1.Induced Endoplasmic Reticulum (ER) Stress And Binding Of Over-expressed ER Specific Chaperone GRP78/BiP With Dimerized Epidermal Growth Factor Receptor In Mammalian Cells Exposed To Low Concentration Of N-methyl-N'-nitro-N-nitrosoguanidine 2.Endopl

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, 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. However, 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 (0.2 μM) can induce nontargeted mutation 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 plasmid replicated in pretieated cells than the spontaneous mutation frequency of the plasmid replicated in control cells. This kind of mutation did not occur immediately after MNNG 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 mutation, whereas the mutation occurs at damaged DNA site. Differential gene expression can be detected inMNNG-treated mammalian cells, which are in a state of inducing untargeted mutagenesis. Further studies showed that DNA replication fidelity is decreased and there is an alteration of DNA polymerase spectrum when mammalian cells are attacked by MNNG. Moreover, this process is accompanied with decreased DNA replication fidelity and altered gene expression profiles including the up-regulation ofDNA polymerase 0(POL- ￡), and and the activation of cAMP-PKA-CREB pathway. In addition, it is clear that the signal to activate certain signal transduction pathways is not from nucleus. Furthermore, we have demonstrated that low concentration MNNG exposure induced comprehensive cellular responses. For example, we have found the clustering of EGFR (epidermic growth factor receptor) and TNFR (tumor necrosis factor receptor) after MNNG treatment. It is even more interesting that the activation of these pathways seems to be independent of DNA damage, because these events can still occur in enucleated cells.Therefore cellular response to the environmental mutagens is not always initiated by DNA damage. Exposure to genotoxic agents would trigger a series of comprehensive and complex responses in cells to counteract the abnormal conditions, for example, through cell receptors and their related signal molecules or other unidentified pathways, which leads to the rapid activation of signal transduction pathways independent of the nuclear damage signal and late alteration of gene expression.In the study of MNNG induced cell receptor clustering, we found EGFR (epidermic growth factor receptor) clustering after MNNG treatment. However, unlike EGF treatment, the autophosphorylation of Y845, Y992, Y106 and Y1073 of EGFR were not observed, and the downstream signaling molecule RAS was not activated. Furthermore, MNNG pretreatment does not interfere the ligand binding of EGFR but can block the autophosphorylation of EGFR and activation of RAS by EGF . One possibility is that MNNG might chemically perturb the EGFR by inducing conformational modifications, probably by its methylation effect on EGFR. Alternatively, some unknown suppressive molecules might be triggered by MNNGand bind to EGFR, leading to the down-regulation of EGFR signaling pathway initiated by EGF.In this study, in order to further investigate the underlying mechanism of blockade of MNNG induced epidermal growth factor receptor pathway, Western blot was used to detect whether EGFR can be phosphorylated in cell lysis exposed to 0.25|imol/L MNNG. We found that although EGFR can be phosphorylated by its ligand in normal cell lysis, it does not occur in MNNG pretreated cell lysis. In this study, we also demonstrated that the EGFR clustering on cell surface induced by low concentration MNNG ( 0.25 and 1 umol/L) was indeed the EGFR dimerization. However, the dimerization does not occur at lOumol/L although clustering did exist. Moreover, unlike EGF treatment, the dimerization initiated by MNNG was irreversible upon mild-acid washing. Besides, in accordance with our previous results, the recruitment of adaptor proteins Grb-2/Shc/Sos 1, which play key roles in activating ensuing RAS-MAPK pathway, was also suppressed.Surprisingly, ER specfic chaperone GRP78/Bip was found in Cell lysates immunoprecipitated with anti-EGFR antibody followed by immunoblotting with anti-GRP78/BiP antibody. ATP treatment resulted in a