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[Cancer Research 62, 1271-1274, March 1, 2002]
© 2002 American Association for Cancer Research

Advances in Brief

The flt-1 Promoter for Transcriptional Targeting of Teratocarcinoma1

Gerd J. Bauerschmitz, Dirk M. Nettelbeck, Anna Kanerva, Andrew H. Baker, Akseli Hemminki, Paul N. Reynolds and David T. Curiel2

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
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
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 transgene-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
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
A variety of gene therapy approaches for cancer have been undertaken based on in situ molecular chemotherapy, where systemically administered prodrugs are locally converted to their toxic counterparts. 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, 4, 5) . These considerations are especially relevant for Ad3 vectors, which exhibit a marked tropism for the liver when administered i.v. (3 , 5) . Thus, strategies to target transgene expression have been explored for Ad vector-based gene therapy approaches for cancer (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 limiting 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, 9, 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
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
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 cultured in the recommended growth medium and maintained in a humidified 37°C atmosphere containing 5% CO2.

Viruses.
Ad5flt-1luc1 and AdCMVluc1 are replication-defective adenoviruses 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 propagated 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 x 1011 VPs/ml, 1.2 x 1010 pfu/ml, and 50 VPs/pfu; and AdCMVluc1, 9.4 x 1011 VP/ml, 1.9 x 1010 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 x 1012 VPs/ml, 2.0 x 1010 pfu/ml, and 100 VPs/pfu; and Ad5CMVLacZ, 5.0 x 1012 VPs/ml, 5.0 x 1010 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 µl 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 µl of lysis buffer (Reporter Lysis Buffer; Promega Corp., Madison, WI) and freeze-thawed once. Twenty µl of these samples were mixed with 100 µl of luciferase assay reagent (Reporter Lysis Buffer; Promega) and measured with Berthold Lumat LB9501. Standardization was accomplished by setting the values obtained with CMV promoter 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 µl 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-ß-D-galactopyranoside solution (containing 40 µl 2% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, 10 µl of 0.3 M potassium ferricyanide, 10 µl of 0.3 M potassium ferrocyanide, and 940 µl 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 x10.

RT-PCR.
RNA of cells was extracted with RNeasy mini prep kit (Qiagen, Valencia, CA) and treated with DNase I (Life Technologies, Inc., Rockville, MD) for 30 min, and RT-PCR of 90 ng of RNA each was performed with the OneStep RT-PCR kit (Qiagen) using the following primers: Flt-1 sense, 5'-TGC TTG AAA CCG TAG CTG G-3'; Flt-1 antisense, 5'-GGT GCC AGA ACC ACT TGA TT-3'; GAPDH sense, 5'-TCC CAT CAC CAT CTT CCA-3'; and GAPDH antisense, 5'-CAT CAC GCC ACA GTT TCC-3'. Preliminary serial dilution assays determined the linear range of amplification for the genes under investigation.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
In 1996, Abu-Jawdeh et al. (13) reported that the mRNA of the flt-1 receptor is strongly expressed in ovarian borderline and malignant neoplasms, although the cell type expressing the receptors was not further characterized. Studies in other tumors have identified both flt-1 and vascular endothelial growth factor expression in malignant cells, suggesting the presence of an autocrine stimulatory loop promoting tumor cell growth in addition to the more commonly recognized effects of vascular endothelial growth factor on tumor angiogenesis. Therefore, we decided to explore the flt-1 promoter as a potential candidate for transcriptional targeting of adenoviral gene therapy for breast or gynecological cancer (14) . Using luciferase as a reporter gene in a replication incompetent adenovirus, three ovarian adenocarcinoma cell lines (Hey, OV-4, and SKOV3.ip1), one ovarian teratocarcinoma cell line (PA-1), three breast cancer cell lines (AU565, GI-101A, and ZR-75-1), and two cervical cancer cell lines (Caski and HeLa) were infected with Adflt-1luc1 and the control vector AdCMVluc1, respectively, with 5, 50, and 500 pfu/cell. Only the teratocarcinoma cell line PA-1 exhibited significant flt-1 promoter activity when compared with CMV (14%; Fig. 1Citation ).



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  		Fig. 1. Adflt-1luc1 induces high marker gene expression in the PA-1 teratocarcinoma cell line but low expression in breast, ovarian, and cervical lines. Cells were infected for 2 h with 5 pfu/cell of Adflt-1luc1 or AdCMVluc1. After 24 h, cells were lysed, and luciferase activity was measured. flt-1 promoter activity is presented as a percentage of CMV promoter activity. Data are from triplicate experiments; bars, SD.

 
We then expanded the analysis to include all commercially available teratocarcinoma cell lines [human ovarian (PA-1), murine ovarian (F9), and human testicular teratocarcinoma (NCCIT/NTERA-2)] and demonstrated activity of the flt-1 promoter in all of these lines (Fig. 2)Citation . The relative values compared with CMV were 10% for NCCIT, 15% for PA-1, 33.5% for F9, and nearly 50% for NTERA-2. Thus, the flt-1 promoter appears to be active in teratocarcinoma, irrespective of whether the cells are of ovarian or testicular origin. Of note, NTERA-2 and F9 exhibited higher relative ratios than the flt-1-positive control cell line human umbilical vein endothelial cells with 32%. Infection with 50 and 500 pfu/cell produced similar results (data not shown).



