Effects Of RGS16 And P38MAPK On The Cell Cycle And Apoptosis Of The Glioma C6 Cells

Regulators of G-protein signaling (RGS) are GTPase-activating proteins (GAP) for activated Galpha subunits. Sst2 is the prototype for the newly recognized RGS family. Overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay. Genetic analysis show that overexpression of the RGS 16 gene dramatically inhibits yeast response to alpha-factor similar to Sst2. Some results show RGS 16 may implicate in the regulation of T cell responses to inflammation because RGS 16 can attenuate galphaq- dependent p38 mitogen-activated protein kinase activation by platelet-activating factor and it was demonstrated that activation of PKC induces RGS 16 expression via TNFalpha in a calcium-sensitive manner. Other results indicate that TNFalpha activates anti-proliferative pathway including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis whereas ERKs do the opposite. Stabilized p53 was activated by p38(MAPK) resulting in G(l) arrest while RGS16 can be induced in response to genotoxic stress and overexpression of p53 protein. Thus to study the mysterious relationship between RGS 16, p38MAPK and p53 and the effects of p38MAPK and RGS16 on the biological characteristics of glioma C6 cells we respectively or collectively transfected pCMV5-p38 and pCMV5-RGS16 into C6 cells by lipofectin in the same time SB-202190 was used to treated the transfected and nontransfected pCMV5-p38 C6 cells. Expression of RGS16 and p38 and phosphorylation of p53 was examined by immunocytochemical method both before and after the transfection.AIM (1) To study the effect of RGS 16 on the proliferation of glioma C6 cells;(2) To study the effect of p38MAPK on the biological characteristics of glioma C6 cells;(3) To study the mechanism of p38MAPK induction of glioma C6 cells to apoptosis and cell cycle inhibition.METHODS pCMV5-RGS16 and pCMV5-p38MAPK was respectivelyor collectively transfected into C6 cells by lipofectin. SB-202190 was used to treated the transfected and nontransfected pCMV5-p38MAPK. C6 cells. The morphological and adhesive changes of the cells were observed under an inverted microscope. Proliferation of C6 cells was measured by JH-thymidine (3H-TdR) assay after gradient transfections of pCMV5-RGS16 and pCMV5. Expression of RGS16 and p38 and phosphorylation of p53 was examined by immunocytochemical method both before and after the transfection. Flow Cytometry was adopted to measure the fraction number changes of the cell cycle phase and to detect apoptosis of C6 cells.RESULTS (1) RGS16 expressed wavely with time. The highest positive rate reached at 13% after 36h and was not detected after 72h. Consistent with the expression of RGS16 .the fraction number of Gl phase reduced from 70.5% to 60.2% and that of S phase increased from 20.9% to 34.9% after 36h; but after 48h that of Gl phase accumulated to 76.2% and that of S phase deduced to 11.4% ;after 72h the cell cycle returned to normal. In contrast, the control group did not express RGS16 and had nearly no changes of the morphology and the fraction number of each phase.(2) 36h later the positive relationship between the proliferation of C6 cells and the gradient transfections of pCMV5-RGS16 was display by 3H-TdR assay. (3) 36 hours later the transfected pCMV5-p38MAPK and/or pCMV5-RGS16 of C6 cells positively expressed p38MAPK and RGS16; Flow cytometry show that transfected p38MAPK induced apoptosis of C6 cells by 33.8% and the fraction number of Gl phase accumulated by 17% and that of S phase reduced by 14%; However, RGS16 didn't induced C6 cells to apoptosis while reduced the fraction number of Gl phase by 10% and accumulated that of S phase by 14%;The collectively transfected pCMV5-p38MAPK and pCMV5-RGS16 group had no obvious effect on the cell cycle of C6 cells but the collectively transfected pCMV5-p38MAPK and pCMVS group induced a apoptosis peak and had nearly similar effect on the cell cycle of C6 cells with transfected pCMV5-p38MAPK group;SB-202190 reduced the fraction number of Gl phase of the nontr...