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1.
iScience ; 26(9): 107530, 2023 Sep 15.
Article in English | MEDLINE | ID: mdl-37664628

ABSTRACT

Ionizing radiation (IR) is a risk factor for acute myeloid leukemia (rAML). Murine rAMLs feature both hemizygous chromosome 2 deletions (Del2) and point mutations (R235) within the hematopoietic regulatory gene Spi1. We generated a heterozygous CBA Spi1 R235 mouse (CBASpm/+) which develops de novo AML with 100% incidence by ∼12 months old and shows a dose-dependent reduction in latency following X-irradiation. These effects are reduced on an AML-resistant C57Bl6 genetic background. CBASpm/Gfp reporter mice show increased Gfp expression, indicating compensation for Spm-induced Spi1 haploinsufficiency. Del2 is always detected in both de novo and rAMLs, indicating that biallelic Spi1 mutation is required for AML. CBASpm/+ mice show that a single Spm modification is sufficient for initiating AML development with complete penetrance, via the "two-hit" mechanism and this is accelerated by IR exposure. Similar SPI1/PU.1 polymorphisms in humans could potentially lead to enhanced susceptibility to IR following medical or environmental exposure.

2.
Lupus Sci Med ; 10(1)2023 02.
Article in English | MEDLINE | ID: mdl-36822800

ABSTRACT

OBJECTIVE: The aim of the study was to investigate whether the IRAK1/JAK2/Flt3 inhibitor pacritinib prevents disease development in the lupus-prone ABIN1[D485N] knock-in mouse. METHODS: ABIN1[D485N] knock-in mice aged 8 weeks were fed for 10 weeks on a diet containing pacritinib. Body weight was monitored, and serum collected at the end to measure pacritinib, autoantibody and immunoglobulin levels. Splenic immune cell populations were analysed, and the kidney, liver and lungs examined for pathology. RESULTS: Pacritinib prevented multiple facets of the lupus phenotype in ABIN1[D485N] knock-in mice, including splenomegaly, expansion of splenic germinal centre B cells, follicular T helper cells, and neutrophils, elevated serum levels of double-stranded DNA antibodies and immunoglobulins, glomerular IgA and lung inflammation. CONCLUSIONS: Pacritinib may be useful for the treatment of multiorgan inflammation in patients with lupus.


Subject(s)
Lupus Erythematosus, Systemic , Animals , Mice , Inflammation , Kidney/pathology , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/pathology , Phenotype
3.
J Pathol ; 258(4): 382-394, 2022 12.
Article in English | MEDLINE | ID: mdl-36073856

ABSTRACT

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Subject(s)
Hamartoma Syndrome, Multiple , Thyroid Neoplasms , Mice , Animals , Male , Female , Humans , Infant , Sirolimus/pharmacology , Sirolimus/therapeutic use , Phosphatidylinositol 3-Kinases/genetics , Longevity , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Hamartoma Syndrome, Multiple/drug therapy , Hamartoma Syndrome, Multiple/genetics , Hamartoma Syndrome, Multiple/pathology , Phosphatidylinositol 3-Kinase/genetics , Phosphoinositide-3 Kinase Inhibitors , Mechanistic Target of Rapamycin Complex 1/genetics , Germ Cells/metabolism , Germ-Line Mutation
4.
Lab Anim ; 56(5): 466-470, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35360986

ABSTRACT

The main objective of a post mortem is to identify, at a macroscopic level, any anatomical features that characterise mutant or sick mice and to describe lesions contributing to morbidity and mortality. Tissues collected for subsequent examination require appropriate handling and preservation to prevent deterioration. Therefore, efficient routine procedures are essential to facilitate histology and to ensure high-quality samples. In addition, optimised techniques minimise data loss from damaged samples reducing the numbers of animals used and supporting the 3Rs principle of reduction. Here, we describe an optimised method for tissue collection in the mouse. Training tips and points for caution are included.


