DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21Cip1/Waf1 axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H2O2. The effect of ROS induction caused the G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H2O2-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21Cip1/Waf1 axis; but inhibiting the canonical NF-κB pathway exacerbated H2O2-induced A549 cell apoptosis. HDF premature ageing occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. P53 knock-down abrogated H2O2-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.

ηος πόλος ηος NF-κB ζηην απόπηωζη κςηηάπων πος εκηίθενηαι ζε παπάγονηερ επαγωγήρ οξειδωηικού ζηπερ και ζε κςηηαποκίνερ». ΦΖΚΑ ΑΛΔΞΑΝΓΡΟ ΒΙΟΛΟΓΟ ΓΗΓΑΚΣΟΡΗΚΖ ΓΗΑΣΡΗΒΖ ΙΩΑΝΝΙΝΑ, 2009 Κεθάιαην Γεύηεξν: Τιηθά-Μέζνδνη 66 2.1 ΤΛΙΚΑ …………………………………………………………………………..67 2.2 Βαηηδνζαηέξ Σεπκζηέξ …………………………………………………………69 2.3 Κοηηανζηή ηαθθζένβεζα ………………………………………………………..74 2.4 Πνμζδζμνζζιυξ ημο ηοηηανζημφ ηφηθμο ιε ηοηηανμιεηνία νμήξ …………79 2.5 Πνμζδζμνζζιυξ απμπηςηζηχκ ηαζ δεοηενεουκηςξ κεηνςηζηχκ ηοηηάνςκ ιε ηοηηανμιεηνία νμήξ ……………………………………………………………80 2.6 Υμνήβδζδ Η 2 Ο 2 ζε ηφηηανα Α549 …………………………………………..80 2.7 Υμνήβδζδ εημπμζζδίμο/VP-16 ζε ηφηηανα Α549 ηαζ MRC5-TERT ……..81 2.8 Υμνήβδζδ ηοημελαιίδζμο (CHX) ζε ηφηηανα Α549 ………………………..81 2.9 Υμνήβδζδ TNFα ζε ηφηηανα Α549…………………………………………..81 2.10 Πνμζδζμνζζιυξ ηςκ εκδμβεκχκ επζπέδςκ ΓΜΟ ζε ηφηηανα Α549 ηαζ MRC5-TERT………………………………………………………………………. 82 2.11 Ακάθοζδ ιε βμκίδζμ ακαθμνάξ NF-ηB θμοζζθενάζδξ…………………... 82 2.12 Έιιεζμξ ακμζμθεμνζζιυξ ηαζ ζοκεζηζαηή ιζηνμζημπία………………. 83 2.13 Απμιυκςζδ ηαζ Ακάθοζδ Νμοηθεσηχκ μλέςκ…………………………... 83 2.14 Απμιυκςζδ ηαζ Ακάθοζδ Πνςηεσκχκ……………………………………. 87 2.15 Απμιυκςζδ μθζημφ RNA…………………………………………………... 95 2.16 Ακηίδναζδ ακηίζηνμθδξ ηνακζηνζπηάζδξ/πμθοιενάζδξ [ Reverse transcriptase/polymerase chain reaction (RTPCR)]…………………………... 96 2.17 Δζζαβςβή βμκζδίςκ ζε ηφηηανα εδθαζηζηχκ…………………………….. 97 Κεθάιαην Σξίην: Απνηειέζκαηα 105 Μέξνο Πξώην: Ο ξόινο ηνπ NF-θB ζηηο απνθξίζεηο ησλ θπηηάξσλ ζην νμεηδσηηθό ζηξεο πνπ επάγεηαη από ην H 2 O 2 ……………………………106 3.1 Η πμνήβδζδ H 2 O 2 είπε ςξ απμηέθεζια ηδ ζοζζχνεοζδ ηςκ εκδμβεκχκ επζπέδςκ ΓΜΟ ηαζ εηηίκδζε ιζα απυηνζζδ ζε αθάαεξ ζημ DNA………… 107 3.2 Η πμνήβδζδ H 2 O 2 ακέζηεζθε ημκ ηοηηανζηυ πμθθαπθαζζαζιυ ηαζ επήβαβε ηδ ζοζζχνεοζδ ηςκ ηοηηάνςκ Α549 ζηδ G2-M θάζδ ημο ηοηηανζημφ ηφηθμο ……………………………………………………………………………………109

DNA damage, such as that experienced by people undergoing chemotherapy, can directly activate NF-κB signalling which in turn can lead to resistance to genotoxic stress. NF-κB signalling is highly regulated by phosphorylation, but the enzymes required for these processes remain largely unknown. Identifying those enzymes responsible for regulating NF-κB activity may yield attractive targets for new clinical therapies, as well as provide the basis for better understanding of signalling network crosstalk. Here we present datasets from two independent RNAi screens using a stable NF-κB reporter U2OS cell line with the aim of identifying enzymes that alter NF-κB activity in response to DNA damage following etoposide and ionising radiation treatments. Although we observed high internal validity and specificity to NF-κB modulation within the screens, there was a striking dissimilarity between the results of the two different screens. These data therefore provide a cautionary lesson regarding the use of RNAi screening but also provide new candidates for kinase and phosphatase regulation of NF-κB activity in response to genotoxic stress.