Research progress in establishment of N-methyl-N'-nitro-N-nitroso-guanidine-induced rat model of Precancerous lesion of gastric cancer / 中国中药杂志

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.

Improvement of Fungal Cellulase Production by Mutation and Optimization of Solid State Fermentation

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.

Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N'-nitro-nitrosoguanidine and reflux of duodenal contents in the rat / Carcinogênese do trato gastrointestinal alto induzida pelo N-methyl-N'-nitro-nitrosoguanidina e pelo refluxo duodenogástrico no rato

PURPOSE: To investigate the combined effects of reflux of duodenal contents through the pylorus and treatment with N-methyl-N'-nitro-nitrosoguanidine (MNNG) on the development of lesions in the glandular stomach, at the gastrojejunal anastomosis and in the forestomach of rats. METHODS: Eighty Male Wistar rats were divided into 4 groups: G1: MNNG + Reflux, G2: Reflux, G3: MNNG and G4: Gastrostomy. MNNG was given in the drinking water (100 mg/ml) for 12 weeks and then two groups (G1 and G2) were submitted to a gastrojejunal anastomosis followed by section of the afferent loop and suture of both stumps to allow reflux of duodenal contents through the pylorus. The animals were sacrificed 18 and 36 weeks after surgery. The lesions obtained in the antral mucosa, at the gastrojejunal anastomosis and in the forestomach were analysed histologically. RESULTS: Duodenal reflux induced proliferative lesions at both glandular and squamous mucosa of the stomach. In the antrum, adenomatous hyperplasia (AH) was observed in 20% and 50% of the animals at the 18th and 36th weeks respectively. Aditionally 85% of the animals presented AH at the gastrojejunal anastomosis and 60% developed squamous hyperplasia at the squamous portion of the stomach. MNNG treatment plus duodenal reflux enhanced the development of malignant tumors at both glandular and squamous mucosa, since there were 30% of antral adenocarcinomas and 45% of squamous carcinomas at the 18th week and the frequency of these malignant tumors rose to 50% in the antrum and 65% in the squamous mucosa at the 36th week. CONCLUSION: The reflux of duodenal contents through the pylorus enhanced the development of proliferative lesions, benign and malignant, in the glandular stomach and in the forestomach of rats.

OBJETIVO: Investigar os efeitos do refluxo duodenogástrico e sua interação com o cancerígeno químico N-methil-N'-nitro-nitrosoguanidina (MNNG) no desenvolvimento de lesões no estômago glandular, anastomose gastrojejunal e no estômago escamoso do rato. MÉTODOS: Foram utilizados 80 ratos Wistar divididos em 4 grupos: G1: MNNG + Refluxo, G2: Refluxo, G3: MNNG e G 4: Gastrostomia. O MNNG foi oferecido na água de beber (100mg/ml) por 12 semanas. A seguir foi feita anastomose gastrojejunal na porção glandular do estômago nos grupos G1 e G2, com secção da alça aferente junto ao estômago e sutura de ambos os cotos para permitir o refluxo do conteúdo duodenal para o estômago pelo piloro. Os animais foram sacrificados 18 e 36 semanas após a cirurgia. As lesões identificadas foram submetidas à exame histopatológico. RESULTADOS: O refluxo duodenogástrico levou ao desenvolvimento de lesões proliferativas no estômago glandular e na porção escamosa. No antro, hiperplasia adenomatosa (HA) foi diagnosticada em 20 e 50% dos animais (G2) na 18ª e 36ª semanas, respectivamente. Na anastomose gastrojejunal 85 por cento dos animais (G2) apresentaram HA e 60% apresentaram hiperplasia escamosa no estômago escamoso, na 36ª semana. No grupo MNNG+Refluxo foram identificados na 18ª semana, 30% adenocarcinomas no antro e 45%carcinomas escamosos. A freqüência destas lesões malignas aumentou, respectivamente, para 50% e 65% na 36ª semana. CONCLUSÃO: O refluxo duodenogástrico potencializou o desenvolvimento de lesões proliferativas benignas e malignas no estômago glandular e em sua porção escamosa, no rato.

Fungal Strain Improvement for Cellulase Production Using Repeated and Sequential Mutagenesis

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.

