In addition, we investigated the cytotoxic effect of a widely used chemotherapeutic agent 5Fu on laryngeal squamous cell cancer cell lines and evaluated the role of p53 in 5Fu treatment. We found that the apoptosis and G1/S cell arrest mediated by 5Fu in laryngeal cancers is p53-independent but p21 WAF1/CIP1-dependent. We further demonstrated the effect of 5Fu on HPV16-associated laryngeal cancer cells. Using cytotoxicity assay and Annexin V staining, we proved that 5Fu induces apoptosis in all of the transfected cells in a dose- and time-dependent manner, suggesting that the process was not prevented by HPV16 E6 or E7. 5Fu induced the accumulation of active pRb and cyclin dependent kinase inhibitor p21WAF1/CIP1 together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. In addition, G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu in all cell lines. Through RT-PCR, 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in transfected UMSCC 12 cells. / Our results suggest that HPV16 E6 and E7 oncoproteins do not prevent 5Fu medicated apoptosis and G1/S cell arrest in laryngeal cancers. The anti-cancer effect of 5Fu is probably decided by the level of p21 WAF1/CIP1 while the sensitivity of laryngeal cancer cells responded to 5Fu treatment is associated with the increase of Bak or/and the decrease in Bcl-2, not with the HPV16 viral proteins and p53 status. 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer. However, the anti-viral effect of 5Fu still needs further investigation. / Our study indicated that (1) the evasion of apoptosis mediated by HPV16 E6 and E7 plays a critical role in laryngeal carcinogenesis; (2) HPV16 E6 or E7 plays an important role in regulating the expression of Bak, Bax and Bcl-2; (3) The degradation of Bak by HPV16 E6 is not caused by interacting with the promoter of Bak; (4) The induction of Bcl-2 is mediated through HPV16 E7; (5) HPV16 transfection does not interfere with the apoptosis and cell cycle arrest mediated by 5Fu in human laryngeal squamous cancer cells. / There is a growing body of evidence that human papillomavirus type 16 (HPV16) is involved in the development of human laryngeal cancer, especially in Chinese population. The two oncoproteins, HPV16 E6 and E7 that target host cell tumor suppressor proteins p53 and Rb respectively, may generate antiapoptotic effects and induce cell immortalization. However, the effect of both oncoproteins on apoptosis in laryngeal cancers is not completely clear. In this study, we demonstrated the possible mechanism of high risk HPV16 in laryngeal carcinogenesis and evaluated the effect of 5Fu on HPV16-positive laryngeal cancer cells. / We employed two human laryngeal cancer cell lines---UMSCC12 (with truncated p53) and UMSCC11A (with mutant but functional p53) in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1, which provided a good foundation for further study on the carcinogenic mechanism of HPV16 E6 or E7 in human laryngeal cancers. Through Annexin V staining and protein stability assay, we found that the transfection of HPV16 E6 and E7 induced fewer spontaneous apoptosis in both UMSCC11A and UMSCC12 cells accompanied with enhanced protein stability of Bcl-2 and increased protein degradation of Bak. Similar results were obtained when E6- and E7-transfected cells exposed to apoptosis stimuli---TNF-alpha/CHX. These results indicate that stable transfection of E6 and E7 in human laryngeal cancer cells on one hand shortened the half-life of Bak protein, and on the other hand, enhanced the steady-state levels of Bcl-2 protein. In order to gain insight into the role of Bak and Bcl-2 in regulating apoptosis in HPV-associated laryngeal cancer cells, we performed transient transfection of Bcl-2 into E6- and E7-transfected cells. It is found that HPV16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer, indicating that the induction of Bcl-2 require the transfection of HPV16 E7. Furthermore, Luciferase assay was performed to investigate whether the viral proteins E6 and E7 altered the stability of Bak through interaction with the promoter of Bak. Negative results were obtained, suggesting that E6 or E7 do not alter the transcription activity of Bak, indicating the degradation of Bak by E6 or E7 may be mediated through other mechanisms. / Liu Han-ching. / "August 2006." / Advisers: C. A. van Hasselt; George G. Chen. