Integration of human papillomavirus is not a necessary mechanism in cervical cancer development.

• Other Authors: Cheung, Lai Ken Jo.
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Cervix uteri > Cancer > Etiology; Papillomaviruses > Pathogenicity; Papillomavirus diseases; Uterine Cervical Neoplasms; Uterine Cervical Neoplasms > etiology; Papillomaviridae > pathogenicity; Papillomavirus Infections
• Online Access: http://library.cuhk.edu.hk/record=b5549521; http://repository.lib.cuhk.edu.hk/en/item/cuhk-327972

子宮頸癌是女性的主要癌症殺手，而人類乳頭瘤病毒 (HPV) 則是子宮頸癌形成的必要條件之一。HPV16型及HPV18型是全球最普遍的高危型HPV；而另一方面，HPV52及HPV58兩型在東亞地區的流行程度比世界其他地區為高。 === 過往有科學研究顯示HPV病毒載量的高低是引致高度癌前病變的重要決定因素，也有研究指出病毒載量與病變的嚴重程度成正比例，但同時亦有研究指兩者並無關係。HPV基因組可以兩種物理形態存在：游離型及整合型。HPV的E2基因可對E6及E7致癌基因產生重要的調節作用，而當HPV病毒與宿主染色體整合後，可使E2基因斷裂，因而令控制E6及E7致癌基因表達的負反饋基制失效。 === 本研究假設高病毒載量及由HPV基因組整合所造成的E2基因斷裂，並非引致子宮頸癌的僅一途徑。本研究分析了在不同程度的子宮頸細胞病變下，HPV16型、18型、52型及58型的病毒載量及基因整合情況。其中，有關HPV16型的研究部份更深入地探討了E6/7 mRNA的轉錄水平、E2和LCR的序列變異及E2結合位點的甲基化情況，最終希望能找出除了病毒基因整合之外的另一種致癌機理。 === 本研究的結果顯示，在不同HPV型所引致的子宮頸細胞病變中，病毒載體及病變程度之間的關係也存有差異；而根據管家基因的數量來為細胞DNA標準化，對準確分析不同程度子宮頸細胞病變的實驗結果至關重要。本研究的一項重要發現是部份侵襲性癌細胞只含有游離型HPV基因組；而在只含游離HPV基因組的侵襲性子宮頸癌樣本中，有三種E6/E7 mRNA的抄錄本水平與只含整合型基因組的樣本相若，反映在只含游離型HPV基因組的侵襲性子宮頸癌樣本中，E6/ E7 mRNA的表達量亦有上調。最重要的是，此表達量的上調並非由基因整合或E2基因斷裂所引致。 === 在只含有游離型病毒基因組的侵襲性子宮頸癌樣本中，E6及E7致癌基因表達上調的另一種機理，很可能是HPV16啟動區內E2結合位點上的CpG位點出現甲基化。這項觀察解釋及支持了當E2蛋白因結合位點甲基化而失去對E6及E7基因轉錄的抑制功能時，E6及E7致癌蛋白仍能保持高水平，而兩種蛋白產生協同作用，令細胞轉型及出現癌變。總結之言，本實驗也肯定了HPV整合並非導致子宮頸癌形成的唯一機理。 === Cervical cancer is a major cause of cancer-related death in women worldwide. Human papillomavirus (HPV) is essential, though not sufficient, to cause cervical cancer. HPV16 and HPV18 are the most prevalent high-risk types worldwide, whereas, HPV52 and HPV58 also show a notable higher prevalence in East Asia than in other parts of the world. === Studies have suggested that HPV viral load is an important determinant for the development of high-grade lesions. While some studies observed a positive correlation between viral load and disease severity, others have reported no association. The HPV genome can exist in two physical forms, episomal or integrated. The E2 gene, encoded by HPV has an important role in the regulation of E6 and E7 viral oncogenes. When HPV integrates into the host chromosome, it may result in disruption of the E2 gene thereby its control on the expression of the E6 and E7. === The hypothesis for this study was that high viral load and disruption of E2 gene associated with integration of HPV into the host genome was not the only pathway leading to cervical cancer development. In this study, the viral load and integration profile for HPV types 16, 18, 52 and 58 among different severity of cervical lesions were analyzed. Further detailed studies were performed on HPV16 with emphases on E6/E7 mRNA transcript levels, E2 and LCR sequence variation and the methylation status of two E2 binding sites. The ultimate aim was to determine what other alternative mechanisms exist apart from viral integration to drive the oncogenicity of HPV that lead to the development of cervical cancer. === The results showed that the relationship between viral load and disease varied between different HPV types and that normalization of cellular DNA input using a housekeeping gene was crucial for accurate interpretation among different cervical lesion grades. A key finding from this study was that a substantial proportion of invasive cervical carcinomas were found to contain the purely episomal form of the HPV genome. The levels of the three E6/E7 mRNA transcripts species in invasive cervical carcinomas containing the pure episomal form of the viral genome were found to be similar to those with pure integrated forms. This observation suggested that invasive cervical carcinoma samples