N-甲基-N'-硝基-N-亚硝基胍诱发的vero细胞JNK/SAPK通路的激活

Activation of JNK/SAPK pathway in vero cells induced by N-methyl-N'-nitro-N-nitrosoguanidine

目的和方法 :为了研究DNA损伤剂N -甲基 -N -硝基 -N -亚硝基胍 (MNNG)诱发vero细胞非定标性突变发生的信号转导机制 ,我们观察了MNNG诱发vero细胞c -JunNH2 -terminalkinase/stressactivatedproteinkinase(JNK/SAPK)通路激活的情况 :分别用Western印迹法和固相激酶活性测定法检测JNK1磷酸化和JNKs激酶活性。结果 :发现用 0 2 μmol/LMNNG和 1mg/L放线菌素酮 (CHM )分别处理vero细胞 2 5h和 1h后 ,都引起细胞抽提液中磷酸化JNK1的比例明显增高。同时通过测定JNKs的底物c -Jun的磷酸化程度 ,发现这两种处理也都可引起JNKs激酶活性显著增高 (分别增高 6 7和 3 0倍 )。结论 :证明 0 2 μmol/LMNNG和 1mg/LCHM分别处理vero细胞 2 5h和 1h都可引起vero细胞内JNK/SAPK通路被激活。

AIM and METHODES: To evaluate the possible signal transduction mechanism of nontargeted mutagenesis in vero cells induced by DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine(MNNG),the activation of c-Jun NH 2-terminal kinase/stress activated protein kinase(SAPK/JNK) pathway in vero cells induced by MNNG was studied. Western Blot analysis and Solid-phase kinase assay were used to measure the phosphorylation of JNK1 and kinase activity of JNKs, respectively. RESULTS: After 0.2 μmol/L, 2.5 h MNNG or 1 mg/L, 1 h cycloheximide (CHM) treatment, the proportion of phosphorylated JNK1 in cell extract increased significantly, simultaneously the kinase activity of JNKs increased dramatically(6.7 and 3.0 folds respectively), as measured by the phosphorylation of c-Jun, a substrate of JNKs. CONCLUSION: Both 0.2 μmol/L 2.5 h MNNG and 1 mg/L 1 h CHM treatment can induce the activation of JNK/SAPK pathway, one of the stress signal transduction pathways, in vero cells.