WWOX被證明是一個腫瘤抑制蛋白，且被報導在攝護腺癌、肺癌、乳癌及胃癌中皆有表現量下降的情形，然而，WWOX在腎細胞癌中的角色仍為未知。WWOX被報導位於細胞中粒線體、高基氏體、粗內質網、溶小體、細胞膜及核中，其在細胞中的表現位置極具爭議性。本篇研究包含有兩個研究子題: (1)WWOX在腎細胞癌是否有表現量下降的情形，並評估WWOX做為診斷及預後指標的可能性，(2)磷酸化對WWOX之調控。為了進行WWOX於腎細胞癌中表現量之研究，我們製備了WWOX多株抗體並確定其專一性，並以此抗體對33個腎細胞癌檢體及138個石蠟包埋檢體分別進行WWOX之西方點墨法及免疫組織化學之研究，比較正常與癌組織中WWOX表現量。我們的結果顯示WWOX表現量下降情形僅存在於亮細胞型(clear cell type)腎細胞癌中(p=0.018)，其表現量下降比例在西方點墨法及免疫組織化學研究中分別為60.7 %及90.7 %，且在亮細胞及亮細胞顆粒性(granular)細胞混合型腎細胞癌中，WWOX的表現量下降和病人存活率有正相關性（p=0.048）。因此，WWOX可以做為亮細胞型腎細胞癌的診斷及預後的指標。此外，我們以生物資訊學方式預測人類型WOX1的磷酸化位置，並進行其磷酸化對表現位置影響之研究，我們的結果顯示PKC會對人類型WOX1的Thr49及Thr102位置進行磷酸化，並調控hWOX1於細胞內之位置。

WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

Abstract
Chinese…………………………………………………………………….…………..…….I
English……………………………………………………………………….……………..II
Abbreviations…………………………………………………………………………..……IV
Introduction……………………………………………………………………….………….1
Material and Methods………………………………………………………………………14
PART. I The role of WWOX in renal cell carcinoma.
Results……………………………………………………….……………………………... 27
Discussion…………………………………………………………………………………....32
Tables and Figures
Table.1 Summary of the results of hWOX1 expression level in different type RCC in immunoblotting study………………………………………………………………………..35
Table.2 Summary of theresult of immunohistochemical study of hWOX1 expression pattern in different type RCC………………………………………………………………………...36
Table.3 Statistics of down-regulation of hWOX1 in clear cell type RCC…………………....37
Fig.1 Induction and Purification of GST-hWOX1 ww protein………………………………38
Fig.2 The Specificity of hWOX1 antibody…………………………………………………..39
Fig.3 The Expression level of hWOX1 in different type RCC examined by immumoblotting
………………………………………………………………………………………………..40
Fig.4 The Expression pattern of hWOX1 in different type RCC examined by immunohistochemistry……………………………………………………………………….41
Fig.5 Down-regulation of hWOX1 in clear cell type RCC in immunoblotting study……….42
Fig.6 Kaplan-Meier estimates of clear cell and clear-granular combined type RCC survival probability stratified onto relationship of hWOX1 expression in normal and tumor part…...44
PART. II The regulation of WWOX by phosphorylation.
Results…………………………………………….………………………………………....46
Discussion………………………………………………………...…………………….……51
Tables and Figures
Table.4 Phosphorylation site prediction of hWOX1…………………………………………54
Fig.7 Scheme of hWOX1 constructs…………………………………………………………55
Fig.8 Expression of hWOX1 fragment in HeLa cells………………………………………..56
Fig.9 Localization of hWOX1 fragments in HeLa cells……………………………………..57
Fig.10 Phosphorylation of PKC at WW domains of hWOX1………………………………..58
Fig.11 PKC phosphorylates hWOX1 at Thr 49 and Thr 102………………………………...59
Fig. 12 Expression of hWOX1 mutants in HeLa cells……………………………………….60
Fig. 13 Localization of hWOX1 mutants in HeLa cells……………………………………...61
Fig.14 hWOX1 T102A mutant reduces the effect of TPA on the nuclear translocalization…63
Fig. 15 Subcellular localization of hWOX1 in cell cycle…………………………………….64
References…………………………………………………………………………….……..66
Appendix…………………………………………………………………………………….73

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