JWA regulates N-methyl-N'-nitro-N-nitrosoguanidine induced malignant transformation in human bronchial epithelial cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inducing human bronchial epithelial (HBE) cells' neoplastic transformation.</p><p><b>METHODS</b>JWA 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>JWA 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.</p>

Long-term Activation of c-Jun N-terminal Kinase through Receptor Interacting Protein is Associated with DNA Damage-induced Cell Death

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.

In vivo interactive effect of garlic oil and vitamin E against stavudine induced genotoxicity in Mus musculus.

Stavudine (Zerit, d4T) is widely used as an anti HIV infection drug. It prevents HIV by altering the genetic material of healthy cells but causes mutations in mitochondrial and nuclear DNA. It also produces clastogenic effects in mice. In the present investigation, comet assay test was applied to evaluate the possible genomic damage caused by stavudine and also the ameliorating effects of garlic oil and vitamin E against its genotoxicity in different organs of mice. Two different doses of garlic oil (low and high dose) and vitamin E were administered to mice separately and in combination for six consecutive days followed by a dose of stavudine. The mice were sacrificed after 24, 48 and 72 h of stavudine administration. Both the antioxidants (vitamin E and garlic oil) separately and in combination reduced the genotoxicity of stavudine. The protective effects of high doses of garlic oil were more pronounced as compared to vitamin E administered group.

Inhibition of N-Methyl N'-nitro-N-nitrosoguanidine (MNNG) induced gastric carcinogenesis by Phyllanthus amarus extract.

Chemopreventive activity of Phyllanthus amarus Schum & Thonn (Euphorbiaceae) extract was studied with regard to N-methyl N'-nitro-N-nitrosoguanidine (MNNG) induced stomach cancer in Wistar rats. Administration of the extract with MNNG significantly reduced the incidence of gastric neoplasms in rats (44%) as well as their numbers. Moreover, elevated levels of enzymes in the stomach were found to be reduced by P. amarus administration. For example, gamma-glutamyl transpeptidase activity was decreased from 20.3 +/- 6.7 mmol/min/mg protein to almost normal levels (2.8 +/- 0.9) by 750 mg/kg body weight of the extract. Similarly glutathione S-transferase activity (1317.6 +/- 211 n mol/min/mg protein) and glutathione reductase (368 +/- 66) levels in the MNNG treated group were found to be lowered to 494.8 +/- 76 and 192 +/- 45, respectively, while reduced glutathione (GSH) was increased from 4.6+/- 0.9 to 8.5+/-1.4 n mol/min/mg protein. AgNOR dots and clusters, indicators of cellular proliferation, which were increased by MNNG treatment, became near to normal in P. amarus treated animals.

The role of JWA in N-methyl-N'-nitro-N-nitrosoguanidine induced human bronchial epithelial cell apoptosis / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human bronchial epithelial (HBE) cell apoptosis.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>MNNG 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.</p><p><b>CONCLUSION</b>That MNNG treatment activates nuclear transcription factor binding to JWA proximal promoter may be involved in intracellular apoptosis associated signal pathway.</p>

A distinct whole nucleus stain pattern of gamma H2AX induced by N-methyl-No-nitro-N-nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To characterize the DNA damage property represented by the distinct whole nucleus stain pattern of gammaH2AX induced by N-methyl-No-nitro-N-nitrosoguanidine (MNNG).</p><p><b>METHODS</b>MNNG-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.</p><p><b>RESULT</b>A 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.</p><p><b>CONCLUSION</b>Although normal gammaH2AX foci were regarded as a biomarker for DSBs, the whole nucleus stain pattern might represent DNA damage other than DSBs.</p>

Biochemical characterization of N-methyl N'-nitro-N-nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger.

Two cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger, among the six isolated by mutagenization with N-methyl N'-nitro-N-nitrosoguanidine (MNNG) at pH 6.4 were selected for the study. Analysis of lipid composition of the mutants and the wildtype indicated that total lipid as well as individual lipids of the cadmium resistant mutants were changed as compared with that of the wildtype. The increased activities of metal-lothionein and reduced activities of D-xylose isomerase and L-phenylalanine ammonia lyase in cell free extract of the cadmium resistant mutants suggested that mutants could allow high concentration of cadmium salt as compared with that of the wildtype. The respiratory activity and intracellular as well as extracellular Cd2+ concentration of the mutants reflected the high tolerance of the Cd mutants to cadmium ion.

