Identification of a novel catalytic inhibitor of topoisomerase II alpha that engages distinct mechanisms in p53wt or p53-/- cells to trigger G2/M arrest and senescence.

We report a novel topoisomerase IIα inhibitor, mercaptopyridine oxide (MPO), which induces G2/M arrest and senescence with distinctly different cell cycle regulators (p21 or p14ARF) in HCT116p 53WT and HCT116 p53-/- cells, respectively. MPO treatment induced defective topoisomerase IIα-mediated decatenation process and inhibition of the enzyme's catalytic activity that stalled entry into mitosis. Topoisomerase IIα inhibition was associated with ROS-mediated activation of ATM-Chk2 kinase axis in HCT116 p53WT cells, but not in HCT116 p53-/- cells displaying early Chk1 activation. Results suggest that E2F1 stabilization might link MPO-induced p53 phospho-activation in HCT116 p53WT cells or p14ARF induction in HCT116 p53-/- cells. Also, interaction between topoisomerase IIα and Chk1 was induced in both cell lines, which could be important for decatenation checkpoint activation, even upon p53 ablation. Notably, TCGA dataset analyses revealed topoisomerase IIα upregulation across a wide array of cancers, which was associated with lower overall survival. Corroborating that increased topoisomerase IIα expression might offer susceptibility to the novel inhibitor, MPO (5 µM) induced strong inhibition in colony forming ability of pancreatic and hepatocellular cancer cell lines. These data highlight a novel topoisomerase IIα inhibitor and provide proof-of-concept for its therapeutic potential against cancers even with loss-of-function of p53.

Assessment of oxidative stress-induced DNA damage by immunoflourescent analysis of 8-oxodG.

Oxidative stress refers to the imbalance between the generation of reactive oxygen species (ROS) and their scavenging by the inherent antioxidant defenses of the cell. The abnormal accumulation of ROS is the underlying pathology in a variety of human diseases such as neurodegenerative phenomena, inflammatory diseases, metabolic disorders, and cancer. The mechanism by which abnormal accumulation of ROS contributes to pathological conditions involves damage or oxidative modification of biomolecules, such as nucleotides, lipids, and proteins. One of the most common targets of ROS is DNA, modifications of which have been associated with cellular transformation and genome instability. There are a number of experimental strategies to assess oxidative modification of DNA bases, such as chromatography-based assays and indirect immunofluorescence. While the former provide quantitative assessment of oxidative modification, the latter is a much simpler assay for qualitative determination of DNA base modification in very small sample sizes. Here, we present a brief background of the various methodologies for the assessment of a specific oxidative DNA modification, 8oxodG, and present a more detailed account of the indirect immunofluorescence assay.