JMJD5 cleaves monomethylated histone H3 N-tail under DNA damaging stress.

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N-tail at the carboxyl side of monomethyl-lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (Kme2), trimethyl-lysine (Kme3), or unmethyl-lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N-tail cleavage in gene expression regulation.

The role of acetylation in TLR4-mediated innate immune responses.

Toll-like receptor 4 (TLR4) plays a critical role in the innate immune response to Gram-negative bacterial infection. A large number of the components involved in TLR4 signaling pathways have been identified over the past ten years. Recent studies focusing on the post-translational modification of TLR4 signaling pathways have begun expanding our knowledge of the impact of lysine acetylation on TLR4 signaling cascades. In this review, we will focus on the potential roles of acetylation in TLR4-mediated innate immune responses.

Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses.

Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25µM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

Temporal gene expression changes induced by a low concentration of benzo[a]pyrene diol epoxide in a normal human cell line.

(+ or -)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), which causes bulky-adduct DNA damage, is well-characterized as the ultimate carcinogen of benzo[a]pyrene (BaP). In this study, we have employed Affymetrix HG-U133 Plus 2.0 microarray and quantitative real-time RT-PCR methods to investigate a temporal transcriptomic response triggered by a low concentration (0.05 microM) of BPDE at 1, 10, and 22 h after exposure in normal human cells. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after the BPDE-caused damage. The dynamic regulation of the genes related with cell cycle progression and cell fate exhibited a tendency from inhibition to survival, which was accordant with the cell cycle arrest and cytotoxicity data induced by the low-dose BPDE exposure. In silico comparison of the genomic data revealed that BPDE and ultraviolet induced a panel of common transcriptional responses, which might be related with a series of similar molecular processes elicited by these two DNA-damaging agents. In conclusion, this whole-genome time-course study has identified a dynamically regulated transcriptional signature after low-dose BPDE exposure, which may help to understand the complex mechanisms of mutagenesis and carcinogenesis induced by BPDE.

Low concentration of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induces alterations of extracellular protein profile of exposed epithelial cells.

7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) exposure induces adduct formation and oxidative damage on DNA, and consequently triggers complicated stress responses, including such responses as signaling pathway activation, cell cycle arrest, DNA repair, translesion DNA synthesis and mutagenesis. In the present study, 2-DE and MALDI-TOF MS were employed to analyze the differential extracellular protein patterns of human amniotic epithelial cells (FL cells) after exposure to 5 nM BPDE and control. As a result, one protein spot that appeared in the culture medium of BPDE treatment group was successfully identified as 14-3-3zeta, and three up-regulated protein spots were identified as annexin A3, annexin V and hydroxypyruvate isomerase homolog. Among them, 14-3-3zeta was further detected in some pleural fluid specimens also. These results demonstrate that BPDE exposure can induce alterations of extracellular protein profiles of exposed cells, which may be served as a starting point for searching candidate biomarkers for benzo[a]pyrene exposure.

Differential nuclear proteomes in response to N-methyl-N'-nitro-N-nitrosoguanidine exposure.

As a model carcinogen in studying the mutagenicity and carcinogenicity of N-nitroso alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can induce DNA damages and many cellular defensive events, such as DNA repair, G2/M-phase arrest and apoptosis. Noticeably, diverse cellular responses were observed to occur following MNNG treatment in our previous studies, including nontargeted mutations (NTM) at undamaged DNA bases, endoplasmic reticulum stress (ER stress) induction and the activation of several signal transduction pathways. In addition, whole cell proteome analysis also revealed that comprehensive and various changes were triggered by this mutagen. However, low-abundance proteins with key functions, such as nuclear proteins, are always underrepresented in proteomic studies. To reduce the complexity of protein samples and monitor subcellular alterations in response to MNNG exposure, nuclear extracts were fractionated from MNNG-treated cells and compared using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Twenty-three differentially expressed protein spots were observed after 0.25 and 1 muM MNNG exposure, and 8 of them were shared between these two treatment groups. When MALDI-TOF MS was used, 17 nuclear proteins affected by MNNG were identified. These proteins were involved in regulation of RNA processing, signal transduction, metabolism, DNA repair, protein biosynthesis and microtubule or cytoskeleton organization. Among them, two nuclear proteins with nucleocytoplasmic shuttling activity, 14-3-3 zeta and hnRNP K, were further demonstrated to undergo different dynamic changes in response to MNNG exposure. These results will aid our understanding of the complicated mechanisms of MNNG-induced cellular responses.

