Gastric cancer(GC), one of the most common malignancies worldwide, seriously threatens human health due to its high morbidity and mortality. Precancerous lesion of gastric cancer(PLGC) is a critical stage for preventing the occurrence of gastric cancer, and PLGC therapy has frequently been investigated in clinical research. Exploring the proper animal modeling methods is necessary since animal experiment acts as the main avenue of the research on GC treatment. At present, N-methyl-N'-nitro-N-nitroso-guanidine(MNNG) serves as a common chemical inducer for the rat model of GC and PLGC. In this study, MNNG-based methods for modeling PLGC rats in related papers were summarized, and the applications and effects of these methods were demonstrated by examples. Additionally, the advantages, disadvantages, and precautions of various modeling methods were briefly reviewed, and the experience of this research group in exploring modeling methods was shared. This study is expected to provide a reference for the establishment of MNNG-induced PLGC animal model, and a model support for the following studies on PLGC.
Animals ; Gastric Mucosa ; Methylnitronitrosoguanidine/toxicity* ; Precancerous Conditions/chemically induced* ; Rats ; Stomach Neoplasms/drug therapy*

Spores of Aspergillus sp. SU14 were treated repeatedly and sequentially with Co60 gamma-rays, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine. One selected mutant strain, Aspergillus sp. SU14-M15, produced cellulase in a yield 2.2-fold exceeding that of the wild type. Optimal conditions for the production of cellulase by the mutant fungal strain using solid-state fermentation were examined. The medium consisted of wheat-bran supplemented with 1% (w/w) urea or NH4Cl, 1% (w/w) rice starch, 2.5 mM MgCl2, and 0.05% (v/w) Tween 80. Optimal moisture content and initial pH was 50% (v/w) and 3.5, respectively, and optimal aeration area was 3/100 (inoculated wheat bran/container). The medium was inoculated with 25% 48 hr seeding culture and fermented at 35degrees C for 3 days. The resulting cellulase yield was 8.5-fold more than that of the wild type strain grown on the basal wheat bran medium.
Aspergillus ; Cellulase ; Dietary Fiber ; Fermentation ; Hydrogen-Ion Concentration ; Magnesium Chloride ; Methylnitronitrosoguanidine ; Polysorbates ; Seeds ; Spores ; Sprains and Strains ; Starch ; Triticum ; Urea

A fungal strain producing a high level of cellulase was selected from 320 fungal isolates and identified as Aspergillus sp. This strain was further improved for cellulase production by sequential treatments by two repeated rounds of gamma-irradiation of Co60, ultraviolet treatment and four repeated rounds of treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The best mutant strain, Aspergillus sp. XTG-4, was selected after screening and the activities of carboxymethyl cellulase, filter paper cellulase and beta-glucosidase of the cellulase were improved by 2.03-, 3.20-, and 1.80-fold, respectively, when compared to the wild type strain. After being subcultured 19 times, the enzyme production of the mutant Aspergillus sp. XTG-4s was stable.
Aspergillus ; beta-Glucosidase ; Cellulase ; Mass Screening ; Methylnitronitrosoguanidine ; Mutagenesis ; Sprains and Strains

OBJECTIVETo investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inducing human bronchial epithelial (HBE) cells' neoplastic transformation.

METHODSJWA overexpression vector and its stable transfection HBE cells were established. The characteristics of transformed HBE cells were determined by methyl thiazolyl tetrazolium (MTT) assay and the soft agar colony formation assay. The expressions of JWA and P53 were detected by Western blot.

RESULTSThe growth rates of the HBE cells which were treated with MNNG were significantly accelerated than the JWA overexpression HBE cells and controlled HBE cells (P < 0.05). The soft agar colony formation of JWA overexpression HBE cells with and without MNNG treatment (8.06% and 10.14%) was significantly lower than that of the normal HBE cells with MNNG treatment (26.80%) (P < 0.01). After exposure of MNNG, the P53 expressions were gradually increased in HBE cells with the increased passages. However, the expression of P53 in JWA over expressed HBE cells showed a different manner. P53 reached an over expression peak at early stage (the first passage), and then with a gradually down-regulated expression spectrum with increased passages of the cells.

CONCLUSIONJWA might be a key molecule and play an important role in MNNG inducing neoplastic transformation in HBE cells through regulation of the expression of P53.

