HRAS is silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property.
Bottom Line: We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1.We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules.In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%.
Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.Show MeSH
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Mentions: Given their critical role in transcription regulation, the two neighboring G-quadruplexes can be targeted by small ligands in order to downregulate HRAS: an oncogene playing a key role in the pathogenesis of urinary bladder cancer (5,6,33,34). These molecules should bind preferentially G4-DNA instead of B-DNA, and consequently repress transcription by increasing the stability of the G-quadruplexes. This should inhibit the unfolding of the quadruplex by MAZ and arrests cell growth. To support these hypotheses, we used an antrathiophenedione (ATPD-1), which was recently found to strongly bind to hras-1 (KD of 0.34 ± 0.07, ΔTM of 12 and 22°C, in 100 mM KCl) and hras-2 (KD of 0.27 ± 0.02, ΔTM of 19°C, in 10 mM KCl) quadruplexes (35). We first treated T24 cancer cells with ATPD-1 for 24 h, then with the expression vectors for further 48 h. At the end of the incubation a dual luciferase assay was carried out. The relative firefly luciferase values obtained with wild-type and mutant plasmids, in absence and presence of 2.5 and 5 μM ATPD-1, are reported in the histogram of Figure 8A. It shows that ATPD-1 completely represses, in a dose-response manner, luciferase of wild-type plasmid pHRAS-luc, bearing both HRAS quadruplex-forming motifs. In contrast, the ligand reduced by ∼50% the luciferase expressed by mutants Mut-1 and Mut-2, which bear only one quadruplex-forming motif. This suggests that only one G-quadruplex is unable to block transcription completely. As we expected, Mut-A and Mut-B being unable to form any G-quadruplex, do not respond to ATPD-1. Instead Mut-C and Mut-D respond to the ligand, as they have one quadruplex-forming motif (hras-1).
Affiliation: Department of Medical and Biological Sciences, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.