (11). These two considerations have suggested its utility as a promoter for use in Ad-based gene therapy applications for cancer, including gynecological malignancies. Our studies imply that the flt-1 promoter is active in a subset of this class, specifically teratocarcinomas, suggesting that this promoter may be useful for gene therapy of a defined subset of cancers based on a common pathobiology. Materials and Methods Cell Culture. Hey, SKOV3.ip1, and OV-4 ovarian adenocarcinoma cell lines were kind gifts from Drs. Judy Wolf, Janet Price (both of M. D. Anderson Cancer Center, Houston, TX), and Timothy J. Eberlein (Harvard Medical School, Boston, MA), respectively. The other cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were cul- tured in the recommended growth medium and maintained in a humidified 37°C atmosphere containing 5% CO 2 . Viruses. Ad5flt-1luc1 and AdCMVluc1 are replication-defective adenovi- ruses with a luciferase reporter gene, driven by the flt-1 or CMV promoters, respectively, in the E1 region (12). The viruses are isogenic and were propa- gated on 293 cells. Purification was done with double CsCl gradients using standard methods and titered for VPs with spectrophotometry. Functional titer (pfu) was determined with plaque assay with an initial overnight infection of 293 cells. The viruses had the following titers: Adflt-1luc1, 6.0  10 11 VPs/ml, 1.2  10 10 pfu/ml, and 50 VPs/pfu; and AdCMVluc1, 9.4  10 11 VP/ml, 1.9  10 10 pfu/ml, and 49 VPs/pfu. For the replication-defective viruses Ad5flt-1LacZ and Ad5CMVLacZ, the reporter gene is LacZ, driven by the identical promoters as described before (11). The viruses are isogenic and had the following titers: Ad5flt-1LacZ, 2.0  10 12 VPs/ml, 2.0  10 10 pfu/ml, and 100 VPs/pfu; and Ad5CMVLacZ, 5.0  10 12 VPs/ml, 5.0  10 10 pfu/ml, and 100 VP/pfu. Luciferase Assay. Cell lines were plated on day 1 at 25,000 cells/well on 24-well plates in 1 ml of GM. On day 2, cells were infected with 5, 50, or 500 pfu/cell for 2 h in 200 l of 2% GM on a rocker. Afterward, cells were washed once with 1 ml of PBS, and 1 ml of GM was added per well. After 24 h, the GM was removed, cells were lysed with 200 l of lysis buffer (Reporter Lysis Buffer; Promega Corp., Madison, WI) and freeze-thawed once. Twenty l of these samples were mixed with 100 l of luciferase assay reagent (Reporter Lysis Buffer; Promega) and measured with Berthold Lumat LB9501. Stand- ardization was accomplished by setting the values obtained with CMV pro- moter as 100% for each cell line. LacZ Staining. Cell lines were plated on day 1 at 50,000 cells/well on 24-well plates in 1 ml of GM. On day 2, cells were infected with 500 pfu/cell for 2 h in 200 l of 2% GM on a rocker. Afterward, cells were washed once with 1 ml of PBS, and 1 ml of GM was added per well. After 24 h, the GM was removed, and cells were washed twice with PBS. Cells were fixed for 15 min with 0.5% glutaraldehyde and washed twice with PBS. Cells were stained for 2.5 h with standard 5-bromo-4-chloro-3-indolyl--D-galactopyranoside so- lution (containing 40 l 2% 5-bromo-4-chloro-3-indolyl--D-galactopyrano- side, 10 l of 0.3 M potassium ferricyanide, 10 l of 0.3 M potassium ferrocyanide, and 940 l of PBS/ml), washed again for 10 min with PBS, and fixed a second time with 10% buffered formalin for 30 min. Pictures were taken by bright field microscopy at 10. Received 12/7/01; accepted 1/10/02. 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 This work was supported by Deutsche Forschungsgemeinschaft Grants BA2076/1-1, BA2076/1-2 (both to G. J. B.), and NE832/1 (to D. M. N.) and grants from the Damon Runyon-Walter Winchell Cancer Research Fund, the Sigrid Juselius Foundation, the Emil Aaltonen Foundation, the Maud Kuistila Foundation, the Finnish Medical Foundation, United States Army Department of Defense Contract PC991018, Grant LF043 from The Lustgarten Foundation, NIH Specialized Program of Research Excellence Grant P50 CA83591, and NIH Grant R01 CA83821. 2 To whom requests for reprints should be addressed, at Division of Human Gene Therapy, Gene Therapy Center, WTI #620, 1824 6th Avenue South, University of Alabama at Birmingham, Birmingham, AL 35294-3300. Phone: (205) 934-8627; Fax: (205) 975-7476; E-mail: david.curiel@ccc.uab.edu. 3 The abbreviations used are: Ad, adenoviral; CMV, cytomegalovirus; VP, viral particle; pfu, plaque-forming unit(s); GM, growth medium; RT-PCR, reverse transcrip- tion-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1271 on July 23, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from