A Cross-Linking Approach to Stabilizing Stimuli-Responsive Colloidal Crystals Engineered with DNA.

Two DNA-cross-linking reagents, bis-chloroethylnitrosourea and 8-methoxypsoralen, are used to covalently cross-link interstrand base pairs in DNA bonds that, in part, define colloidal crystals engineered with DNA. The irreversible linkages formed increase the chemical and thermal stability of the crystals and do not significantly affect their long-range order, as evidenced by small-angle X-ray scattering data. The post-modified crystals are stable in environments that the pre-modified structures are not, including solvents that encompass a broad range of polarities from ethanol to hexanes, and in aqueous media at pH 0 and 14. Interestingly, the cross-linked DNA bonds within these crystals still retain their flexibility, which is reflected by a solvent-dependent reversible change in lattice parameter. Since these organic cross-linking reagents, in comparison with inorganic approaches (use of silver ions or SiO2), have marginal effects on the composition and properties of the crystals, they provide an attractive alternative for stabilizing colloidal crystals engineered with DNA and make them potentially useful in a broader range of media.

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice.

ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest.

Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry.

Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.

In vitro and in vivo evaluation of 2-chloroethylnitrosourea derivatives as antitumor agents.

AIM: To evaluate potential of Naphthal-NU, Napro-NU and 5-Nitro-naphthal-NU, 2-chloroethylnitrosourea compounds with substituted naphthalimide in the pre-clinical studies. MATERIALS AND METHODS: In vitro cytotoxicity of three nitrosoureas was determined in human and mouse tumor cell lines by MTT assays. In vivo anti-tumor potential was evaluated in Sarcoma-180 (S-180) and Ehrlich's carcinoma (EC) solid tumors. Apoptosis in S-180 cells was analyzed by using Annexin V-Propidium Iodide (PI). Histological analysis of liver and kidney was performed at optimum dose (50 mg/kg). Expression status of CD4(+), CD8(+) and CD25(+) cells in treated mouse were also examined. RESULTS: Significant tumor growth retardation by the compounds was noted in early and advanced disease groups, as the life span of drug treated mice increased considerably. Drug induced killing was observed by induction of apoptosis. Naphthal-NU and 5-Nitro-naphthal-NU were effective to normalize the tumor induced structural abnormalities of liver and kidney. The compounds have no immunotoxic effect on CD4(+) and CD8(+) T cells and down regulate CD4(+)CD25(+) regulatory T cells. CONCLUSION: Overall data holds promise for the antitumor activity with lower toxicity of the compounds that can be utilized for the treatment of human malignant tumors.

Caspase-9 is required for normal hematopoietic development and protection from alkylator-induced DNA damage in mice.

Apoptosis and the DNA damage responses have been implicated in hematopoietic development and differentiation, as well as in the pathogenesis of myelodysplastic syndromes (MDS) and leukemia. However, the importance of late-stage mediators of apoptosis in hematopoiesis and leukemogenesis has not been elucidated. Here, we examine the role of caspase-9 (Casp9), the initiator caspase of the intrinsic apoptotic cascade, in murine fetal and adult hematopoiesis. Casp9 deficiency resulted in decreased erythroid and B-cell progenitor abundance and impaired function of hematopoietic stem cells after transplantation. Mouse bone marrow chimeras lacking Casp9 or its cofactor Apaf1 developed low white blood cell counts, decreased B-cell numbers, anemia, and reduced survival. Defects in apoptosis have also been previously implicated in susceptibility to therapy-related leukemia, a disease caused by exposure to DNA-damaging chemotherapy. We found that the burden of DNA damage was increased in Casp9-deficient cells after exposure to the alkylator, N-ethyl-nitrosourea (ENU). Furthermore, exome sequencing revealed that oligoclonal hematopoiesis emerged in Casp9-deficient bone marrow chimeras after alkylator exposure. Taken together, these findings suggest that defects in apoptosis could be a key step in the pathogenesis of alkylator-associated secondary malignancies.

Defective craniofacial development and brain function in a mouse model for depletion of intracellular inositol synthesis.

myo-Inositol is an essential biomolecule that is synthesized by myo-inositol monophosphatase (IMPase) from inositol monophosphate species. The enzymatic activity of IMPase is inhibited by lithium, a drug used for the treatment of mood swings seen in bipolar disorder. Therefore, myo-inositol is thought to have an important role in the mechanism of bipolar disorder, although the details remain elusive. We screened an ethyl nitrosourea mutant mouse library for IMPase gene (Impa) mutations and identified an Impa1 T95K missense mutation. The mutant protein possessed undetectable enzymatic activity. Homozygotes died perinatally, and E18.5 embryos exhibited striking developmental defects, including hypoplasia of the mandible and asymmetric fusion of ribs to the sternum. Perinatal lethality and morphological defects in homozygotes were rescued by dietary myo-inositol. Rescued homozygotes raised on normal drinking water after weaning exhibited a hyper-locomotive trait and prolonged circadian periods, as reported in rodents treated with lithium. Our mice should be advantageous, compared with those generated by the conventional gene knock-out strategy, because they carry minimal genomic damage, e.g. a point mutation. In conclusion, our results reveal critical roles for intracellular myo-inositol synthesis in craniofacial development and the maintenance of proper brain function. Furthermore, this mouse model for cellular inositol depletion could be beneficial for understanding the molecular mechanisms underlying the clinical effect of lithium and myo-inositol-mediated skeletal development.

