| Summary: | 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
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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.
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