肝癌為全世界致死率第二高之癌症。約70-80%之肝癌屬於肝細胞癌 (Hepatocellular carcinoma, HCC) 並且其中近六成與慢性B型肝炎病毒感染有關，而B型肝炎病毒之大表面蛋白 (HBV large surface antigen, LHBs) 則被視為促使肝細胞癌發生之致癌蛋白。在本研究中，我們發現LHBs的對於腫瘤抑制蛋白p53的表現有負調控之作用。此負調控肇因於降低腫瘤抑制蛋白p53之穩定性並同時促進其負調控因子Mdm2之表現。我們也發現在帶有LHBs之肝細胞中，p53的負調控現象會伴隨著較高DNA損傷以及減低細胞對與DNA損傷之反應，且在此細胞中進一步誘導DNA損傷並無法活化腫瘤抑制因子p53以及其下游分子p21。而LHBs的表現也造成了細胞出現多核 (multinucleation) 之情形，顯示負調控p53使LHBs細胞無法維持其正確之染色體倍數。利用Nutlin-3干擾p53-Mdm2之間交互作用可恢復細胞中p53表現量但不具活性，反之抑制細胞中Sirtuins之去乙醯化活性可回復p53表現量及其生物功能。為了釐清LHBs如何調控p53蛋白之穩定性，我們鑑定出一屬於BAG蛋白質家族之伴護子調節蛋白，Bag2，能與LHBs結合而影響其原本做為p53伴護子之功能。實驗結果顯示在LHBs細胞中大量表現Bag2可恢復部分p53的表現。總結上述結果，LHBs利用兩種不同機制來弱化腫瘤抑制因子p53之功能，分別為藉由與Bag2結合來影響其保護p53之功能以及將p53去乙醯化使p53失去生物活性。這些發現對於LHBs如何誘發肝癌的致病機轉提供了新的機轉。

Liver cancer is the second frequent cause of cancer death worldwide. About 70-80% of liver cancer belongs to hepatocellular carcinoma (HCC) and nearly 60% of HCC are associated chronic hepatitis B virus (HBV) infection. HBV large surface antigen (LHBs) has been considered as a potential oncoprotein in hepatocarcinogenesis. In this study, we found that the presence of LHBs down-regulates tumor suppressor p53 in hepatocytes. This down-regulation is associated with decreased stability of p53 and increased Mdm2 expression. We also found that p53 down-regulation accompanies with intrinsic DNA damages and reduced of DNA damage response in hepatocytes. Chemical induced DNA damages fails to activate p53 and its downstream p21 in LHBs-expressing cells. Moreover, we detected a significant increase in multinucleation in LHBs-expressing cells, indicating a loss of ploidy control. The disruption of p53-Mdm2 interaction with Nutlin-3 is not sufficient to restore p53 expression in LHBs-expressing cells, whereas the inhibition of Sirtuin-mediated p53 deacetylation successfully rescues p53 expression and its biological function. To clarify how does LHBs regulates p53 stability, we identified BAG family molecular chaperone regulator 2 (Bag2), a binding partner of LHBs which acts as a chaperon protein of p53. Overexpression of Bag2 partially restored p53 expression level in LHBs-expressing cells. Together these results indicate that LHBs attenuates p53 functionality via two independent mechanisms, compete with p53 for binding to chaperon protein Bag2 and inactivate p53 by deacetylation. These findings therefore highlight the pathologic function of LHBs in HBV-mediated hepatocarcinogenesis.

Abstract 3
中文摘要 4
1. Introduction 5
1.1 HBV-mediated hepatocarcinogenesis 5
1.2 Tumor suppressor p53 7
1.3 p53 loss of function and aneuploidy 8
1.4 Sirtuins and cancer 9
2. Hypothesis and specific aims 11
3. Material and methods 12
3.1 Cell culture 12
3.2 Plasmid construction 12
3.3 RNA extraction and reverse transcriptase-polymerase chain reaction 12
3.4 Western blotting 13
3.5 Immunoprecipitation 13
3.6 Immunofluorescence staining (IF) 14
3.7 Affinity tagged protein pulldown assay 14
3.8 Cell proliferation assay 14
3.9 Recombinant p53-HA stability analysis 14
3.10 Time-lapse live cell imaging 15
3.11 RNA interference 15
3.12 Flow Cytometry Analysis 15
4. Results 16
4.1 The down-regulation of p53 in LHBs-expressing cell was mediated by the post-translational regulation 16
4.2 LHBs protein accelerate p53 degradation through promote Mdm2 expression 16
4.3 LHBs-expressing cell carries intrinsic DNA damages 17
4.4 LHBs promotes proliferation of multinucleated hepatocytes 17
4.5 p53 activity can be rescued in LHBs-expressing cells by inhibiting Sirtuin 18
4.6 BAG family molecular chaperone regulator 2 (Bag2), binds to LHBs protein in LHBs-expressing cell 19
4.7 Bag2 associate with p53 and restore p53 level in LHBs-expressing cell 19
4.8 Sirtuins-mediated p53 deacetylation destabilize p53 through dissociate from Bag2 20
5. Discussion 21
5.1 How is Mdm2 regulated by LHBs? 21
5.2 How is Sirtuins activated by LHBs? 21
5.3 The role of Bag2 in LHBs-expressing cells 22
5.4 The role of LHBs in HCC progression 23
6. Figures 25
Figure 1. Large hepatitis surface antigen (LHBs) attenuates p53 expression in hTERT-immortalized hepatic progenitor cell NeHepLxHT. 25
Figure 1. Continued 26
Figure 2. Protein stability of p53 is down-regulated in LHBs-positive cells. 27
Figure 3. LHBs promotes Mdm2-mediated proteasomal degradation of p53. 28
Figure 3. Continued 29
Figure 4. Cell proliferation of LHBs-expressing cells is independent of p53-Mdm2 interaction. 30
Figure 5. LHBs-expressing cell responses poorly to Etoposide. 31
Figure 5. Continued 32
Figure 6. LHBs promotes multinucleation in hepatocytes. 33
Figure 6. Continued 34
Figure 7. The LHBs expressing cells have a higher mitotic index. 35
Figure 8. Ectopic expressing p53 increases p21 expression in LHBs expressing cells. 36
Figure 9. Inhibition of Sirtuins increases the expressions of p53 and p21. 37
Figure 9. Continued 38
Figure 10. LHBs interacts with BAG family molecular chaperone regulator 2 (Bag2) in hepatocyte. 39
Figure 11. Overexpression Bag2 and knockdown LHBs rescued p53 in LHBs-expressing cells. 40
Figure 12. Inhibition of Sirtuin by Tenovin-6 increases the interaction between Bag2 and p53 in LHBs-expressing cells. 41
Figure 13. Knockdown LHBs and treatment of Tenovin-6 restored the protective effect of Bag2 on p53. 42
Figure 14. Cell proliferation in LHBs cells were inhibited upon Tenovin-6 treatment. 43
Figure 15. The consequence of LHBs expression in DNA damage response. 44
7. Reference 45
8. Tables 51
9. Appendices 54

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