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AIM:The goal of this study was to characterize the AFPreceptor,its possible signal transduction pathway and itsproliferative functions in human hepatoma cell line Bel 7402.METHODS:Cell proliferation enhanced by AFP was detectedby MTT assay,~3H-thymidine incorporation and S-stagepercentage of cell cycle analysis.With radioactive labeled ~(125)I-AFP for receptor binding assay;cAMP accumulation,proteinkinase A activity were detected by radioactive immunosorbentassay and the change of intracellular free calcium([Ca~(2+)]_i)was monitored by scanning fluorescence intensity under TCS-NT confocal microscope.The expression of oncogenes N-ras,p53,and p21~(ras)in the cultured cells in vitro were detected byNorthern blotting and Western blotting respectively.RESULTS:It was demonstrated that AFP enhanced theproliferation of human hepatoma Bel 7402 cell in a dosedependent fashion as shown in MTr assay,~3H-thymidineincorporation and S-phase percentage up to 2-fold.Twosubtypes of AFP receptors ware identified in the cells withKds of 1.3×10~(-9)mol.L~(-1)and 9.9×10~(-8)mol.L~(-1)respectively.Pretreatment of cells with AFP resulted in a significantincrease(625%)in cAMP accumulation.The activity ofprotein kinase A activity were increased up to 37.5,122.6,73.7 and 61.2% at treatment time point 2,6,12 and 24hours.The level of intracellular calcium ware elevated afterthe treatment of alpha-fetoprotein and achieved to 204% at 4min.The results also showed that AFP(20 mg.L~(-1))couldupregulate the expression of N-ras oncogenes and p53 andp21~(ras)in Bel 7402 cells.In the later case,the alteration were 81.1%(12h)and 97.3%(12h)respectively compared with control.CONCLUSION:These results demonstrate that AFP is apotential growth factor to promote the proliferation of humanhepatoma Bel 7402 cells.Its growth-regulatory effects aremediated by its specific plasma membrane receptorscoupled with its transmembrane signaling transductionthrough the pathway of cAMP-PKA and intracellular calciumto regulate the expression of oncogenes.
AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its profoliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, ~ 3H-thymidine incorporation and S-stagepercentage of cell cycle analysis.With radioactive labeled ~ (125) I-AFP for receptor binding assay; cAMP accumulation, proteinkinase A activity were detected by radioactive immunosorbentassay and the change of intracellular free calcium ([Ca ~ (2 +)] _i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N-ras, p53, and p21 ~ (ras) in the cultured cells in vitro were detected by Northern blotting and Western blot respectively .RESULTS: It was initially for that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTr assay, ~ 3H-thymidine incorporation and S-phase percentage up to 2-fold. wosubtypes of AFP receptor ware id entified in the cells with Kds of 1.3 × 10 -9 mol·L -1 and 9.9 × 10 -8 mol·L -1 respectively.Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. Activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24hours. The level of intracellular calcium ware elevated afterthe treatment of alpha- fetoprotein and achieved to 204% at 4 min. These results also showed that AFP (20 mg.L -1) couldupregulate the expression of N-ras oncogenes and p53 and p21 ras in Bel 7402 cells. In the later case , the alteration were 81.1% (12h) and 97.3% (12h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is apotential growth factor to promote the proliferation of human hepatoma 7402 cells. Its growth-regulatory effects are byiated by its specific plasma membrane receptorscoupled with its transmembrane signaling transductionthrough the pathway of cAMP-PKA and intracellular calciumto regu late the expression of oncogenes.