Recent studies have shown that activation of the PI3K/AKT pathway confers protection against cell cycle arrest and apoptosis induced by some DNA-damaging agents. Aim of the present study was to investigate whether AKT is implicated in cellular responses to the methylating agent temozolomide (TMZ). The mismatch repair (MMR)-proficient cell line TK6 and its MMR-deficient subline MT1 were incubated with 12.5 ?M TMZ (a drug concentration previously shown to induce G2 arrest and apoptosis in TK6 but not in MT1 cells) for 72 h. AKT phosphorylation at Ser473, total amount of AKT and phosphorylation of the AKT target GSK-3 at Ser9 were evaluated every 24 h. An increase in the levels of phosphorylated AKT and GSK-3 was observed at all the time points analyzed in TMZ-treated TK6 but not in drug-treated MT1 cells, indicating that AKT is activated in response to TMZ and that this event requires a functional MMR system. No changes in total amount of AKT were observed in both TK6 and MT1 cells exposed to TMZ. A clone of the MMR-proficient cell line MCF-7 was stably transfected with the pUSEamp-Akt1 vector, encoding a dominant negative mutant form of AKT1, or with the empty vector as a control. The transfected cell were then exposed to TMZ, in the presence of 10 ?M O6-benzylguanine to prevent repair of methyl adducts at O6-guanine, and analyzed for proliferation and colony formation efficiency. MCF-7 cells transfected with mutant AKT were significantly more susceptible to TMZ-induced inhibition of cell growth and colony formation than control cells. Moreover, treatment of control cells with SH-5, a selective inhibitor of AKT, increased their sensitivity to TMZ. Our results suggest that strategies combining TMZ with inhibitors of the AKT pathway may enhance the antitumor effects of the drug. Supported by the Italian Ministry of Health
AKT is activated in response to temozolomide and confers protection against drug-induced cytotoxicity
2005
Abstract
Recent studies have shown that activation of the PI3K/AKT pathway confers protection against cell cycle arrest and apoptosis induced by some DNA-damaging agents. Aim of the present study was to investigate whether AKT is implicated in cellular responses to the methylating agent temozolomide (TMZ). The mismatch repair (MMR)-proficient cell line TK6 and its MMR-deficient subline MT1 were incubated with 12.5 ?M TMZ (a drug concentration previously shown to induce G2 arrest and apoptosis in TK6 but not in MT1 cells) for 72 h. AKT phosphorylation at Ser473, total amount of AKT and phosphorylation of the AKT target GSK-3 at Ser9 were evaluated every 24 h. An increase in the levels of phosphorylated AKT and GSK-3 was observed at all the time points analyzed in TMZ-treated TK6 but not in drug-treated MT1 cells, indicating that AKT is activated in response to TMZ and that this event requires a functional MMR system. No changes in total amount of AKT were observed in both TK6 and MT1 cells exposed to TMZ. A clone of the MMR-proficient cell line MCF-7 was stably transfected with the pUSEamp-Akt1 vector, encoding a dominant negative mutant form of AKT1, or with the empty vector as a control. The transfected cell were then exposed to TMZ, in the presence of 10 ?M O6-benzylguanine to prevent repair of methyl adducts at O6-guanine, and analyzed for proliferation and colony formation efficiency. MCF-7 cells transfected with mutant AKT were significantly more susceptible to TMZ-induced inhibition of cell growth and colony formation than control cells. Moreover, treatment of control cells with SH-5, a selective inhibitor of AKT, increased their sensitivity to TMZ. Our results suggest that strategies combining TMZ with inhibitors of the AKT pathway may enhance the antitumor effects of the drug. Supported by the Italian Ministry of HealthI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
