MNNG Treatment Research Articles

MRE11A: a novel negative regulator of human DNA mismatch repair

BackgroundDNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3′–5′ exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear.MethodsInitially, short-term and long-term survival assays were used to measure the cells’ sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used.ResultsWe show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity.ConclusionsOur findings reveal that MRE11A is a negative regulator of human MMR.Graphical

Trypanosoma brucei and Trypanosoma cruzi DNA Mismatch Repair Proteins Act Differently in the Response to DNA Damage Caused by Oxidative Stress.

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites' MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei.

Leptin enhances N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)-induced tumour growth in gastric mucosa of male Sprague-Dawley rats

Individuals who are obese are at a greater risk of developing gastric cancer. They are however also hyperleptinaemic. Chronic leptin treatment has been shown to upregulate numerous cancer-causing genes in the stomach of male Sprague-Dawley rats. It is however unclear if leptin enhances the effect of gastric carcinogens in vivo. This study was therefore done to investigate the effect of leptin on gastric carcinogenesis in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-four, 6-week old male Sprague-Dawley rats were divided equally into three groups: G1 served as age-matched controls; G2 was treated with MNNG in drinking water ad libitum (200mgL-1); G3 was given leptin and MNNG. Rats were euthanized after 40weeks of treatment and their stomachs were removed for histopathology, microarray, and RT-qPCR analysis. Fisher's exact test and one-way ANOVA were used to analyse the data. Fifty percent of the MNNG-treated rats developed gastric hyperplasia (p < 0.05), but there was no significant change in any carcinogenic genes. Concurrent MNNG and leptin treatment however induced hyperplasia, dysplasia, hypertrophy, and adenocarcinoma in 75% (6/8) of the rats; with upregulation of microRNAs, olfactory receptors, Hey2 (transcription factor), Tmed2 (vesicular trafficking), and Lcn11 (cell proliferation) genes. It appears that leptin enhances MNNG- induced tumour growth in stomachs of Sprague-Dawley rats and its role in gastric cancer requires further scrutiny.

ATR–CHK1–E2F3 signaling transactivates human ribonucleotide reductase small subunit M2 for DNA repair induced by the chemical carcinogen MNNG

BackgroundN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent and an environmental carcinogen, causes DNA lesions and even carcinomas. DNA damage responses induced by MNNG activate various DNA repair genes and related signaling pathways. The present study aimed to investigate the regulatory mechanisms of human RR small subunit M2 (hRRM2) in response to MNNG. ResultsIn this study, we demonstrated that the RRM2 gene was transactivated by MNNG exposure more strongly than the other small subunit, p53R2. The upregulated RRM2 translocated to the nucleus for DNA repair. Further study showed that E2F3 transactivated RRM2 expression by directly binding to its promoter after MNNG exposure. The transactivation was enhanced by the upregulation of NFY, which bound to the RRM2 promoter adjacent to the E2F3 binding site and interacted with E2F3. In response to MNNG treatment, E2F3 accumulated mainly through its phosphorylation at S124 and was dependent on ATR–CHK1 signaling. In comparison, p53R2 played a relatively weaker role in the MNNG-induced DNA damage response, and its transcription was regulated by the ATR–CHK2–E2F1/p53 pathway. ConclusionsWe suggest that MNNG-stimulated ATR/CHK1 signaling stabilizes E2F3 by S124 phosphorylation, and then E2F3 together with NFY co-transactivate RRM2 expression for DNA repair. General significanceWe propose a new mechanism for RRM2 regulation to maintain genome stability in response to environmental chemical carcinogens.

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.

Repeated dose liver and gastrointestinal tract micronucleus assays using N-methyl-N′-nitro-N-nitrosoguanidine in young adult rats

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) is a direct-acting mutagen that induces tumors in the glandular stomach, but not in the liver or colon, of rats after oral administration. To evaluate the performance of repeated dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats, MNNG was administered by oral gavage to male CD (SD) rats aged 6 weeks at doses of 0 (vehicle; 2.5% DMSO aqueous solution), 3.125, 6.25, 12.5, and 25mg/kg/day once daily for 14 and 28 days, and the MN frequencies were examined in the hepatocytes, glandular stomach cells, and colonic cells. The MN induction in immature erythrocytes in the bone marrow of these animals was also simultaneously evaluated. The frequencies of micronucleated (MNed) glandular stomach cells were significantly increased in all MNNG treatment groups in a dose-dependent manner in both repeated dose studies. In contrast, the frequencies of MNed hepatocytes and colonic cells were not significantly increased compared to the vehicle control. In the bone marrow, a small but significant increase in the frequency of MNed immature erythrocytes was observed only at the highest dose in the 28-day study. Since a clear positive result in the glandular stomach agrees with the tissue specificity of tumor induction by this chemical, the MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may be useful for detecting genotoxic compounds that are short-lived in vivo, such as MNNG.

PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca2 +-permeable AMPA receptors

An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG, 100μM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD+ and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca2+ permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.

OP0018 mi-RNA-150 and seven regulators of mesenchymal stem cells action on colon cancer inflammatory tumour microenvironment

Background This study is the first step toward use of mesenchymal stem cells (MSCs) in therapy to alleviate the effects of radiation exposure in patients. MSCs improve repair in various organs and tissues, in particular after irradiation. However, before using MSCs in a cancer environment, for instance after radiotherapy, it must be established that MSC injections do not promote malignant cells proliferation. Methods A rat model of colorectal carcinogenesis close to human cancer was used as preclinical model. The carcinogen N-methyl-N ′ -nitro-N-nitroso-guanidine (MNNG) was instilled in rats by four intrarectal deposits within 2 weeks, and two MSC injections (10 7 cells per rat) 2 and 4 weeks after the end of carcinogen treatment. Tumours were then examined 32 and 52 weeks after initiating MNNG treatment. Another cohort of rats entered a survival experiment. Findings MSC treatment reduced cancer significantly, lowering the number of adenomas and adenocarcinomas and extending the lifespan of rats. The anti-cancer effect of MSCs was mediated by their immunological properties. In adenocarcinoma of MSC-treated rats, CD68+ monocyte/macrophage infiltration was lowered and CD3+ lymphocytes increased. MSCs induce macrophages to turn into regulatory cells involved in phagocytosis, inhibiting the production of proinflammatory cytokines. MSCs decrease NK cells and rTh17 cell activities, Treg recruitment, CD8+ lymphocytes, and endothelial cell number and restore Th17 cell activity. MiRNA mi-150 and miRNA-7 are the key effectors. Mi-150 inhibits tumour invasion and mi-RNA-7 negatively regulates the EGFR/AKT pathway, promoting cell death. Interpretation MSC infusion has a durable action on colon cancer development by modulating the immune component of the tumour microenvironment. For the first time, two mi-RNA were identified as responsible for the anti-cancer effect of MSCs. We propose that in this model of colorectal cancer early bone-marrow-derived MSC injections, by a long-term effect, reoriented tumour immune response and induced regression of colonic carcinogenesis.

FOXO1-dependent DNA damage repair is regulated by JNK in lung cancer cells

DNA damage or mutation in cells contributes to tumorigenesis. The transcription factor FOXO1 modulates the expression of genes involved in DNA damage repair, cell cycle arrest and apoptosis. The transcriptional activity of FOXO1 is fundamentally regulated by post-translational modification and subcellular localization. H1299 lung cancer cells were treated with the alkylating agent MNNG, and the cell viability and DNA damage were separately determined by MTT and comet assay. Using immunofluorescence and western blotting, we observed the subcellular localization of FOXO1 and measured the relevant protein expression levels, respectively. To examine cell cycle arrest and apoptosis, flow cytometry analysis was preformed. The interaction between FOXO1 and JNK was analyzed through immunoprecipitation. Our results showed that cell viability was reduced at 24 h after MNNG treatment, and appeared to recover to some degree at 48 h. The increased expression and nuclear export of FOXO1 emerged at 4 h after the treatment. Nuclear FOXO1 played a pivotal role in cell cycle arrest, apoptosis and DNA damage repair by upregulating p27(Kip1), Bim and GADD45 gene expression, respectively. AKT-dependent S256 phosphorylation of FOXO1 and the S473 phosphorylation of AKT were both enhanced following DNA damage. Moreover, our studies revealed that FOXO1 directly interacted with JNK, and the inhibition of the JNK activity led to decreased expression of FOXO1 target genes. These findings suggest for the first time that FOXO1 is a promising candidate substrate for JNK, and the FOXO1-dependent DNA damage repair may be regulated positively by the JNK pathway in H1299 lung cancer cells.

