低浓度MNNG诱发非DNA损伤依赖的细胞信号通路

Activation of nucleus-independent signals triggered by N-methyl-N'-nitro-N-nitrosoguanidine

目的 :研究基因组 DNA损伤与细胞信号通路激活的关系以及低浓度 N-甲基 - N’-硝基 - N-亚硝基胍(MNNG)对细胞膜表面受体的影响。方法 :采用 ELISA法来检测蛋白激酶 A(PKA)的活性 ,不连续梯度密度离心法完成细胞脱核 ,以及免疫荧光法和激光共聚焦显微镜观察细胞表面受体聚集情况。结果 :0 .2 μmol/ L MNNG能在脱核细胞中引起 PKA活性升高 2 .3倍 ,并可在 5 min内引起 vero细胞表面生长因子受体和肿瘤坏死因子 α受体的聚集 ;在脱核细胞中 ,表面受体聚集现象与在完整细胞中一样。结论 :低浓度 MNNG对 PKA的激活和诱导细胞表面受体聚集均不依赖于基因组 DNA的损伤 ,表明细胞信号转导通路的起源可能位于细胞膜或者细胞质中。

Objective: To study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage. Methods: Vero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy. Results: In enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor α was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells. Conclusion: The results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.