Redox modulation of NF-kappaB nuclear translocation and DNA binding in metastatic melanoma. The role of endogenous and gamma-glutamyl transferase-dependent oxidative stress.
OBJECTIVE
The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity--expressed by a number of malignancies, including melanoma--can act as a basal source of superoxide, hydrogen peroxide and other prooxidants.


METHODS
In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme--through the promotion of redox processes--may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kappaB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies).


RESULTS
Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status.


CONCLUSIONS
Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.
Dominici
Silvia
S
Department of Experimental Pathology, University of Pisa Medical School, Pisa, Italy.
Visvikis
Athanase
A
Pieri
Lisa
L
Paolicchi
Aldo
A
Valentini
Marta A
MA
Comporti
Mario
M
Pompella
Alfonso
A
D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D014178:Translocation, Genetic; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D004273:DNA, Neoplasm; D016328:NF-kappa B; D006861:Hydrogen Peroxide; D008156:Luciferases; D005723:gamma-Glutamyltransferase