Cell Transformation, Neoplastic ; drug effects ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Heat-Shock Proteins ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Methylnitronitrosoguanidine ; toxicity ; Tumor Suppressor Protein p53 ; metabolism

Activation of c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is an important cellular response that modulates the outcome of the cells which are exposed to the tumor necrosis factor (TNF) or the genotoxic stress including DNA damaging agents. Although it is known that JNK is activated in response to genotoxic stress, neither the pathways to transduce signals to activate JNK nor the primary sensors of the cells that trigger the stress response have been identified. Here, we report that the receptor interacting protein (RIP), a key adaptor protein of TNF signaling, was required to activate JNK in the cells treated with certain DNA damaging agents such as adriamycin (Adr) and 1-beta-D-arabinofuranosylcytosine (Ara-C) that cause slow and sustained activation, but it was not required when treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and short wavelength UV, which causes quick and transient activation. Our findings revealed that this sustained JNK activation was not mediated by the TNF (tumor necrosis factor) receptor signaling, but it required a functional ATM (ataxia telangiectasia) activity. In addition, JNK inhibitor SP-600125 significantly blocked the Adr-induced cell death, but it did not affect the cell death induced by MNNG. These findings suggest that the sustained activation of JNK mediated by RIP plays an important role in the DNA damage-induced cell death, and that the duration of JNK activation relays a different stress response to determine the cell fate.
Cell Death ; DNA ; DNA Damage ; Doxorubicin ; Humans ; JNK Mitogen-Activated Protein Kinases ; Methylnitronitrosoguanidine ; Necrosis ; Protein Kinases ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human bronchial epithelial (HBE) cell apoptosis.

METHODSThe cell growth inhibition rate was detected by MTT, the cell apoptosis was measured by Hoechst staining, the expression of JWA protein was detected by Western blot, and the potential binding protein of JWA proximal promoter was detected by Southwestern assay.

RESULTSMNNG treatment of HBE cells for 24 hours induced apoptosis with significant dose-effect relationship and in this course the expression of JWA protein was elevated. The 2.0 microg/ml MNNG treated cells for 24 hours activated nuclear transcription factor expression that specifically bound to JWA proximal promoter.

CONCLUSIONThat MNNG treatment activates nuclear transcription factor binding to JWA proximal promoter may be involved in intracellular apoptosis associated signal pathway.

Apoptosis ; Bronchi ; cytology ; drug effects ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Epithelial Cells ; drug effects ; metabolism ; Heat-Shock Proteins ; biosynthesis ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; physiology ; Methylnitronitrosoguanidine ; toxicity ; Signal Transduction ; Transcription Factors ; metabolism

OBJECTIVETo characterize the DNA damage property represented by the distinct whole nucleus stain pattern of gammaH2AX induced by N-methyl-No-nitro-N-nitrosoguanidine (MNNG).

METHODSMNNG-induced gammaH2AX foci formation in human amnion FL cells was observed by immunofluorescent microscopy. DNA double-stranded breaks (DSBs) were detected by neutral comet assay. General DNA damages were detected by alkaline comet assay.

RESULTA distinct whole nucleus stain pattern of gammaH2AX was induced by high concentration MNNG (10 mg/L). 1 mg/L MNNG also induced this type of stain pattern in a small fraction of cells, although the effect was transient. Neutral comet assay did not detect any significant DSBs formation in this type of cells, while alkaline comet assay revealed the presence of DNA damage.

CONCLUSIONAlthough normal gammaH2AX foci were regarded as a biomarker for DSBs, the whole nucleus stain pattern might represent DNA damage other than DSBs.

Amnion ; cytology ; Cell Nucleus ; drug effects ; metabolism ; Comet Assay ; DNA Breaks, Double-Stranded ; drug effects ; DNA Damage ; Histones ; analysis ; Humans ; Methylnitronitrosoguanidine ; pharmacology ; Microscopy, Fluorescence ; Phosphoproteins ; analysis

OBJECTIVETo study the effect drug contained canine serum, prepared by gastric perfusion with Sanchi extract (SE), in inhibiting proliferation and promoting apoptosis of cultured precancerous gastric cells by cell culture.

