title: Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy.
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.
authors.affiliation: Brain Tumor Research Center of the Department of Neurological Surgery, University of California, San Francisco, California 94143-0520, USA.
authors.forename: Dongfang
authors.initials: D
authors.lastname: Wang
authors.forename: Hangjun
authors.initials: H
authors.lastname: Ruan
authors.forename: Lily
authors.initials: L
authors.lastname: Hu
authors.forename: Kathleen R
authors.initials: KR
authors.lastname: Lamborn
authors.forename: Eileen L
authors.initials: EL
authors.lastname: Kong
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
authors.forename: Dennis F
authors.initials: DF
authors.lastname: Deen
mesh_terms: D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics / Q000627:therapeutic use*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics; D005437:Flucytosine / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism / Q000627:therapeutic use*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000628:therapy*; D011355:Prodrugs / Q000627:therapeutic use*; D013539:Simian virus 40; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014410:Tumor Stem Cell Assay; D015003:Yeasts
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011355:Prodrugs; D014157:Transcription Factors; D004921:Erythropoietin; D005437:Flucytosine; D043525:Cytosine Deaminase; D005472:Fluorouracil