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  		Fig. 2. Adflt-1luc1 indicates high marker gene expression in all teratocarcinoma cell lines tested. PA-1 is a human ovarian teratocarcinoma cell line, NCCIT and NTERA-2 are human testicular teratocarcinoma cell lines, and F9 is a murine ovarian teratocarcinoma cell line. Cells were infected for 2 h with 5 pfu/cell of Adflt-1luc1 or AdCMVluc1. After 24 h, cells were lysed, and luciferase activity was measured. flt-1 promoter activity is presented as a percentage of CMV promoter activity. Data are from triplicate experiments; bars, SD.

 
Theoretically, luciferase activity correlates more with promoter activity than the number of infected cells. Thus, we used ß-galactosidase (LacZ) as a reporter gene to evaluate the percentage of teratocarcinoma cells in which flt-1 promoter-driven transgene expression was achieved (11) . Comparing the flt-1 promoter with CMV promoter-driven reporter expression, specificity of flt-1 promoter for teratocarcinoma cells was demonstrated. When teratocarcinoma cells were infected, the percentage of cells expressing the reporter was virtually equal with flt-1 promoter and with CMV promoter-driven viruses. Of note, the negative cell line BEAS-2B showed no visible transgene expression after infection with Ad5flt-1LacZ (Fig. 3)Citation . These results suggest that the flt-1 promoter is active in most teratocarcinoma cells.



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  		Fig. 3. Ad5flt-1LacZ is expressed in a high proportion of teratocarcinoma cells but not in lung epithelial cells (BEAS-2B). Cells were infected for 2 h with 500 pfu/cell of Adflt-1LacZ or AdCMVLacZ. After 24 h, cells were fixed and stained with LacZ. Pictures were taken by bright field microscopy at x10.

 
Transcriptional activity of cell-specific promoters typically correlates with the level of expression of the corresponding endogenous gene; thus, we hypothesized that the activity of the flt-1 promoter would correlate to the relative levels of flt-1 mRNA expressed in the tested cell lines. Such correlations could in theory be used to tailor transcriptionally targeted vectors to individual patient tumors based on determinations of the endogenous level of promoter activity in biopsy samples. flt-1 and GAPDH mRNA levels of the cell lines were assessed by RT-PCR. Whereas similar levels of expression were seen for the housekeeping GAPDH gene in all cell lines used, flt-1 mRNA was only detected in the teratocarcinoma cell lines (Fig. 4)Citation . No flt-1 mRNA could be detected, even with 45 cycles PCR for the ovarian adenocarcinoma cell lysates. This correlation thus confirms that the flt-1 vector has good transcriptional fidelity in Ad vectors and suggests that assays of flt-1 mRNA could prove useful when deciding upon specific gene-based tumor therapy.



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  		Fig. 4. flt-1 is selectively expressed in teratocarcinoma cell lines. Cells were lysed, and RNA was extracted. RNA was treated with deoxyribonuclease I, and RT-PCR was performed with flt-1 and GAPDH primers, respectively. PA-1, NTERA-2, and NCCIT are teratocarcinoma cell lines. BEAS-2B is a lung epithelial cell line. Hey, OV-4, and SKOV-3.ip1 are ovarian adenocarcinoma cell lines.

 
Our results demonstrate endogenous flt-1 expression in teratocarcinomas but not in ovarian adenocarcinoma cells. In addition, these data suggest that the expression of flt-1 mRNA reported previously in ovarian adenocarcinoma tumor blocks (13) was likely caused by non-carcinoma cells, such as endothelial cells of the tumor vessels (Fig. 4)Citation . In contrast to our findings are the results of Masood et al. (15) , who found a variety of cancer cell lines positive for flt-1 expression with RT-PCR, including the ovarian adenocarcinoma cell line Hey. A possible reason for the discrepancy could relate to the cells (e.g., passage number) or be associated with the sensitivity of the RT-PCR assays used.

A question currently unanswered is the biological reason for flt-1 promoter activity in teratocarcinoma cells. Because teratocarcinomas are derived from undifferentiated, pluripotent early embryonal cells and contain cells from endo-, meso-, and ectoderm (16) , lack of differentiation could be a possible reason for the expression of a promoter normally active in mature endothelium. In fact, previous studies suggested cross dependence of flt-1 gene expression and differentiation status (17 , 18) . Therefore, we induced artificial differentiation of teratocarcinoma cells with retinoid acid and cyclic AMP or valproic acid (19 , 20) , followed by infection with a luciferase-expressing adenovirus. Although we could validate artificial differentiation by observing changes in CMV-driven gene expression and microscopic appearance of the cells, neither experiment demonstrated an association between flt-1 expression and artificially induced differentiation (data not shown). However, both models are distinct from natural differentiation processes and thus may offer limited insight in this context.

In summary, we showed endogenous flt-1 expression in teratocarcinoma cell lines but not in ovarian, breast, and cervical carcinoma cell lines. Although teratocarcinoma are rare tumors and usually treatable with chemotherapy, these findings are of potential interest because they represent the first time a promoter has been demonstrated to be active in teratocarcinoma, independent of the tissue type from which it originates. The flt-1 promoter is active irrespective if the teratocarcinoma cell is derived from ovarian or testicular tissue. Any promoters that retain fidelity when placed in the Ad genome achieve good levels of transgene expression and have "liver off" phenotype are potentially of clinical utility. The close correlation of transgene expression and flt-1 mRNA shown here is further evidence that this promoter has the required characteristics. These data suggest that flt-1 could be useful for transcriptionally targeting teratocarcinoma, and that further evaluation in other flt-1-positive tumors is warranted.


   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 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. Back

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{at}ccc.uab.edu. Back

3 The abbreviations used are: Ad, adenoviral; CMV, cytomegalovirus; VP, viral particle; pfu, plaque-forming unit(s); GM, growth medium; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 12/ 7/01. Accepted 1/10/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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