Transgenic Mouse Mutation Research Articles

Homozygous R136S mutation in PRNP gene causes inherited early onset prion disease

BackgroundMore than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date.MethodsWe describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro PrPSc propagation studies were performed using recombinant-adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease.ResultsThe index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated PrPSc fragment, consistent with a Gerstmann-Sträussler-Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of PrPSc studies failed to show disease transmissibility.ConclusionIn conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be “probably damaging”, heterozygous carriers are protected, at least from an early onset providing evidence for a potentially recessive pattern of inheritance in human prion diseases.

Keratin 8 mutations in transgenic mice predispose to lung injury.

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62-Cys (Gly62 replaced with Cys) or Ser74-Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62-Cys mice and K8 Ser74-Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74-Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.

The effect of variable troponin C mutation thin filament incorporation on cardiac muscle twitch contractions

One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k−Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k−Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SummaryA challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.

De Novo T790M Mutation in an L858R Epidermal Growth Factor Receptor Mutant-Associated Lung Adenocarcinoma

Simple SummaryLung adenocarcinomas caused by (L858R) somatic mutation in the epidermal growth factor receptor (EGFR) show good therapeutic response to tyrosine kinase inhibitors. These tumors develop resistance to therapy mainly after acquired T790M mutation. However, whether the T790M mutation occurred before or after therapy is unknown. To demonstrate this, we developed mice with tetracycline-inducible lung-specific expression of the full-length genomic DNA of the human epidermal growth factor receptor containing an L858R mutation or both L858R and T790M mutations and evaluated de novo T790M mutation in untreated transgenic mice carrying a single L858R EGFR mutation. The results showed that lung tumors spontaneously acquire T790M mutation without any drug-related selective pressure.Background: Lung cancer is the leading cause of mortality for cancer worldwide. A point mutation in exon 21 of the epidermal growth factor receptor resulting in the substitution of arginine for leucine at position 858 (L858R) is a frequent cause of lung adenocarcinoma. Tyrosine kinase inhibitors are effective for treating patients with lung cancer associated with mutant epidermal growth factor receptors but most tumors become resistant shortly after treatment. The substitution of methionine for threonine at position 790 (T790M) on exon 20 is the most frequently acquired mutation leading to resistance to tyrosine kinase inhibitors. Whether the T790M mutation occurred after tyrosine kinase inhibitor therapy or it already existed before therapy is unclear. Methods: Here, we developed mice with tetracycline-inducible lung-specific expression of the full-length genomic DNA of the human epidermal growth factor receptor containing an L858R mutation or both L858R and T790M mutations and evaluated de novo T790M mutation in untreated transgenic mice carrying a single L858R EGFR mutation. Results: The L858R mutation-associated lung adenocarcinoma acquired de novo T790 mutation without previous therapy. Conclusions: The results of this study suggest that lung tumors may spontaneously acquire T790M mutations without any drug-related selective pressure.

Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Passenger Mutations Confound Interpretation of All Genetically Modified Congenic Mice

Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.

Mutagenic Effects of Perfluorooctanesulfonic Acid in gpt Delta Transgenic System Are Mediated by Hydrogen Peroxide.

Perfluorooctane sulfate (PFOS), a persistent organic pollutant, has recently been closely linked with an increased risk of tumorigenesis. However, PFOS has yielded negative results in various tests of genotoxicity. The present study aimed to investigate the mutagenic response to PFOS in the gpt delta transgenic mouse mutation system and to illustrate the contribution of hydrogen peroxide (H2O2) to PFOS genotoxicity. Mutations at the redBA/gam loci were determined by Spi(-) assay both in vitro and in vivo. DNA damage was measured by phosphorylated histone H2AX (γ-H2AX) and mouse bone marrow micronucleus (MN) testing. Our data showed that PFOS induced concentration-dependent increases in γ-H2AX foci and in mutation frequencies at redBA/gam loci in transgenic mouse embryonic fibroblast cells, which were confirmed by the formation of MNs in the bone marrow and the observations of mutation induction in the livers of gpt delta transgenic mice. Concurrent treatment with catalase, an efficient H2O2 scavenger, significantly decreased the formation of γ-H2AX foci and the mutation yields induced by PFOS. In addition, the generation of H2O2 was found to be closely related to the abnormal peroxisomal β-oxidation caused by PFOS. These finding might provide new mechanistical information about genotoxic effects of PFOS.

