Demonstration of Quadruplex DNA in fixed normal ocular lens tissues: cell differentiation and cell death studies

Abstract

Purpose : G-quadruplex DNA (G4-DNA) is recognized in telomeres, and non-telomeric genomic DNAs. They regulate normal biological processes (e.g., recombination, transcription, replication) and pathologies. The lens provides a tissue to examine differentiation, cell cycle, mitosis, and cell death [terminal differentiation (denucleation)]. Therapeutically targeting G4-DNA might prove helpful in treating cataracts, and learning more about the mechanisms that regulate cell death. Previous research shows that there is an increase in ss-DNA, and a gradual decline of canonical B-DNA and Z-DNA in terminally differentiating fiber cells in adult lens. We hypothesize that G4-DNA will be minimal within the central zone epithelium (quiescent), with a gradual increase in the germinative zone and differentiating cells, and then a gradual decline within the nucleated secondary fiber cells.

Methods : Lewis rat eyes were fixed in 10% NBF or Davidson’s for 48 hrs. Tissues were processed to obtain paraffin-embedded 3 µm sections. Colorimetric/fluorescence staining was performed with an anti-G4-DNA MAb and conjugated 2° antibody to locate staining sites and quantify staining intensities. Microscopic procedures were performed using a Leitz DMRB microscope. Quantification of staining was determined as mean optical density (MOD) units (Quantimet 500+ image-analysis system). Results were expressed as the mean ± standard deviation, and the statistical significance was measured at p<0.05.

Results : Our group successfully demonstrated the presence of G4-DNA in lens tissue. Tissues fixed in Davidson resulted in superior morphology/immunostaining. The highest MODs were seen in mitotic cells. Staining was also identified at weaker MODs within the anterior epithelium and secondary nucleated fibers.

Conclusions : We conclude that G4-DNA is located within the lens and may play a role in regulating epithelium, lens fibers, mitosis, and cell death. Antigen Retrieval suggests that G4-DNA is covered by many G4-DNA binding proteins. G4-DNA gradually declined within the secondary fiber cells, similar to B-DNA and Z-DNA, indicating the destruction of DNA in denucleating fibers. All DNAs examined are undergoing destruction during denucleation. Knowledge of G4-DNA in lens cells and fibers may shed light on the molecular biology of cataracts and cancerous tissues.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.