Detection of Reactive Oxygen Species in Cells Undergoing Oncogene-Induced Senescence

Abstract

Reactive oxygen species (ROS) derive from molecular oxygen and present higher reactivity. ROS designation comprehends free radicals such as superoxide radical (O₂°⁻), hydroxyl radical (OH°⁻); but also nonradical molecule like hydrogen peroxide (H₂O₂). ROS play a critical role in several physiological functions like proliferation and signalling pathways. Thanks to cellular (oxidant/antioxidant) systems, ROS level is tightly regulated to avoid excessive damage to biological macromolecules (proteins, lipids, and DNA). An imbalance of redox equilibrium can lead to persistent oxidative stress favoring senescence, inflammation, and carcinogenesis.

Oncogene activation can induce severe or irreparable DNA damage and causes proliferative arrest named senescence. Senescence acts as a tumorigenesis barrier, and its bypass can promote transition between normal homeostasis and neoplastic transformation. The mechanisms through which oncogenes induce senescence remain unclear but involve increased cellular level of Reactive Oxygen Species. Among ROS, H₂O₂ is of particular interest because the hydrogen peroxide is more stable, can diffuse actively or freely through the cellular membranes, and can generate locally the hydroxyl radicals by iron-mediated Fenton reaction. Interestingly, growing data support the role of H₂O₂ in the propagation of the stressful effects of senescent cells to their neighbors through the bystander effect. In this protocol, we present our routinely used methodology to detect extracellular H₂O₂ using the Amplex red/horseradish peroxidase assay. This highly sensitive method detects specifically H₂O₂, and offers the possibility to quantify it using the H₂O₂ standard curve.

To illustrate this method of detection of extracellular H₂O₂ in cells undergoing oncogene-induced senescence, we compare two human cell lines: BCPAP (from human papillary thyroid carcinomas carrying BRAF^V600E mutation) and HTori-3.1 cell line (immortalized human thyroid epithelial cells) because expression of BRAF^V600E in human thyroid cells triggers senescence.