Qualification and comparison of Big Blue® transgenic mouse and rat mutation assays with n-ethyl-n-nitrosourea (ENU) and benzo(a)pyrene (BaP)

Qualification and comparison of Big Blue® transgenic mouse and rat mutation assays with n-ethyl-n-nitrosourea (ENU) and benzo(a)pyrene (BaP)

Integration of cII and Pig-a mutation and micronucleus endpoints into the Big Blue® transgenic mouse mutation assay: Results for benzo(a)pyrene (BaP) and n-ethyl-n-nitrosourea (ENU)

Integration of cII and Pig-a mutation and micronucleus endpoints into the Big Blue® transgenic mouse mutation assay: Results for benzo(a)pyrene (BaP) and n-ethyl-n-nitrosourea (ENU)

Annonacin, a natural lipophilic mitochondrial complex I inhibitor, increases phosphorylation of tau in the brain of FTDP-17 transgenic mice

Both genetic and environmental factors likely contribute to the neuropathology of tauopathies, but it remains unclear how specific genetic backgrounds affect the susceptibility towards environmental toxins. Mutations in the tau gene have been associated with familial tauopathies, while annonacin, a plant-derived mitochondrial inhibitor, has been implicated in an environmental form of tauopathy. We therefore determined whether there was a pathogenic synergy between annonacin exposure and the expression of the R406W-tau mutation in transgenic mice. We found that annonacin exposure caused an increase in the number of neurons with phosphorylated tau in the somatodendritic compartment in several brain areas in R406W+/+ mice as opposed to mice that had only the endogenous mouse tau (R406W−/−). Western blot analysis demonstrated a concomitant increase in total tau protein without increase in tau mRNA, but reduced proteasomal proteolytic activity in R406W+/+, but not R406W−/− mice, upon annonacin-treatment. Phosphorylated tau levels exceeded the increase in total tau protein, along with increased levels of different tau kinases, foremost a striking increase in the p25/p35 ratio, known to activate the tau kinase Cdk5. In summary, we observed a synergistic interaction between annonacin exposure and the presence of the R406W-tau mutation, which resulted in reduced degradation, increased phosphorylation and redistribution of neuronal tau.

Overexpression Of Leukemia Associated CblY371H Mutation In Transgenic Mice Causes Dosage Dependent Embryonic Lethality

Juvenile Myelomonocytic Leukemia (JMML) is a devastating childhood cancer which is rapidly fatal with infiltration of myeloid cells into multiple organs (Hess, Zutter, Castleberry, & Emanuel, 1996). Based on the observation that uniparental disomy was found in the chromosomal region 11q in JMML patient samples, about 15% of patient samples were found to contain a mutation in c-Cbl (Loh et al., 2009). Moreover, mutant Cbl was also found to be a tumor suppressor gene where a germline mutation results in the predisposition for developing JMML (Niemeyer et al., 2010). The c-Cbl gene encodes a multifunctional adaptor protein which contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif which contain E3 ligase activity, and a C-terminal ubiquitin-associated domain. Previous mutations in myeloid malignancies have been described where mutations occur in the RING finger domain or the linker domain (Caligiuri et al., 2007; Dunbar et al., 2008). Interestingly, a hotspot for mutations at residue 371 exists in JMML patients where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H (Loh et al., 2009). This residue belongs in the linker region of the CBL protein, and it was previously observed that Y371 mediates the binding of c-Cbl to the p85 subunit of PI3 kinase (Blaydes et al., 2001). In vitro, CblY371H mutation does indeed destroy its ligase function resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene was silenced (Niemeyer et al., 2010). However, how mutant Cbl gives rise to JMML and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we tested the oncogenicity of the CblY371H mutation in transgenic mice. Of the 6 founder transgenic mice, we chose L5 line for further analysis because it had the highest level of expression. As expected, overexpression of CblY371H by itself in wild type mice had no phenotype since inactivation of wild type Cbl allele is seen in patients. We, then, generated transgenic mice which were heterozygous for both the BAC transgene and the cbl null mutation (CblY371H; Cbl+/-) and then bred them to Cbl heterozygous knockout mice (Cbl+/-). Because homozygous knockout mice have reduced fertility, we preferentially utilized heterozygous mice for the breeding (El Chami et al., 2005). Interestingly, out of the first 118 progeny genotyped where 1/8 of the pups is expected to be the desired genotype (CblY371H;Cbl-/-), no pups were of the desired genotype suggesting embryonic lethality. Because mice which lack both c-cbl and cbl-b are embryonic lethal, we reasoned that high expression that is achieved from the L5 line was causing a dominant negative affect on cbl-b function resulting in an embryonic lethal phenotype. We then tested if a lower level of the transgene expression would result in viable pups. We used the K5 line which was the second highest expressing transgenic line. We were now able to generate the desired genotype (CblY371H; Cbl-/-) although at less than expected numbers. Thus far, we have generated 4 mice which have the correct genotype which are being monitored for disease. We also monitored the heterozygous mice that carried the transgene (CblY371H; Cbl+/-) which were also asymptomatic without any obvious phenotype. Further studies are being done to characterize the hematopoietic phenotype in these mice by isolating fetal liver cells followed by transplantation into lethally irradiated mice and will be discussed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

