Quantifying ROS levels using CM-H2DCFDA and HyPer.

At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).

Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells.

The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress-associated pathologies. The pro-oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics. Here, we present a protocol that can be used: (a) for isolation of mitochondrial, cytosolic, and mitochondrial-associated membrane fractions from adherent cells lines; (b) to perform p66Shc detection with specific antibodies in order to monitor its translocation between different cellular compartments in response to the oxidative stress; and (c) to modulate the p66Shc pathway with the use of pharmacological approaches or gene-silencing methods.

The interplay between p66Shc, reactive oxygen species and cancer cell metabolism.

The adaptor protein p66Shc links membrane receptors to intracellular signalling pathways and has the potential to respond to energy status changes and regulate mitogenic signalling. Initially reported to mediate growth signals in normal and cancer cells, p66Shc has also been recognized as a pro-apoptotic protein involved in the cellular response to oxidative stress. Moreover, it is a key element in processes such as cancer cell proliferation, tumor progression, metastasis and metabolic reprogramming. Recent findings on the role of p66Shc in the above-mentioned processes have been obtained through the use of various tumor cell types, including prostate, breast, ovarian, lung, colon, skin and thyroid cancer cells. Interestingly, the impact of p66Shc on the proliferation rate was mainly observed in prostate tumors, while its impact on metastasis was mainly found in breast cancers. In this review, we summarize the current knowledge about the possible roles of p66Shc in different cancers.

Oncogenic and oncosuppressive signal transduction at mitochondria-associated endoplasmic reticulum membranes.

The different mechanisms employed by proto-oncogenes and tumor suppressors to regulate cell death pathways are strictly linked to their localization. In addition to the canonical control of apoptosis at a transcriptional/nuclear level, intracellular zones are emerging as pivotal sites for the activities of several proapoptotic and antiapoptotic factors. Here, we review the function of the endoplasmic reticulum-mitochondria interface as a primary platform for decoding danger signals as well as a structural accommodation for several regulator or effector proteins.