AbstractsBiology & Animal Science

The adenovirus E4orf4 protein induces cell death independently of p53 in human cancer cells but not normal primary cells through a mechanism that remains to be elucidated. Previous studies have suggested that E4orf4 induces an arrest or a delay in mitosis, a fate that is heavily dependent on E4orf4's interaction with the Bα regulatory subunit of protein phosphatase 2A. In this study, a protocol using 0.01% FBS media was developed to synchronize human lung carcinoma (H1299) cells in G0/G1. Time-lapse microscopy was performed using this G0/G1 synchronization as well as S phase arrest and release in mCherry-H2B H1299 cells to look at both morphological changes by phase contrast and cell cycle progression by immunofluorescence. It was found that cells expressing E4orf4 display an overall increased mobility, rounding up, and demonstrate a slow transit through mitosis, a delay and often failure in cytokinesis, and those cells that survive take a prolonged period of time to re-enter another round of the cell cycle. In fact, when E4orf4 was expressed in G0/G1 arrested cells, most cells failed to initiate DNA synthesis, as shown in parallel flow cytometry studies by our group (Cabon, Sriskandarajah et al, 2013). Studies in this thesis using time-lapse microscopy confirmed that in such cells no further nuclear division was evident. However, when E4orf4 was expressed in cells blocked in S phase and then released, cells completed DNA synthesis but often remained tetraploid and by time-lapse microscopy, demonstrated difficulties completing cytokinesis successfully. Studies in this thesis also addressed the issue of whether the cell death induced by E4orf4 occurs in G1 arrested diploid or tetraploid cells. Cell sorting experiments followed by flow cytometry revealed that E4orf4 induced cell death occurred in both tetraploid and diploid cells. Thus my work has confirmed that E4orf4 toxicity is characterized by difficulty in transiting though mitosis to successful completion of cytokinesis. Il a été démontré que la protéine adénovirale E4orf4 induit la mort cellulaire indépendamment de p53 dans des cellules cancéreuses humaines mais non dans des cellules primaires normales par un mécanisme d'action qui cependant reste largement inconnu. Des études ont suggéré que l'intéraction entre E4orf4 et la sous-unité régulatrice Bα de la protéine phosphatase 2A résulte en l'induction d'un arrêt ou un délai dans la progression mitotique.Dans cette thèse, un protocole de synchronisation de cellules de carcinome pulmonaire humaines (H1299, en culture à 0.01%FBS)) en G0/G1 a été développé. Grâce à cette synchronisation combinée à un arrêt puis libération de la phase S du cycle cellulaire de cellules H1299 exprimant un mCherry-H2B, une étude microscopique par immunofluorescence ainsi que par contraste de phase a permis d'investiguer les changements morphologiques ainsi que la progression à travers le cycle cellulaire.Il a été constaté que les cellules exprimant E4orf4 affichaient une mobilité globale accrue ansi qu'une morphologie arrondie. …