1、MNNG诱发表面受体聚集与DNA损伤 2、FRET技术的建立

【作者】 王政；

【导师】 余应年；

【作者基本信息】 浙江大学 ， 生理学， 2003， 硕士

【摘要】 化学致癌物可以引起DNA损伤和非DNA损伤效应。非定标突变是指发生在非损伤DNA部位的突变，可被以上两种损伤效应所诱发。 然而机体能主动地对环境的刺激作出反应；突变的生成也并非完全是被动的过程。当受到外源DNA损伤因子的攻击时，大肠杆菌E.coli能启动SOS反应以应对不良环境，提高对致损伤刺激的耐受能力，减少损伤的发生。而在出现SOS反应的细胞中还观察到了在复制过程中产生的、发生在没有外源损伤DNA上的突变事件，被称为SOS非定标性突变。可诱导的DNA聚合酶在其中起到了关键作用。但对真核细胞，尤其是哺乳类动物细胞中非定标性突变的情况目前还研究不多，发生机制也有待阐明。我们实验室的工作证明0.2μM的MNNG处理过的哺乳类细胞中也存在非定标性突变。已经证明：哺乳类细胞非定标突变发生机制依赖于由细胞信号转导通路介导的基因表达改变。 我们发现，低剂量MNNG处理后的细胞反应包括DNA复制保真度下降，DNA聚合酶谱发生改变，转录因子NF-κB、AP-1和CREB被激活，JNK/SAPK被激活，还可激活cAMP-PKA-CREB通路。而且PKA的激活不依赖于基因组DNA的损伤，因为在脱核处理细胞中PKA的激活程度与在完整细胞中是完全一致的。虽然这种不依赖于DNA损伤的信号通路激活方式有悖于以前的经典认识，但在UV诱发的UV反应中和另一种烷化剂MMS诱发的细胞反应中，获得了相似的证据：UV可诱发细胞表面受体EGFR和TNFR的快速聚集，并且这些表面受体的激活参与了广泛的UV反应如JNK激活和细胞凋亡。为了研究MNNG引浙江大学硕士学位论文 王政起的细胞信号转导改变与DNA损伤的关系及信号的起源，我们以免疫荧光法用激光共聚焦显微镜观察了低剂量MNNG处理后短时间内VerO细胞表面EGF－R和ThFRI聚集情况。发现在正常Vero细胞中，低剂量MNNG处理5分钟即可见表面受体的聚集，15分钟后聚集更加明显，45分钟时，聚集簇增大成块状；该现象与受体跟相应配体结合后引起的受体聚集情况类似，在DMSO和空白对照组中，均未见受体聚集发生。 进一步地，我们将 Vero细胞在含 20pM细胞松弛素 B的 Ficoll 70不连续密度梯度管中超速离心得到去核的胞质部分，重新贴壁后给予MNNG处理，以观察脱核细胞表面受体状况。结果显示：在脱核Vero细胞中，MN’NG可同样引起细胞表面受体EGFR和TNFR的聚集，且在我们观察的时间范围内，表面受体的聚集情况与完整细胞一致，提示细胞膜表面受体的聚集不需要基因组DNA损伤的参与，而很可能是细胞内信号转导通路的起源。 不论在正常还是脱核哺乳动物细胞中，低剂量MNNG均可在短时间内引起细胞表面受体的聚集，且该聚集现象表现为非核依赖性。而配体诱发的受体聚集被证明是其激活的重要表现，因此，低剂量MN－NG可能通过细胞表面受体的途径影响细胞内信号转导通路，但其下游信号途径的激活或改变尚有待进一步证明。本实验提示了在低剂量MNNG引起的非定标突变中，可能有非核依赖的信号转导通路参与，且该信号的起源可能位于细胞膜或是细胞浆中。更多还原

【Abstract】 The association between chemical exposure and the development of specific human cancers has been recognized for a long time, and such examples include amine dyes and bladder cancer, UV and skin cancer, benzene and leukemia, and cigarette smoking and lung cancer. These agents are called carcinogens, and most of the known human carcinogens are genotoxic, which means they can produce alterations in the DNA of the host. The rest are called epigenetic carcinogens, include those substances that promote cancer in ways other than direct DNA damage.Since society has made human health and safety an important issue, considerable resources are being expended to understand the mechanisms of carcinogenesis. N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is a potent human carcinogen which can be found in cigarette smoke. It is a monofunctional alkylating agent that targets the cellular DNA and induce severe genotoxic stress to the cell, leading to chromosomal aberrations, sister chromatid exchanges and point mutation. Furthermore, We have found that low concentration of MNNG (0.2 uM) treatment does not cause significant cytotoxic effect, but rather induces a phenomenon called untargeted mutation (UTM), in which the mutations occur at undamaged DNA sites. Moreover, this process includes the activation of JNK pathway and changes in gene expression at both transcription and translation levels.Although the mechanism by which the signaling cascades initiated by the DNA damaging agents is not fully understood, accumulating evidences show that parts ofcellular responses are independent of nucleus genomic DNA damaging, one of which is that UV exposure induces rapid tyrosine phosphorylation of EGF receptor (EGFR) and the clustering and activation of TNF receptor (TNFR).Taken the fact that cell membrane receptors are involved in the initiation of lots of transduction pathways, in this study we tried to find out if EGFRs and TNFRs might play a role in the upstream signal of the cellular response induced by MNNG.Our data shows that, similar to the cell response caused by UV, the treatment of low concentration MNNG (0.2 M) induced rapid clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor (TNF ). It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations 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 damaging property.更多还原