Since high-risk Human Papillomavirus (HPV) plays a critical role in cervical carcinogenesis, it is essential to improve our understandings on the role of HPV-related molecular markers in cervical neoplasia. The aim of this study was to investigate the expression and prognostic significance of selected HPV-related markers, including HPV16/18 E2 binding sites (E2BS) methylation, Pak 1, c-FLIP, Notch 1 and Brd4 expressions, as well as the potential functions of Pak 1 in cervical neoplasia. First, the differential expressions of these markers among clinical samples of normal cervical epithelium, low-grade and high-grade cervical intraepithelial neoplasia (CINs) and cervical cancer were studied. Methylation status of E2BS 1, 2 and 4 was determined by pyrosequencing. Expressions of the target proteins were assessed by immunohistochemistry. Both HPV16/18 E2BS 1&2 and E2BS4 methylation progressively increased from normal cervix through CINs to cancer. More importantly, HPV16 E2BS1&2 (for transcriptional repression of E6/E7 oncoproteins) became more heavily methylated than E2BS4 (for transcriptional activation of E6/E7) in cervical cancer, favouring the differential binding of E2 protein to E2BS4. Pak 1, c-FLIP, Notch 1 and Brd4 were all overexpressed in cervical cancer. Their expressions increased progressively from normal cervix to low-grade +/- high-grade CINs. Pak 1 and c-FLIP expression was positively correlated with HPV18 E6 and HPV16 E7 expression respectively. Notch 1 expression was inversely correlated with HPV16 E7 and HPV18 E6 expressions. Brd4 expression was positively correlated with HPV16 E2 and inversely correlated with HPV16 E7 expressions. The prognostic significance of the molecular markers was investigated by correlation with clinical parameters. Heavier methylation at E2BS1&2 relative to E2BS4 was associated with better overall and disease-free survival in cervical cancer patients. HPV16 E2BS1&2 hypermethylation, weak cytoplasmic Brd4 expression, strong c-FLIP or Notch 1 expression were associated with higher risk of recurrent abnormal smears after treatment of CINs. The role of Pak 1 in cervical cancer was further explored by comparing its functions between cervical cancer cell lines with and without transient knock-down of Pak 1 by siRNAs. It was demonstrated that the significant functions of Pak 1 in cervical cancer were on promoting cell proliferation and inhibiting apoptosis. Transient HPV16 E6 inhibition showed no effect on total / phosphorylated Pak 1 expressions. Lastly, the function of Pak 1 on the regulation of cervical cancer cell radiosensitivity was also investigated. Pak 1 inhibition increased cell sensitivity in response to irradiation by enhancing apoptosis and inhibiting cell proliferation. Conclusively, differential methylation status at HPV16/18 E2BS, as well as Pak 1, c-FLIP, Notch 1 and Brd4 proteins contribute to cervical carcinogenesis, and are potential prognostic markers in cervical cancer and CIN patients. Pak 1 inhibitor may be a potential adjunctive agent to improve radiotherapy. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

Thesis (Ph.D.)--Kent State University, 2007. / Title from PDF t.p. (viewed July 8, 2009). Advisor: Angelo L. DeLucia. Keywords: human papilloma virus, mammalian lignans, p53, E6 oncogene. Includes bibliographical references (p. 133-149).