An N-ethyl-N-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess.

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, may be due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh(-120/+) mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh(-120/+) mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh(-120/+) mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh(-120/+) mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Protective effect of spin-labeled 1-ethyl-1-nitrosourea against oxidative stress in liver induced by antitumor drugs and radiation.

This study was carried out to investigate possible protection effect of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (SLENU), synthesized in our laboratory, against oxidative liver injuries induced in mice treated by antitumor drugs: doxorubicin (DOX), bleomycin (BLM), or gamma irradiation (R). Specifically, alterations in some biomarkers of oxidative stress, such as lipid peroxidation products measured as malondialdehyde (MDA) levels and activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied in liver homogenates isolated from tumor bearing C57 black mice after i.p. treatment with solutions of DOX (60 mg/kg), BLM (60 mg/kg), or after total body gamma-irradiation with a single dose of 5 Gy. The same biomarkers were also measured after i.p. pretreatment of mice with SLENU (100 mg/kg). Statistical significant increased MDA levels and SOD and CAT enzymes activities were found in the liver homogenates of tumor bearing mice after alone treatment with DOX or gamma-irradiation compared to the control mice, while these parameters were insignificantly increased after BLM administration compared to the same controls.

ENU-induced mutation in the DNA-binding domain of KLF3 reveals important roles for KLF3 in cardiovascular development and function in mice.

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3(H275R) ). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3(H275R) mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3(H275R) mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon.

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.

An accurate nonlinear QSAR model for the antitumor activities of chloroethylnitrosoureas using neural networks.

The quantitative structure-activity relationship (QSAR) studies are investigated in a series of chloroethylnitrosoureas (CENUs) acting as alkylating agents of tumors by neural networks (NNs). The QSAR model is described inaccurately by the traditional multiple linear regression (MLR) model for the substitution of CENUs at N-3, whose characteristics play key roles in the biological activity. A nonlinear QSAR study is undertaken by a three-layered NN model, using molecular descriptors that are known to be responsible for the antitumor activity to optimize the input variables of the MLR model. The results demonstrate that NN models present the relationship between antitumor activity and molecular descriptors clearly, and they yield predictions in excellent agreement with the experiment's obtained values (R(2)=0.983). The R(2) value is 0.983 for the 5-8-1 NN model, compared with 0.506 for the MLR model, and the nonlinear model is able to account for 98.3% of the variance of antitumor activities.

Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

Codon 12 region of mouse K-ras gene is the site for in vitro binding of transcription factors GATA-6 and NF-Y.

Codon 12 of the K-ras gene is a generally recognized example of a mutational hot spot. By the approach of gel retardation and specific antibodies, a double-stranded oligonucleotide corresponding to the codon 12 region of the mouse K-ras gene (from 20 to 50 bp with respect to the exon 1 start) was found to be a site for cooperative binding of the transcription factors GATA-6 and NF-Y. GATA-6 and NF-Y were selectively activated with lung carcinogens 3-methylcholanthrene and nitrosoethylurea in mice of strains susceptible to lung tumorigenesis but not in animals of resistant strains. The interaction of GATA-6 and NF-Y with the codon 12 region of the K-ras gene is suggested to be involved in the mechanism of lung carcinogenesis.

Optimization of ENU mutagenesis of Caenorhabditis elegans.

Chemical mutagenesis of Caenorhabditis elegans has relied primarily on EMS to produce missense mutations. The drawback of EMS mutagenesis is that the molecular lesions are primarily G/C --> A/T transitions. ENU has been shown to produce a different spectrum of mutations, but its greater toxicity to C. elegans makes it a difficult mutagen to use. We describe here methods for minimizing ENU toxicity in C. elegans. Methods include preparing ENU stocks in absolute ethanol and storing stock solutions for not more than 2 weeks at -20 degrees C. To maintain reasonable brood sizes of mutagenized animals, mutagenic solutions should not exceed 1.0mM ENU. We provide data which suggest ENU is degraded or altered to more toxic products in aqueous solution, but less so in solvents such as absolute ethanol.

Probing RNA structure with chemical reagents and enzymes.

This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.