Article Figures & Data
Figures
- Figure 1.
A, schematic diagram showing the location of hypoxia response element (HRE) and the binding sites for transcription factor Sp1 in the proximal promoter region of the VEGF gene. Runs of guanines and cytosines are enclosed in boxes. B, models for the repression of VEGF gene transcription by G-quadruplex–interactive agents. C, oligonucleotides used in this study.
- Figure 2.
Determination of the intramolecular G-quadruplex structures formed by the G-rich sequence of the VEGF promoter region in the presence of 100 mmol/L KCl. A, top, EMSA of VEGF-Pu20 preincubated under the conditions specified in the figure using 16% native polyacrylamide gel. Bottom, DMS footprinting of each band (1–5) from EMSA. AG and TC, sequencing lanes; lanes 1 to 5, bands from EMSA. B, top, EMSA of VEGF-Pu20m preincubated under the conditions specified in the figure using 16% native polyacrylamide gel. Bottom, DMS footprinting of each band (1–3) from EMSA. AG and TC, sequencing lanes; lanes 1 to 3, bands from EMSA. The vertical bars to the left of lane 5 (A) and lane 3 (B) correspond to DMS-protected guanine repeats, and the sequences to the right show the protected guanines (○). C, summary of DMS footprinting of VEGF-Pu20 and VEGF-Pu20m in the presence of 100 mmol/L KCl. The protected guanines from DMS are indicated by open circles, and guanine residues hypermethylated by DMS are indicated by arrowheads. D, determination of the structures of G-quadruplexes formed from VEGF-Pu26, Pu24, Pu21, and Pu29 in the presence of 100 mmol/L KCl. Top, EMSA of VEGF-Pu26, Pu24, Pu21, and Pu29 (left to right) preincubated under the conditions specified in the figure using 16% native polyacrylamide gel. Bottom, DMS footprinting of each band from EMSA. AG and TC, sequencing lanes; each lane corresponds to the bands from corresponding EMSA.
- Figure 3.
EMSA, DMS footprinting of VEGF-Pu20, and CD spectrum of the sequence myc-1245. A, sequence of the wild-type (WT) and mutant (myc-1245) NHE III1 in the C-MYC promoter. B, EMSA of the sequence myc-1245 preincubated under the conditions specified in the figure using a 16% native polyacrylamide gel. C, DMS footprinting of each band from EMSA. D, CD spectra of the myc-1245 (solid line) in comparison with that of VEGF-Pu20 (dashed line). The CD data were obtained with a 5 μmol/L strand concentration in the presence of 100 mmol/L KCl at 25°C.
- Figure 4.
Molecular modeling structures for G-quadruplexes formed by VEGF-Pu20 in comparison with the known folding pattern of myc-1245. Top, alignment of both sequences. Middle, schematic illustration (left) and molecular modeling structure (right) of the VEGF parallel quadruplex. Bottom, schematic illustration (left) and molecular modeling structure (right) of the myc-1245 parallel quadruplex. The structure of the human c-myc parallel quadruplex was used as a starting structure (35). Necessary replacements and deletions were done. Loop geometries were obtained as in the case of the human telomeric DNA parallel quadruplex (PDB code 1KF1; ref. 34). Modeling was done using the Biopolymer module within the Insight II modeling software and charges, and potential types were assigned using the Amber force field within Insight II (Accelerys). Structures were minimized using 2,500 steps of Discover 3 (Accelerys) minimization as previously described (42).
- Figure 5.
Taq DNA polymerase stop assay showing the stabilization of G-quadruplex structures by KCI and TMPyP2, TMPyP4, and Se2SAP. A, sequence of the ssDNA template annealed with the primer used in the DNA polymerase pause assay. B, KCl-dependent pausing of DNA polymerase DNA synthesis at the 3′ side of the fourth guanine run in the DNA template containing the VEGF-Pu20 sequence. C, structures of TMPyP2, TMPyP4, and Se2SAP. D, left, stabilization of G-quadruplex structure formed by VEGF-Pu20 with the addition of increasing concentrations of TMPyP2, TMPyP4, and Se2SAP. Arrows, positions of the full-length product (F) of DNA synthesis, the G-quadruplex pause sites (S), and the free primer (P). Lanes A, G, T, and C, dideoxysequencing reactions with the same template as the size marker for the precise arrest sites. Right, percentage of the major arrest products of each sample to the total product was plotted against drug concentration.
- Figure 6.
Effect of G-quadruplex–interactive agents on VEGF mRNA synthesis in the MDA-MB231 (A), HEC1A (B), A498 (C), and 786-O (D). Human cancer cells were treated with TMPyP2 (P2), TMPyP4 (P4), and Se2SAP at various concentrations for indicated times, and VEGF mRNA and β-actin mRNA were measured by reverse transcription PCR. Less than 30% reduction in the number of cells occurred over 3 d after treatment with 50 μmol/L of TMPyP4, TMPyP2, and Se2SAP (data not shown). Two products were obtained after reverse transcription PCR, which correspond to c-DNAs of VEGF121 and VEGF165.
Volume 7, Issue 4