ADAM10 Missense Mutations Potentiate β-Amyloid Accumulation by Impairing Prodomain Chaperone Function

The generation of Aβ, the main component of senile plaques in Alzheimer's disease (AD), is precluded by α-secretase cleavage within the Aβ domain of the amyloid precursor protein (APP). We identified two rare mutations (Q170H and R181G) in the prodomain of the metalloprotease, ADAM10, that cosegregate with late-onset AD (LOAD). Here, we addressed the pathogenicity of these mutations in transgenic mice expressing human ADAM10 in brain. In Tg2576 AD mice, both mutations attenuated α-secretase activity of ADAM10 and shifted APP processing toward β-secretase-mediated cleavage, while enhancing Aβ plaque load and reactive gliosis. We also demonstrated ADAM10 expression potentiates adult hippocampal neurogenesis, which is reduced by the LOAD mutations. Mechanistically, both LOAD mutations impaired the molecular chaperone activity of ADAM10 prodomain. Collectively, these findings suggest that diminished α-secretase activity, owing to LOAD ADAM10 prodomain mutations, leads to AD-related pathology, strongly supporting ADAM10 as a promising therapeutic target for this devastating disease.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Identification of Potential Regulatory Elements in the 5′ and 3′ UTRs of 12 Translationally Regulated mRNAs in Mammalian Spermatids by Comparative Genomics

To facilitate identifying translational control elements by studies of mutations in transgenic mice, a database of orthologous 5' and 3' ends of 12 messenger RNA (mRNA) species from 13 to 23 mammals that undergo delayed translational activation in spermatids was constructed for the Acev2, Akap3, Akap4v2, Gapdhs, Odf1, Prm1, Prm2, Prm3, Smcp, Spata18, Tnp1, and Tnp2 mRNAs. This database, available here, was searched for conserved sequences in conserved positions and known translational control elements. Numerous potential mRNA-specific elements were identified, including upstream open reading frames, conserved sequences upstream and downstream of the poly(A) signal, and noncanonical and multiple poly(A) signals. RNA electrophoresis mobility shift assays demonstrate that Y-box proteins bind 30 of the 36 permutations of the degenerate Y-box recognition sequence (YRS), [UAC][CA]CA[UC]C[ACU], and this information was used to identify hundreds of YRSs in the untranslated region (UTR) database. Collectively, these findings suggest that the distal ends of both UTRs are particularly well conserved, implying that translation of each mRNA is regulated by mechanisms involving the poly(A) binding protein and the closed loop. In addition, the 5' flanking regions of all 12 genes have conserved, gene-specific sequences and configurations of elements that resemble the binding site of the testis-specific isoform of cyclic AMP response element modulator, and all 12 genes lack retrogene paralogues, demonstrating the efficacy of mechanisms that limit the proliferation of retroposons in the male germ line. This study illustrates the power of comparative genomics in identifying novel hypothetical regulatory elements for analysis with biochemical and in vivo genetic approaches.

