| Summary: | 碩士 === 臺北醫學大學 === 細胞及分子生物研究所 === 93 === Abstract
Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. In previous study, our laboratory constitutively overexpressed active Notch1 in K562 cells to explore the effects of Notch1 signaling on hematopoietic cell growth and to investigate the underlying molecular mechanisms. The yeast-two hybrid system was used to search the Notch1 receptor intracellular domain (N1IC)-associated protein. The nuclear Ⅱ-tubulin was found as a candidate of N1IC-associated proteins. The presence of Ⅱ-tubulin in nuclei is correlated with cancerous state of cells. However, the mechanisms that the function of Ⅱ-tubulin locating in the nucleus and orchestrate the multiple molecules insults required for progression of hematopoietic cells still are not clear.
To investigate the relationship between Notch signaling andⅡ-tubulin, the expression plasmids of Ⅱ-tubulin with an nuclear localization signal (NLS)sequence were constructed. This NLS tag did not increase the nuclear localization of Ⅱ-tubulin . The exogenous Ⅱ-tubulin protein expression was knocked down by the cotransfecton of Ⅱ-tubulin-expressing construct and RNAi expression plasmids with several target sequences of Ⅱ-tubulin. Furthermore, SiRNAs against Ⅱ-tubulin suppressed the CBF-1 mediated transactivation activity of N1IC by reporter assay.
On the other hand, we showed that overexpression of Notch1 was able to inhibit the growth of hematopoietic cells in vitro and in vivo. The overexpressed active Notch1 in K562 cells decreased expression CDK2, CDK4 and the E2F1 protein. Up-regulation of retinoblastoma protein expression was observed. Furthermore, the constitutively active Notch1 could inhibit the growth of K562 cells in nude mice and cell cycle distribution analysis showed an increase in G0/G1 phase.
Taken together, these results suggested that nuclearⅡ-tubulin enhanced the CBF-1 mediated transactivation activity of N1IC and the classical NLS-dependent pathway does not promote the nucleus localization of Ⅱ-tubulin protein to modulated Notch signaling. We also demonstrated that Notch1 signaling results in growth inhibition of hematopoietic cells both in vitro and in vivo, affecting Rb/E2F pathway that control cell proliferation.
|
|---|