disassociation of EGFR from BiP, and the EGF induced phosphorylation status of EGFR can be revitalized upon the addition of ATP. Therefore it is concluded that ER stress participates in MNNG induced down regulation of EGFR pathway.ER is the main organelle for protein synthesis and protein folding in eukaryotic cells, the labyrinth of the endoplasmic reticulum (ER) interweaves the cytosol and connects to the nucleus, mitochondria, and the plasma membrane. In the lumen of the ER, the essential function of lipid synthesis, Ca2+ storage, folding, and maturation of proteins take place. Therefore, the tight regulation and maintenance of ER homeostasis is vital. Disturbance of the Ca2+ homeostasis during hypoxia, or imbalance between the demand and capacity of the protein-folding apparatus, initiates an adaptive response of the cell, termed the unfolded protein response (UPR, ER stress response). As a result, ER-localized chaperones, particular GRP78/BiP, which belongs to glucose-starved regulated protein family, are induced. The effect ofGRP78/BiP is protein-specific and depends on the stable association of GRP78/BiP with its target. Such a specificity of GRP78/BiP may be important for protecting cells under various stressors, such as 2-deoxyglucose, glucosamine, and tunicamycin. Il can bind to the misfolded protein, thus the protein synthesis is slowed down, and a protein degrading system is initiated. At the same time, activation of transcription factor ATF6 and transmembrane protein kinase PERK induces GADD153/CHOP expression, thus inhibits Bcl-2 expression and leads to cell apoptosis. This becomes particularly evident under conditions of stress, whereas the unfolded protein response protects cells from problems arising in the ER. However, if the ER stress cannot be alleviated. it can activate caspase-12, which play a role in the initiation of cell death.We also found that MNNG can induce ER stress or unfolded protein response (UPR) which is characterized by induced expression of ER-stress response proteins, such as GRP78/BiP and GADD153/CHOP, and also by induced activation of ER-localized caspase-12. Thus, it is suggested that in MNNG-exposed cells. ER stress was activated and may participate in the blockage of EGFR-signaling pathway by forming a stable complex of EGFR/BiP. This is the first report to show the involvement of ER stress in MNNG-induced stress responses and downregulation of EGFR signal pathway.Detection of tyrosine phosphorylated EGFR in vitro by using SDS-PAGE and western blot. FL cells were treated with DMSO (solvent control), different concentrations of MNNG (0.25uM, luM ) for 30 min, followed by cell lysis. Subsequently the lysates were incubated with 100 ng/ml EGF for 30 min, respectively. After electrophoresis, the tyrosine phosphorylation levels of EGFR were determined by immunoblotted with anti-tyrosine phosphorylated EGFR antibody.Results: EGF stimulation activated the tyrosine phosphorylation of EGFR in lysates prepared from DMSO treated cells, however, it did not occur in lysates prepared from MNNG pre-treated cells. The observation indicates that MNNG treatment either induced conformation change in EGFR, or induced some suppressive molecules which may bind to EGFR, thus leading to a defect in EGFR phosphorylation.Studies on dimerization of EGFR by cross-linking of EGFR, SDS-PAGE and western blot. For dimerization study, FL cells were treated with DMSO, EGF or different concentrations of MNNG (0.25piM, luM, lO^M) for 30 min, then were either washed with PBS or incubated with 0.2 M sodium acetate, to remove the surface bound ligand.The first procedure subjected all cellular EGFR to cross-linking. Cells were permeabilized by incubating in CMF-PBS containing 0.012% digitonin, then were solubilized in lysis buffer. A membrane non-permeable regant BS was then added to cross link EGFR in the total pool of cell lysates. In the second procedure, cross-linking of surface EGFR dimmer was conducted by incubating the cells with BS3, followed by cell lysis.Cell lysates obtained after each type of cross-linking procedure were separated by SDS-PAGE on 3-10% linear gradient gel and transferred to nitrocellulose membrane. The bands corresponding to the monomeric and dimeric form