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This dissertation, Mechanism of Apoptosis by Histone Deacetylase and Proteasome Inhibitors in Epstein-Barr Virus-positive Lymphoblastoid and Burkitt Cells by Po-ling, Yeung, 楊寶玲, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Mechanism of apoptosis by histone deacetylase and proteasome inhibitors in Epstein-Barr virus-positive lymphoblastoid and Burkitt cells Submitted by Yeung Po Ling for the degree of Master of Philosophy at the University of Hong Kong in Oct 2015 Epstein-Barr virus (EBV) drives the development of post-transplant lymphoproliferative disorder (PTLD) via the concerted action of the latent EBV proteins. We reported that Wp-restricted Burkitt lymphoma (BL) and lymphoblastoid cells (LCLs; in-vitro cell model of PTLD) have higher drug resistance to histone deacetylase inhibitor (HDACi) than latency I-restricted BL cells and the combination of HDACi and proteasome inhibitor (Pi) can overcome this drug resistance and synergistically induce apoptosis of Wp-restricted BL cells and LCLs. Since EBNA-3 proteins, EBNA-3A, -3B and 3C, are expressed in both Wp-restricted BL cells and LCLs but not in latency I-restricted BL cells, we hypothesize that combined HDACi/Pi may counteract the survival and cell cycle regulatory function of the EBNA-3 proteins in Wp-restricted BL cells and LCLs. The effects of FDA-approved HDACis (suberoylanilide hydroxamic acid [SAHA] and Romidepsin) and Pis (Bortezomib and Carfilzomib) were first tested in Wp- restricted BL cells and LCLs. The combination of SAHA or Romidepsin with Bortezomib or Carfilzomib could all up-regulate p16 and p21, which are down- regulated by the EBNA-3 proteins, and synergistically induce apoptosis in the tested cells in a reactive oxygen species (ROS)-dependent manner. To investigate which EBNA-3 protein(s) might be counteracted by combined HDACi/Pi, we tested the effects of the drugs in 8 BL (BL31) cell lines containing pairs of individual EBNA-3 knockout (KO) and revertant (Rev) cells, including those of EBNA-3A, -B and -C and BL31 (with wt EBV) and parental BL31 (EBV-negative). MTT assay showed that combined HDACi/Pi synergistically induced cell death in EBV-positive cells but not in EBV-negative cells and induced significantly greater synergistic killing in 3C Rev than 3C KO cells. Such differential response was not observed in either 3A or 3B Rev and KO cells. Furthermore, higher percentage of sub- G1 population and stronger proteolytic cleavage of apoptotic markers including PARP and caspase-3 as well as up-regulation of p21 were found in EBNA-3C expressing cells when compared to 3C KO cells, under the same treatment condition. In contrast, p16 was not detected, suggesting p21 instead of p16 might play a major role in mediating the synergism between the drugs. Finally, combined HDACi/Pi mediated G2/M arrest in 3C KO cells but not in EBNA-3C expressing cells and produced enhanced suppression of the growth of EBNA-3C expressing but not 3C KO BL31 xenografts in SCID mice, supporting that EBNA-3C might be the major counteracting target of the drug combination. The combined HDACi/Pi regimen was further tested in spontaneous LCLs (sLCLs) derived from paediatric PTLD patients. Significant increase in percentage of sub-G1 population without G2/M arrest and up-regulation of cleaved PARP and caspase-3 as well as p21 (but not p16) were also observed in the sLCLs, upon treatment with combined HDACi/Pi than either drug alone. In summary, combined HDACi/Pi induces potent apoptosis in Wp-restricted BL and LCLs through counteracting the

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