cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .

The Effect of cAMP on Expression of Several Genes and CREB DNA Binding Activity in Transformed C_3H_ 10 T_(1/2) Cells

In orden to investigate the mechanism of cAMP in inhibition of transformed cell proliferation.The activities of PKC(protein kinase C)and PKA(protein kinase A)in transformed C 3H 10 T 1/2 cells treated by db\|cAMP(1 mmol/L)were analysed.Furthermore,the correlation between the gene expression of c\|myc 、CaM(calmodulin)\,p53\,Rb and the DNA binding activity of CREB(cAMP\|response element binding protein)in transformed cells treated by db\|cAMP was examined.The results demonstrated that the transformed cells displayed the most rapid growth rate,while the growth rate of transformed cells treated by db\|cAMP(1 mmol/L)significantly decreased.The activity of PKC was markedly reduced and the activity of PKA was significantly enhanced in transformed cells after treatment with db\|cAMP for 30 min.The gel mobility shift assay was carried out by using double stranded oligonucleotide containing the consensus sequence for the CRE DNA binding site.The enhancement of DNA binding activity of CREB in transformed C 3H 10 T 1/2 cells after treatment with db\|cAMP(1 mmol/L) for 24 h was observed.However,the expression of c myc and CaM was inhibited and the expression of p53 and Rb was enhanced in transformed C 3H 10 T 1/2 cells treated by db\|cAMP(1 mmol/L) for 2 h.The results suggest the inhibition of proliferation in transformed C 3H 10 T 1/2 cells after treatment with db\|cAMP is related to the inhibition of gene expression of CaM and c myc and enhancement of expression of tumor suppressor genes p53 and Rb,but this has no relation to the DNA binding activity of CREB.