Elevated Levels of G-Quadruplex Formation in Human Stomach and Liver Cancer Tissues

Abstract

Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

“…This antibody revealed punctate fluorescence staining primarily in the nucleus after treatment with RNase ( Fig. 4C), which is consistent with published results (7,10). Interestingly, upon treatment of cells with H 2 O 2 , there is an increase in punctate G4DNA fluorescent foci appearing in the cytoplasm and a reduction of foci in the nucleus (Fig.…”

“…We applied a G-quadruplex structure-specific antibody, BG4 (7,10,51), to determine whether levels of endogenous G4DNA would increase in the cytoplasm with oxidative stress. Cells were treated with H 2 O 2 and harvested, and the cytosol was isolated.…”

“…Cells were further permeabilized with a buffer containing 0.2% Triton X-100, 2 mg/ml chicken ovalbumin, and 0.02% sodium azide in PBS. Before staining with BG4, cells were treated with 0.1 mg/ml RNase A for 30 min as described (7)(8)(9)(10) so that only DNA quadruplexes would be detected. After staining with antibodies, coverslips were mounted with 2.5 mg/ml DABCO (1,4-diazobicyclo[2.2.2]octane) and 0.1 g/ml DAPI in 25% PBS, 75% glycerol.…”

“…Antibodies to G4DNA have been used to visualize G4DNA in cells (7)(8)(9)(10). Small molecules designed to interact specifically with G4DNA have been found to alter gene expression in a G4DNA-selective manner (11,12).…”

Recent Developments in G-Quadruplex Probes

“…In recent years, a number of structure-specific antibodies for DNA and RNA G-quadruplexes have been developed for the imaging of G-quadruplexes in cells or tissues (Biffi et al, 2014a(Biffi et al, , 2014bHenderson et al, 2013;Lam et al, 2013). Antibodies bind to G-quadruplexes with affinities in the nanomolar range (compared with typically low-to submicromolar affinities for small molecules), and the use of such antibodies has provided arguably the strongest evidence for the in vivo existence of G-quadruplexes in mammalian cells to date.…”