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Involvement of AP-2 binding sites in regulation of human beta-glucuronidase

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Abstract

The lysosomal hydrolase β-glucuronidase (β-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 β-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 β-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 β-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 β-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 β-glucuronidase by A23187. Our studies provide a basis for the understanding of the transcriptional regulation of the human β-glucuronidase gene and could be useful for the optimisation of glucuronide prodrug therapy.

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Abbreviations

β-gluc:

β-Glucuronidase

MEM:

Minimal essential medium

DMEM:

Dulbecco’s modified Eagle’s medium

SD:

Standard deviation

AP-2:

Activating protein-2

PMT:

Prodrug mono-therapy

nt:

Nucleotide

NFκB:

Nuclear factor κB

Neg. con.:

Negative control

PLB:

Passive lysis buffer

HBS:

HEPES-buffered saline

COX-2:

Cyclooxygenase 2

MnSOD:

Manganese superoxide dismutase

TPA:

12-O-tetradecanoylphorbol-13-acetate

Sp1:

Specificity protein 1

HMR 1826:

N-[4-β-glucuronyl-3-nitrobenzyloxycarbonyl]doxorubicin

E. coli :

Escherichia coli

bp:

Base pair

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Acknowledgements

We are grateful to Birke Kalb and Bärbel Uecker for their excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Kr 945/4-3, 4-4, 7-1), Bonn, Germany.

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Authors and Affiliations

  1. Department of Pharmacology and Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Ernst Moritz Arndt-University, Friedrich-Loeffler-Strasse 23d, 17487, Greifswald, Germany

    Christiane Kunert-Keil, Bernhard Sperker, Sandra Bien, Markus Grube & Heyo K. Kroemer

  2. Institute of Biochemistry, Ernst Moritz Arndt-University, Sauerbruchstrasse, 17487, Greifswald, Germany

    Gabriele Wolf

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  1. Christiane Kunert-Keil
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  2. Bernhard Sperker
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  3. Sandra Bien
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  4. Gabriele Wolf
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  5. Markus Grube
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  6. Heyo K. Kroemer
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Correspondence to Heyo K. Kroemer.

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Kunert-Keil, C., Sperker, B., Bien, S. et al. Involvement of AP-2 binding sites in regulation of human beta-glucuronidase. Naunyn-Schmiedeberg's Arch Pharmacol 370, 331–339 (2004). https://doi.org/10.1007/s00210-004-0989-3

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