Proteomic Analysis Of Cellular Responses To N-methyl-N'-nitro-N-nitrosoguanidine In The Dose-related And Time-course Study

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is widely used in research laboratory as a model carcinogen in studying the mechanism of N-nitroso alkylating agent induced mutagenesis and carcinogenesis. It can generate adducts with DNA and proteins, which can lead to point mutations, chromosomal aberrations, or even cell death. Among the alkylation types of MNNG induced DNA damage, O~6-methylguanine (O~6MeG) is the predominant mutagenic one. It also appears to be involved in tumor initiation, particularly in gastric carcinogenesis. Environmental mutagens can also induce mutations at undamaged sites and result in the so-called nontargeted mutation. In our laboratory, a unique mutation detection system using a shuttle vector plasmid has been established to demonstrate that a low concentration of MNNG (0.2μM) can induce nontargeted mutation in mammalian cells. Furthermore, we have demonstrated that low concentration MNNG exposure induced comprehensive cellular responses. For example, DNA replication fidelity is decreased and there is an alteration of DNA polymerase spectrum when mammalian cells are attacked by MNNG. Moreover, we have found the clustering of EGFR (epidermic growth factor receptor) and TNFR (tumor necrosis factor receptor), though the Ras/MAPK pathway is not activated, and the activation of endoplasmic reticulum stress and cAMP-PKA-CREB and JNK/SAPK pathways after MNNG treatment.For further understanding the global cellular responses to chemical carcinogens,we have investigated the differentially expressed proteins in mammalian cells exposed to low concentration MNNG treatment with the high-throughput proteomic techniques. In that study, we found that the expression of more than 60 proteins was affected, and 36 of them have been identified successfully. These proteins take part in a wide variety of cellular processes and may contribute to the complicated cellular responses. As for the protein pattern of cellular response will change in waves over time and will not be fixed in its later developed events, it's really necessary to study the dose-related and time-course responses.Main results:1. In this study, we performed a proteomic analysis of whole cellular proteins from human amnion epithelial cells after exposing to MNNG at three different doses. 0.25 μM was chosen as the low-dose, 1 μM as the middle one and 10 μM as the high one. More than 80 proteins were affected by MNNG treatment, and 71 proteins among them were identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. These proteins take part in a wide variety of cellular processes including regulation of transcription, signal transduction, metabolism, DNA repair and cell cycle, etc. We found that only a few differentially expressed proteins existed in more than one treatment groups, while most of them altered with a dose-independent manner. It reflected the complication of cellular responses and the misunderstanding of considering the high-dose response only as an amplification of low-dose exposure. In all of the three treatment groups, there was a rough trend that the number of up-regulated proteins decreased, and down-regulated proteins increased with the increase of concentrations. We suspect this was because of the enhanced cytotoxic effect of the increased concentrations of MNNG rather than a random phenomenon.2. We carried out the time-course proteomic study with the middle-dose (1μM) MNNG treatment for the relatively lower cytotoxic effect and more evident cellular responses. Cells were collected at 3 h, 12 h and 24 h after MNNG exposing, respectively. 78 differentially expressed proteins were screened out and 64 of themwere identified by MS. To probe the affected proteins as more as possible, we applied a solid-phase extraction (SPE) device after the traditional enzyme digestion process, and it turned out to be quite efficient as increasing the protein detection sensitivity by MS from 82% to 90%. The proteins identified also participate in diverse functional groups, such as regulation of cell cycle, metabolism and signal transduction etc., and mostly assembled in the 3 h and 12 h treatment groups, which indicated the main cellular stress responses might happened around 3-12 h after MNNG exposing.3. A protein identified as the nuclear isoform of dUTP pyrophosphatase (DUT-N) has exhibited a decreased intracellular content, while its appearance in the culture medium was detected after low dose MNNG treatment by an independent proteomic study and verified by Western blot. It may serve as a new candidate biomarker for monitoring the exposure of human populations to environmental carcinogens.4. Since cell nuclear play an important role in many cell responses, we also conducted a nuclear proteomic analysis with 0.25 μM and 1 μM MNNG treatment to further probe the affected nuclear proteins might masked by relatively high abundant cytoplasmic proteins in whole cell lysate proteomic study. We adopted the more accurate "fluorescence two-dimensional differential gel electrophoresis (2D-DIGE)" technology which developed on the traditional 2D-PAGE, and screened out 24 differentially expressed proteins. Among the 18 proteins identified eventually, most of them existed in both treatment groups and belong to 14-3-3, hnRNPs and cytoskeleton protein families, which reflected the common influences in cell nuclear with the different MNNG exposing doses.5. Verification of differentially expressed proteins by Western blot analysis: From the candidates, RanGAP1, 14-3-3 β and 14-3-3 ε were selected for Western blot analysis. The expression change of the selected protein was consistent with the 2-DE and silver-staining results, which demonstrated that the proteomic analysis of cellular responses to MNNG was convincing.Main conclusion:1. Proteomic approaches were carried out to evaluate the dose- and time- related cellular responses to MNNG treatment, and 85 and 78 proteins turned out to be affected, respectively. The identified proteins are involved in a variety of cellular processes, which indicated the comprehensive and complex changes happened in cells after MNNG attack.2. Only a few proteins altered in a dose- or time-dependent manner. Therefore, the biological responses of high dose exposure can't be considered only as an amplification of low dose response, and MNNG may activate a series of independent pathways to regulate the cell response machinery for different exposure times.3. The appearance of DUT-N in culture medium and down-regulation in cell lysate serve itself as a new candidate biomarker for monitoring the exposure of human populations to environmental carcinogens.4. In the nuclear proteomic analysis with 0.25 μM and 1 μM MNNG treatment, most of the differentially expressed proteins belong to 14-3-3, hnRNPs and cytoskeleton families, which reflected the common influences in cell nuclear with the different MNNG exposing doses.