N-ethyl-N-nitrosourea Research Articles

Stress Response Gene Egr-1 As Tumor Suppressor in BCR-ABL Mediated Leukemia

Abstract 33Chronic Myelogenous Leukemia (CML) is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. In the late 1990s a Tyrosine Kinase Inhibitor (TKI), imatinib mesylate (Gleevec, Novartis) started to be effectively used on CML patients. However, imatinib, therapy suppresses rather than eliminates the disease, and resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. The transcription factor early growth response 1 (Egr-1) was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There's evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). Therefore, we chose to investigate if Egr-1 can act as a suppressor of BCR-ABL driven leukemia. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, we investigated the stem cell compartment of both Egr-1 WT and Egr-1−/− BM, by determining the percentage of stem cells (Lin−Sca+c-Kit+, LSK), before infection with BCR-ABL; no statistically significant difference in the percentage of LSK cells was observed between Egr-1 WT and Egr-1−/− BM. Thus, the BM stem cell compartment of the Egr-1−/− mice does not offer a quantitiative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. Furthermore, when BM of transplanted mice was analyzed we observed an increased population of lineage negative cells in Egr-1−/− BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1−/− BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Additional data from our animal model, as well as analysis of human leukemia samples will be presented, further corroborating that Egr-1 functions as a suppressor of BCR-ABL driven CML. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics for CML, as well as for other leukemic diseases. Disclosures:No relevant conflicts of interest to declare.

A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis

AbstractAbstract 608Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia. Disclosures:No relevant conflicts of interest to declare.

Functional annotation and ENU

Functional annotation of every gene in the mouse genome is a herculean task that requires a multifaceted approach. Many large-scale initiatives are contributing to this undertaking. The International Knockout Mouse Consortium (IKMC) plans to mutate every protein-coding gene, using a combination of gene trapping and gene targeting in embryonic stem cells. Many other groups are performing using the chemical mutagen ethylnitrosourea (ENU) or transpon-based systems to induce mutations, screening offspring for phenovariants and identifying the causative mutations. A recent paper in BMC Research Notes by Arnold et al. presents data from an ENU-based mutagenesis project that provides not only some of the first phenotype-genotype information for a large number of genes, but also a trove of information, all publicly available, that demonstrates the specificity and efficiency of ENU mutagenesis.

Repair of diverse diabetic defects of β‐cells in man and mouse by pharmacological glucokinase activation

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of β-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of β-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse β-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons.

Insensitivity of the in vitro cytokinesis-block micronucleus assay with human lymphocytes for the detection of DNA damage present at the start of the cell culture

The cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes is a well-established assay in genotoxicity testing and human biomonitoring. For both approaches, human lymphocytes are stimulated by phytohaemagglutinin (PHA) and cultured for about 72 h; 44 h after PHA stimulation, cytochalasin B (CytB) is added and micronuclei (MN) are scored in binucleated cells. The main difference between these two applications is the way lymphocytes are exposed to mutagens. In order to maximise the probability of detecting a mutagen, the OECD guideline 487 recommends starting the exposure to the test substance at 44-48 h after PHA stimulation. In human biomonitoring, blood samples are taken from subjects exposed to environmental mutagens in vivo and lymphocytes with induced DNA damage at the start of the cell culture are investigated with regard to potentially increased MN frequencies in binuclear lymphocytes. We compared the sensitivity of these two protocols by either treating lymphocyte cultures for 2h with known DNA-damaging mutagens at the start of the culture or 42 h after PHA stimulation. The mutagens used were methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-ethylurea (ethyl nitrosourea; ENU), styrene oxide (SO), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and mitomycin C (MMC). All substances induced MN under the conditions of the standard in vitro CBMN assay but only MMC clearly induced MN in lymphocytes exposed at the start of the culture. All mutagens (except MMC, a known crosslinker) were tested by the comet assay with blood cultures exposed at the start of the culture and clearly induced DNA migration. The nuclear division index (NDI) indicated that damaged lymphocytes proliferated well in these experiments. The lack of increased MN frequencies despite increased damage levels and good proliferation suggests that the CBMN assay is rather insensitive for the detection of mutagens/clastogens when damage is induced at the start of the blood cultures. Potential consequences for the interpretation of human biomonitoring studies are discussed in this article.

Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0mg/kg), control (2mg/kg) and supplemented (6mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

Topoisomerase IIβ regulates base excision repair capacity of neurons

Topoisomerase IIβ (TopoIIβ), an enzyme involved in DNA rearrangements, is predominantly present in brain and its levels are shown to decrease with age. This study characterizes the function of TopoIIβ in regulating BER (base excision repair) activity. TopoIIβ deficient granule neurons (CGNT̄) show greater sensitivity to N-ethyl N-nitroso urea (ENU)-mediated DNA damage. The cell-free extracts of TopoIIβ knockdown cells (ECGNT̄) show a significant decrease in G-U BER activity during ENU-treatment as well as during recovery, suggesting that TopoIIβ promotes G-U BER activity. Since G-U BER activity is not affected in the presence of ICRF-193, catalytic inhibitor of TopoIIβ, the activity of enzyme per se may not be participating in BER activity. Further characterization of the activities of BER enzymes present in ECGNT̄ shows that uracil DNA-glycosylase (UDG) and ligase (LIG) activities decrease significantly in both ENU treatment and recovery. Supplementation of TopoIIβ to ECGNT̄ does not restore ligation activity and ICRF-193 does not influence the LIG activity. These results suggest a role, at least an indirect one, of TopoIIβ in the repair of ENU-mediated strand breaks via BER pathway including the activities of UDG and LIG.

Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis

Natural killer (NK) cells are bone marrow (BM)-derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases.

Folate supplementation limits the tumourigenesis in rodent models of gliomagenesis

A hallmark of cancer is the paradoxical co-presence, in the same tumour, of local and global DNA hypomethylation together with the regional hypermethylation of certain genes. Due to the oncogenic role of these different DNA methylation alterations, two therapeutic strategies are possible: the use of DNA methylating agents (DMA, such as folate) to inhibit global or local DNA hypomethylation or the use of DNA hypomethylating agents (DHA, such as 5-aza-2-deoxycytidine) to abrogate the accumulation of hypermethylated genes.Here we explored the use of folate to treat gliomas in a mouse model, using tumours induced by either PDGF-B or Ras/Akt overexpression, or by ethylnitrosourea (ENU) treatment. Under all conditions the volume of tumours were significantly less in folate treated mice than in untreated mice. Quantitative methylated DNA immunoprecipitation (qMeDIP) and quantitative methylated specific PCR (qMSP) analysis of methylation status showed that folate treatment, increased the methylation level of DNA repeat elements in tumour and in colorectal tissue and that of MGMT and specific oncogenes (PDGF-B or survivin) in tumours (but not in colorectal tissue), but had no effect on the expression of tumour suppressor genes (p53, PTENorbax) in tumours or in colorectal tissue. This suggests that folate has anti-neoplastic effects in gliomas and that no preneoplastic or neoplastic alterations were observed in unaffected colorectal tissue in response to the potential tumourigenic effects of folate.Collectively, our data support the proposal to include folate as a promising adjuvant in the design of anti-glioma therapeutic protocols in clinical studies.

Gsdma3 Mutation Causes Bulge Stem Cell Depletion and Alopecia Mediated by Skin Inflammation

