在台灣，感染B型肝炎病毒為肝癌發生的一個重要致病因子，B型肝炎病毒的大表面蛋白和其所引起的肝癌是有正相關的，但大表面蛋白在細胞週期中所扮演的角色目前仍舊不明。首先，我們發現了大表面蛋白在人類逆轉錄酶永生肝細胞株中會促進有絲分裂蛋白，包括 Polo-like kinase-1（Plk1），Cyclin-dependent kinase-1（Cdk1）和Cyclin B的大量表現。此外，我們藉由活細胞攝影技術，發現了大表面蛋白會促進肝細胞有絲分裂。接著，我們以 BrdU incorporation assay 也證明了大表面蛋白會促進肝細胞在DNA複製完全前進入有絲分裂。一般而言，正常的肝細胞若抑制topoisomerase-II，則完全無法進入有絲分裂，但在表現大表面蛋白的肝細胞株中，卻只有些微延遲有絲分裂進入的時間，這意味著表現大表面蛋白可能可以弱化肝細胞的 G2/M檢查點。我們接著發現抑制Plk1可以減少 Cdk1-tyr15其抑制型磷酸化的表現，進而得以延遲有絲分裂的進入，因此，我們推測弱化G2/M檢查點可能是由於大量表現Plk1進而活化Cdc25而來。最後，我們更進一步發現在表現大表面蛋白的肝細胞株中，熱休克蛋白(HSP)，包括HSP70和HSP72都大量表現，倘若抑制了HSP70家族蛋白的活性，不僅可以減少Aurora B，Plk1和Cdk1的表現，還可以延遲有絲分裂的進入和細胞增殖。綜觀而言，從我們的研究中，我們推測大表面蛋白可以藉由HSPs和有絲分裂激酶來弱化G2/M檢查點，更進一步得以促進細胞有絲分裂。

Viral infection of hepatitis B virus (HBV) is a major etiologic factor of hepatocellular carcinoma (HCC) in Taiwan. The expression of viral large surface protein (LHBs) is involved in HBV-related hepatocarcinogenesis. To investigate the role of LHBs in cell cycle progression, we showed that several essential mitotic regulators, including Polo-like kinase-1 (Plk1), Cyclin-dependent kinase-1 (Cdk1) and Cyclin B, were upregulated by LHBs proteins in human telomerase reverse transcriptase (hTERT)-immortalized hepatocytes. With the application of live cell imaging, we confirmed that hepatocytes carrying LHBs proteins displayed early mitotic entry. Furthermore, we demonstrated that LHBs-positive cells entered mitosis prior to the completion of DNA replication, as indicated by BrdU incorporation assay. Inhibition of topoisomerase-II completely blocked mitotic entry in control cells, but only caused a slight mitotic delay in LHBs-positive cells, suggesting that the G2/M checkpoint is attenuated in the presence of LHBs. To explore the underlying mechanism leading to this attenuation, we found that the inhibition of Plk1 decreased the phosphorylation on tyrosine-15 of Cdk1, and thereby delayed the mitotic entry. Thus, we speculate that Plk1 overexpression attenuate the G2/M checkpoint through activating of Cdc25. In searching for potential upstream regulators of these mitotic kinases, we found several heat shock proteins (HSP), including HSP70 and HSP72, were overexpressed in LHBs-expressing cells. Inhibitions of HSP70 family not only reduced the expression levels of Aurora B, Plk1 and Cdk1, but also delayed the mitotic entry and overall cell proliferation in LHBs-expressing cells. Together, these results suggest that LHBs promotes early mitotic entry through the attenuation of G2/M checkpoint, which is mediated by the HSP-dependent induction of essential mitotic kinases.