人類乳頭瘤病毒 (HPV) 是引起宮頸癌的必要條件。在高危型HPV中，以HPV16在癌症樣本中最為常見，其全球盛行率達50%以上。近年來，用以辨認HPV16變異子譜系的序列特徵已經建立。雖然這個系統建基於全球的HPV16變異株，但是它只包含了四個亞洲地區。為了改善這個系統於亞洲樣本的準確性，是次研究收集了更多亞洲地區的序列。 / 是次研究提供了在香港和韓國收集的HPV16樣本的系統發生史及序列變異 (LCR、E6 和 E7)。此外，是次研究也檢測了HPV16變異株的在兩地的分佈和致癌風險。 / 是次研究從香港和韓國收集了329個HPV16呈陽性的宮頸樣本。利用LCR、E6、E7 和整合的LCR-E6基因序列以極大似然法來構建HPV16變異株的系統發生樹。序列變異會按照系統發生樹之拓撲結構來分類並詳細描述。卡方檢驗或費雪精確性檢定用於分析HPV16變異株在兩地的分佈和致癌風險。 / 是次研究結果顯示用以辨認HPV16變異子譜系的序列特徵需加以改善。我們建議採用A7287C/T作為亞洲子譜系的序列特徵，以替代原有的A7287C。有關HPV16變異株的地理分佈，亞洲和歐洲的變異株在香港 (亞洲變異株: 70%，歐洲變異株: 25.3%) 和韓國 (亞洲變異株: 61.2%，歐洲變異株: 20.2%) 均十分普遍。另外，1和2型亞美變異株在香港和韓國的分佈有著明顯差別 (1型亞美變異株: 2% 與12.4%，P < 0.001; 2型亞美變異株: 0% 與2.8%，P = 0.04)。 / 另外，是次研究發現亞洲子譜系於韓國民族中呈較高致癌風險 [比值比 (95% 置信區間) 2.02 (1.03-3.99)]。在進化支中，E6的第五進化支[2.44 (1.27-4.74)]和E7的第三進化支[2.02 (1.03-3.99)]也於韓國民族中呈較高致癌風險。在SNP中，E6 T178G [2.17 (1.11-4.23)]、兩個E7的SNPs (A647G [1.73 (0.88-3.42)]、T846C [2.27 (1.16-4.49)]) 和9個LCR SNPs (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] 和 C24T [2.36 (1.20-4.66)])於韓國民族中也呈較高致癌風險。這些進化支和SNPs都與亞洲子譜系有關聯。在香港方面，兩個LCR SNPs (A7289C [1.89 (0.92-3.87)] 和 T7781C [2.07 (0.92-4.71)])呈較高致癌風險。 / 是次研究發現的高危SNPs和進化支需要進一步的大型流行病學研究和生物化學實驗來核實。這些序列特徵可作為生物標誌物以檢測出與HPV有關的早期宮頸病變。 / Human papillomavirus (HPV) is necessary for the development of cervical cancer. Of those high-risk HPV types, HPV16 is the most common type detected in cervical cancer and accounts for a prevalence of greater than 50% worldwide. Recently, a sequence signature-based system for identifying the sub-lineages of HPV16 variants has been established. Although this system was developed from HPV16 variants collected worldwide, only four Asian regions were included. To improve the accuracy of this sub-lineage classification system for Asian samples, more sequence data from Asian regions were included in the current study. / The current study provided data on the phylogeny and the sequence variation of Long control region (LCR), E6 and E7 open reading frames (ORFs) of HPV16 isolates collected in Hong Kong and Korea. The distribution of HPV16 variants between two regions and the risk association of HPV16 variants with cervical cancer development were also examined. / A total of 329 HPV16-positive cervical samples were collected from Hong Kong and Korea. The phylogenetic trees were constructed for the LCR, E6, E7 and concatenated LCR-E6 sequences using the maximum likelihood method. The sequence variation of each region was delineated and grouped according to the tree topology. The distribution and risk association of HPV16 variants were examined using the chi-square test or Fisher’s exact test as appropriate. / The results showed that the previously described sequence signatures for classifying sub-lineages of HPV16 variants required further improvement, especially for the Asian sub-lineage. We proposed A7287C/T as a signature SNP of the Asian sub-lineage rather than A7287C as suggested by Cornet et al. In regard to the distribution of HPV16 variants, the Asian (As) and European (Eur) variants were commonly found in Hong Kong (As: 70%, Eur: 25.3%) and Korea (As: 61.2%, Eur: 20.2%). Furthermore, Asian American-1 and 2 (AA1 and AA2) variants were found to distribute significantly different between Hong Kong and Korea (AA1: 2% versus 12.4%, P < 0.001; AA2: 0% versus 2.8%, P = 0.04). / A key finding was that variants of the Asian sub-lineage carried a higher oncogenicity among