of the EGFR were then immunodetected on the membrane using polyclonal EGFR antibody.Results: Using chemical cross-linking after mild permeabilization, we did detect dimers of EGFR in the total pool of cellular protein prepared from the cells treated with either 100 ng/ml EGF, 0.25 or 1 uM MNNG, In addition, a mild acid (pH 4.5) wash of cells resulted in a rapid monomerization of surface EGFR dimers in EGF-treated cells, however, the dimeric form of EGFR remained after mild acid washing in MNNG treated cells, indicating that low concentration of MNNG-induced EGFR dimerization on cell surface is mild acid washing resistant.Immunoblot analysis of adaptor proteins with their association with EGFR upon MNNG treatment FL cells were treated with DMSO (solvent control), different concentrations of MNNG (0.25uM, luM ) for 30 min. For immunoblot analysis, cells were lysed and fractionated by SDS-PAGE and then analyzed by incubating in polyclonal anti-EGFR, anti-Grb2, anti-She, or anti-Sosl antibody, respectively.Results: There was neither Grb2 nor Sosl complexes bound to the receptor protein in MNNG exposed cells, while she had a slight association with EGFR.Immunofluorescent analysis of relocalization of Grb-2, She and Sosl upon MNNG treatment FL cells were treated with DMSO, EGF, or MNNG (0.25 uM) for 30 min. The relocalization of Grb-2, She and Sosl was observed by using immunofluorescent microscopy. In cells treated with EGF, relocalization of she. Grb2 and sosl were observed as the formation of bright dots or patches, while in MNNG treated cells no such changes were detected. Grb2, she and sosl showed a diffuse distribution throughout the cytosol.Detection of ER stress protein GRP78/BiP and its interaction with EGFR by using immunoprecipitation and immunoblot Cell lysates were immunoprecipitated with anti-EGFR antibody followed by immunoblotting with anti-GRP78/BiP antibody. Results showed that GRP78/BiP bound specifically to EGFR upon MNNG treatment but not by EGF. Therefore, we inferred that MNNG might chemically change the comformation of EGFR, expose the binding site for GRP78, and lead to the blockage of EGFR tyrosine phosphorylation.EGFR autophosphorylation status is recovered by EGF upon the addition of ATP in vitro in MNNG pretreated cells MNNG pre-treated cells were lyzed and the lysates were incubated with lmM ATP and 100 ng/ml EGF for 30 min. Tyrosine phosphorylation levels of EGFR were determined by Western blot analysis. Results: Treatment of the lysates with ATP results in a recovery of the EGF-induced phosphorylation of EGFR, however, it did not occur in lysates without ATP. The observation indicated that treatment of the complex with ATP results in a release of EGFR from BiP, and a reactivation of EGFR phosphorylation.Detection of up-regulation of ER stress specific proteins upon MNNG by using immunobnlot analysis To determine if MNNG can induce ER stress, several ER stress specific proteins, such as GRP78/BiP, CHOP, and caspase-12 were examined.Results: Using tunicamycin as positive control, we detected increased GRP78/BiP and CHOP expression, as well as activated caspase-12 with corresponding antibodies by immunoblot analysis in MNNG treated cells. Taken together, these data provide strong evidence for the induction of ER stress by MNNG treatment.Innovative conclusion was drawn from the experiements:1 Endoplasmic reticulum (ER) stress participates in MNNG-induced down-regulation of EGFR signaling transduction. It was demonstrated that the ER specific chaperone glucose-regulated protein 78 (GRP78/BiP) formed a stable complex with EGFR in MNNG-treated cell and blocks its autophosphorylation. We supposed that MNNG might chemically perturb the conformation of EGFR and expose the binding site of some amino acid residues for GRP78/BiP, which leads to the blockage of EGFR phosphorylation. ER stress participates in the blockage of EGFR signaling pathway by forming a stable complex of EGFR/BiP, and inhibits the downstream Ras-MAPK pathway. Treatment of the lyates with ATP results in a disassociation of EGFR from GRP78/BiP, and a recovery of the EGF induced phosphorylation status of EGFR.2 MNNG 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 of environmental alkylating agents.