麹菌の育種によるみその品質向上を目指して

The aim of this study was the development of miso with high quality and value by mutation breeding of koji molds with superior characteristics. We selected mutants generated by MNNG treatment of koji molds, and obtained two kinds of applicable strains for miso production. One is a high vitamin B2-producing strain with high neutral protease activity (R2 strain), which is useful for improving the color and taste of miso. The other is a high lipase-producing strain (NT12 strain), which increases miso functionality. We compared each mutant to BF-1, the commercial koji mold used as the control strain, during both laboratory-scale and pilot-scale miso fermentation. The koji manufactured from the R2 mutant showed high protease activity and contained 2∼3 times more vitamin B2 that manufactured from BF-1. In addition, the miso from the R2 strain showed higher x and y color values than that from BF-1, although the Y value (%) was somewhat lower. It also contained high contents of both formol and water-soluble nitrogen, and its color brightness and umami taste were evaluated well. On the other hand, the koji from the NT12 mutant showed lipase activity about 3 times higher than that from BF-1. The resulting miso using the NT12 strain was high in fatty acid ethyl esters, and showed higher inhibitory effects on the growth of tumor cells in vitro compared to BF-1.

Reactive oxygen species mediate Epstein-Barr virus reactivation by N-methyl-N'-nitro-N-nitrosoguanidine.

N-nitroso compounds (NOCs) and Epstein-Barr virus (EBV) reactivation have been suggested to play a role in the development of nasopharyngeal carcinoma (NPC). Although chemicals have been shown to be a risk factor contributing to the carcinogenesis of NPC, the underlying mechanism is not fully understood. We demonstrated recently that N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) enhances the genomic instability and tumorigenicity of NPC cells via induction of EBV reactivation. However, the mechanisms that trigger EBV reactivation from latency remain unclear. Here, we address the role of ROS in induction of EBV reactivation under MNNG treatment. EBV reactivation was induced in over 70% of EBV-positive NA cells and the promoter of Rta (Rp) was activated after MNNG treatment. Inhibitor experiments revealed ATM, p38 MAPK and JNK were activated by ROS and involved in MNNG-induced EBV reactivation. Significantly, ROS scavengers N-acetyl-L-cysteine (NAC), catalase and reduced glutathione inhibited EBV reactivation under MNNG and H2O2 treatment, suggesting ROS mediate EBV reactivation. The p53 was essential for EBV reactivation and the Rp activation by MNNG. Moreover, the p53 was phosphorylated, translocated into nucleus, and bound to Rp following ROS stimulation. The results suggest ROS play an important role in initiation of EBV reactivation by MNNG through a p53-dependent mechanism. Our findings demonstrate novel signaling mechanisms used by NOCs to induce EBV reactivation and provide a novel insight into NOCs link the EBV reactivation in the contribution to the development of NPC. Notably, this study indicates that antioxidants might be effective for inhibiting N-nitroso compound-induced EBV reactivation and therefore could be promising preventive and therapeutic agents for EBV reactivation-associated malignancies.

AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.

A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

A continuous cell line derived from the pupal ovary of Spodoptera exigua was established by treating primary cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Three days after treating cells with 3.0 μg/ml of MNNG, the cells formed a monolayer and were initially subcultured 60 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Spex 12, consisted of a mixture of three types of cells, including spherical, spindle-shaped, and oval cells. The population doubling time of the cell line during its logarithmic growth phase was found to be 71 h. DNA amplification fingerprinting polymerase chain reaction analysis confirmed that the new cell line originated from S. exigua. Susceptibility of IOZCAS-Spex 12 cells to infection by certain nucleopolyhedroviruses was investigated. The results showed that the cell line was highly susceptible to infection by S. exigua nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, slightly susceptible to infection by Spodoptera litura nucleopolyhedrovirus, and not susceptible to infection by Helicoverpa armigera nucleopolyhedroviruses or Hyphantria cunea nucleopolyhedroviruses. The results of this study suggest that MNNG treatment may overcome existing limitations to obtaining continually proliferating cells and may open up the possibilities for immortalizing isolated insect cells.