METHODSThe precancerous model cells (MC) used in the experiment were prepared through transforming eternalized human gastric mucosa epithelial cells GES-1 by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). After once gastric perfusion of SE extract to dogs, the canine serum gotten before and at different time points after medication was used for test. The inhibitory effect of the drug serum obtained at different time points on MC after acting for 72 hrs was detected by 3-(4,5)-dimethy thioazol-2-yl-2,5-diphenyl-tetrazoliumbromide (MTT) method to find the optimal time point for drug serum preparation, that were 2 hrs and 6 hrs after medication. Then the cell apoptosis promoting effect after acting for 72 hrs of the drug serum obtained at the optimal time points was determined by flow cytometry.

RESULTSThe drug serum obtained at 2-hr and 6-hr after medication showed the highest inhibitive effect on MC cells, reaching 45.3% and 42.4% respectively, as compared with the effect of blank serum, the difference was significant (P<0.01). They could evidently promote the MC cell apoptosis, the apoptosis rate also showed significant difference to that of the blank serum (P < 0.05). Under their action, the proportion of MC cells in G0/G1 phase was obviously decreased (P < 0.05) while that in the G2/M phase significantly increased (P <0.05). However, the change of cells in S phase was not uniform.

CONCLUSIONThe drug contained canine serum gotten 2 hr and 6 hr after SE feeding shows the optimal MC proliferation inhibitive effect and significant apoptosis promoting effect. Besides, it could significantly decrease the proportion of MC cells in G0/G1 phase and significantly increase that in G2/M phase, this effect might be one of the mechanisms of ES in inhibiting MC cell proliferation and promoting its apoptosis.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Araliaceae ; Cell Proliferation ; drug effects ; Cell Transformation, Neoplastic ; Cells, Cultured ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Embryo, Mammalian ; Gastric Mucosa ; cytology ; Ginsenosides ; pharmacology ; Humans ; Methylnitronitrosoguanidine ; Precancerous Conditions ; pathology ; Stomach Neoplasms ; pathology

OBJECTIVETo elucidate the potential molecular mechanism responsible for the early time of tumor promotion, gene expression profile was studied in the transformed BALB/c 3T3 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).

METHODSThe two-stage cell transformation model was established by using the initiator of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoter of TPA. Cell proliferation was measured by trypan blue staining and cell cycle analysis was carried out by flow cytometry assay. A cDNA microarray representing 1 152 genes was used to investigate the gene expression profiles of BALB/c 3T3 cells exposed to TPA at 4 h and 24 h respectively.

RESULTSTPA could effectively inhibit cell proliferation and induce the G1 and S cell cycle arrested in the early time. Moreover 19 genes were found differentially expressed at least twofold in the TPA treated cells as compared with the control cells, 9 of them were upregulated and 10 downregulated. Most of the differentially expressed genes were involved in cell proliferation, differentiation or apoptosis, and related to ras or p53 signal transduction pathway.

CONCLUSIONTPA could influence the transcriptional expression of some genes related to cell cycle modulation and ultimately result in the cell growth arrest.

Animals ; Apoptosis ; drug effects ; genetics ; BALB 3T3 Cells ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Transformation, Neoplastic ; drug effects ; genetics ; Flow Cytometry ; Gene Expression ; drug effects ; Gene Expression Profiling ; Methylnitronitrosoguanidine ; pharmacology ; Mice ; Oligonucleotide Array Sequence Analysis ; methods ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology

BACKGROUNDMutations in mitotic checkpoint genes have been detected in several human cancers, which exhibit chromosome instability. We wanted to know whether mutation of hBub1 could occur in transformed human embryo lung fibroblasts (HELF) cells induced by a chemical carcinogen.

METHODSHELF cells were transformed by N-methyl-N'-nitro-N-nitrosoguaridine (MNNG), and three flasks of transformed HELF cells (named as T1, T2, and T3) were selected as amplifiers, and mutations of hBub1 in these transformed cells were analyzed by PCR-SSCP and sequencing.

RESULTSIt was found that any one of three transformed cell lines exhibited aneuploidy with a low mitotic checkpoint function. Subsequent PCR-SSCP and sequence analysis showed an AGT to CGT or ATT mutation at codon 80 in hBub1 gene in T1 cells with a resultant change in amino acid sequence.

CONCLUSIONOur study demonstrated that the mitotic checkpoint genes could be targets of MNNG.

Cell Line, Transformed ; Chromosome Aberrations ; Down-Regulation ; Fibroblasts ; drug effects ; Humans ; Lung ; cytology ; Methylnitronitrosoguanidine ; toxicity ; Mitosis ; drug effects ; Mutation ; Protein Kinases ; genetics ; Protein-Serine-Threonine Kinases