Effect Of Polyamine On The Growth And Signal Transduction Of PC12 Cells

[Objective] The present study is to exam the effects of spermidine and the polyamine synthesis inhibitor, α -difluoromethyl ornithine (DFMO), on the growth of PC12 cells and to investigate the characteristics and correlation between MAPKs pathway and ODC/ polyamines system.[Method] Pheochromocytoma cells (PC 12) in logarithm period were taken in the experiment. The cells were divided into two groups according to the different treatment, group SPD (with 1.25. 2.5, 5, 10 mmol/L SPD respectively ) and group DFMO (with 2, 4, 61 8 mmol/L DFMO respectively). In the four days, PC 12 cells were observed everyday. The morphological changes of PC 12 cells were investigated with light microscope. Cell growth curve was made with trpanblau staining. The cell viability was examed by MTT. The cell cycle was monitored with FCM (flow cytometry). When PC12 cells grow in SPD and DFMO of different time courses and doses, phosphorylation of ERK1/2 and p38 were examed with western blotting.[Result] In this study showed morphologically on the 4th day that (1) expose to 5 and 10 mmol/L SPD, the cell bodies are bigger and the length of neurite longer than in control group, (2) no marked changes were found in 1.25,2.5 mmol/L SPD, (3) expose to 4 and 6 mmol/L DFMO. the cell bodies became round and the neurite missing; and (4) expose to 2 and 8 mmol/L DFMO, the length of the neurite were shorter than in control group. In the cell growth curve can be found that cell growth was promoted by SPD, was inhibited by DFMO, and no marked changes can be seen in DFMO with SPD. The results of MTT test revealed that during 1, 2, 3, 4 days respectively, (1) SPD of different doses(1.25, 2.5, 5, 10 mmol/L) could promote the proliferation of PC12 cells, and the cells populations increased by 49.1% at concentration of S mmol/L on the 4th day, (2) DFMO of 2-8 mmol/L could suppress the proliferation of PC 12 cells during 4 days, and the cell populations decreased by 40.3% at concentration of 6 mmol/L on the 4th day, And (3) there were in dose and time-dependent manner. FCM showed that the cell cycle of the PC 12 cells could be inhibited in S period at a concentration of 6 mmol/L DFMO on the 4th day. During 3 days, especially on the 3rd day, western-blot tests showed that the phosphorylation of ERK1/2 increased gradually in 1.25, 2.5, 5 mmol/L SPD-treated PC12 cells; the phosphorylation of ERK1/2 of PC 12 cells incubated in 2, 4, 6 mmol/L DFMO culture media decreased gradually; the phosphorylation of p38 decreased by degrees in 1.25, 2.5. 5mmol/L SPD-treated PC12 cells; Thephosphorylationofp38 enhanced by degrees in 2, 4, 6mmol/LDFMO-trcatedPC12 cells.(Conclusion] In this study, it was suggested that the growth and proliferation of PC12 cells can be promoted by SPD or suppressed by DFMO at a certain concentration. With the concentration of SPD increasing and time prolonging, the phosphorylation of ERK1/2 enhanced gradually, the activation p38 decreased by degrees. However, ERK1/2 activation decreased and p38 activation increased in DFMO-treated PC 12 cells. These results indicate that ERK1/2 and p38 of MAPKs signal transduciton pathway plays an important role in the growth and death of nerve cells regulated by ODC/polyamines system.