Primary cicatricial alopecias (PCAs) are a group of permanent hair loss disorders, of which the pathogenesis is still poorly understood. The alopecia and excoriation (AE) mouse strain is a dominant mutant generated from ethyl nitrosourea mutagenesis. AE mice exhibit a progressive alopecia phenotype similar to that seen in PCAs, resulting from a point mutation in the gasdermin A3 gene. Mutant mice begin to show alopecia on the head from postnatal day 22 and experience complete hair loss by the age of 6 months, along with hyperkeratosis and catagen delay. The results of a histological examination showed that bulge stem cells in AE skin are gradually depleted, as indicated by decreased keratin 15 and CD34 expression, and reduced bromodeoxyuridine label-retaining cells in the AE bulge. In addition, AE mice display an inflammatory condition in the skin from postnatal day 7, including elevated tumor necrosis factor-α and monocyte chemotactic protein-1 mRNA levels and significantly increased macrophages and dendritic cell number. Immune privilege in the bulge was also compromised in AE skin. Consistently, after treatment with the immunosuppressive agent, cyclosporine A, immune privilege collapse, stem cell destruction, and alopecia phenotype of AE mice were all rescued. Collectively, our data demonstrate that immune-mediated destruction of bulge stem cells plays a crucial role in the pathogenesis of alopecia in AE mice, and this strain might be an interesting model for PCAs, especially for lichen planopilaris.

Abstract 17947: Large Scale Mouse Mutagenesis Screen Using Fetal Echocardiography Indicates Genetic Contribution to Hypoplastic Left Heart Syndrome and Wide Spectrum of Other Congenital Heart Defects

Introduction: Large scale forward genetic screens with ethyl-nitroso-urea (ENU) mutagenesis can provide a non-gene biased approach to elucidate the genetic etiology of congenital heart disease (CHD). We are using noninvasive fetal echocardiography to elucidate the genetic etiology of CHD with scanning of 100,000 mouse fetuses to achieve 5 fold genome coverage. Methods: C57BL/6J mice (G0) are ENU mutagenized and the resulting G1 males and their G2 daughters are backcrossed to generate G3 fetuses for ultrasound scanning. Primary screening used the clinical Acuson Sequoia ultrasound system with a 15 MHz transducer. Fetuses with CHD were further scanned at 40 MHz with the Vevo 2100 ultra-high frequency biomicroscope (UBM). CHD diagnoses were susbsequently confirmed by necropsy, microCT, and/or histopathology. Results: Ultrasound scanning of >20,000 G3 fetuses from >600 G1 pedigrees revealed 4% of fetuses with cardiac defects. A wide spectrum of CHD was observed, including DORV, TGA, PTA, pulmonary/aortic atresia, coarctation, arch anomalies, atrioventricular septal defects (AVSD) and other defects. The most common CHD found was ventricular septal defect. In contrast, hypoplastic left heart syndrome was found in only one mutant line (Figure). We also recovered 10 lines with laterality defects. Heterotaxy mutants exhibited complex CHD, but no heart disease was seen with situs inversus totalis. Overall, the UBM had higher detection sensitivity for cardiac situs (P<0.01), AVSD (P<0.01) and outflow tract malalignment (P<0.01), with 98.3% of the cardiac situs, 91% of the AVSD and 70% of the outflow defects identified by UBM. Conclusions: Our studies showed the integration of conventional ultrasound with the UBM for fetal mouse cardiovascular phenotyping can maximize the recovery of CHD mutants. The wide spectrum of CHD observed in our large scale screen indicates the importance of genetic contribution in all varieties of structural heart disease.

Harnessing mutant donor plants for microspore culture in Indian mustard [Brassica juncea (L.) Czern and Coss

Haploid mutagenesis offers several advantages over conventional (seed) approach. However, its potential has not been utilised for Brassica juncea, an important oilseed. In this study, mutant donor plants of three Indian B. juncea genotypes, generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU), were used for microspore culture. The response of mutant donor plants was about 100 times lower than non-mutant controls; a total of 9,411 embryos were produced from the EMS treated donor plants, while microspores isolated from ENU treated donors did not yield any embryos. The lethality of induced mutations demonstrated itself mainly as the induction of abnormal embryos (80%), failure of germination (70%) and failure of plantlet development (70%). Nine doubled haploid (DH) mutant lines and three non-mutant DH lines obtained through this approach were tested for agronomic and biochemical variation over two growing seasons. High variability was observed and stable mutants were recovered for reduced height (125 vs. 168 cm for the control), appressed pod character, altered fatty acid composition higher protein proportion in de-oiled meal (48%) and a lower glucosinolate content in de-oiled meal (59.5 μM/g) relative to controls. The approach demonstrates that despite severe reduction in efficiency of the DH line production, valuable mutants can be recovered from mutated donor plants.