Early whole-genome transcriptional response induced by benzo[a]pyrene diol epoxide in a normal human cell line.

(+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage. In this study, we investigated early transcriptional signatures induced by various concentrations (0.005, 0.05, and 0.5 microM) of this carcinogen in a normal human cell line (FL human amnion epithelial cells) using the whole-genome Affymetrix HG-U133 Set microarray. The numerous identified genes were involved in multiple functions and higher doses of BPDE elicited more robust expression changes. The disturbance of genes involved in cell cycle regulation, growth and apoptosis was correlated with the S and G(2)/M phase cell cycle arrest and cytotoxic phenotypes induced by different levels of BPDE. Bioinformatic analysis showed that several transcription factors and their related stress signaling pathways might partly account for the transcriptional signature induced by BPDE. Additionally, gene ontology analysis of the microarray data showed down-regulation of transport, cytoskeleton and DNA repair by 0.5 microM BPDE exposure. In conclusion, this genomic analysis helps to understand the mechanism of cellular response to BPDE.

Identification of temporal differentially expressed protein responses to microcystin in human amniotic epithelial cells.

The increased distribution of microcystins (MCs) has become a global issue. However, the MC response machinery has not been completely studied and elucidated until now. Our previous dose-related analysis has revealed that different concentrations of mirocystin-RR (MC-RR) can trigger comprehensive and various alterations in the protein expression profile using a proteomic approach. To further dissect the kinetics of cellular responses associated with MC-RR cytotoxic effects, the present study has set out to investigate the temporal protein expression pattern at three time points (3, 12, and 24 h after exposure). The relative intensity has changed significantly in MC-RR-treated FL cells, as compared with the control, in a total of 127 protein spots. One hundred and two proteins have been further identified with high confidence by peptide mass fingerprinting. These proteins can be categorized into diverse functional classes such as signal transduction, apoptosis, protein degradation, cell cycle, cell differentiation, transporter, and so forth. The wide range of different proteins involved suggests that MC-RR has profound effects on the biological response and toxic consequences of the affected cells. The identification of p53 and its potential targets confirms a known role for p53 in the MC-RR response. Moreover, it is noteworthy that MC-RR can up-regulate the expression of the PP2A A subunit and down-regulate the expression of a number of proteins implicated in the ubiquitin-proteasome pathway. Therefore, the present expression data provide a global view of dynamic changes in cell responses to MC-RR and, more importantly, generate novel hypotheses regarding MC-RR-responsive mechanisms.

Translation initiation proteins, ubiquitin-proteasome system related proteins, and 14-3-3 proteins as response proteins in FL cells exposed to anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide.

Benzo[a]pyrene (B[a]P) is an ubiquitous environmental carcinogen produced during incomplete combustion of organic substances. Anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), is the most carcinogenic form of the ultimate metabolites of B[a]P. The goal of this study was to investigate the responses of human amniotic epithelial FL cells to BPDE at different time intervals after exposure and to find potential biomarkers involved in these responses. Cells were treated with 0.05 microM BPDE for 2 h and incubated for another 3, 12, and 24 h to obtain protein extracts which were resolved by 2-DE and visualized by silver staining. Sixty-four spots were up-regulated while 66 were down-regulated following BPDE exposure. These altered spots were excised from the gels and analyzed by MALDI-TOF-MS. The analysis led to the identification of 84 proteins affected by BPDE. These proteins were involved in regulation of transcription, cell cycle, apoptosis, transport, signal transduction, metabolism,and so forth. Among them, subunits of eukaryotic translation initiation factor 3 (EIF3) including EIF3S2, EIF3S3, EIF3S12, and EIF5A, component proteins of ubiquitin-proteasome system (ubiquitin carboxyl-terminal esterase L3, proteasome beta 4 subunit, and proteasome beta 3 subunit) and 14-3-3 proteins (14-3-3 zeta and epsilon) have not been previously associated with a response to BPDE exposure. All these results aid our understanding of the mechanism of BPDE induced cell defensive responses and hazardous effects as well as providing the possibility of the establishment of potential biomarkers.

Identification of early responsive genes in human amnion epithelial FL cells induced by N-methyl-N'-nitro-N-nitrosoguanidine using oligonucleotide microarray and quantitative real-time RT-PCR approaches.