Korean population [odds ratio (95% confidence interval) = 2.02 (1.03-3.99)]. In clade level, E6 clade 5 [2.44 (1.27-4.74)] and E7 clade 3 [2.02 (1.03-3.99)] were found to carry a higher oncogenicity among Korean population. In SNP level, E6 T178G [2.17 (1.11-4.23)], two SNPs of E7 ORF (A647G [1.73 (0.88-3.42)] and T846C [2.27 (1.16-4.49)]) and nine SNPs of LCR (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] and C24T [2.36 (1.20-4.66)]) were also found to carry a higher oncogenicity among Korean population. Those clades and SNPs were linked to the Asian sub-lineage. In contrast, only two SNPs of LCR (A7289C [1.89 (0.92-3.87)] and T7781C [2.07 (0.92-4.71)]) were found to associate with a higher oncogenicity among Hong Kong population. / The risk associations of SNPs, clades of the HPV16 Asian sub-lineage revealed by the current study should be verified by large-scale epidemiological studies and biochemical experiments. These signatures may serve as biomarkers for early detection of HPV-related cervical neoplasia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ma, Tsz Ue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-156). / Abstracts also in Chinese. / Abstract of Thesis --- p.I / 論文摘要 --- p.V / Acknowledgements --- p.VIII / Contents --- p.X / Figures --- p.XIII / Tables --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- History of Human Papillomavirus --- p.2 / Chapter 1.2 --- Biology of Human Papillomavirus --- p.4 / Chapter 1.2.1 --- Genome Organization and Protein Functions --- p.4 / Chapter 1.2.1.1 --- E5 Protein --- p.7 / Chapter 1.2.1.2 --- E6 Protein --- p.8 / Chapter 1.2.1.3 --- E7 Protein --- p.9 / Chapter 1.2.2 --- Life Cycle of Human Papillomavirus --- p.10 / Chapter 1.2.3 --- Taxonomy of Human Papillomavirus --- p.12 / Chapter 1.3 --- Cervical Cancer --- p.16 / Chapter 1.3.1 --- Natural History --- p.16 / Chapter 1.3.2 --- Risk Factors --- p.17 / Chapter 1.4 --- Epidemiology of Cervical Cancer --- p.19 / Chapter 1.4.1 --- Global Disease Burden --- p.19 / Chapter 1.4.2 --- Disease Burden in Hong Kong --- p.21 / Chapter 1.4.3 --- Disease Burden in South Korea --- p.22 / Chapter 1.5 --- Human Papillomavirus Type 16 --- p.23 / Chapter 1.6 --- Background and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.31 / Chapter 2.2 --- Study Samples --- p.35 / Chapter 2.2.1 --- HPV16-Positive Samples --- p.35 / Chapter 2.2.2 --- Samples with Unknown HPV Status --- p.36 / Chapter 2.3 --- Laboratory Methods --- p.39 / Chapter 2.3.1 --- DNA Extraction --- p.39 / Chapter 2.3.2 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.2.1 --- PGMY09/11 PCR --- p.40 / Chapter 2.3.2.2 --- HPV16-Specific PCR --- p.42 / Chapter 2.3.3 --- Genotyping of HPV --- p.48 / Chapter 2.3.4 --- Purification of PCR Products --- p.51 / Chapter 2.3.5 --- Sequencing Reaction --- p.52 / Chapter 2.4 --- Data Analysis --- p.54 / Chapter 2.4.1 --- Sequence Edit and Alignment --- p.54 / Chapter 2.4.2 --- Sequence Variation of HPV16 Variants --- p.56 / Chapter 2.4.3 --- Construction of Phylogenetic Tree --- p.56 / Chapter 2.4.4 --- Distribution and Comparison of HPV16 Variants in Hong Kong and Korea --- p.57 / Chapter 2.4.5 --- Distribution of HPV16 Variants in Normal and Cancer Samples and Risk Association Study --- p.58 / Chapter Chapter 3 --- Results --- p.59 / Chapter 3.1 --- Study Samples --- p.60 / Chapter 3.1.1 --- HPV16-Positive Samples --- p.60 / Chapter 3.1.2 --- Samples with Unknown HPV Status --- p.61 / Chapter 3.2 --- Sub-lineage Identification of HPV16 Variants --- p.63 / Chapter 3.2.1 --- Based on the Phylogenetic Analysis in the Current Study --- p.63 / Chapter 3.2.1.1 --- Concatenated LCR-E6 Phylogenetic Tree --- p.63 / Chapter 3.2.1.2 --- LCR Phylogenetic Tree --- p.66 / Chapter 3.2.1.3 --- E6 Phylogenetic Tree --- p.69 / Chapter 3.2.2 --- Based on the Single Nucleotide Polymorphisms Proposed by Cornet et al. --- p.74 / Chapter 3.2.2.1 --- Single Nucleotide Polymorphisms of LCR Sequence --- p.74 / Chapter 3.2.2.2 --- Single Nucleotide Polymorphisms of E6 Open Reading Frame --- p.78 / Chapter 3.3 --- Sequence Variation of HPV16 Variants --- p.82 / Chapter 3.3.1 --- LCR Sequence --- p.82 / Chapter 3.3.2 --- E6 Open Reading Frame --- p.91 / Chapter 3.3.3 --- E7 Open Reading Frame --- p.95 / Chapter 3.4 --- Distribution of HPV16 Variants in Hong Kong and Korea --- p.100 / Chapter 3.4.1 --- Sub-lineage Level --- p.100 / Chapter 3.4.2 --- Clade Level of E6 Open Reading Frame --- p.101 / Chapter 3.4.3 --- Clade Level of E7 Open Reading Frame --- p.102 / Chapter 3.4.4 --- Single Nucleotide Polymorphisms Level --- p.105 / Chapter 3.4.4.1 --- LCR Sequence --- p.105 / Chapter 3.4.4.2 --- E6 Open Reading Frame --- p.107 / Chapter 3.4.4.3 --- E7 Open Reading Frame --- p.108 / Chapter 3.5 --- Risk Association and distribution of HPV16 Variants in normal and Cancer samples --- p.112 / Chapter 3.5.1 --- Sub-lineage Level --- p.112 / Chapter 3.5.2 --- Clade Level of E6 Open Reading Frame --- p.114 / Chapter 3.5.3 --- Clade Level of E7 Open Reading Frame --- p.115 / Chapter 3.5.4 --- Single Nucleotide Polymorphisms Level --- p.122 / Chapter 3.5.4.1 --- LCR Sequence --- p.122 / Chapter 3.5.4.2 --- E6 Open Reading Frame --- p.125 / Chapter 3.5.4.3 --- E7 Open Reading Frame --- p.126 / Chapter Chapter 4 --- Discussion --- p.132 / Chapter 4.1 --- HPV16 Variant Sub-lineages --- p.133 / Chapter 4.2 --- Comparison of HPV16 variants between Hong Kong and Korea --- p.137 / Chapter 4.3 --- Risk Association of HPV16 Variants --- p.138 / Chapter 4.4 --- Strength and Weakness --- p.144 / Chapter 4.5 --- Implications for Future Work --- p.146 / References --- p.148

子宮頸癌是全球女性中第三常見的癌症。人類乳頭瘤狀病毒（HPV）已被證實為引致子宮頸癌的主要因素。目前已發現了150多種HPV。HPV-52在世界上較為少見，但在亞洲，特別是東亞地區，卻相當流行。 / 本回顧性研究收集了303個HPV-52陽性的子宮頸樣本，其中185個來自香港，118個來自韓國首爾。我們通過對HPV基因組中E6、E7、L1和LCR區域進行擴增和測序，以檢測HPV-52變異株的序列多樣性和致癌風險。 / L1-LCR-E6-E7串聯片段佔據了HPV-52基因組全長的41%。由191條該種序列構建的系統發育樹顯示，HPV-52變異株進化成四個世系。原型系A進化系在香港和首爾都很少見，只占全部樣本的3.7%。B進化系（89.5%）則是最普遍的HPV-52病毒系。E6的最大序列差異為1.6%，L1（2.3%），E7（3.4%）和LCR（4.8%）依次增大。因此，E6作為最保守的基因組區域可作為HPV-52通用引物PCR的靶點，而E7更適宜作為特定變異株的PCR靶點。此外，在短片段序列中發現了可識別HPV-52進化系和進化枝的單核苷酸突變。它們可用於擴增斷裂的DNA片段或大規模實驗中。再者，進化壓力分析顯示E6、E7和L1三個編碼區域都經歷了強烈的淨化選擇作用。 / HPV-52進化系和常見變異株在香港和首爾的分佈情況沒有顯著差異。但E6中的nt 356G>A、nt 378A>C和nt 467C>A (N122K) 核苷酸突變只出現在香港樣本，而L1的nt 6239G>A以及LCR的nt 7395G>A和nt 7911A>C核苷酸突變只在首爾樣本中發現。HPV-52 E6的N122K突變對子宮頸癌有較高的致癌風險（P-value = 0.002）。E6中的nt 378A>C (P-value = 0.014) 同義突變, 以及LCR中的nt 7665G>A (P-value < 0.001)和nt 94G>A (P-value = 0.007)突變，亦與高致癌風險相關。LCR中的nt 7911A>C (P-value = 0.007)和nt 19T>C (P-value = 0.008) 突變則對子宮頸癌的發展有較低風險。HPV-52 E7或L1中的突變與子宮頸癌的發展無明顯關係。上述結果需要通過進一步研究證實。針對HPV-52序列變異的病毒學和作用機理的深入研究是必要的。 / Cervical cancer is the third most common cancer in women worldwide. It has been proven that human papillomavirus (HPV) is the primary causative agent of cervical cancer. To date, more than 150 HPV types have been characterized. HPV-52 is rare around the world but frequently detected in Asia, especially East Asia. / This retrospective study analyzed 303 cervical samples that 185 were collected from Hong Kong, and 118 were collected from Seoul, Korea. All samples were positive for HPV-52. HPV gene regions of E6, E7, L1 and LCR were amplified and sequenced to determine sequence diversity and risk association of HPV-52 variants between the two cities. / The 191 concatenated L1-LCR-E6-E7 sequences that comprised 41% of the whole HPV-52 genome displayed four distinct clusters. The prototype-like lineage A was rare in both cities, only found in 3.7% of all samples. Lineage B (89.5%) was found to be the most prevalent lineage. The maximum sequence divergence of E6 was 1.6%, followed by L1 (2.3%), E7 (3.4%) and LCR (4.8%). E6 being the most conserved region could be a target for HPV-52 consensus PCR, and E7 could be a target for variant-specific PCR. Besides, several single-nucleotide substitutions diagnostic for HPV-52 lineage and clade classification were identified within a few short fragments. They might be useful when handling fragmented DNA and being a more feasible approach in large-scale studies. Moreover, analysis of evolutionary pressure indicated that all the three encoding regions, E6, E7 and L1, underwent strong purifying selection. / No significant difference in the distribution pattern of HPV-52 lineages and common variants between Hong Kong and Seoul was observed. But nucleotide substitutions nt 356G>A, nt 378A>C and nt 467C>A (N122K) were only found in Hong Kong samples; whereas nt 6239G>A, nt 7395G>A and nt 7911A>C were exclusively found in samples from Seoul. A significantly higher risk for cervical cancer was found for the HPV-52 E6 variant N122K (P-value = 0.002). A synonymous substitution of E6, nt 378A>C (P-value = 0.014), as well as two nucleotide substitutions of LCR, nt 7665G>A (P-value < 0.001) and nt 94G>A (P-value = 0.007), were also associated with a significant increase in risk for cervical cancer. Two substitutions found to confer a lower risk for cervical cancer were nt 7911A>C (P-value = 0.007) and nt 19T>C (P-value = 0.008), both of which located at LCR. No significant associations between HPV-52 E7 or L1 variants and cervical cancer development were observed. Further studies are needed to confirm these findings, and in-depth investigations into the virological and functional implications of HPV-52 sequence variations are warranted. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Chuqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-137). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of contents --- p.vii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xii / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- History of Human Papillomavirus --- p.2 / Chapter 1.2 --- Biology of Human Papillomavirus --- p.4 / Chapter 1.2.1 --- Genome structure --- p.4 / Chapter 1.2.2 --- Protein function --- p.6 / Chapter 1.2.3 --- Latent and lytic life cycle --- p.9 / Chapter 1.2.4 --- Classification --- p.10 / Chapter 1.3 --- Epidemiology of Human Papillomavirus --- p.14 / Chapter 1.3.1 --- Global burden --- p.14 / Chapter 1.3.2 --- Transmission --- p.18 / Chapter 1.3.3 --- Clinical