Defining EMS and ENU dose–response relationships using the Pig-a mutation assay in rats

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose–response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose–response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose–response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose–response relationships, further confirming the possibility of a threshold dose–response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose–response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose–response relationship.

High Throughput Congenital Heart Disease Phenotyping in Mice with Noninvasive Fetal Echocardiography

Fetal ultrasound imaging is extensively used clinically for congenital heart disease (CHD) diagnosis. We previously showed fetal echocardiography using the Acuson Sequoia clinical ultrasound system with a 15 MHz transducer was highly effective in recovering mutants with CHD in a mouse ethylnitrosourea (ENU) mutagenesis screen. Building on this study, we initiated a new mouse ENU screen for mutations causing CHD, with the goal of scanning 100,000 mouse fetuses for 5X genome coverage. ENU mutagenized mice are bred in a G2XG1 backcross breeding scheme to generate G3 fetuses for ultrasound scanning at E14.5–15.5. Litters with Acuson findings are secondarily scanned with the higher resolution Vevo2100 ultrasound system using a 30 MHz transducer. To confirm the CHD diagnosis, fetuses/newborn pups are further analyzed using a combination of microCT, necropsy and/or histopathology with episcopic fluorescence image capture. Thus far, ultrasound scanning showed 4% of the G3 fetuses (n=3099) from 60% of the G1 pedigrees (n=89) exhibit cardiac defects, a similar yield to our previous screen. The most common cardiac defects include atrial/ventricular septal defects, outflow tract alignment defects, and hypertrophy. This new forward genetic screen promises to be highly productive in yielding new insights into the genetic etiology of CHD. Funded by U01HL098180

Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals.Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow® flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays.A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays.All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint.The data generated from these investigations demonstrate the suitability of the multi-endpoint design.

The anti-tumor effects of calorie restriction are correlated with reduced oxidative stress in ENU-induced gliomas

The anti-tumor effects of calorie restriction (CR) and the possible underlying mechanisms were investigated using ethylnitrosourea (ENU)-induced glioma in rats. ENU was given transplacentally at gestational day 15, and male offspring were used in this experiment. The brain from 4-, 6-, and 8-month-old rats fed either ad libitum (AL) or calorie-restricted diets (40% restriction of total calories compared to AL rats) was studied. Tumor burden was assessed by comparing the number and size of gliomas present in sections of the brain. Immunohistochemical analysis was used to document lipid peroxidation [4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA)], protein oxidation (nitrotyrosine), glycation and AGE formation [methylglyoxal (MG) and carboxymethyllysine (CML)], cell proliferation activity [proliferating cell nuclear antigen (PCNA)], cell death [single-stranded DNA (ssDNA)], presence of thioredoxin 1 (Trx1), and presence of heme oxygenase-1 (HO-1) associated with the development of gliomas. The results showed that the number of gliomas did not change with age in the AL groups; however, the average size of the gliomas was significantly larger in the 8-month-old group compared to that of the younger groups. Immunopositivity was observed mainly in tumor cells and reactive astrocytes in all histological types of ENU-induced glioma. Immunopositive areas for HNE, MDA, nitrotyrosine, MG, CML, HO-1, and Trx1 increased with the growth of gliomas. The CR group showed both reduced number and size of gliomas, and tumors exhibited less accumulation of oxidative damage, decreased formation of glycated end products, and a decreased presence of HO-1 and Trx1 compared to the AL group. Furthermore, gliomas of the CR group showed less PCNA positive and more ssDNA positive cells, which are correlated to the retarded growth of tumors. Interestingly, we also discovered that the anti-tumor effects of CR were associated with decreased hypoxia-inducible factor-1α (HIF-1α) levels in normal brain tissue. Our results are very exciting because they not only demonstrate the anti-tumor effects of CR in gliomas, but also indicate the possible underlying mechanisms, i.e. anti-tumor effects of CR observed in this investigation are associated with reduced accumulation of oxidative damage, decreased formation of glycated end products, decreased presence of HO-1 and Trx1, reduced cell proliferation and increased apoptosis, and decreased levels of HIF-1α.

