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研究生：	林彥伶 Lin, Yang-Ling
論文名稱：	NDPK-A 和SGK-1在人類母纖維細胞瘤所扮演的角色 The role of NDPK-A and SGK-1 in neuroblastoma
指導教授：	張玲 Chang, Christina Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	111
中文關鍵詞：	人類神經母細胞瘤、核苷二磷酸激-A 、血清和醣皮質素調控激酶-1 、細胞存活、賀爾蒙壓力、高張壓力
外文關鍵詞：	neuroblastoma, NDPK-A, SGK-1, cell viability, hormonal stress, hypertonic stress
相關次數：	點閱：109 下載：2
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人類神經母細胞瘤是兒童中最常見的癌症之一，導因主要是神經細胞在發育時不正常的分化以及失調的死亡。處在惡性階段的神經母細胞瘤病人，發現其核苷二磷酸激-A (nucleoside diphosphate kinase A; NDPK-A)有放大和過度表現的現象。先前我們實驗室的研究結果發現核苷二磷酸激-A 的促進人類神經母細胞瘤癌的轉移且也的增進細胞的存活力。根據實驗室cDNA microarray的結果，NDPK-A可以增加血清和醣皮質素調控激酶-1 (serum-and glucocorticoid- regulated protein kinase-1; SGK-1) 在人類神經母細胞瘤細胞株NB69的表現。過去的研究發現在荷爾蒙或高張壓力環境下SGK-1具有促進細胞存活的能力。因此本研究的假說是NDPK-A在人類神經母細胞瘤中所促進細胞存活的能力是藉由影響SGK-1的表現來調控。在實驗過程中，我利用10uM濃度的人工合成的糖皮質素dexamethasone讓細胞處在荷爾蒙的壓力下三天，實驗結果顯示表現外生型NDPK-A的NB69細胞存活力相較於控制組增加了1.5-2倍。在高濃度的氯化鈉的高張壓力下，NDPK-AWT及NDPK-AS120G同樣也增加了1.5-2倍細胞存活力。然而，失去酵素活性的NDPK-AH118F卻喪失此功能。當NB69細胞同時表現NDPK-A與SGK-1野生型或突變型時，在荷爾蒙壓力及高張壓力下不具激酶活性的SGK-1會抑制野生型NDPK-A所調控的細胞存活，而野生型或持續活化的SGK-1突變則無法。總結來說，在人類神經母細胞瘤中，NDPK-A經由SGK-1的路徑而促進細胞的存活。

Neuroblastoma is a common childhood cancer, due to neuronal differentiation arrest and deregulated cell death. Amplification and overexpression of nucleoside diphosphate kinase A (NDPK-A) has been detected in advanced neuroblastoma. Our laboratory previously demonstrated that NDPK-A promotes neuroblastoma metastasis in part by enhancing the survival of neuroblastoma cells. According to our cDNA microarray data, a high level of NDPK-A appears to up-regulate serum- and glucocorticoid-regulated protein kinase-1 (SGK-1) in human neuroblastoma NB69 cells. SGK-1 is known to play a role in promoting cell survival under hormonal and hypertonic stresses. In this study, therefore, we hypothesize that overexpression of NDPK-A, as detected in advanced neuroblastoma, increases the survival of neuroblastoma cells via SGK-1. When NB69-derived stable transfectants were under hormonal stress by treating with 10 μM dexamethasone for 72 h, a high level of ectopic NDPK-A increased cell viability by 1.5-2 folds, relative to the vector-transfected control. Under hypertonic stress when the cells were treated with high concentrations of NaCl for 72 h, both ectopic wild type NDPK-AWT and metastasis-associated NDPK-AS120G mutant increased cell viability by ~1.5 and 2 folds respectively. However, this was not the case for an enzymatically inactive NDPK-AH118F mutant. I further generated NB69-derived stable transfectants that co-expressed NDPK-A and SGK-1 variants. Under hypertonic and hormonal stresses, dominant negative and not the wild type or constitutively active SGK1 inhibited NDPK-A mediated cell survival. Our findings indicate that NDPK-A enhanced the viability of neuroblastoma cells via upregulating SGK-1.

