| 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 early development and function of many tissues and organs. Notch signaling affects cell specification, proliferation, and apoptosis during cell development. Notch genes encode highly conserved type I transmembrane glycoprotein, which can be activated via direct interaction with transmembrane ligands expressed on the surface of neighboring cells. Upon ligand binding, Notch can be cleaved and release an intracellular domain (ICN). ICN translocates into nuclie, associate with transcriptional factors known as Su(H)/CBF1, regulate the expressions of target genes, and successively modulate the development and growth of cells including tumor cells, immune cells and hematopoietic cells et al. Over-expression of constitutively active ICN by gene transfection in target cells also results in "activated " Notch phenotype. Previous studies demonstrated that Notch signaling could display different effects on tumor cells: promoting carcino-genesis or inhibiting tumor cell proliferation. Notch signal might play different roles in different cells. In this study, we observed the effects of Notch1 signaling on the growth and apoptosis of hepatocarcinoma cells, thus to explore the mechanisms of Notch signaling as well as the crosstalk of Notch signaling with some other signal transduction events. By calcium phosphate precipitation mediated gene transduction of 293T package cells, we prepared recombinant retrovirus MSCV-ICN/GFP expressing bothconstitutively active Notch1 intracellular domain and GFP reporter protein as well as control recombinant retrovirus MSCV-GFP expressing only GFP. The recombinant retroviruses were used to infect human hepatocarcinoma cells SMMC7721 to over-express ICN. As detected by FACS, 45-65% cells expressed GFP one day after infection. However, in the case of human hepatocarcinoma cell Hep3B, only 20% cells expressed GFP. We speculated that different hepatocarcinoma cell lines might be differently susceptible to the infection of retroviruses. After infected, proliferation of SMMC7721 cells and Hep3B hepatocarcinoma cells were analyzed by MTT. Compared with the cells expressing GFP, the growth of SMMC7721 cells transiently expressing ICN was inhibited significantly. Cell cycle distribution analysis by FACS showed that SMMC7721 cells in G0/G1 phase were significantly increased after infection of MSCV-ICN/GFP recombinant retrovirus. These results suggested that Notch1 signaling inhibits the growth of human hepatocarcinoma cells and induces G0/G1 cell cycle arrest. However, only slight effects could be observed in Hep3B cells, that might be due to the lower infection efficiency of MSCV-ICN/GFP in Hep3B cells. In order to investigate whether stably expressed ICN displays the same effects on the growth of SMMC7721 cells, we isolated several subclones of the SMMC7721-ICN/GFP cells stably expressing both ICN and GFP as well as several subclones of the SMMC7721-GFP cells stably expressing GFP. Data from colony formation, [(3)H]thymidine incorporation, and MTT assay demonstrated the growth of SMMC7721-ICN/GFP cells was inhibited markedly as compared with that of SMMC7721-GFP cells. Analyzed by FACS, SMMC7721-ICN/GFP cells consistently showed a higher fraction of cells in G0/G1 phase and a lower fraction of cells in S phase as compared with SMMC7721-GFP cells. These results showed that either transient or stable expression of ICN could induce growth inhibition and G0/G1 cell cycle arrest in hepatocarcinoma cells. We also detected the production of IL-6 in the supernatants of SMMC7721 cells. As compared with that of SMMC7721-GFP, the secretion of IL-6 by SMMC7721-ICN/GFP cells was significantly increased.Cell proliferation and death are closely linked with progression of the cell cycle, which is regul...
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