| Monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a widely spread environmental mutagen and carcinogen that targets DNA and proteins to generate adducts. Among the adducts, O6-methyl-guanine is the predominant mutagenic lesion, which is involved in tumor initiation, particularly in gastric carcinogenesis and the mutagenesis takes place at the site of damaged base. However, environmental mutagens can also induce mutations at undamaged sites and result in the so-called nontargeted mutagenesis. This kind of mutation may be not only being triggered by the signals originated from DNA damage, but also by the signals of extra-nuclear origin through the epigenetic pathway including the activation of various signal transduction pathways and alterations of gene expressions.The previous studies in our laboratory have demonstrated that low concentration MNNG treatment can induce nontargeted mutagensis in mammalian cells, and the mutagenesis is at least partly caused by the gene expression changes initiated with the activation of several signaling transduction pathways. Comprehensive cellular responses was found in human amnion FL cells following exposure to low concentration of MNNG, such as the lowering of DNA replication fidelity resulted from alteration of DNA polymerase profile; activation of a lot of transcription factors, such as API, CREB, NF-KB etc; clustering of EGFR (epidermal growth factor receptor) and TNFR (tumor necrosis factor receptor) and activation of cAMP-PKA-CREB and JNK/SAPKsignal pathways. It is even more interesting that the activation of some pathways, i.e., the activation of cAMP-PKA-CREB and clustering of cellular surface receptor EGFR seem to be independent of DNA damage, because these events can still occur in enucleated cells. We also have performed a proteomic analysis of FL cellular proteins responsive to low-concentration MNNG treatment by a combination of 2DE and MALDI-TOF mass spectrometry. More than 60 protein spots showed significant changes at their expression level. The identified proteins are involved in a variety of cellular process including several zinc finger proteins relevant to transcription regulation, such as zinc finger 198, 263, 14, 224, zf6, novel protein similar to transcriptional represser CTCF, and kruppel-like zinc finger protein; two members of the ADAMs (a disintegrin and metalloprotease domain) family; two members of integrin family; several proteins involved in the signal transduction, cell-cycle control, chromatin remodeling and transcription repression; and also some proteins of cell skeleton and some with unknown functions.Protein expression could be regulated at several levels. However, the most important one is at the transcriptional initiation. In this study, we tried to find out if the eight MNNG-induced co-expressive proteins identified by mass spectrometry are co-regulated by any common regulatory pathway. Phylogenetic footprinting and TRANSFAC Position Weight Matrix (PWM) searching program were used to predict the common transcription factor binding sites among the conservative promoter regions of these genes. The predicted results were validated with Electrophoresis mobility shift assay (EMSA). Establishment of orthologous relationships between human and mouse genesThe mRNA sequences of the genes, i.e., ZNF198, ITGBL1, ADAM17, KNG, ITGB2, PRKCBP1, F9 and ADAM28 which encoding the eight MNNG-induced proteins identified by mass spectrometry were extracted from the GenBank database. Each of the human mRNA sequences was BLASTed against mouse mRNA sequences and the top hit was BLASTed back to the original human mRNA database. If the top human mRNA hit is the same as the original human mRNA, we assumed an orthologous relationship between the human and mouse genes. To minimize theredundancy, only human mRNA in GenBank RefSeq section was used to establish anyhuman-mouse ortholog relationships.Transcript mapping and construction of ortholog promoter relationshipTo ensure that the 5' end of a mRNA is close to t...
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