Research On Mechanism Of Apoptosis Of U2-os Osteosarcoma Cell Line Induced By BFGF

ObjectiveCells will receive mutiple stimulation of external signals during growth and differentiation. Among these signals, growth factors have important effects on cell life. At the same time, growth factors have relation to genesis of tumor. Fibroblast growth factor (FGF) can promote proliferation of tissues derived from mesoderm or neuroectoderm and many tissues and cells can secrete FGF. Growth factors can act on many kinds of cells as mitotic promoter and deprival of growth factors can cause cease of cell growth or cell death. Thus growth factors are usually thought relative to cell growth and differentiation. But many facts show that some growth factors can also induce apoptosis, for example, epidermal growth factor (EGF) can induce apoptosis of A431, MDA -MB -468 cell line, bFGF have the same effect on MCF - 7 cell line and E wings'sarcoma. So the nomenclature of growth factors has lost its traditional meanings, growth factors have double regulative effects on cell growth.All of the processes during cell life are precisely controlled; apoptosis is included, which has important role in cell differentiation and development. Many cell signal transduction pathways have been discovered in the regulation of apoptotic induction and reduction. Mito-gen - activated protein kinase ( MAPK) is a group of particular Ser/Thr protein kinases which are activated when its conservative Thr and Tyr residues are both phosphorylated by upstream MAPK kinases. Active MAPKs translocate into nucleus and thus regulate gene expression. Now, three main parallel pathways of MAPK family have been discovered : ERK1/2, JNK and p38. They can induce different gene expression and thus cause different cell reaction. It is believed that ERK mainly transduces growth promoting signals and the other two mainly tranduce growth inhibiting signals.Therefore, this project intend to explore mechanism of apoptosis mediated by protein tyrosine kinase (PTK) pathway by study of the role of ERK1/2, JNK and p38 in apoptosis of U 2 - os cell line induced by bFGF.Materials and Methods1. Use trypan blue exclusion method and MTT colorimetric method to detect U 2 - os cell growth inhibition caused by treatment of bFGF.2. Use PI staining of fixed cells and FACS analysis to determine type of U 2 - os cell death induced by bFGF.3. Use active anti -phosphorylated ERK1/2, JNK, p38 antibodies and SDS - PAGE, western blot to measure activity changes of ERK1/2, JNK and p38 during apoptosis induced by bFGF.Results1. Effects of different concentration of bFGF treatment on U 2 -os cell growth.As the concentration of bFGF increases, trypan blue exclusion method shows that percent of dead U 2 - os cells increases accordingly and MTT colorimetric method shows that U 2 - os cell activity decreases.2. Effects of different time of bFGF treatment on U 2 - os cell growth.As the time of bFGF treatment increases, trypan blue exclusion method shows that percent of dead U 2 - os cells increases accordingly and MTT colorimetric method shows that activity of U 2 - os cells decreases. There appears obvious apoptotic peak on DNA content curve of FACS analysis. And as the time of bFGF treatment increases, percent of apoptotic cells increases.3. Effects of different time of bFGF treatment on ERK1/2, JNK and p38 activity.As the time of bFGF treatment increases, ERK1/2 activity decreases, JNK activity increases abruptly and keeped on higher level and p38 activity increases a little.Conclusion1. bFGF can promote death of U 2 - os osteosarcoma cell line, at the same time, the type of cell death is apoptosis. And the proapoptot-ic effect of bFGF is concentration and time dependent.2. The proapoptotic effects result from the reduction of ERK, activation of JNK during treatment of bFGF and p38 also has some role in this process.