Abstract 3566: Effect of afatinib on lung cancer burden induced by an exon 19 EGFR mutation in transgenic mice

Abstract Afatinib (BIBW2992) is an irreversible dual inhibitor of EGFR and HER2. Afatinib was reported to have anti-tumor effect against L858R/T790M EGFR lung cancer model (Oncogene 27, 2008). Kinase inhibitors had different dissociation constant for binding deletion mutation and point mutation (Nat Biotechnol 26, 2008). Thus, the effect of afatinib on lung cancer harboring deletion or deletion/T790M EGFR double mutations should be clarified because it is possible that afatinib may affect differently tumors with deletion mutations compared to tumors with L858R or L858R/T790M point mutations. Our present study was carried out to investigate the preclinical efficacy of afatinib in lung cancer harboring exon19 EGFR del mutations. Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR were established from parental PC-9 cells harboring exon 19 del mutation (delE746-A750) in our laboratory (Cancer Res 67, 2007). The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effect of afatinib and gefitinib. Protein expression was determined by Western blotting. The mean IC50s of afatinib were 0.00032 µM for PC-9 cells and 0.164 µM for RPC-9 cells and were less than those of gefitinib for PC-9 (0.0033 µM) and RPC-9 (11.45 µM), respectively. Phosphorylated EGFR, AKT and ERK of PC-9 were suppressed by treatments with afatinib (1 nM) and with gefitinib (100 nM). In RPC-9 cells, afatinib was 100-fold less active (100 nM) compared to parental cells while gefitinib was ineffective (10000 nM). In addition, 1 nM and 100 nM of afatinib completely inhibited phosphorylation of HER2 in PC-9 and RPC-9, respectively. We generated transgenic mice expressing the delE748-A752 mutant of mouse EGFR, which is equivalent to the delE746-A750 mutant, driven by the SP-C promoter (Cancer Sci 99, 2008). The mice invariably developed multi-focal pulmonary adenocarcinomas of various sizes at 5 to 6 weeks of age and died of lung cancer about 2 months later, if left untreated. To investigate the effect of afatinib on lung tumors induced by the activating EGFR mutation, the transgenic mice were treated with oral afatinib (5 mg/kg/day), gefitinib (5 mg/kg/day) or vehicle alone from 11 to 15 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the afatinib-treated group, gefitinib-treated group and vehicle group was 0.12 ± 0.13, 1.25 ± 0.60 and 9.00 ± 1.5 respectively. There were significant differences of the tumor numbers between afatinib-treated group and vehicle group (P < 0.01), while afatinib tended to reduce the tumors compared to gefitinib (P = 0.06). In addition, afatinib was more effective than gefitinib in pathological specimens as the tumors in afatinib-treated group almost disappeared. In conclusion, our data suggest that afatinib may be more effective for treatment of lung cancer induced by the EGFR mutation in exon 19 than gefitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2011-3566

Identification of elements in the Smcp 5′ and 3′ UTR that repress translation and promote the formation of heavy inactive mRNPs in spermatids by analysis of mutations in transgenic mice

The sperm mitochondria-associated cysteine-rich protein (Smcp) mRNA is transcribed in step 3 spermatids, and is stored in free mRNPs until translation begins ∼6 days later in step 11. To identify sequences that control the timing of Smcp mRNA translation, mutations in both UTRs were analyzed in transgenic mice using green fluorescent protein (GFP), squashes of seminiferous tubules, and quantification of polysomal loading in adult and 21 dpp testes in sucrose and Nycodenz gradients. GFP fluorescence is first detected in step 9 spermatids in lines harboring a transgene containing the Gfp 5' UTR and Smcp 3' UTR. Unexpectedly, this mRNA is stored in large, inactive mRNPs in early spermatids that sediment with polysomes in sucrose gradients, but equilibrate with the density of free mRNPs in Nycodenz gradients. Randomization of the segment 6-38 nt upstream of the first Smcp poly(A) signal results in early detection of GFP, a small increase in polysomal loading in 21 dpp testis, inactivation of the formation of heavy mRNPs, and loss of binding of a Y-box protein. GFP is first detected in step 5 spermatids in a transgene containing the Smcp 5' UTR and Gfp 3' UTR. Mutations in the start codons in the upstream reading frames eliminate translational delay by the Smcp 5' UTR. Collectively, these findings demonstrate that Smcp mRNA translation is regulated by multiple elements in the 5' UTR and 3' UTR. In addition, differences in regulation between Smcp-Gfp mRNAs containing one Smcp UTR and the natural Smcp mRNA suggest that interactions between the Smcp 5' UTR and 3' UTR may be required for regulation of the Smcp mRNA.

