| N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a monofunctional SN1 alkylating agent, widely used in research laboratory as a model carcinogen in studying the mechanism of N-nitroso alkylating agent induced mutagenesis and carcinogenesis. MNNG can generate adducts with DNA and proteins and induce DNA damages which can lead to chromosomal aberrations, mutations, and cell death. Among the adducts, O~6-alkyl guanine is the predominant mutagenic lesion because of its mispairing properties. This lesion also appears to be involved in many tumor initiations.In our laboratory, a unique mutation detection system using a shuttle vector plasmid has been established to demonstrate that the nontargeted mutation-can be induced by low concentration of MNNG (0.2 μM) in mammalian cells: the mammalian cells were exposed to 0.2 μM MNNG for 2.5 h, then a shuttle plasmid pZ189 carrying supF tRNA gene was transfected into cells after 24h culture. We found a 5-fold higher mutation frequency of the supF tRNA gene in the plasmid replicated in pretreated cells than the spontaneous mutation frequency of that replicated in control cells. This kind of mutation did not occur immediately afterMNNG exposure. Time-course analysis showed that the frequency of MNNG induced nontargeted mutation increased gradually, reached the peak at 12 h after MNNG treatment, and then declined. The specific nontargeted mutation spectrum is different from that of targeted one, whereas the mutation occurs at the site of damaged base.Comprehensive cellular responses were found in cells following exposure DNA damage agents as soon as DNA damaged and modified. These cellular responses included two parts, induced respectively by DNA damage dependent and independent pathways to counteract the abnormal conditions. Wide 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/SAPK signal pathways, as well as activation of endoplasmic reticulum stress. 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 found widely cellular proteins responsive in FL cells treated with low-concentration MNNG by a combination of 2DE and MALDI-TOF mass spectrometry. In addition, more than 30 differential expressed sequence tags (EST) have been isolated by mRNA differential display. Among them, 2 fragments showed association with nontargeted mutation by MNNG. MNNG also induced cellular responses include endoplasmic reticulum stress, that is, except for DNA damage and alteration of receptor signal transduction system, ER stress also plays a role in cellular responses originated from exposure ofenvironmental alkylating agents. Therefore, cellular response to the environmental mutagens is not always initiated by DNA damage. Therefore exposure to genotoxic agents would trigger a series of comprehensive and complex cellular responses evoked by the signals originated from nucleus (genomic DNA damage), cell receptors and their related signal molecules and endoplasmic reticulum of cells to counteract the abnormal conditions, and thus leads to the rapid activation of signal transduction pathways and finally the alteration of gene expression. It is important to study the profiles of gene expression, which will help understand the global cellular stress responses to chemical carcinogens, and further elucidate the mechanisms ofnontargeted mutagenesis.The rapid development of biological techniques has given us the possibility tounderstand the changes more comprehensively. Nowadays, many high-throughput methods including gene expression microarray assay, real-time RT-PCR analysis and bioinformatics are under way in this laboratory for the study of cellular responses with some all kinds of carcinogens. Genechip technology is an ideal technique for studying the global gene expression changes of cell. Coupled with the completion of the human genome project, it became possible to measure the relevant levels of all 30,000-40,000 human genes in a single experiment. The applications of these methods to chemical carcinogens research may help us to further understanding the molecular mechanisms involved in cellular responses to chemical carcinogens exposure, to monitoring the environmental pollutions and their effect, and to finding the potential biomarkers for chemical carcinogens induced stress response, as well as giving a clue for biomarker te search for monitoring the exposure risk of chemical carcinogens.In this study, to further probe the molecular mechanisms involved in cellular responses to MNNG treatment, and to find potential biomarkers induced by MNNG, we analyzed the expression profiles of human amnion epithelial cells in response to MNNG using Gene Chip? Human Genome U133 of Affymetrix Inc. The study ofglobal gene expression changes of human amnion epithelial cells induced by MNNG exposure includes two parts, i.e., the dose-response relationship and time-response relationship. In the study of the dose-response relationship, the expression level of 281 genes were found to be affected in human amnion epithelial cells exposed to different concentration of MNNG. There were 70, 112, and 146 genes in cells exposed to 0.2uM, l.OuM, and IO.OuM MNNG, respectively. On the other hand, in the study of the time-response relationship, 2408 genes were affected in human amnion epithelial cells exposed to l.OuM MNNG in 3 different time intervals after exposure. Among them, 1425 genes were affected in 3h, 643 genes in 12h and 675 genes in 24h after exposure respectively. There are totally 422 genes of expression changes more than 2 folds, 200, 151, and 145 genes respectively in the order of the above 3 different intervals after exposure. These differential expression genes take part in a wide variety of cellular processes including regulation of transcription, signal transduction, cell cycle, cytoskeleton organization, protein synthesis, immune response, metabolism, etc.At the same time, low sensitive and high false positive rate are currently the biggest question of gene chip technology with advantage of high throughput. Therefore, the results of genechip experimentation must be validated by other experimental methods, for example Northern hybridization and PCR. Nowadays Real-time quantitative PCR has many advantages, for instance, high automalization, low pollution and high throughput etc. Those advantages satisfy the need of gene expression quantitation. In this study, we validated a part of genes responsive to MNNG exposure as screened out by microarray technique using TaqMan? Low Density Array of AB Inc. 23 of responsive genes have validated by real-time PCR technology, 12 and 11 genes from the studies of dose-response and time-response relationships, respectively. Among them, 5 genes were found to be up-regulated and18 genes down-regulated. These genes involved in different biological, i.e., ID1, ZNF302, NFAT5, EGR1 and CEBPG worked as transcription regulators, CCNE2, CYR61, 1IL8, IGF1R as participators in cell cycle, signal transduction and immune response control et al.Therefore, gene chip technology coupled with high throughput Real-time PCR is an effective technique to screen the responsive genes of mammal cells exposed to MNNG. The cellular responses to MNNG exposure are wide, complex, and can be triggered by signals originated from different compartments of the exposed cells. The significance of these genes in the genesis of MNNG induced cellular defensive response and hazardous effect remains to be elucidated, the results may also give a clue for biomarker search for monitoring the risk of MNNG Exposure. |
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