containing the episomal form of the HPV genome were also mediated by the up-regulated E6/E7 mRNA expression. More importantly, this up-regulation in E6/E7 mRNA expression did not depend on integration and disruption of the E2 gene. === The alternative mechanism that up-regulated of the expression of E6 and E7 oncogene found in invasive cervical carcinoma samples harbouring the episomal form of the viral genome was likely to be a consequence of methylation of CpG sites in the two E2 binding sites at the promoter region of HPV16. This observation explained and supported that the repressive role of E2 on E6 and E7 transcriptional regulation was abolished due to methylation of the E2 binding sites, and that a sustained level of the E6 and E7 oncoproteins was maintained, working in synergy in cell transformation and in carcinogenesis. These observations confirmed the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer. === 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. === Cheung, Lai Ken Jo. === Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references (leaves 233-248). === Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Abstract also in Chinese. === Acknowledgements --- p.I === Abstract of thesis --- p.IV === 論文摘要 --- p.VII === Publications --- p.IX === Contents --- p.X === Figures --- p.XV === Tables --- p.XVIII === Abbreviations --- p.XIX === Chapter Chapter 1 --- Introduction --- p.1 === Chapter 1.1 --- Cervical Cancer --- p.2 === Chapter 1.1.1 --- Cervical Cytology Screening --- p.3 === Chapter 1.1.2 --- Classification System for Cervical Squamous Cell Dysplasia --- p.4 === Chapter 1.1.3 --- Histological Grading of Cervical Lesions --- p.6 === Chapter 1.1.4 --- Development of Cervical Cancer --- p.6 === Chapter 1.2 --- Structure of HPV --- p.7 === Chapter 1.1.1 --- HPV Genome Organization --- p.8 === Chapter 1.1.2 --- The E1 Protein --- p.10 === Chapter 1.1.3 --- The E2 Protein --- p.10 === Chapter 1.1.4 --- The E4 Protein --- p.13 === Chapter 1.1.5 --- The E5 Protein --- p.13 === Chapter 1.1.6 --- The E6 Protein --- p.14 === Chapter 1.1.7 --- The E7 Protein --- p.14 === Chapter 1.1.8 --- The L1 Protein --- p.15 === Chapter 1.1.9 --- The L2 Protein --- p.16 === Chapter 1.1.10 --- The Long Control Region --- p.17 === Chapter 1.3 --- HPV and Cervical Cancer --- p.19 === Chapter 1.3.1 --- HPV is an Etiological Cause of Cervical Cancer --- p.19 === Chapter 1.3.2 --- Establishment of HPV Infection --- p.20 === Chapter 1.3.3 --- Regulation and Control of HPV Viral Gene Transcription --- p.23 === Chapter 1.3.4 --- Viral Oncogene Expression by Alternative RNA Splicing --- p.23 === Chapter 1.3.5 --- DNA Methylation in Viral Oncogene Expression --- p.24 === Chapter 1.3.6 --- The Roles of E6 and E7 Protein in Cervical Carcinogenesis --- p.26 === Chapter Chapter 2 --- Controversies and Hypothesis --- p.33 === Chapter 2.1 --- Controversies in Mechanism of Cervical Carcinogenesis --- p.34 === Chapter 2.1.1 --- Viral Integration and Risk of Cervical Cancer Development --- p.34 === Chapter 2.1.2 --- Viral Load and Risk of Cervical Cancer Development --- p.35 === Chapter 2.2 --- Hypothesis of Study --- p.37 === Chapter 2.2.1 --- Study Design --- p.38 === Chapter Chapter 3 --- Materials and Methods --- p.41 === Chapter 3.1 --- Patient Recruitment and Sample Preparation --- p.42 === Chapter 3.1.1 --- Study subject recruitment --- p.42 === Chapter 3.1.2 --- Collection of cytology samples --- p.43 === Chapter 3.1.3 --- Collection of cervical biopsy samples --- p.44 === Chapter 3.2 --- Nucleic Acid Extraction and Preparation --- p.44 === Chapter 3.2.1 --- Extraction of DNA from cervical cytology samples --- p.44 === Chapter 3.2.2 --- Extraction of DNA from cervical biopsy samples --- p.45 === Chapter 3.2.3 --- Extraction of RNA from