臺灣博碩士論文加值系統

自1990年DNA-PK催化次體 (DNA-PK catalytic subunit; DNA-PKcs) 被發現以來，許多實驗顯示了此分子在細胞內參與多種重要的功能，包括在DNA傷害修復、 V(D)J重組、DNA複製、細胞週期檢查點的調控和細胞凋亡等。 但由於缺乏適當的研究工具，以至DNA-PKcs在這些作用機制內所扮演的角色和作用模式，至今仍是非常的不清楚。 因此，我們備製了數種DNA-PKcs的專一性抗體，包括能辨識於特定胺機酸上發生磷酸化的DNA-PKcs，也就是其磷酸激脢活性被啟動後的分子。 這使得我們第一次能以活體內的方式，觀察DNA-PKcs在不同生理狀況中被活化的情形，而近一步了解其在生理上扮演的意義。 由臨床病理組織切片的觀察，發現DNA-PKcs的磷酸化與惡性肝細胞癌的進程有高度密切的關係。 再者，我們發現在DNA複製期間， DNA-PKcs會磷酸化並存於複製後的中心體(centrosome)上，且對微小管的形成表現出正面調控的角色。 這些新發現，不但提供了細胞質可如何與DNA複製協調合作的可能機制，並賦予DNA-PKcs在細胞週期調控和腫瘤細胞的進行中所代表的關鍵位置。 本研究的第二部分運用具磷酸化特異性的抗體 P*N 鑑定一個新的核仁蛋白hNuPARP。 結果顯示此蛋白具有聚合腺苷二磷酸激酶的活性，並可被Cdk2/CyclinE磷酸化，而hNuPARP的磷酸化不論在細胞週期行進、或進入生長休止階段的過程中，均呈現受到高度調控的現象。 由於磷酸化hNuPARP與RNA聚合酶I並存於核糖體DNA轉錄點與核仁組織區中，推測此蛋白可能參與於核糖體DNA組織的調控機制中。

Since its first description in 1990, DNA-PK catalytic subunit has been one of the most attractive and intriguing molecules in the research field. The bulk of studies has implicated its participation in a wide array of cellular functions, including DNA damage repair, V(D)J recombination, DNA replication, cell-cycle checkpoint and apoptosis. Nevertheless, the role and mode of action of DNA-PKcs in these pathways still remain obscure, mainly because of its difficulty in manipulation, as well as the lack of reagents for tracking the active molecule in vivo. We have raised several antibodies against different portions of DNA-PKcs, and most importantly, antibodies that can recognize specifically phosphorylated DNA-PKcs, which represents the active kinase in in vivo. These antibodies enable us to describe for the first time the behavior of activated DNA-PKcs in physiological conditions, as well as during cell cycle progression. Our clinicopathological survey revealed a strong correlation between loss of DNA-PKcs phosphorylation and malignant tumor progression. Furthermore, we detected the presence of phosphorylated DNA-PKcs on duplicated centrosomes, and described its positive role in microtubule nucleation. These intriguing observations could offer the key to unravel the role of DNA-PKcs in coordination of DNA replication, control of chromosome integrity, and tumor progression. In second part, we report the identification of a novel nucleolar protein hNuPARP that possesses intrinsic poly ADP-ribosylation activity. Our preliminary studies suggest that hNuPARP may be the major epitope for the P*N antibody, a putative substrate of the Cdk2/cyclinE kinase, that is suppressed in growth arrested cells and associates with the RNA polymerase I machinery.

I. Introduction 1
Part I DNA-PKcs 2
Part II P*N epitope 4
II. Materials and Methods
Cell culture 6
Western blot analysis 6
DNA-PK kinase assay 6
Immunohistochemical staining 7
Immunofluorescence staining 7
Isolation and analysis of centrosomes 8
Microtubule nucleation test 8
Nucleolar isolation 9
In vitro kinase assays 9
Chromosome spreads 10
Nuclease treatment 10
In situ run-on transcription assay 10
PARP activity assay 11
III. Results
Part I: DNA-PKcs 12
Production and characterization of anti-DNA-PKcs antibodies 12
T2609 is a DNA-PKcs autophosphorylatable site 13
Expression of DNA-PKcs proteins in fetal and adult tissues 14
DNA-PKcs T2609 phosphorylation in HCC 15
Clinicopathological significance of DNA-PKcs T2609 Phosphorylation in HCC 15
Correlation of DNA-PKcs T2609 phosphorylation with AFP elevation and p53 and beta-catenin mutations 16
Association of DNA-PKcs T2609 phosphorylation with low-grade,
low-stage HCC, and infrequent high AFP elevation and early tumor recurrence: significance of beta-catenin and p53 mutations 17
Intracellular localization of DNA-PKcsT2609 phosphorylation 18
Identification of DNA-PKcs as a centrosomal protein 19
DNA-PKcs may be involved in microtubule nucleation 19
Part II: hNuPARP
Characterization of the P*N epitope 20
P*N epitope co-fractionates with purified nucleoli. 21
The interphase epitope for P*N is sensitive to Cdk inhibitors and is likely to be hNuPARP 22
Association of the P*N epitope with PolI transcription modules 23
The P*N epitope is not required for initiation of rDNA transcription and ribosome biogenesis 24
hNuPARP is a PARP that ADP-ribosylates itself 25
IV. Discussion
Part I 26
Chromosome instability and stability versus p53 and B-catenin mutations in HCC: role of DNA-PKcs-Ku complex 26
Future directions: linking DNA-PKcx activity to prediction of radiotherapy outcomes 28
DNA-PKcs is a centrosomal protein 29
Part II 31
V. Tables and Figures
Part I
TABLE 1. DNA-PKcs T2609 phosphorylation in 421 unifocal resected
primary hepatocellular carcinoma 34
TABLE 2. DNA-PKcs T2609 phosphorylation in relation to -fetoprotein level, and mutations of beta-catenin and p53 genes in hepatocellular carcinoma 35
TABLE 3. Clinicopathological correlation of DNA-PKcs T2609 phosphorylation and beta-catenin mutation in unifocal hepatocellular carcinoma 36
TABLE 4. Correlation of DNA-PKcs T2609 phosphorylation and p53
mutations with clinicopathological features of unifocal surgically resected hepatocellular carcinoma 37
Figure 1. The DNA-PKcs protein 38
Figure 2. Characterization of the anti-DNA-PKcs antibodies 40
Figure 3. T2609 is a DNA-PKcs autophosphorylatable site 42
Figure 4. Detection of DNA-PKcs T2609 phosphorylation in fetal tissues 43
Figure 5. Phosphorylation of DNA-PKcs at T2609 in different adult human tissues and in HCC samples 44
Figure 6. Cell cycle-dependent distribution of DNA-PKcs 46
Figure 7. DNA-PKcs is a component of the centrosome 48
Figure 8. Inhibition of microtubule nucleation by anti-DNA-PKcs antibodies 50
Part II
Figure 1. The hNuPARP protein 52
Figure 2. Characterization of the P*N antibody in mammalian cell lines 54
Figure 3. P*N recognizes a Cdk-phosphorylated nucleolar protein that is likely to be hNuPARP 56
Figure 4. Association of the P*N epitope with PolI transcription modules 58
Figure 5. The P*N epitope is not required for Initiation of rDNA transcription 60
Figure 6. Suppression of the P*N epitope in growth - arrested/differentiated cells 61
Figure 7. Association of the P*N epitope with PolI is DNA dependent 62
Figure 8. hNuPARP possess poli-ADP rybosylation activity 63
VI. References 65

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