Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effectson cellular H2O2 production, cell surface protein thiol oxidation and NF-kappa B activation status

The metabolism of glutathione by membrane-bound gamma-glutamyl transpeptida se (GGT) has been recently recognized as a basal source of hydrogen peroxid e in the extracellular space. Significant levels of GGT activity are expres sed by malignant tumours, and in melanoma cell lines they were found to cor relate with the malignant behaviour. As hydrogen peroxide and other oxidant s can affect signal transduction pathways at several levels, the present st udy was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent proo xidant reactions on known redox-sensitive cellular targets, i.e. protein th iols, the nuclear transcription factor NF-kappa B and p53, Two melanoma Me6 65/2 cell clones, exhibiting traces of (done 2/21) or high (clone 2/60) GGT activity, were studied, The occurrence of GGT-dependent production of hydr ogen peroxide was apparent in 2/60 cells, in which it was accompanied by lo wer levels of cell surface protein thiols, In 2/60 cells, GGT expression wa s also associated with higher levels of NF-kappa B activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activ ity in 2/60 cells resulted in progressive activation or inactivation of NF- kappa B, respectively. An analysis of the p53 gene product indicated lack o f protein expression in 2/60 cells, whereas a mutant protein was highly exp ressed in 2/21 cells, Taken together, these results indicate that the expre ssion of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to mod ify protein thiols at the cell surface level. In addition, GGT expression r esults in an up-regulation of the transcription factor NF-kappa B, which co uld explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.