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A novel method for the determination of basal gene expression of tissue-specific promoters: An analysis of prostate-specific promoters

Cancer Gene Therapy volume 8, pages 927–935 (2001)Cite this article

Abstract

Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter “leakiness” using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50 <75 ng) was found for the CMV promoter in all cell lines, which is in agreement with the dual luciferase assay findings. Unlike the dual luciferase findings, however, the DT-tox176 assay showed protein inhibition of CN65 (PSA promoter/enhancer) and PSE–hK2 (PSA enhancer and basal human kallikrein 2 promoter) in HEK293 and DLD cells indicating “leakiness” of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT-tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay. Cancer Gene Therapy (2001) 8, 927–935

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Acknowledgements

The authors acknowledge the helpful advice of Donald Coffey, William Nelson, Jonathan Simons, Theodore DeWeese, Dale vander Putten, and Thomas Gardner during the course of these experiments. We also thank Donna Klinedinst for technical assistance, and Helen Kelley for secretarial assistance. Funding came from CaPCure (Santa Monica, CA), The Robert Wood Johnson Foundation (Princeton, NJ), and the Dutch Cancer Foundation (Amsterdam, the Netherlands).

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Authors and Affiliations

  1. Brady Urologic Institute, Johns Hopkins Medical School, Baltimore, 21287, Maryland, USA

    Henk G van der Poel, John McCadden, Mark Kruszewski, Fernando Ferrer, Michael Carducci & Ronald Rodriguez

  2. Urological Research Laboratory, Nijmegen, The Netherlands

    Gerald W Verhaegh & Jack A Schalken

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  1. Henk G van der Poel
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  2. John McCadden
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  3. Gerald W Verhaegh
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  5. Fernando Ferrer
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  6. Jack A Schalken
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  7. Michael Carducci
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  8. Ronald Rodriguez
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Correspondence to Ronald Rodriguez.

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van der Poel, H., McCadden, J., Verhaegh, G. et al. A novel method for the determination of basal gene expression of tissue-specific promoters: An analysis of prostate-specific promoters. Cancer Gene Ther 8, 927–935 (2001). https://doi.org/10.1038/sj.cgt.7700385

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