title: Involvement of AP-2 binding sites in regulation of human beta-glucuronidase.
abstract: The lysosomal hydrolase beta-glucuronidase (beta-gluc) can be used for the bioactivation of non-toxic glucuronide prodrugs of anticancer agents. The enzyme is present at high levels in many tumours and hence may lead to an enhanced drug targeting by tumour-selective release of the active anticancer drug. Individual expression and regulation of this enzyme is one factor modulating the bioactivation of glucuronide prodrugs. Nevertheless, in contrast to murine beta-gluc, which is inducible by androgens, the human enzyme has been regarded as an unregulated housekeeping gene due to a lacking TATA box and high G+C contents within the putative promotor sequence. Despite these facts, we were able to demonstrate downregulation of human beta-gluc expression by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. However, cis-acting elements responsible for this regulation have not yet been identified. We therefore characterised the 5'-untranslated region of the human beta-gluc gene using transient transfection assays with promotor-luciferase constructs in HepG2 cells and cloned fragments between 3,770 bp and 107 bp. A23187 reduced the beta-gluc promotor activity. This effect disappeared using fragments smaller than 356 bp. Using site-directed in vitro mutagenesis and gel-electrophoretic-mobility shift assays, we found evidence of an involvement of transcription factor activating protein-2 (AP-2) binding sites on the regulation of human beta-glucuronidase by A23187. Our studies provide a basis for the understanding of the transcriptional regulation of the human beta-glucuronidase gene and could be useful for the optimisation of glucuronide prodrug therapy.
authors.affiliation: Department of Pharmacology and Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Ernst Moritz Arndt-University, Friedrich-Loeffler-Strasse 23d, 17487 Greifswald, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Kunert-Keil
authors.forename: Bernhard
authors.initials: B
authors.lastname: Sperker
authors.forename: Sandra
authors.initials: S
authors.lastname: Bien
authors.forename: Gabriele
authors.initials: G
authors.lastname: Wolf
authors.forename: Markus
authors.initials: M
authors.lastname: Grube
authors.forename: Heyo K
authors.initials: HK
authors.lastname: Kroemer
mesh_terms: D001665:Binding Sites / Q000502:physiology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D005966:Glucuronidase