cervical cytology samples --- p.45 === Chapter 3.2.4 --- Extraction of RNA from cervical biopsy samples --- p.46 === Chapter 3.3 --- Detection and Genotyping of Human Papillomavirus --- p.46 === Chapter 3.4 --- Determination of Viral Load using Real-Time Polymerase Chain Reaction --- p.47 === Chapter 3.4.1 --- Optimization of HPV16, 18, 52 and 58 E7 real-time PCR --- p.48 === Chapter 3.4.2 --- Optimization of housekeeping gene real-time PCR --- p.50 === Chapter 3.4.3 --- Determination of HPV16, 18, 52 and 58 viral load --- p.50 === Chapter 3.5 --- Determination of HPV Genome Physical Status --- p.53 === Chapter 3.5.1 --- HPV E2 gene primer design --- p.53 === Chapter 3.5.2 --- Optimization of HPV16, 18, 52 and 58 E2 Real-time PCR --- p.56 === Chapter 3.5.3 --- Determination of the HPV genome physical status --- p.59 === Chapter 3.6 --- Evaluation of Housekeeping Genes for Normalization of Viral Gene Expression --- p.62 === Chapter 3.6.1 --- Optimization of housekeeping gene real-time PCR --- p.62 === Chapter 3.6.2 --- Quantitation of RNA and DNase treatment --- p.66 === Chapter 3.6.3 --- cDNA synthesis from the extracted RNA --- p.67 === Chapter 3.6.4 --- Detection of five housekeeping gene levels from cervical cytology samples by real-time PCR --- p.67 === Chapter 3.6.5 --- Data analyses --- p.68 === Chapter 3.7 --- Quantitation of HPV16 mRNA Transcripts --- p.69 === Chapter 3.7.1 --- Preparation of RNA from CaSki cells --- p.69 === Chapter 3.7.2 --- Amplification of mRNA transcripts from CaSki cells --- p.69 === Chapter 3.7.3 --- Amplification of artificial mRNA transcript E6*II --- p.73 === Chapter 3.7.4 --- Gel purification of mRNA transcript amplicons --- p.73 === Chapter 3.7.5 --- Cloning of E6 mRNA transcripts --- p.74 === Chapter 3.7.6 --- Confirmation of the mRNA transcript inserts --- p.74 === Chapter 3.8 --- Quantitation HPV16 E6 mRNA Transcript Levels Using Real-Time PCR --- p.79 === Chapter 3.8.1 --- mRNA transcript primer and probe design --- p.79 === Chapter 3.8.2 --- Optimization of real-time PCR for the detection of mRNA transcripts --- p.82 === Chapter 3.8.3 --- Determination of mRNA transcript levels from invasive carcinomas --- p.83 === Chapter 3.8.4 --- Normalization of mRNA transcript expression with a housekeeping gene --- p.84 === Chapter 3.9 --- Sequence Variation of the HPV16 E2 and Long Control Region --- p.84 === Chapter 3.9.1 --- Identification of sequence variation of the E2 gene --- p.84 === Chapter 3.9.2 --- Identification of sequence variation of the long control region --- p.87 === Chapter 3.1 --- Detection of Methylation Status of E2BS1 and E2BS2 on the LCR using Pyrosequencing --- p.87 === Chapter 3.10.1 --- Bisulfite DNA conversion --- p.87 === Chapter 3.10.2 --- Amplification of E2 binding site regions on the LCR --- p.88 === Chapter 3.10.3 --- Purification of PCR product prior to pyrosequencing --- p.92 === Chapter 3.10.4 --- Quantitation of methylation using pyrosequencing --- p.92 === Chapter Chapter 4 --- Results --- p.93 === Chapter Hypothesis 1 --- p.94 === Chapter Results of Study Part: 1 --- p.95 === Chapter 4.1 --- Human Papillomavirus Type 16 Viral Load and Genome Physical Status --- p.96 === Chapter 4.1.1 --- E7 viral load --- p.96 === Chapter 4.1.2 --- Viral genome physical status --- p.100 === Chapter 4.1.3 --- E2 disruption site --- p.105 === Chapter 4.2 --- Human Papillomavirus Type 18 Viral Load and Genome Physical Status --- p.107 === Chapter 4.2.1 --- E7 viral load --- p.107 === Chapter 4.2.2 --- Viral genome physical status --- p.110 === Chapter 4.2.3 --- E2 disruption site --- p.113 === Chapter 4.2.4 --- Infection status --- p.116 === Chapter 4.2.5 --- Adeno/adenosquamous carcinoma versus squamous cell carcinoma --- p.119 === Chapter 4.3 --- Human Papillomvirus Type 52 Viral