Contents
英文摘要………………………………………………………………….…iii
中文摘要………………………………………………………………….….v
I.	Introduction
I.1.	Neuroblastoma	1
I.2.	Nucleoside diphosphate kinase (NDPK)-A	1
I.3.	Serum- and glucocorticoid-regulated protein kinase-1 (SGK-1)	2
I.4.	Hypertonic stress	4
I.5.	Glucocorticoid mediated hormonal stress	4
II.	Hypothesis and specific aims
II.1.	Hypothesis	6
II.2.	Specific aims	6
III.	Materials and methods
III.1.	Cell lines and culture	7
III.2.	Calcium phosphate transfection	8
III.3.	Construction of SGK-1 expression plasmids	8
III.4.	Addition of the hygromycin selection marker via recombination	8
III.5.	Site-directed mutagenesis	9
III.6.	Generation of stable cell lines	10
III.7.	Immunoblotting	10
III.8.	MTT assay (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetra- zolium bromide assay)	12
III.9.	Hyperosmotic and hormonal stresses	12
III.10.	Generation of knockdown NDPK-A in HeLa with the measurement of NDPK-A and SGK-1protein level	13
III.11.	Time course of endogenous SGK-1 expression under hypertonic and hormonal stresses	13
III.12.	Cell sub-G1 percentage analysis by flow cytometry under hypertonic and hormonal stresses	14
IV.	Experimental results
IV.1.	Constructions of SGK-1 variants expressing plasmids	15
IV.2.	The effects of NaCl-depended hypertonic stress on the cell viability of cells over-expressed wild type NDPK-A	15
IV.3.	The effects of NaCl-depended hypertonic stress on the cell viability when knockdown NDPK-A on HeLa	16
IV.4.	The relative cell viability on hypertonic stress when cells over-expressed NDPK-A variants	17
IV.5.	The percentage of sub-G1 on NB69 and HeLa cells under hypertonic stress by cell cycle analysis	17
IV.6.	The percentage of sub-G1 on NB69-derived NDPK-A variants transfectants under hypertonic stress by cell cycle analysis	18
IV.7.	Endogenous SGK-1 protein expression level when NB69 cells under hypertonic stress by time-dependent matter	18
IV.8.	Endogenous SGK-1 protein expression level in the cells which over-expressed NDPK-A variants after hypertonic stress treatment	19
IV.9.	The relative cell viability on hypertonic stress treatment when cells over-expressed SGK-1 variants	19
IV.10.	The relative cell viability on hypertonic stress treatment when cells over-expressed NDPK-AWT and SGK-1 variants together	20
IV.11.	The relative cell viability on hypertonic stress treatment when cells over-expressed NDPK-AS120G and SGK-1 variants together	20
IV.12.	The relative cell viability on hypertonic stress treatment when cells over-expressed NDPK-AH118F and SGK-1 variants together	21
IV.13.	The effect of hormonal stress by dexamethasone on the cell viability of cells over-expressed wild type NDPK-A	22
IV.14.	The effect of hormonal stress on the cell viability when knockdown NDPK-A  in HeLa	22
IV.15.	The relative cell viability on hormonal stress when cells over-expressed NDPK-A variants	23
IV.16.	The percentage of sub-G1 on NB69 and HeLa cells under hormonal stress by cell cycle analysis	23
IV.17.	The percentage of sub-G1 on NB69-derived NDPK-A variants transfectants under hormonal stress by cell cycle analysis	23
IV.18.	Endogenous SGK-1 protein expression level when NB69 cells under hormonal stress by time-dependent matter	24
IV.19.	Endogenous SGK1 protein expression level in the cells which over-expressed NDPK-A variants after hormonal stress	24
IV.20.	The relative cell viability on hormonal stress when cells over-expressed SGK-1 variants	25
IV.21.	The relative cell viability on hormonal stress when cells over-expressed NDPK-AWT and SGK-1 variants together	25