Abstract 3
摘要 4
1. Introduction 5
1.1 HBV and HCC 5
1.2 Ground glass hepatocytes (GGHs) and Pre-S mutants 6
1.3 G2/M checkpoint mechanism controls mitotic entry 8
1.4 Heat shock protein and mitosis 10
2. Hypothesis and specific aims 12
3. Materials and methods 13
4. Results 16
4.1 Pre-S2-LHBs enhances mitotic regulators on transgenic mice 16
4.2 LHBs promotes mitotic regulators on hTERT-immortalized hepatocytes 16
4.3 LHBs enhances mitotic index on immortalized hepatocytes 17
4.4 The induction of mitotic regulators is cell cycle independent 17
4.5 LHBs proteins promotes early mitotic entry 17
4.6 LHBs-positive cells enter mitosis before the completion of DNA replication 18
4.7 LHBs-positive cells attenuate G2/M checkpoint 18
4.8 Activities of ATR/ATM checkpoint kinases are intact in LHBs-positive cells 19
4.9 Inhibitions of Plk1 and Cdk1 delay the mitotic entry of LHBs-positive hepatocytes 19
4.10 Plk1 promotes dephosphorylation of Cdk1 in LHBs-positive cells 20
4.11 Early mitotic entry in LHBs-positive cells is independent of FoxM1 and HIF-1 20
4.12 LHBs promotes the expression of heat shock proteins HSC70, HSP72 and HSP90 23
4.13 LHBs increases cell sensitivity to inhibitors targeting the mitotic progression 23
5. Discussion 25
5.1 What are the roles of HSP70 and HSP90 in LHBs-positive cells? 25
5.2 Do FoxM1 and HIF-1α play a role in mitotic entry in LHBs-positive cells? 26
5.3 How is G2/M checkpoint attenuated in LHBs-positive cells? 27
6. Figures 29
Figure 1. LHBs increased the expressions of essential mitotic regulators on hTERT immortalized hepatocytes. 29
Figure 2. LHBs enhances mitotic index on hTERT-immortalized hepatocytes. 30
Figure 3. The induction of mitotic regulators is cell cycle independent in LHBs-positive cells. 31
Figure 4. LHBs-positive hepatocytes displayed early mitotic entry. 32
Figure 5. LHBs-positive cells entered mitosis prior to the completion of DNA replication. 33
Figure 6. LHBs-positive cells attenuate G2/M checkpoint. 34
Figure 7. Activities of ATR/ATM checkpoint kinases are intact in LHBs-positive cells. 35
Figure 8. Delayed the mitotic entry on LHBs-positive hepatocytes upon inhibitions of Plk1 and Cdk1. 36
Figure 9. Plk1 promotes dephosphorylation of Cdk1 in LHBs-positive cells. 37
Figure 10. Overexpression of FoxM1 in LHBs-positive cells. 38
Figure 11. PI3K, mTOR, MAPK and AMPK are not involved in the induction of mitotic regulators and FoxM1 in LHBs-positive cells. 39
Figure 12. The depletion of FoxM1 levels did not reduce the expressions of Plk1, Cdk1 and Aurora B 40
Figure 13. Echinomycin reduced the expression levels of mitotic regulators 41
Figure 14. Echinomycin abolished the mitotic entry in LHBs-expressing cells. 42
Figure 15. The depletion of HIF-1α did affect mitotic regulator expression in LHBs-positive cells 43
Figure 16. LHBs promoted the expression of heat shock proteins (HSC70, HSP72 and HSP90) in LHBs-positive cells. 44
Figure 17. Inhibition of HSP70/90 reduced expressions of mitotic regulators in LHBs-positive cells. 45
Figure 18. Delayed mitotic entry by VER-155008. 46
Figure 19. Hepatocytes carrying LHBs proteins displayed higher sensitivity to inhibitors targeting cell progression but were more resistant to ER stress. 47
Figure 20. Current working model illustrating the role of LHBs in regulating mitotic entry through attenuating the G2/M checkpoint. 48
7. References 49
8. Tables 56
Table 1. Reagents and kits 56
Table 2. List of the drugs 57
Table 3. List of antibodies 57
Table 4. Buffer and solution preparation 58
9. Appendices 59
Appendix 1. The expression of mitotic regulators in different cancers 59
Appendix 2. The table of cell cycle associated proteins which regulated by HSP90 59
Appendix 3. Pre-S2-LHBs enhances mitotic regulators in vivo 60

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