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular DNA damage and other comprehensive alterations that lead to chromosomal aberrations, mutations, tumor initiation, and cell death. However, the molecular mechanism of MNNG-induced cellular stress remains unclear. We undertook a genome-wide analysis of early transcriptional responses of human FL amnion epithelial cells to three relatively low doses of MNNG (0.2, 1.0, and 10.0 microM). Using Affymetrix GeneChip HG-U133 Set oligonucleotide microarrays, a total of 281 genes were found to change their expression after exposure to all three doses of MNNG compared with the vehicle control, DMSO. Of these, 70, 112, and 146 genes showed different expression levels following treatment with low, medium, and high-dose MNNG, respectively. A subset of these genes were selected for further confirmation using quantitative real-time RT-PCR with ABI TaqMan((R)) low-density arrays, and the differential expression of 33 genes was validated. The results demonstrate that low doses of MNNG can induce various changes in gene expression at an early stage of exposure. The responsive genes are involved in multiple cellular biological processes including transcription regulation, signal transduction, cell cycle regulation, cytoskeleton organization, protein synthesis, immune responses, and metabolism. The cell cycle progression was down-regulated, in which several genes were validated involved, including the cell cycle regulators (CDK6, STK6, CENPA, and CCNF), the transcription factor ID-1, and the calcium signaling molecules (CAMK2G and NFAT5). The possible roles of the responsive genes and their related pathways in MNNG-induced cellular responses are discussed. This study helps to complete the picture of how cells respond to environmental chemical exposure via transcriptional regulation.

Proteomic analysis of different temporal expression patterns induced by N-methyl-N'-nitro-N-nitrosoguanidine treatment.

We have previously shown that N-methyl- N'-nitro- N-nitrosoguanidine (MNNG), a well-known DNA alkylating agent and carcinogen, can induce multiple cellular responses with dynamic characteristics, including such responses as nontargeted mutations (NTM) at undamaged bases in DNA, up-regulation of low fidelity DNA polymerases, clustering of epidermal growth factor receptor (EGFR) and interference with its downstream signaling pathway. A dose-related analysis also revealed that different concentrations of MNNG can trigger diverse proteome changes associated with different cytotoxic effects. To further understand the dynamic cellular responses and hazardous effects caused by environmental carcinogen, a proteomic time-course study of whole cellular proteins from human amniotic epithelial cells after MNNG treatment was performed. Analysis at three different time points (3, 12 and 24 h after exposure) revealed that the major changes were taking place around 3 and 12 h after exposure. Using MALDI-TOF MS coupled with a micro solid-phase extraction (SPE) device, 90% ( n = 70) differentially expressed proteins were identified. Functional assignment revealed that many important pathways were affected, including the protein biosynthesis pathway and Ran GTPase system. We also carried out a network analysis of these proteins and the data suggest a central role for some key regulators in different pathways.

Proteomic analysis of cellular responses to different concentrations of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide in human amniotic epithelial FL cells.

Benzo(a)pyrene is an ubiquitous environmental carcinogen produced during incomplete combustion of organic substances, and anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), is the most carcinogenic form of its ultimate metabolites. The goal of this study was to examine the response of human amniotic epithelial FL cells to increasing concentrations of BPDE and to find potential biomarkers involved in this cellular response. Human amniotic epithelial FL cells were incubated with 0.005, 0.05, and 0.5 microM BPDE to obtain protein extracts which were resolved by two-dimensional electrophoresis (2-DE) and visualized by silver staining. More than 60 protein spots significantly changed after BPDE exposure. Among these, 2 spots were detected only in the exposed group, and 36 spots were up-regulated, while 27 spots were down-regulated. These altered spots were excised from the gels and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The analysis led to the identification of 46 proteins affected by BPDE. These proteins were involved in regulation of transcription, cell cycle, cell proliferation, transport, signal transduction, metabolism, and so forth. However, no single protein changed in a dose-dependent manner in all three concentrations. Therefore, the changes of proteomic profiles cannot be considered as only an amplification of low-dose response in the case of high-dose exposure and the cellular responses to different doses of DNA damaging agent may be quite different. These results will aid our understanding of the mechanism of BPDE-induced cell response and provide the possibility of the establishment of potential biomarkers.

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry.

