tetra(4-n-methylpyridyl)porphine and dimethyl-sulfate

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.

Topics: Base Sequence; Breast Neoplasms; Cell Hypoxia; Circular Dichroism; DNA Footprinting; Electrophoretic Mobility Shift Assay; Endometrial Neoplasms; Female; G-Quadruplexes; Humans; Kidney Neoplasms; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Porphyrins; Potassium Chloride; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Selenium Compounds; Sequence Homology, Nucleic Acid; Sulfuric Acid Esters; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A