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Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy

Cancer Gene Therapy volume 12, pages 276–283 (2005)Cite this article

Abstract

One important feature of human solid tumors is the presence of a hypoxic microenvironment. Under hypoxia, genes that contain a hypoxia-response element (HRE) can be activated by the binding of hypoxia-inducible factor-1. To reach the goal of selectively killing tumor cells in a hypoxic microenvironment using a gene therapy approach, we developed a cytosine deaminase (CD) gene construct (pH9YCD2) that contains an HRE gene enhancer. CD is an enzyme that catalyzes the conversion of noncytotoxic 5-fluorocytosine (5-FC) to the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU). Yeast CD was cloned into an SV40 promoter-based mammalian expression vector, and an HRE enhancer was inserted in front of the promoter. Human glioblastoma U-87 MG cells were transfected with pH9YCD2. Western blots revealed that CD was strongly expressed under hypoxic conditions (0.3–1% O2), whereas only minor CD expression was seen under normoxic conditions. To confirm that the expressed CD enzyme retains catalytic activity, we performed a 5-FC/5-FU-conversion assay in which 5-FC was incubated with the lysates of pH9YCD2-transfected cells. The percentage of conversion from 5-FC to 5-FU was 63% under hypoxia versus 13% under normoxia. In vitro, cell viability and colony-forming efficiency assays demonstrated that the gene construct was able to significantly kill glioblastoma cells in a hypoxia-dependent manner. In addition, 5-FC treatment of hypoxic pH9YCD2-transfected cells produced a marked bystander effect, which could be a distinct advantage for gene therapy. If this construct exhibits antitumor efficacy in vivo, it may have promise as an antitumor agent in humans.

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Acknowledgements

We thank Dr Philippe Erbs, Transgene SA, Strasbourg Cedex, France, for providing technical assistance for the cytosine deaminase activity assay. We are also grateful to Sharon Reynolds, Department of Neurological Surgery, University of California at San Francisco, for editorial assistance. This work was supported by NIH Grants CA-85356 (to DFD), NS-42927 (to DFD) and CA-85878 (to AR).

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Authors and Affiliations

  1. Brain Tumor Research Center of the Department of Neurological Surgery, University of California, San Francisco, California, 94143-0520, USA

    Dongfang Wang, Hangjun Ruan, Lily Hu, Kathleen R Lamborn, Eileen L Kong & Dennis F Deen

  2. Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan, 48109, USA

    Alnawaz Rehemtulla

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  1. Dongfang Wang
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Correspondence to Dennis F Deen.

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Wang, D., Ruan, H., Hu, L. et al. Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy. Cancer Gene Ther 12, 276–283 (2005). https://doi.org/10.1038/sj.cgt.7700748

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