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1569. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 245-274). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lo, Chun Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 71-80). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.iv / List of figures --- p.vi / Abbreviations --- p.vii / Contents --- p.viii / Chapter Chapter One --- General Introduction --- p.Page / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Human papillomavirus infection at the larynx --- p.2 / Chapter 1.2.1 --- Biology of human papillomavirus --- p.4 / Chapter 1.2.2 --- HPV E6 protein --- p.5 / Chapter 1.2.3 --- HPV E7 protein --- p.7 / Chapter 1.3 --- Apoptosis --- p.9 / Chapter 1.3.1 --- Apoptosis signaling pathways --- p.11 / Chapter 1.4 --- Transcription factor: Nuclear factor -kB --- p.14 / Chapter 1.4.1 --- Overview of the NF-kB signaling pathway --- p.14 / Chapter 1.4.2 --- Regulation of NF-kB signaling --- p.16 / Chapter 1.4.3 --- Roles of NF-kB in cancers --- p.19 / Chapter 1.5 --- Pteris semipinnata L extract: ent-11 -hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) --- p.21 / Chapter 1.6 --- Objectives --- p.22 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cell culture --- p.24 / Chapter 2.2 --- Cell proliferation analysis --- p.24 / Chapter 2.3 --- Western Blotting --- p.26 / Chapter 2.3.1 --- Total protein extraction --- p.26 / Chapter 2.3.2 --- Nuclear and cytoplasmic protein extraction --- p.26 / Chapter 2.3.3 --- Quantification of protein concentration --- p.27 / Chapter 2.3.4 --- Sodium dodecyl sulfate - polyacylamide gel electrophoresis (SDS-PAGE) and protein transfer --- p.28 / Chapter 2.3.5 --- Immunoblotting --- p.29 / Chapter 2.4 --- NF-kB Luciferase Assay --- p.29 / Chapter 2.5 --- Annexin V apoptosis assay --- p.31 / Chapter 2.6 --- mRNA expression analyses --- p.33 / Chapter 2.6.1 --- RNA extraction --- p.33 / Chapter 2.6.2 --- Reverse Transcription --- p.33 / Chapter 2.6.3 --- Polymerase Chain Reaction --- p.34 / Chapter 2.7 --- Antibodies --- p.35 / Chapter Chapter Three --- Results / Chapter 3.1 --- "Anti-proliferation effect of 5F on laryngeal cancer cells UMSCC11A, UMSCC12 and HEp-2 cells" --- p.36 / Chapter 3.2 --- Suppression by 5F in HEp-2 of mRNA and protein expression levels in HPV18 E7 while the expression level of HPV18 E6 was not altered --- p.38 / Chapter 3.3 --- Quantification of 5F-induced apoptosis in laryngeal cancer cells by Annexin V assay --- p.40 / Chapter 3.4 --- Morphological changes in laryngeal cancer cells induced by 5F --- p.41 / Chapter 3.5 --- "Cleavage of poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 induced by 5F in UMSCC11 A, UMSCC12 and HEp-2 cell lines" --- p.47 / Chapter 3.6 --- "Down-regulation of TNF-α-induced NF-kB subunit p65 and p50 nuclear translocations in UMSCC11 A, UMSCC12 and HEp-2 by 5F" --- p.47 / Chapter 3.7 --- Dose-dependent inhibition of 5F on NF-kB transcriptional activity measured by luciferase assay --- p.53 / Chapter 3.8 --- Partial inhibition of TNF-α induced kBα degradation by 5F in UMSCC11A but not in UMSCC12 and HEp-2 --- p.56 / Chapter 3.9 --- Cell proliferation inhibition and apoptosis induction by Bay (11-7082) in laryngeal cancer cells --- p.56 / Chapter 3.10 --- Differential basal nuclear translocation of p65 and p50 in laryngeal cancer cell lines --- p.57 / Chapter 3.11 --- 5F regulated NF-kB target gene expression --- p.58 / Chapter Chapter Four --- Discussions --- p.64 / Reference --- p.71 / Appendix / Appendix 1 Map of pLuc- NF-kB plasmid --- p.81