Μελέτη του ρόλου του NF-kB στην απόπτωση κυττάρων που εκτίθενται σε παράγοντες επαγωγής οξειδωτικού στρες και σε κυτταροκίνες (Doctoral thesis)
NF-ηB is a redox sensitive transcription factor, however, its activation and its role in human lung tumor cells in response to oxidative stress remains elusive. Here, we addressed the role of NF-ηB signaling in H2O2 or VP-16 treatment human lung tumor epithelial cells A549. To this end we genetically manipulated the NF-ηB signaling pathways either using a super repressor of NF-ηB, IηBαSR, or by knocking down its activating IKK kinases, IKKα and IKKα.First, we showed that treatment of A549 cells with H2O2 or VP-16 resulted in the accumulation of endogenous reactive oxygen species, initiated a DNA damage checkpoint response leading to inhibition of cell proliferation and induction of apoptosis, only after treatment with H2O2, dose-dependently. H2O2 and VP-16 treatment had differential effects on the expression of proteins involved in the regulation of cell cycle progression cyclin D1 and Cdc6, f E2F1 and p53-p21Cip/Waf1 axis. Also, H2O2 treatment, but not VP-16, induced apoptosis, which was accompanied by a reduction in Bcl-2 expression, conformational changes of Bax, activation of caspase-3 and nuclear accumulation of proteolyticaly cleaved form of PARP-1. H2O2, but not VP-16, stimulated the nuclear translocation and accumulation of IKKα, but not of IKKα, and of IηBα and in addition mediated the phosphorylation of cytoplasmic IηBα, dose-dependently. Nuclear translocation of IKKα correlated with the phosphorylation of histone H3 at serine 10. Further, H2O2 mediated the phosphorylation of p65 and nuclear translocation of p-p65 and stimulated NF-ηB transcriptional activity and VP-16 mediated the phosphrylation of p65 and nuclear translocation of both p65 and p-p65.While forced overexpression of NF-ηB super repressor, IηBαSR, altered the expression of cell cycle regulatory proteins and sensitized A549 cells to H2O2-mediated apoptosis, constitutive activation of NF-ηB, using a constitutively active IKKα T-loop mutant IKKαca suppressed H2O2-mediated apoptosis. Importantly, while knocking-down IKKα-mediated canonical NF-ηB signaling sensitized A549 cells to H2O2-mediated early and late apoptosis, ablation of IKKα produced, mainly, evidence of late apoptosis. These data suggested that both H2O2 and VP-16 activated NF-ηB, but H2O2 through IKKα-mediated phosphorylation of RelA/p65 and VP-16 through the canonical pathway and that interfering with NF-ηB signaling pathways sensitized tumor cells to apoptosis.In addition, we showed that both H2O2 and VP16 effected differentially the expression of the replication licensing factor, Cdc6. Our data suggested that the IηΒ kinase α (ΙΚΚα) plays a major role in the regulation of Cdc6.
|Institution and School/Department of submitter:||Πανεπιστήμιο Ιωαννίνων. Σχολή Ιατρικής. Τμήμα Ιατρικής Τομέας Μορφολογικός - Κλινικοεργαστηριακός Εργαστήριο Ανατομίας - Ιστολογίας - Εμβρυολογίας|
|Keywords:||Απόπτωση,Οξειδωτικό στρες,Κυτταροκίνες,Δραστικές μορφές οξυγόνου (Δ.Μ.Ο.),Αναστολή κυτταρικού κύκλου,Υπεροξείδιο υδρογόνου,Χημειοθεραπεία|
|Appears in Collections:||Διδακτορικές Διατριβές|
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