Gene expression profiles in early stage of BALB/c 3T3 cells' transformation promoted with 12-O-tetradecanoylphorbol-13-acetate / 中华预防医学杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>TPA 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.</p><p><b>CONCLUSION</b>TPA could influence the transcriptional expression of some genes related to cell cycle modulation and ultimately result in the cell growth arrest.</p>

Cell proliferation inhibitive and apoptosis promoting effects of sanchi extract on GES-1 cell after being transformed by MNNG / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Down-regulation of mitotic checkpoint in transformed human embryo lung fibroblasts induced by N-methyl-N'-nitro-N-nitrosoguaridine / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Mutations 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.</p><p><b>METHODS</b>HELF 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.</p><p><b>RESULTS</b>It 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.</p><p><b>CONCLUSION</b>Our study demonstrated that the mitotic checkpoint genes could be targets of MNNG.</p>

Ethanolic neem leaf extract protects against N-methyl -N'-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in Wistar rats.

We evaluated the effects of ethanolic neem leaf extract on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats. The extent of lipid peroxidation and the status of the antioxidants superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in the stomach, liver and erythrocytes were used as biomarkers of chemoprevention. Animals were divided into four groups of six animals each. Rats in group 1 were given MNNG (150 mg/kg bw) by intragastric intubation three times with a gap of 2 weeks in between the treatments. Rats in group 2 administered MNNG as in group 1, in addition received intragastric intubation of ethanolic neem leaf extract (200 mg/kg bw) three times per week starting on the day following the first exposure to MNNG and continued until the end of the experimental period. Group 3 animals were given ethanolic neem leaf extract alone, while group 4 served as controls. All the animals were killed after an experimental period of 26 weeks. Diminished lipid peroxidation in the stomach tumour tissue was associated with enhanced antioxidant levels. In contrast to tumour tissue, enhanced lipid peroxidation with compromised antioxidant defences was found in the liver and erythrocytes of tumour bearing animals. Administration of ethanolic neem leaf extract significantly reduced the incidence of stomach tumours, modulated lipid peroxidation and enhanced antioxidant status in the stomach, liver and blood. From the results of our study, we suggest that ethanolic neem leaf extract may exert its chemopreventive effects by modulating lipid peroxidation and enhancing the antioxidant status in the stomach, liver and erythrocytes.

Proteomic analysis of cellular responses to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine in human amnion FL cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the protein profile after treatment of low concentration of N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human FL cells.</p><p><b>METHODS</b>After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images were analyzed with 2D analysis software. The differentially expressed protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULT</b>More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). There were 18 protein spots detected only after MNNG treatment, while 13 protein spots were detected only in the control cells. Moreover, the levels of another 31 proteins were either increased or decreased in MNNG-treated FL cells. And some of the proteins were identified by MALDI-TOF-MS.</p><p><b>CONCLUSION</b>There are significant alterations of protein profile after MNNG attack.</p>

Activation of nucleus-independent signals triggered by N-methyl-N'-nitro-N- nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.</p><p><b>METHODS</b>Vero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.</p><p><b>RESULT</b>In enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.</p><p><b>CONCLUSION</b>The results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.</p>

Activation of transcription factors induced by low concentration of N-methyl-N'-nitro-N-nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of MNNG on some of the transcription factors such as NF- kappaB, CREB, AP-1 and c-Myc.</p><p><b>METHODS</b>The activities of these transcription factors were measured by transient transfection assay of SEAP vectors.</p><p><b>RESULT</b>The expressions of AP-1, CREB and NF- kappaB driven reporter genes were elevated for about 1.3, 1.4 and 1.3 times in MNNG-treated cells, respectively, as compared to untreated controls. However, the exposure of MNNG had no effect on the activity of c-Myc.</p><p><b>CONCLUSION</b>The activation of certain transcription factors might be involved in the process of untargeted mutation induced by low concentration of MNNG treatment.</p>

Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).</p><p><b>METHODS</b>Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system.</p><p><b>RESULT</b>Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB. Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01).</p><p><b>CONCLUSION</b>Human mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.</p>

Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).</p><p><b>METHODS</b>A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.</p><p><b>RESULT</b>The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.</p><p><b>CONCLUSION</b>POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.</p>