Proteomic analysis of cellular responses to low concentration N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in human amnion FL cells

Jin, Jinghua; Yang, Jun; Gao, Zhihua; Yang, Yu

doi:10.1002/em.20001

Cited by 19 publications

(10 citation statements)

References 26 publications

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“…2-DE and image analysis were conducted by using the protocol established previously in our laboratory (9,10). Briefly, approximately 250 g of extracted protein was placed in 350 l (final volume) of a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% Bio-Lyte (pH 3 to 10), and a trace of bromophenol blue and applied to linear IPG Readystrips (17 cm; pH 4 to 7; Bio-Rad) by in-gel rehydration for 12 h at 20°C.…”

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“…A total of six gels, three containing extracts of mock-infected cells and three containing extracts of M. pneumoniae-infected cells, were symmetrically assembled into a Protean Plus Dodeca cell (Bio-Rad) and run simultaneously in an effort to ensure reproducibility. Finally, the gels were stained by using an improved silver-staining method as described previously (9,10). The silver-stained 2-DE gels were scanned with a GS-800 calibrated imaging densitometer (Bio-Rad) at a resolution of 150 dots per inch.…”

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confidence: 99%

“…The resulting data were exported to Microsoft Excel, and a statistical analysis was performed with Student's t test. A P value of Ͻ0.05 was considered significant (9,10).…”

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Mycoplasma pneumoniae Infection Induces Reactive Oxygen Species and DNA Damage in A549 Human Lung Carcinoma Cells

Sun

Wang

et al. 2008

Infect Immun

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Mycoplasma pneumoniae is a frequent cause of community-acquired bacterial respiratory infections in children and adults. In the present study, using a proteomic approach, we studied the effects of M. pneumoniae infection on the protein expression profile of A549 human lung carcinoma cells. M. pneumoniae infection induced changes in the expression of cellular proteins, in particular a group of proteins involved in the oxidative stress response, such as glucose-6-phosphate 1-dehydrogenase, NADH dehydrogenase (ubiquinone) Fe-S protein 2, and ubiquinol-cytochrome c reductase complex core protein I mitochondrial precursor. The oxidative status of M. pneumoniae-infected cells was evaluated, and the results revealed that M. pneumoniae infection indeed caused generation of reactive oxygen species (ROS). It was further shown that M. pneumoniae infection also induced DNA double-strand breaks, as demonstrated by the formation of ␥H2AX foci. On the other hand, an ROS scavenger, N-acetylcysteine, could inhibit the ROS generation, as well as decrease ␥H2AX focus formation. This is the first report showing that M. pneumoniae infection can directly induce DNA damage, at least partially, through the generation of ROS, and thus this report strengthens the powerful application of proteomics in the study of the pathogenesis of M. pneumoniae.Mycoplasma pneumoniae is one of the smallest self-replicating organisms capable of cell-free existence. As a human pathogen, M. pneumoniae is a major cause of community-acquired respiratory infections in children and adults, which can lead to tracheobronchitis and primary atypical pneumonia (17). Besides causing diseases in the respiratory system, M. pneumoniae has been implicated in several extrapulmonary complications arising from infection; for example, it is a factor in the development of arthritis, cardiovascular diseases, and neurotropic infections (32,34).Considerable progress in our understanding of M. pneumoniae pathogenesis has been made over the years. The activation of the host immune response and direct invasion of cells are believed to contribute to this pathogenesis (32, 37). It has been shown that attachment of M. pneumoniae to the respiratory epithelium is a prerequisite for disease (22). The close interaction between M. pneumoniae and host cells protects the bacterium from removal by the host's mucociliary clearance mechanism and allows it to proliferate and produce metabolites, which in turn can cause cytotoxic effects (32, 34). Simultaneously, M. pneumoniae attachment induces the cells' inflammatory reaction and the host's immune response. For example, upregulation of interlukin-2, -4, -5, -6, -10, -12, and -18 and interferon has been detected in bronchoalveolar lavages, blood, and lungs of M. pneumoniae-infected patients (34). Recently, it was found that the M. pneumoniae protein MPN372, which contains key amino acids similar to the pertussis toxin S1 subunit, might be responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in human...

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Mycoplasma pneumoniae Infection Induces Reactive Oxygen Species and DNA Damage in A549 Human Lung Carcinoma Cells

Sun

Wang

et al. 2008

Infect Immun

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“…The sensor and transducer signaling in DDR largely rely on the protein-protein interactions and alterations in protein activity by protein post-translational modifications. Current studies on the transcriptomics, a genome-wide RNA transcript expression level, and proteomics have tremendously expanded our knowledge on the DDR (Daub 2012;Jin et al 2004). They presented that hundreds of additional proteins are targets of checkpoint kinases and more than a thousand genes are differentially expressed upon DNA damage as a result of transcription factor/microRNA regulation (Derks et al 2014).…”

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Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

et al. 2014

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Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol d) results in an inter-conversion between heterotetramer (Pol d4) and heterotrimer (Pol d3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNAdamaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HUtreated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

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“…NMethyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, is a potent carcinogen and is able to induce various cellular responses, including the activation of intracellular signal transduction pathways and changes in protein expression profile [22][23][24]. One unique response induced by MNNG treatment is the clustering of TNFR and EGFR in both African green monkey kidney Vero cells and human amnion FL cells [24,25].…”

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

Huang

Yang

Shen

et al. 2005

Biochemical and Biophysical Research Communications

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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 MNNGinduced 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. KeywordsSphingolipids; Epidermal growth factor receptor; Lipid raft; Lipidomics Receptor clustering or oligomerization is a prerequisite for the activation of cellular signaling pathways for many types of receptors, including receptor tyrosine kinases (RTKs, such as epidermal growth factor receptor, EGFR), death receptors (such as tumor necrosis factor receptor (TNFR) and Fas/CD95), platelet-derived growth factor receptor (PDGFR), the TCR-CD3 complex, B cell receptor (BCR), CD2, CD28, CD40, CD44, FcεRI, FcgRII, integrins, Lselectin, and probably more [1][2][3][4][5][6][7][8][9]. Upon ligand binding, these receptors can cluster in specific cell membrane regions termed lipid rafts, which are rich in sphingolipids and cholesterols [10][11][12]. Intensive studies have revealed that ceramide, the structural backbone of sphingolipids, plays vital role in this clustering process [13,14]. It has been demonstrated that acid sphingomyelinase (ASM), an enzyme responsible for hydrolyzing sphingomyelin to generate ceramide at the outer leaflet of the cell membrane, could be activated within a few seconds to minutes following ligand binding to receptors; a translocation of ASM also occurs simultaneously [7,15,16]. These events are central in the transformation of rafts in unstimulated, resting cells into active signaling domains transmitting stimuli from the outside into cells [3,13].Besides ligand-induced receptor clustering, accumulating evidences indicate that many stress stimuli can also induce the clustering of various surface receptors. Among the different stimuli, UV pro...

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Proteomic analysis of cellular responses to low concentration N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in human amnion FL cells

Cited by 19 publications

References 26 publications

Mycoplasma pneumoniae Infection Induces Reactive Oxygen Species and DNA Damage in A549 Human Lung Carcinoma Cells

Mycoplasma pneumoniae Infection Induces Reactive Oxygen Species and DNA Damage in A549 Human Lung Carcinoma Cells

Comparative proteomics analysis of global cellular stress responses to hydroxyurea-induced DNA damage in HeLa cells

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

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