Effects Of Activation Of Notch1 On Cell Proliferation And Cross-talk Between Notch1 And NF-κB In Cervical Cancer Cells

Cell development is a complex process involving intricate interplay of cell proliferation, migration, growth, differentiation and death. Recently, the members of Notch family have been found to play critical roles in the regulation of cell-fate decisions that affect the development and function of many tissues and organs. Notch signaling affects cell specification, proliferation, and apoptosis during cell development. However, recent studies demonstrated that Notch1 signaling could display different effects on tumor cells: promoting carcinogenesis or inhibiting tumor cell proliferation. Although the effects of the Notch1 signaling are cell-type specific and context-dependent, the exact mechanisms remain poorly characterized. To highlight some of these points, we focus on cervical cancer in particular, and show how, in principle,Notch1 signaling shows a tumor-suppressive property. By constructing a cervical cancer cell model and a murine model (nude mouse) of cervical cancer cells stably expressing constitutively active Notch1, we observed the effects of expression of constitutive active Notch1, exogenous ICN (intracellular domain of Notch), on the cell proliferation in vitro and in vivo, cell cycle and apoptosis of cervical cancer cells, thus to explore the mechanisms of Notch signaling, as well as the cross-talk of Notch signaling with another important signal pathway, NF-κB (nuclear factor-κB).ICN, active form of Notch1, was inserted into retrovirus expression vector pCLNRX. The vector pCLNRX-ICN and packaging vector pCL-10A1 were co-transfected into 293 package cells by lipofectamineTM 2000, so do pCLNRX and pCL-10A1. The recombinant retrovirus was packaged by 293 cells. The virus-containing supernatant was collected, and the titer of the recombinant viruses was tested on NIH 3T3 cells. Stable expression of exogenous ICN resulted in Notch1 constitutive activation in cervical cancer HeLa cells. We generated clones stably expressing ICN and/or the neomycin (G418) resistance by culture of the infected cells in the presence of G418.After infected, proliferation of HeLa cells in vitro was observed by MTT assay. The proliferation curves showed the proliferation of HeLa-ICN cells (HeLa subclones stably expressing exogenous ICN) was inhibited significantly compared with control HeLa cells and HeLa-G418 cells (HeLa subclones stably expressing G418 resistance gene by transfecting the empty vector alone), respectively. Cell proliferation inhibition is closely linked with cell cycle arrest and apoptosis. FACS (fluorescence activated cell sorter) showed that stable transfection of exogenous ICN resulted in significant accumulation of cells in G2-M phase, which was accompanied with a significant decrease in the percentage of cells in the S phase compared with control cells. Cell cycle distribution analyzed by FACS indicated that activated Notch1 induced G2-M cell cycle arrest and inhibited the proliferation of HeLa cells. Cell proliferation and death are closely linked with progression of the cell cycle, which is regulated by a complex network involving cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinases inhibitors (CKIs). Cyclin-dependent kinase 1 (Cdk1) is a key factor in the regulation of the G2-M checkpoint. The expression of Cdk1 protein was analyzed by Western blot. The expression of Cdk1 protein was down-regulated in HeLa-ICN cells, which might be involved in the induction of G2-M cell cycle arrest.And cellular nuclei were stained with Hoechst 33342 dye under the fluorescence microscope in order to study the changes in nuclear morphology. The stable transfection with exogenous ICN increased chromatin condensation and fragmentation compared with control cells, suggesting the occurrence of apoptotic changes in the cells. Apoptosis induction by exogenous ICN was further studied by a double staining method using FITC-conjugated Annexin V and PI. The results of Annexin V/PI assays revealed that activated Notch1 increased the percentage of HeLa cells in early apoptotic state. The total apoptotic rate of HeLa-ICN cells (38.5%) was increased significantly compared with that of control HeLa cells(2.6%)and HeLa-G418 cells(7.7%). The above results suggested that constitutively overexpressed active Notch1 via stable transfection with exogenous ICN could