db-cAMP对转化细胞增殖抑制及其对CaM水平和PKⅡ活性的影响

THE EFFECTS OF db-cAMP ON CaM LEVEL,PK Ⅱ ACTIVITY AND INHIBITION OF TRANSFORMED CELLS PROLIFERATION

目的 探讨双丁酰 环核苷酸 (db cAMP)对转化细胞增殖及细胞表型作用的机理。 方法 以流式细胞光度术 (flowcytometry ,FCM)、软琼脂集落形成、放射免疫、Northern印迹和激酶活性分析等方法观察db cAMP对转化的C3H1 0 T1 2 小鼠成纤维细胞增殖、细胞表型、钙调素 (calmodulin ,CaM)表达及蛋白激酶Ⅱ (proteinkinaseⅡ ,PKⅡ )活性的影响。 结果 db cAMP(1mmol L)使C3H1 0 T1 2 转化细胞增殖及软琼脂集落形成能力受到显著抑制 ,转化细胞中CaM的表达及PKⅡ活性明显高于正常细胞 ,经db cAMP处理后也受到明显抑制。

Objective To study the mechanism of the effects of db cAMP on the inhibition of cell proliferation and phenotypic changes in transformed cells. Methods The progression of cell cycle was analyzed by the flow cytometry.The transforming phenotype of C 3H 10 T 1/2 cells was observed by the soft agar colony formation.Northern blotting and radioimmunoassay were used to analyze the level of CaM expression.The activity of PKⅡ was determined through γ 32 p ATP incorporation assay. Results The proliferation of transformed C 3H 10 T 1/2 cells was inhibited markedly by 1mmol/L db cAMP.The db cAMP treated transformed cells were accumulated at G 1 phase.After treatment with db cAMP,the soft agar colony formation efficient in transformed cells was decreased evidently,simultaneously,cells altered in morphology,from rounded to flattened shape.In contrast to normal cells,CaM level and PKⅡ activity increased in transformed cells.However,both CaM level and PKⅡ activity in transformed cells treated by db cAMP decreased obviously.Conclusion\ The results indicate that not only alteration of CaM expression,but also the changes of the PKⅡ activity are involved in account for the cell transformation and induced differentiation of transformed cells.It is possible that the role for CaM in the cell transformation and induced differentiation may be mediatied by the effect of PKⅡ. \;[