IV.22.	The relative cell viability on hormonal stress when cells over-expressed NDPK-AS120G and SGK-1 variants together	25
IV.23.	The relative cell viability on hormonal stress when cells over-expressed NDPK-AH118F and SGK-1 variants together	26
V. Discussion……………………………………………………………………………...27
VI. References	31
VII. Table
Table.1.Primer pairs for cloning the wild type and mutant SGK-1 expression plasmids…..	37
VIII.	Figures
Figure 1.The wild type and mutant of SGK-1 expressing plasmids. .	38
Figure 2.	Ectopic NDPK-AWT increases the cell viability of NB69 cells under hypertonic stress.	40
Figure 3.	Knockdown of endogenous NDPK-A in HeLa cells reduces cell viability under hypertonic stress.	41
Figure 4.	NDPK-AWT and NDPK-AS120G, but not NDPK-AH118F increase cell viability under hypertonic stress.	42
Figure 5.Hypertonic stress increases sub-G1 subpopulation in NB69 cells in a time-dependent manner.	43
Figure 6.Hypertonic stress increases sub-G1 subpopulation in HeLa cells in a time-dependent manner.	44
Figure 7.	NDPK-A variants decrease sub-G1 subpopulation under hypertonic stress in NB69 derivatives. 	46
Figure 8.Hypertonic stress affects expressions of endogenous SGK-1 in NB69 cells………	47
Figure 9.	A high level of NDPK-A variants increases endogenous SGK-1 expression under hypertonic stress..	48
Figure 10. SGK1WT and SGK1CA, but not SGK1DN, increase cell viability under hypertonic stress. .	49
Figure 11.Ectopic SGKDN decreases NDPK-AWT mediated cell viability under hypertonic stress.	50
Figure 12. Ectopic SGK1 variants fail to enhance NDPK-AS120G mediated cell viability under hypertonic stress.	51
Figure 13. Ectopic SGKWT and SGKCA enhance the viability of NB69 derivatives that express inactive NDPK-AH118 under hypertonic stress.  	52
Figure 14.NDPK-AWT increases the cell viability of NB69 cells under hormonal stresses…...	53
Figure 15.Knockdown of endogenous NDPK-A in Hela cells did not reduce cell viability under hormonal stress.	54
Figure 16. NDPK-AWT, NDPK-AS120G and NDPK-AH118F increase cell viability under hormonal stress.	55
Figure 17.Hormonal stress fails to increase the death of NB69 cells.	56
Figure 18.Hormonal stress fails to increase the death of HeLa cells.	57
Figure 19.NDPK-AH118F renders NB69 cells more vulnerable to ethanol and not dexamethasone treatment.	59
Figure 20.Hormonal stress affects expression of endogenous SGK-1 in NB69 cells…………	60
Figure 21. A high level of NDPK-A variants increases endogenous SGK-1 expression under hormonal stress.	61
Figure 22.SGK1WT and SGK1CA, but not SGK1DN, increased cell viability under hormonal stress.	62
Figure 23.	Ectopic SGKDN decreases NDPK-AWT mediated cell viability under hormonal stress..	63
Figure 24.	Ectopic SGK1 variants fail to enhance NDPK-AS120G mediated cell viability under hypertonic stress.	64
Figure 25.	Under hormonal stress, the ectopic SGKWT, SGKCA and SGKDN do not affect NDPK-A-mediated cell viability significantly…….	65
XI. Abbreviation………………………………………………………………………….66
X.	Appendixes
Appendix I. Reagents	67
Appendix II. MAPs and sequence for SGK-1 expression plasmids	70
1. pIRES-4xHA-RFP-Hyg……………………………………………………….…….70
2. pIRES-SGKWT-4xHA-RFP-Hyg……………………………………………………77
3. pIRES-SGKDN-4xHA-RFP-Hyg………………………………………….….……..84
4. pIRES-SGKCA-4xHA-RFP-Hyg…………………………………………...…….....91
5. pIRES-SGKT256A-4xHA-RFP-Hyg……………………………………….….…......98
6. pIRES-SGKS422A-4xHA-RFP-Hyg…………………………………….….….……105

                                    

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