course --- p.19 / Chapter 1.3.4 --- Prevention --- p.23 / Chapter 1.4 --- Human Papillomavirus Type 52 --- p.25 / Chapter 1.5 --- Objectives --- p.26 / Chapter Chapter Two --- Materials and Methods --- p.27 / Chapter 2.1 --- Study Design --- p.28 / Chapter 2.2 --- Study population --- p.29 / Chapter 2.3 --- DNA extraction --- p.31 / Chapter 2.4 --- Polymerase chain reaction --- p.32 / Chapter 2.4.1 --- Long-fragment PCR approach --- p.33 / Chapter 2.4.2 --- Short-fragment PCR approach --- p.40 / Chapter 2.4.3 --- Purification of PCR products --- p.46 / Chapter 2.5 --- Nucleotide sequencing --- p.47 / Chapter 2.6 --- Data analysis --- p.48 / Chapter 2.6.1 --- Phylogenetic analysis --- p.48 / Chapter 2.6.2 --- Statistical analysis --- p.49 / Chapter Chapter Three --- Results --- p.50 / Chapter 3.1 --- Phylogeny of HPV-52 --- p.53 / Chapter 3.1.1 --- Concatenated sequence of L1-LCR-E6-E7 --- p.53 / Chapter 3.1.2 --- E6 gene --- p.56 / Chapter 3.1.3 --- E7 gene --- p.59 / Chapter 3.1.4 --- L1 gene --- p.62 / Chapter 3.1.5 --- Long control region --- p.67 / Chapter 3.2 --- Nucleotide sequence variation of HPV-52 --- p.70 / Chapter 3.2.1 --- E6 gene --- p.70 / Chapter 3.2.2 --- E7 gene --- p.73 / Chapter 3.2.3 --- L1 gene --- p.75 / Chapter 3.2.4 --- Long control region --- p.81 / Chapter 3.3 --- Geographical distribution of HPV-52 variants --- p.86 / Chapter 3.4 --- Risk association of HPV-52 variants --- p.96 / Chapter Chapter Four --- Discussion --- p.105 / Chapter 4.1 --- Strengths and weaknesses --- p.107 / Chapter 4.2 --- Phylogeny of HPV-52 variants --- p.109 / Chapter 4.2.1 --- Variant lineage classification system of HPV-52 --- p.109 / Chapter 4.2.2 --- Sequence variability of HPV-52 --- p.110 / Chapter 4.2.3 --- Evolutionary pressure on HPV-52 --- p.111 / Chapter 4.3 --- Nucleotide sequence variations of HPV-52 --- p.113 / Chapter 4.3.1 --- E6 gene --- p.113 / Chapter 4.3.2 --- E7 gene --- p.114 / Chapter 4.3.3 --- L1 gene --- p.116 / Chapter 4.3.4 --- Long control region --- p.117 / Chapter 4.4 --- Conclusions --- p.121 / References --- p.124 / Appendices --- p.138

published_or_final_version / Public Health / Master / Master of Public Health

The bovine papilloma virus type 2 (BPV-2) L2 open reading frame (ORF) was cloned into a λ pL promoter expression vector. This clone was shown to express a fusion protein which comprised 75% of the BPV-2 ORF linked to the first 13 N-terminal amino acids of the λ cIl gene product. Antisera was generated against this fusion protein and subsequently used to identify the L2 gene product as a 64,000 dalton protein in BPV-2 virions. It was also demonstrated that the L2 viral protein was present in full caps ids, but only in very limited amounts in empty caps ids. Densitometer analysis indicated that the L2 protein comprised only 8% of the total L1 + L2 "Coomassie blue stainable" protein in full capsids. The antisera was also used to demonstrate that the BPV-2 L2 gene product is antigenically related to the BPV-1 L2 gene product. Finally, an attempt was made to determine the location of the L2 gene product within the capsid structure. Hemagglutination inhibition and enzyme-llnked-immunosorbent- assay data both indicate that the L2 protein is exposed on the surface of the capsid. Immune electron microscopy data was inconclusive in determining the location of the L2 gene product.