Leukemic stromal hematopoietic microenvironment negatively regulates the normal hematopoiesis in mouse model of leukemia

Leukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term. The mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry. Long-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells. Stromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.

Phenotypic Alteration of Bone Marrow HSC and Microenvironmental Association in Experimentally Induced Leukemia

Leukemia is a heterogeneous disorder of bone marrow (BM) failure syndrome where normal hematopoiesis gets altered due to transformation of either the normal hematopoietic cell or the hematopoietic microenvironment or both. Scientists have tried for decades to understand leukemia development in the context of therapeutic strategies. The existence of "leukemic stem cells" and their possible role in leukemogenesis have only recently been identified and it has changed the perspective with regard to new approaches for treating the disease. However the relationship between leukemic stem cells (LSCs) and leukemogenesis requires further investigation. In this present study, we have experimentally induced leukemia in mice by means of N-N' Ethylnitrosourea (ENU) to investigate the alterations in normal bone marrow cellular phenotype and associated changes in the stromal hematopoietic microenvironment under the event of leukemic disease progression. We have identified a significant decrease in the normal HSC phenotype in terms of Sca1 and c-kit receptor expression and subsequent sharp increase in certain leukemic cell specific receptor expression like CD123, CXCR4 and CD44 in the leukemic bone marrow. The decreased HSC receptor (Sca1 and c-kit) expression profile with concurrent increase in the expression of leukemic cell specific receptors (CD123, CXCR4, CD44) by the bone marrow cells of leukemic mice may account for the possible transformation of the normal hematopoietic cells that is necessary for the disease initiation and progression. Some of these receptors like CXCR4 and CD44 are also known to play an important role in maintaining leukemic cells and their complex crosstalk with the surrounding stromal microenvironment. Thus up-regulation in CXCR4 and CD44 receptor expression essentially pointed towards the stroma dependent surveillance of the leukemic bone marrow cells in leukemia. Leukemic bone marrow cells documented a rapid generation of stromal feeder layer in culture. The rapid stroma generation further supported the fact that leukemic stromal microenvironment gets altered in possible ways to support leukemic cell generation and fueling leukemogenesis. The study presented here, has tried to hint at exploring new therapeutic strategies by not only identifying the expression profile of cell surface receptors unique to cells involved in leukemic progression but also targeting the specific components of the stromal microenvironment that would facilitate therapeutic management of the disease.

MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR→MyD88→AREG/EREG→EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.

Screening of Ethylnitrosourea Mice With Fatty Acid Oxidation Disorders by a Candidate Gene Approach After Proteome Analysis

Background/Purpose Ethylnitrosourea (ENU) is an alkylating agent and primarily induces point mutations such as AT to TA transversions and AT to GC transitions. Due to its high mutagenicity, ENU mouse mutagenesis enables the generation and identification of mouse mutants with aberrance in various phenotypes and to identify novel genes relevant for the expression of the phenotype. The purpose of this study was to investigate the candidate genes involved in fatty acid oxidation disorders by the proteomic approach. Methods We screened ENU mice from 39 families from previously published data and identified two mutant mice that had a striking elevation in blood C4-OH short chain fatty acids compared with ENU controls. Total mitochondrial proteins were extracted from the gastrocnemius for two-dimensional electrophoresis, and two downregulated proteins, adenylate kinase isoenzyme 1 (AK1) and adenosine-5′-triphosphate (ATP) synthase D chain (ATP5H), were identified in the mutant mice through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results After genomic polymerase chain reaction and direct sequencing of Ak1 and Atp5h , no variation was found in both gene sequence analyses. Conclusion Proteomic profiling can be a useful approach for detecting dynamic protein expression in ENU-induced mice. It is important to further clarify mechanisms of the mutant C4-OH disorder responsible for this expression.