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Research Articles: Therapeutics, Targets, and Development

The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex–interactive agents

¹ Department of Pharmacology and Toxicology, College of Pharmacy, ² Department of Biochemistry and Molecular Biophysics, ³ Arizona Cancer Center, and ⁴ BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona

Requests for reprints: Daekyu Sun, BIO5 Institute, Room 102, 1657 East Helen Street, Tucson, AZ 85721. Phone: 520-626-0323; Fax: 520-626-4824. E-mail: sun{at}pharmacy.arizona.edu

Abstract

Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (–85 to –50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex–interactive agents, TMPyP4 and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design. [Mol Cancer Ther 2008;7(4):880–9]

Grant support: National Cancer Institute/NIH grants CA109069 and CA94166.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 5/07; revised 1/ 8/08; accepted 1/21/08.