Human Papillomavirus (HPV) is a small, double stranded DNA tumour virus. Infection with HPV normally results in formation of warts. Certain types of HPV, such as type -16 and -18, are shown to have a causal role in the development of uterine cervical cancer, and are so called high risk type HPV. Recently, a role of HPV in breast cancer has been suggested, although a causal role for HPVs in human breast cancer is yet to be demonstrated. The first part of this study investigates the association of HPV with human breast cancer. The results demonstrate that 48% of breast cancers that occurred in Australian women are HPV positive and they are mainly variants of HPV-18. Further analysis shows that HPV positive breast cancer patients are significantly younger than HPV negative patients, suggesting infection with HPV increases the risk of breast cancer development. This is coincidental with increased risk of HPV infection in sexually active young women and provides evidence that HPV has a role in breast cancer development. The second part of this project investigates the mechanisms by which high risk type HPV oncogenic protein E6, transforms primary human foreskin keratinocytes (natural host cells of HPV). HPV E6 is always expressed in HPV positive cervical carcinoma and results in the degradation of the cellular tumour suppressor protein p53. It is generally believed that HPV E6 contributes to HPV transformation by degradation of p53 protein which leads to cellular immortalisation ? an early step in tumorigenic transformation. Subsequent studies, however, indicate that HPV E6 possesses other functions (such as induction of telomerase activity) which may also be involved in cellular immortalisation. The results of my investigations demonstrate: 1) that degradation of p53 protein is required but is insufficient to immortalise primary cells; 2) that HPV E6 induced telomerase activity is coincidental with an increase in cell culture passage number; 3) that multiple functions of high risk type HPV E6 protein are required for cellular immortalisation. This finding suggests HPV infection is associated with early onset of breast cancer and that multiple functions of high risk type HPV E6 protein are involved in cellular immortalisation. Further study in both of these areas should provide alternative diagnostic markers, leading to prevention and treatment strategies for HPV positive breast cancer and other cancers.

The majority of human papillomavirus (HPV) types cause cutaneous and mucosal disease. Persistent infection with high-risk HPV types is the primary risk factor for the development of cervical cancer. The ability of the virus to persist is contributed to by numerous immune evasion mechanisms. We previously demonstrated that the HPV type 16 (HPV16) E6 protein, down-regulates epithelial (E)-cadherin expression and that the associated Langerhans cells (LC) depletion may contribute to impaired immune recognition by the host. The aims of this study were firstly to establish if E6 down-regulation of E-cadherin is conserved amongst all HPV types, secondly to determine if the reduced E-cadherin expression correlates with reduced LC density in HPV-infected tissues, thirdly, to identify a region of E6 responsible in E-cadherin regulation and fourthly to establish if down-regulation of cell surface E-cadherin also occurs in another DNA tumour virus, adenovirus (Ad). E6 protein from a range of HPV types representing the α, β and γ genera was expressed in HCT116 cells and the effect on cell surface E-cadherin expression was measured by flow cytometry. In addition, a series of tissues infected with HPV types representative of HPV of α, β, [nu] and γ genera were stained to confirm E-cadherin regulation in vivo and to determine the functional significance of E-cadherin expression in relation to LC localisation. In order to identify the region of the E6 protein that was important for E-cadherin regulation, a series of HPV16 E6 mutants were tested for their ability to regulate E-cadherin. Finally, the effects of Ad on cell surface E-cadherin were examined by measuring E-cadherin expression in Ad infected HCT116 cells. E6 down-regulation of E-cadherin was conserved in α, [nu] and γ genera but was lost in β-HPV types, correlating with the ability of the virus to persist. In vivo analysis of patient tissues confirmed this pattern of E-cadherin regulation by E6 types and showed a direct association between loss of E-cadherin and LC depletion, suggesting that E-cadherin regulation by E6 is the cause of depletion of LC in infected tissue. Mutational analysis of HPV16 E6 led to the identification of a putative E-cadherin regulatory region with a conserved motif, H/L/V-[phi]-X-X-X-X-R. A potential mechanism used by E6 to regulate cell surface E-cadherin involved down-regulation of p21[waf1/cip1] (p21) via a p53-independent pathway. Finally, study of Ad showed a similar ability of the virus to regulate E-cadherin, indicating conservation in another DNA tumour virus. This research shows that E-cadherin regulation by E6 is directly associated with LC depletion and viral persistence. The data presented here suggest that LC depletion by HPV is widely conserved in HPV types that cause persistent disease. E-cadherin regulation contributes to this effect through a specific regulatory region of the protein and manipulation of levels of cellular p21. These data may provide a foundation for the development of therapeutics for HPV that aim to overcome immune evasion by the virus.

Thesis (M.A.)--University of Central Florida, 2009. / Adviser: Fernando Rivera. Includes bibliographical references (p. 55-64).