Endogenous oxidative stress in human melanoma cells induces distinct redox forms of TNFR1 receptor with distinct ligand binding ability and apoptotic signaling.

S Dominici PhD, A Paolicchi MD PhD, M Franzini PhD, E Lorenzini PhD, MD De Tata PhD, A Pompella MD PhD

Dipartimento di Patologia Sperimentale BMIE, Università di Pisa – Via Roma 55, Pisa, Italy

AIM. Oxidation/reduction reactions are a mechanism for regulation of important functions of the cell, including the proliferation/apoptosis balance. Several biomolecules involved in signal transduction and regulation of gene expression are sensitive to prooxidants, e.g. reactive oxygen species (ROS), and a true “redox regulation” has been described for many of them. Low, basal levels of prooxidants are produced in the cell by several sources, e.g. respiration, NADPH oxidase, xantine oxidase. Our previous work allowed to identify membrane gamma-glutamyltransferase (GGT) as one additional source (Bioch. Pharm. 64, 1029, 2002). ROS and other free radicals are in fact originated the cell surface during the GGT-mediated metabolism of extracellular glutathione (GSH). Cell surface receptors of the TNFR superfamily are known to possess cysteine-rich regions in their extracellular domains. This study was aimed thus to verify whether different conditions of GGT activity might be reflected in redox changes of TNFR1 redox status and function. METHODS. Me665/2 human melanoma clones, expressing varying levels of GGT activity, were studied, in conditions of GGT stimulation (addition of substrates) vs. specific inhibition with the boronate analogue L-2-amine-4-boronobutanoic acid (ABBA). Thiol redox status of TNFR1 protein was analyzed by immunoblot after derivatization of cell surface reduced thiols with the thiol reagent maleimide-polyethylene glycole (MalPEG), by analyzing the molecular weigth shifts produced with MalPEG. Ligand binding capacity of TNFR1 was determined by using ¹²⁵I-labeled recombinant TNF-alpha. Triggering of apoptosis was investigated in cells exposed to TNF-alpha and cycloheximide, by determining caspase activation with an ELISA assay. RESULTS. At least five distinct redox forms of TNFR1 could be identified with MalPEG. Forms containing more reduced thiols were prevalent in the 2/21 clone, expressing low levels of GGT activity, as compared to GGT-rich 2/60 cells. Accordingly, GGT stimulation in 2/21 clone resulted in a shift from reduced to oxidized TNFR1 forms. At the same time, GGT stimulation resulted in a significant increase in ¹²⁵I-TNF-alpha binding to the cells, while the opposite was observed after GGT inhibition. A higher resistance of cells with ‘oxidized’ TNFR1 to TNF-alpha-induced apoptosis was also observed. CONCLUSIONS. Prooxidant reactions initiated at the cell surface during GGT-mediated GSH catabolism appear able to modulate cell sensitivity to TNF-alpha challenge, by altering the binding affinity of TNFR1 through a redox modulation of its thiol residues. As expression of GGT is frequent in human neoplasms, and is often increased at more advanced stages of progression, the processes described might concur to the onset of drug resistance, particularly in the case of (prooxidant) drugs acting through the cellular apoptotic machinery.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 998 (Signaling pathways - Part III).