A novel Escherichia coli‐derived mutation detected with the Big Blue® cII mutant selectable assay

Transgenic mouse mutation detection systems allow investigation of the origins and mechanisms of mutation associated with exogenous and endogenous mutagen exposures in individual tissues and cell types. In the past, selection assays for transgenic mutants have been contaminated with nonmurine-derived mutations and assay validation is critical to ensure murine in vivo origins of mutations. This is critical in studies of spontaneous mutations and extrapolation to endogenous mammalian genes. Herein, we provide one measure of the contribution of Escherichia coli (E. coli)-derived mutations to the Big Blue(R) cII transgene mutant selection assay. We report the first direct evidence of an E. coli-derived cII mutation identified among mutations recovered in the cII selective assay. An E. coli transposable 5 (Tn5) element IS50R inverted repeat (1,534 bp) was identified at base pair 414 in the cII transgene and the insertion generated a 9 bp target site duplication typical of this type of transposition. The bacterial transposition occurred only once in the assay of 25 x 10(6) plaque forming units and sequencing of 1,177 cII mutants. The observed frequency of this type of mutation is 4 x 10(-8) in retrieved lambda phage and 8.5 x 10(-4) in harvested cII mutants and thus a very rare occurrence in typical analyses of spontaneous in vivo mutations. Given that the frequency of transposition is equal to, or an order of magnitude higher, than the frequency of point mutations in E. coli, this article provides excellent validation for the murine origins of mutations detected using the cII mutant selection assay.

Genotoxic responses to titanium dioxide nanoparticles and fullerene in gpt delta transgenic MEF cells.

BackgroundTitanium dioxide (TiO2) nanoparticles and fullerene (C60) are two attractive manufactured nanoparticles with great promise in industrial and medical applications. However, little is known about the genotoxic response of TiO2 nanoparticles and C60 in mammalian cells. In the present study, we determined the mutation fractions induced by either TiO2 nanoparticles or C60 in gpt delta transgenic mouse primary embryo fibroblasts (MEF) and identified peroxynitrite anions (ONOO-) as an essential mediator involved in such process.ResultsBoth TiO2 nanoparticles and C60 dramatically increased the mutation yield, which could be abrogated by concurrent treatment with the endocytosis inhibitor, Nystatin. Under confocal scanning microscopy together with the radical probe dihydrorhodamine 123 (DHR 123), we found that there was a dose-dependent formation of ONOO- in live MEF cells exposed to either TiO2 nanoparticles or C60, and the protective effects of antioxidants were demonstrated by the nitric oxide synthase (NOS) inhibitor, NG-methyl-L-arginine (L-NMMA). Furthermore, suppression of cyclooxygenase-2 (COX-2) activity by using the chemical inhibitor NS-398 significantly reduced mutation frequency of both TiO2 nanoparticles and C60.ConclusionOur results provided novel information that both TiO2 nanoparticles and C60 were taken up by cells and induced kilo-base pair deletion mutations in a transgenic mouse mutation system. The induction of ONOO- may be a critical signaling event for nanoparticle genotoxicity.

Investigation of the Low-Dose Response in the In Vivo Induction of Micronuclei and Adducts by Acrylamide

Acrylamide is an industrial chemical used in polymer manufacture. It is also formed in foods processed at high temperatures. It induces chromosome aberrations and micronuclei (MN) in somatic cells of mice, but not rats, and mutations in transgenic mice. This study evaluated the low-dose MN response in mouse bone marrow and the shape of the dose-response curve. Mice were treated orally with acrylamide for 28 days using logarithmically spaced doses from 0.125 to 24 mg/kg/day, and MN were assessed in peripheral blood reticulocytes (RETs) and erythrocytes by flow cytometry. Liver glycidamide DNA adducts and acrylamide and glycidamide N-terminal valine hemoglobin adducts were also determined. Acrylamide produced a weak MN response, with statistical significance at 6.0 mg/kg/day, or greater, in MN-RETs and at 4.0 mg/kg/day or greater in MN normochromatic erythrocytes (NCEs). The MN responses at the lower doses were indistinguishable from the concurrent and historical controls. The adducts increased at a much different rate than the MN. When the MN-NCE values were compared to administered dose, the response was consistent with a linear model. However, when hemoglobin or DNA adducts were used as the dose metric, the response was significantly nonlinear, and models that assumed a threshold dose of 1 or 2 mg/kg/day provided a better fit than a linear model. The MN-RET dose-response had greater variability than the MN-NCE response and was consistent with linearity and with a threshold at 1 or 2 mg/kg/day, regardless of the dose metric. These data suggest a threshold for acrylamide in the MN test.