The novel function of β-catenincontributing to Tcf4 stability in PC3 cell
碩士 === 高雄醫學大學 === 醫藥暨應用化學研究所碩士班 === 95 === Two functions of β-catenin have been proposed to influence cell behavior. One is the component of cell adhesion complex as a connecting molecule between E-cadherin and α-catenin, and the other is the coactivator of Tcf/Lef family of transcription factors .T...
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ndltd-TW-095KMC055370012015-12-11T04:04:17Z http://ndltd.ncl.edu.tw/handle/88841639876373518971 The novel function of β-catenincontributing to Tcf4 stability in PC3 cell β-catenin在前列腺癌細胞株PC3內影響轉錄因子Tcf4蛋白穩定性之新功能的探討 kuo-chen jung 容國貞 碩士 高雄醫學大學 醫藥暨應用化學研究所碩士班 95 Two functions of β-catenin have been proposed to influence cell behavior. One is the component of cell adhesion complex as a connecting molecule between E-cadherin and α-catenin, and the other is the coactivator of Tcf/Lef family of transcription factors .The β-catenin /Tcf pathway plays a critical role in embryo development , stem cell renew, and also contributes to carcinogenesis. Mutations in the components controlling β-catenin toward proteasomal degradation including APC and axin, or β-catenin itself have been identified in several cancer types including prostate, skin, colon and liver. Therefore, the mutated β-catenin might be a key point to cause tumor formation. To determine the function of β-catenin participate in Tcf-4 signaling in prostate cancer, we used PC3 cell that contains Tcf-4 transcription factor with over-expression of wild type and mutated form of flag-tag β-catenin as model. This mutated form is β-Catenin (T41A, S45A). By using this model, we have identified a new role of β-catenin by increasing the amount of Tcf4 in the nuclear. Further evidence indicated that this increasing is due to β-catenin enhancing stability of Tcf4. Significantly, this increasing amount of Tcf4 was consistent with Tcf4-driving transcription activity. To know β-catenin effect on the DNA binding activity of Tcf4, we performed electrophoretic mobility shift assay by using the nuclear extracts from the PC3 cells transfected with the vector control, wild type or mutated form of β-catenin. We found no specific-binding among these nuclear extracts. This phenomenon was also demonstrated by using the nuclear extracts from the 293 cells transfected with the control vector or mutated form of β-catenin. Furthermore, we concentrated β-catenin with anti-flag antibody-conjugated agarose and performed the same assay. Interestingly, the specific β-catenin-Tcf4-DNA complex appeared in the same position as the non-specific binding by using the crude nuclear extracts. It suggested that the specific β-catenin-Tcf4-DNA complex was hidden in the non-specific binding band in the assay using the crude nuclear extract. With over-expression of β-catenin, no super-shift band was observed. This implied that β-catenin is essential for Tcf4 to interact with its specific DNA sequence. In 5 addition, we examined whether mutated form of β-catenin can enhance the cell survival under serum-free condition. The results indicated that the PC3 cells overexpressing mutated form of β-catenin has higher survival rate than that of the control cells under serum-free condition. Chihuei Wang 王記慧 2007 學位論文 ; thesis 35 zh-TW |
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碩士 === 高雄醫學大學 === 醫藥暨應用化學研究所碩士班 === 95 === Two functions of β-catenin have been proposed to influence cell
behavior. One is the component of cell adhesion complex as a
connecting molecule between E-cadherin and α-catenin, and the
other is the coactivator of Tcf/Lef family of transcription factors .The
β-catenin /Tcf pathway plays a critical role in embryo development ,
stem cell renew, and also contributes to carcinogenesis. Mutations
in the components controlling β-catenin toward proteasomal
degradation including APC and axin, or β-catenin itself have been
identified in several cancer types including prostate, skin, colon and
liver. Therefore, the mutated β-catenin might be a key point to
cause tumor formation. To determine the function of β-catenin
participate in Tcf-4 signaling in prostate cancer, we used PC3 cell
that contains Tcf-4 transcription factor with over-expression of wild
type and mutated form of flag-tag β-catenin as model. This mutated
form is β-Catenin (T41A, S45A). By using this model, we have
identified a new role of β-catenin by increasing the amount of Tcf4
in the nuclear. Further evidence indicated that this increasing is due
to β-catenin enhancing stability of Tcf4. Significantly, this increasing
amount of Tcf4 was consistent with Tcf4-driving transcription activity.
To know β-catenin effect on the DNA binding activity of Tcf4, we
performed electrophoretic mobility shift assay by using the nuclear
extracts from the PC3 cells transfected with the vector control, wild
type or mutated form of β-catenin. We found no specific-binding
among these nuclear extracts. This phenomenon was also
demonstrated by using the nuclear extracts from the 293 cells
transfected with the control vector or mutated form of β-catenin.
Furthermore, we concentrated β-catenin with anti-flag
antibody-conjugated agarose and performed the same assay.
Interestingly, the specific β-catenin-Tcf4-DNA complex appeared in
the same position as the non-specific binding by using the crude
nuclear extracts. It suggested that the specific β-catenin-Tcf4-DNA
complex was hidden in the non-specific binding band in the assay
using the crude nuclear extract. With over-expression of β-catenin,
no super-shift band was observed. This implied that β-catenin is
essential for Tcf4 to interact with its specific DNA sequence. In
5
addition, we examined whether mutated form of β-catenin can
enhance the cell survival under serum-free condition. The results
indicated that the PC3 cells overexpressing mutated form of
β-catenin has higher survival rate than that of the control cells
under serum-free condition.
|
author2 |
Chihuei Wang |
author_facet |
Chihuei Wang kuo-chen jung 容國貞 |
author |
kuo-chen jung 容國貞 |
spellingShingle |
kuo-chen jung 容國貞 The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
author_sort |
kuo-chen jung |
title |
The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
title_short |
The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
title_full |
The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
title_fullStr |
The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
title_full_unstemmed |
The novel function of β-catenincontributing to Tcf4 stability in PC3 cell |
title_sort |
novel function of β-catenincontributing to tcf4 stability in pc3 cell |
publishDate |
2007 |
url |
http://ndltd.ncl.edu.tw/handle/88841639876373518971 |
work_keys_str_mv |
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