inhibit the cell growth by inducing G2-M cell cycle arrest and apoptosis in human cervical cancer cells. Based on the cervical cancer cell model stably expressing constitutively active Notch1, we examined the tumorigenecity of stably transfected HeLa-ICN, HeLa-G418 or control HeLa cells in vivo by inoculating these tumor cells into nude mice. Although the tumours resulting from injection of HeLa-ICN cells were still detectable, the size of the tumors was much smaller than those of both the control groups. The results indicated that stable expression of the constitutively active Notch1 might suppress growth of the human cervical cancer cells in vivo.NF-κB is believed to play a critical role in enhancing cancer cell survival by inhibiting apoptosis, so inhibition of NF-κB activation sensitizes cervical cancer cells to apoptosis. And increased NF-κB activity has been demonstrated in cervical cancer. Here, NF-κB activity was measured in terms of its DNA-binding activity by EMSA (electrophoretic mobility shift assay). NF-κB activity of HeLa-ICN cells was inhibited significantly compared with that of control HeLa cells and HeLa-G418 cells. To show that the retarded band visualized by EMSA in HeLa-ICN cells was indeed NF-κB, we incubated nuclear extracts from HeLa-ICN cells with antibody to either the p50 or p65 subunit of NF-κB. When nuclear extracts from HeLa-ICN cells were incubated with antibodies to the p50 or p65 subunit of NF-κB, only the bands loaded with antibodies to NF-κB p50 were shifted to higher molecular masses, suggesting that direct or indirect inhibition of NF-κB p50 by Notch1 is the mechanism of inhibition of NF-κB activation in HeLa cells.Cellular expression and localization of NF-κB p50 in HeLa cells was determined by further study. Consistent with these observations, a significant decrease in NF-κB p50 protein levels in the nuclear fraction was observed in HeLa-ICN cells when compared with HeLa-G418 cells. However, the cellular total protein levels of NF-κB p50 remained unchanged. Further studies by immunochemistry showed the NF-κB p50 expression decreased in the nuclei of HeLa-ICN cells, as compared with control cells. These results indicate that inhibition of nuclear translocation of NF-κB p50 may contribute, at least partially, to NF-κB inhibition induced by activated Notch1 in HeLa cells. IκBα(inhibitor of NF-κBα) is the key inhibitor of NF-κB. Consistently, an increase in the cytosolic levels of the IκBαprotein was observed in HeLa-ICN cells when compared with HeLa-G418 cells, suggesting that up-regulation of IκBαmay contribute, at least partially, to inhibition of NF-κB activation.Since NF-κB controls cell growth and differentiation through transcriptional regulation of its responsive genes, such as cyclin D1, Bcl-2 and c-Myc, which are involved in apoptosis and proliferation of cervical cancer cells, we analyzed the cellular total protein expressions of Bcl-2, cyclin D1 and c-Myc. Western blot analysis showed that HeLa-ICN cells expressed significantly less cyclin D1 and Bcl-2, when compared with control HeLa cells or HeLa-G418 cells. The results were consistent with a decrease in NF-κB activity. However, the cellular total protein levels of c-Myc remain unchanged after stable transfection with ICN, indicating activated Notch1 may not affect cellular total protein levels of c-Myc in HeLa cells.Overall, besides induction of cell cycle arrest, our results indicate that, induction of apoptosis may contribute to inhibition of cell growth in cervical cancer HeLa cells; Furthermore, a cross-talk between Notch1 and NF-κB signaling pathways may contribute, at least partially, to cell cycle arrest and apoptosis induced by Notch1 activation in human cervical cancer cells. That is to say, inhibition of the activity and translocation of NF-κB may contribute to induction of apoptosis by activated Notch1. Our results suggest that down-regulation of NF-κB, Cdk1, cyclin D1 and Bcl-2 may contribute partially to cell cycle arrest, apoptosis and growth inhibition induced by Notch1 activation in human cervical cancer cells. These results have confirmed the role of Notch1 signaling in the growth inhibition of cervical cancer cells in vitro and in vivo. The study has also provided new evidence for the mechanisms of Notch1 signaling and the novel models of cervical cancer therapy, Notch1 as a target for signal transduction interference therapy in human cervical cancers.