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins.

dUTP Pyrophosphatase, its appearance in extracellular compartment may serve as a potential biomarker for N-methyl-N'-nitro-N-nitrosoguanidine exposure in mammalian cells.

The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a model chemical widely used for studying the molecular events induced by the widespread environmental N-nitroso alkylating carcinogen. Many studies have focused on understanding MNNG-induced mutagenesis and carcinogenesis. However, the search for specific indicators of MNNG exposure is still underway. In this study, we analyzed the proteins in culture medium of human amnion epithelial cells (FL cells) exposed to MNNG by 2-DE followed by MALDI-TOF MS, in the hope of finding a specific protein marker suitable for MNNG risk assessment. Image visualization and statistical analysis indicated that 12 spots appeared and 4 spots up-regulated after MNNG exposure. Most of them were identified by MS. These proteins include nuclear isoform of dUTP pyrophosphatase (DUT-N), phosphoglycerate mutase 1, heparan sulfate proteoglycan perlecan, etc., which are involved in multiple cellular functions. Interestingly, 2-DE and MS analyses of cell lysate exposed to MNNG revealed that DUT-N was down-regulated. The appearance of DUT-N in culture medium and its down-regulation in cell lysate was confirmed by Western blot. These data suggest that these proteins, especially DUT-N, could be used as candidate biomarkers for monitoring MNNG exposure.

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.

Previously we have shown that alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can induce the clustering of epidermal growth factor receptor (EGFR) in human amnion FL cells. However, the biological consequence of MNNG-induced clustering is different from that of epidermal growth factor (EGF)-induced clustering. In addition, MNNG strongly blocks the autophosphorylation of EGFR in response to its ligand, we speculate it might be due to the altered conformation of EGFR by MNNG alkylation, or the binding of some unknown suppressive molecules to EGFR, which could lead to the down-regulation of EGFR pathway. In this study, we further demonstrated that EGFR could not be phosphorylated by EGF in lysates prepared from MNNG-pretreated cell. In addition, it was found that the clustering of EGFR induced by low concentration (

Proteomic profiling for cellular responses to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine.

Alkylating agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) can induce DNA damages which can lead to chromosomal aberrations, mutations, and cell death. Previous reports from our laboratory have found that low concentration of MNNG can induce nontargeted mutations (NTM) at undamaged bases in DNA, clustering of epidermal growth factor receptor (EGFR) and interference of EGFR mediated signaling, as well as activation of endoplasmic reticulum stress. Thus, the cellular responses to MNNG exposure are very complex, and can be triggered by signals originated from different compartments of the exposed cells. To further probe the molecular mechanisms involved in cellular responses to MNNG treatment, and to find potential biomarkers for MNNG induced stress condition, we performed proteomic analysis of whole cellular proteins from human amnion epithelial cells after exposing to MNNG at 3 different doses. 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, metabolism, cytoskeleton organization, cell cycle, cell proliferation, signal transduction, transportation, etc. The significance of these proteins 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 exposure risk of MNNG.

Proteomic analysis of cellular response to microcystin in human amnion FL cells.

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.

Sphingolipids are involved in N-methyl-N'-nitro-N-nitrosoguanidine-induced epidermal growth factor receptor clustering.

Previously we have found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR). Since sphingolipids, especially ceramide, have been suggested as major players in ligand-induced receptor clustering, their involvement in this ligand-independent, chemical-induced receptor clustering was evaluated. It was shown that MNNG-induced EGFR clustering occurred primarily at lipid rafts, as nystatin, which can disrupt lipid raft structure, significantly decreasing MNNG-induced EGFR clustering. Lipidomic studies revealed that MNNG treatment induced profound changes in sphingolipids metabolism, which were not the same as those induced by EGF treatment. Acid sphingomyelinase (ASM) is responsible for hydrolyzing sphingomyelin to generate ceramide, and it was demonstrated that MNNG treatment caused ASM distribution changing from diffused state to concentrated area of cells, which colocalized with lipid rafts. Nystatin treatment also abolished the redistribution of ASM. In addition, blockage of ceramide production by ASM inhibitor imipramine interrupted MNNG-induced receptor clustering. Taken together, these data suggested that sphingolipids are involved in MNNG-induced receptor clustering; however, the specific species involved may be different from those involved in EGF-mediated receptor clustering.

N-methyl-N'-nitro-N-nitrosoguanidine interferes with the epidermal growth factor receptor-mediated signaling pathway.

Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.