ABSTRACT
Seven cytosolic glutathione S-transferase (GST) classes have been characterized in humans:
, , , , , e . The omega class (GSTO) is the most recently identified. Despite
belonging to the GSTs superfamily, GSTOs lack classic glutathione transferase activities, while showing many other relevant functions such as thioltransferase and dehydroascorbate reductase activities, mono- and dimethylarsonicreductase activity, calcium transport modulation, post-translational activation of interleukin-1. In addition the murine GSTO has been characterized as a stress protein. In humans two different forms of GSTO have been characterized, the GSTO1 and the GSTO2.
Several lines of evidence correlate the overexpression of GSTO1 with the onset of drug resistence of cancer cells. In the laboratory where I carried out this thesis, it has been shown that the overexpression of the GSTO1 in HeLa cells is directly correlated to the acquisition of the resistant phenotype against cisplatin toxicity. The purpose of my thesis has been therefore to study the mechanisms of such chemioresistance. In order to investigate the mechanisms involved, a HeLa cell line stably transfected with GSTO1 complementary DNA (HeLa/GSTO1+) was used; while such line expresses a notable amount of GSTO1, control line (transfected with the only empty vector, HeLa/cont) expresses a negligible quantity of protein. We have investigated if there were differences in drug uptake between transfected and control HeLa cells, but the intracellular content of cisplatin was the same in the two cell lines. One of the most important defence mechanism against cisplatin is represented by the bond of the drug with GSH. Since it is known that in some instances stable transfection involves an increase of the GSH levels, we have verified if it was this the case, but also the content of GSH was the same in the two cell lines. Subsequently, by means of the Comet Assay, we have verified if there were differences in the level of DNA crosslinks, but again meaningful differences were not observed. We have finally verified if there were differences in the extent of to apoptosis following cisplatin treatment. Hoechst method showed that 35% of the transfected cells which overexpress the GSTO1 were apoptotic, as opposed to 80% of control cells.
In subsequent experiments we have tried to acquire information on the antiapoptotic mechanism of GSTO1 verifying, through western blotting, the levels of activation of MAP kinases and AKT. Two hours after cisplatin treatment, the phosphorilation levels of AKT and ERK1/2 result increased in transfected cells in comparison to control cells, while no changes were observed as far as p38 and JNK activation. On the contrary phosphorilation levels of JNK are very elevated 10 and 20 hours after cisplatin treatment in control cells, while they remain low in transfected cells that express high levels of GSTO1. GSTO1 seems therefore to partecipate in the resistence mechanisms against cisplatin treatment, by increasing the levels of ERK1/2 and AKT phosphorilation, usually anti-apoptotic kinases, and decreasing those of JNK, that represents, instead, one of the principal pro-apoptotic pathway.