Abstract 2542: EXO1 is required for SN1 methylator-induced DNA damage response

Abstract SN1 methylating agents are therapeutics that methylate the O6 position of guanine (O6meG) forcing mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes O6meG:T mispairs and activates DNA damage response (DDR). Exonuclease I (EXO1) functions during MMR by excising the damaged strand; however, how MMR mechanistically contributes to DDR activation and if EXO1 is required to activate MMR-dependent response remains unknown. Here we show that Exo1 knockdown in mouse embryonic fibroblasts (MEFs) results in decreased G2/M arrest and checkpoint response (ie, Cdc2/Chk1 phosphorylation), and increased cell proliferation and viability following exposure to the SN1 methylator MNNG. Moreover, ectopic expression of full-length human hEXO1 restored normal response to MNNG. We next examined gamma-H2AX foci formation as a surrogate marker of MMR-dependent DNA repair. 12h after MNNG treatment, 40% of MEFs scored positive for multiple gamma-H2AX foci and this population further increased with prolonged treatment. In contrast, Exo1 depleted MEFs displayed a significantly diminished number of gamma-H2AX foci-positive cells following MNNG. Increased association of Msh2/Msh6 with chromatin was observed in MNNG treated MEFs; however, Exo1 depletion resulted in no upregulation of MMR proteins within the chromatin fraction. Furthermore, MNNG-induced Msh2/Chk1 association was disrupted upon Exo1 depletion. Taken together, we conclude that Exo1 is required to activate DDR following SN1 methylator exposure. Data gathered suggests that Exo1 functions in activating MMR-dependent DNA repair, promoting assembly of MMR complexes with alkylated chromatin, and enhancing the association of MMR components with critical checkpoint mediators. Experiments designed to determine if Exo1 functions in a structural (ie, scaffolding) or catalytic (ie, nucleolytic) manner during SN1 methylator-induced DDR are currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2542. doi:1538-7445.AM2012-2542

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) Triggers MSH2 and Cdt2 Protein-dependent Degradation of the Cell Cycle and Mismatch Repair (MMR) Inhibitor Protein p21Waf1/Cip1

p21(Waf1/Cip1) protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.

BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation

Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.

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 0.25 μM 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.

Oxidative stress initiates DNA damager MNNG-induced poly(ADP-ribose)polymerase-1-dependent parthanatos cell death

The alkylating agent N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG) can cause excess DNA strand breaks that lead to poly(ADP-ribose)polymerase-1 (PARP-1) overactivation and cell death (parthanatos). However, the detail mechanism of MNNG-induced parthanatos was not well-investigated. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the signaling pathways of MNNG-induced parthanatos. We found that MNNG-induced cell death accompanied by rapid PARP-1 activation, c-Jun N-terminal kinase (JNK) activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1 min and peaking at 5–15 min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30 min and time-dependently increasing up to 6 h after MNNG treatment was generated by mitochondria. The antioxidant, NAC can abrogate all phenomena caused by MNNG. Results indicate that the calcium rise was downstream of early ROS production, and was involved in PARP-1 and JNK activation. Moreover, the PARP inhibitor was able to reduce MNNG-induced late-phase ROS production, calcium elevation, and cell death. Results further indicated the involvement of RIP1 in sustained ROS production and calcium increase. We characterized the interactive roles of ROS, calcium, JNK, and RIP1 in MNNG-induced cell death. We found that in addition to the alkylating property previously demonstrated, ROS production triggered by MNNG results in enhanced DNA damage and PARP-1 activation. Moreover, intracellular calcium elevation and ROS production have mutual amplification effects and thus contribute to PARP-1-mediated parthanatos.

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

BackgroundIn this study the effect of myenteric denervation induced by benzalconium chloride (BAC) on distribution of fibrillar components of extracellular matrix (ECM) and inflammatory cells was investigated in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rats were divided in four experimental groups: non-denervated (I) and denervated stomach (II) without MNNG treatment; non-denervated (III) and denervated stomachs (IV) treated with MNNG. For histopathological, histochemical and stereological analysis, sections of gastric fragments were stained with Hematoxylin-Eosin, Picrosirius-Hematoxylin, Gomori reticulin, Weigert's Resorcin-Fuchsin, Toluidine Blue and Alcian-Blue/Safranin (AB-SAF).ResultsBAC denervation causes an increase in the frequency of reticular and elastic fibers in the denervated (group II) compared to the non-denervated stomachs (group I). The treatment of the animals with MNNG induced the development of adenocarcinomas in non-denervated and denervated stomachs (groups III and IV, respectively) with a notable increase in the relative volume of the stroma, the frequency of reticular fibers and the inflammatory infiltrate that was more intense in group IV. An increase in the frequency of elastic fibers was observed in adenocarcinomas of denervated (group IV) compared to the non-denervated stomachs (group III) that showed degradation of these fibers. The development of lesions (groups III and IV) was also associated with an increase in the mast cell population, especially AB and AB-SAF positives, the latter mainly in the denervated group IV.ConclusionsThe results show a strong association in the morphological alteration of the ECM fibrillar components, the increased density of mast cells and the development of tumors induced by MNNG in the non-denervated rat stomach or denervated by BAC. This suggests that the study of extracellular and intracellular components of tumor microenvironment contributes to understanding of tumor biology by action of myenteric denervation.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.