Load and Genome Physical Status --- p.120 === Chapter 4.3.1 --- E7 viral load --- p.120 === Chapter 4.3.2 --- Viral genome physical status --- p.123 === Chapter 4.3.3 --- E2 disruption site --- p.126 === Chapter 4.3.4 --- Infection status --- p.129 === Chapter 4.4 --- Human Papillomavirus Type 58 Viral Load and Genome Physical Status --- p.131 === Chapter 4.4.1 --- E7 viral load --- p.131 === Chapter 4.4.2 --- Viral genome physical status --- p.133 === Chapter 4.4.3 --- E2 disruption site --- p.134 === Chapter 4.4.4 --- Infection status --- p.137 === Chapter 4.5 --- Summary of Study Part 1: --- p.140 === Chapter Hypothesis 2 --- p.141 === Chapter Results of Study Part 2: --- p.142 === Chapter 4.6 --- Housekeeping Gene mRNA Expression Level --- p.143 === Chapter 4.6.1 --- Expression levels across different grades of cervical lesion --- p.143 === Chapter 4.6.2 --- Expression stability of housekeeping genes --- p.145 === Chapter 4.7 --- Summary of Study Part 2: --- p.149 === Chapter Results of Study Part: 3 --- p.150 === Chapter 4.8 --- HPV16 mRNA Transcript Expression Level --- p.151 === Chapter 4.8.1 --- HPV16 viral genome physical status --- p.151 === Chapter 4.8.2 --- HPV16 E2 disruption site --- p.151 === Chapter 4.8.3 --- Expression level of E6/E7 mRNA transcripts --- p.155 === Chapter 4.8.4 --- Expression level of E6/E7 mRNA transcripts and viral genome physical status --- p.157 === Chapter 4.8.5 --- Expression level of E6/E7 mRNA transcripts and E2 gene disruption status --- p.161 === Chapter 4.9 --- Summary of Study Part 3: --- p.163 === Chapter Hypothesis 3 --- p.165 === Chapter Results of Study Part 4: --- p.166 === Chapter 4.1 --- HPV 16 E2 Gene Sequence Variation --- p.167 === Chapter 4.10.1 --- Sequence variation of E2 gene --- p.167 === Chapter 4.10.2 --- Sequence variation and viral genome physical status --- p.168 === Chapter 4.10.3 --- Sequence variation in the E2 binding sites --- p.169 === Chapter 4.10.4 --- Sequence variations of E2 in HPV16 cancer derived cell lines --- p.170 === Chapter 4.11 --- HPV16 Long Control Region Sequence Variation --- p.174 === Chapter 4.11.1 --- Sequence variation of LCR --- p.174 === Chapter 4.11.2 --- Sequence variation and viral genome physical status --- p.175 === Chapter 4.11.3 --- Sequence variation in E2 binding sites --- p.176 === Chapter 4.11.4 --- Sequence variation of LCR in HPV16 cancer derived cell lines --- p.176 === Chapter 4.12 --- Summary of Study Part 4: --- p.183 === Chapter Hypothesis 4 --- p.185 === Chapter 4.13 --- Methylation Status of E2 Binding Sites --- p.187 === Chapter 4.13.1 --- Proportion methylation in E2 binding sites --- p.187 === Chapter 4.13.2 --- Methylation in invasive carcinomas according to the viral genome physical status --- p.191 === Chapter 4.14 --- Summary of Study Part 5: --- p.195 === Chapter Chapter 5 --- Discussion --- p.196 === Chapter 5.1 --- Viral Load --- p.197 === Chapter 5.2 --- Viral Integration --- p.200 === Chapter 5.2.1 --- HPV16 Viral Load and Physical Status --- p.201 === Chapter 5.2.2 --- HPV18 Viral Load and Physical Status --- p.204 === Chapter 5.2.3 --- HPV52 Viral Load and Physical Status --- p.207 === Chapter 5.2.4 --- HPV58 Viral Load and Physical Status --- p.210 === Chapter 5.2.5 --- Viral Load and Physical Status Summary --- p.214 === Chapter 5.3 --- HPV16 E6/E7 mRNA Transcript and Genome Physical Status --- p.215 === Chapter 5.4 --- HPV16 E2 Sequence Variation and Genome Physical Status --- p.218 === Chapter 5.5 --- HPV16 LCR Sequence Variation and Genome Physical Status --- p.222 === Chapter 5.6 --- Methylation of HPV16 E2 Binding Sites and Genome Physical Status --- p.225 === Chapter 5.7 --- Conclusions --- p.230 === Chapter 5.8 --- Implication of Current Findings and Future Work --- p.231 === References --- p.233