Subject(s)
Tissue Banks , Animals , Mice , Autopsy/methods , Disease Models, Animal
5.
EMBO J ; 41(8): e109700, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35274759

ABSTRACT

HOIL-1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL-1's E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and α1:4-linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100-fold by the interaction of Met1-linked or Lys63-linked ubiquitin oligomers with the RBR domain of HOIL-1. HOIL-1 also transferred pre-formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.


Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Animals , Glucans , Glucose , Glycogen/metabolism , Mammals , Mice , NF-kappa B/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
6.
PLoS One ; 17(2): e0263151, 2022.
Article in English | MEDLINE | ID: mdl-35157702

ABSTRACT

The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4+ and CD8+ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44high/CD62Llow expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2-3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.


Subject(s)
Autoimmune Diseases/pathology , Receptors, Interleukin-1 Type I/metabolism , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptors/metabolism , Animals , Autoimmune Diseases/genetics , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Dermatitis/genetics , Dermatitis/pathology , Gene Knock-In Techniques , Mice , Mice, Knockout , Phenotype , Pneumonia/genetics , Pneumonia/pathology , Signal Transduction
7.
Lupus Sci Med ; 8(1)2021 11.
Article in English | MEDLINE | ID: mdl-34836923

ABSTRACT

OBJECTIVE: We have reported previously that the IRAK4 inhibitor PF06426779 given to ubiquitin-binding-defective ABIN1[D485N] mice at 6 weeks of age prevents the major facets of lupus that develop 10 weeks later. The present study was undertaken to investigate whether PF06426779 could reverse the lupus phenotype when administered to 13-week-old ABIN1[D485N] mice that had already developed symptoms of lupus. METHODS: Splenomegaly, the number of splenic neutrophils, TFH and Germinal Centre B (GCB) cells, serum levels of immunoglobulins, the extent of kidney, liver and lung pathology, and glomerular IgA and IgM were measured after feeding 13-week-old ABIN1[D485N] and wild-type mice for another 10 weeks with R&M3 diet with and without PF06426779 (4 g/kg). RESULTS: Following drug treatment, spleen size and weight, splenic neutrophil numbers, and serum IgA and glomerular IgA levels of ABIN1[D485N] mice returned to those seen in wild-type mice. The rise in splenic TFH and GCB numbers, the increase in kidney and liver pathology, and the concentrations of serum IgG1, IgG2A and IgE between 13 and 23 weeks were suppressed. There was no reduction in the level of anti-self double-stranded DNA, anti-self nuclear antigens or IgM during the drug treatment. CONCLUSIONS: The results demonstrate the therapeutic potential of IRAK4 inhibitors for the treatment of lupus and raise the possibility of monitoring efficacy by measuring decreases in the serum levels of IgA. Our results support the view that there may be a closer connection between lupus and IgA nephropathy than realised previously.


Subject(s)
Interleukin-1 Receptor-Associated Kinases , Lupus Erythematosus, Systemic , Animals , Humans , Kidney/pathology , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/pathology , Mice , Phenotype
8.
Dis Model Mech ; 14(10)2021 10 01.
Article in English | MEDLINE | ID: mdl-34477842

ABSTRACT

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder.


Subject(s)
Down Syndrome/pathology , Amyloid beta-Peptides/metabolism , Anemia/complications , Animals , Bone Development , Disease Models, Animal , Down Syndrome/genetics , Down Syndrome/physiopathology , Erythropoiesis , Evoked Potentials, Auditory, Brain Stem , Gene Expression Regulation , Genes, Duplicate , Hearing , Heart Function Tests , Hippocampus/pathology , Locomotion , Memory/physiology , Mice, Inbred C57BL , Otitis Media/complications , Otitis Media/pathology , Otitis Media/physiopathology , Phenotype , Prediabetic State/complications , Prediabetic State/pathology , Prediabetic State/physiopathology , Respiration , Sleep/physiology , Spleen/pathology , Splenomegaly/complications
9.
Cell Rep Med ; 2(4): 100227, 2021 04 20.
Article in English | MEDLINE | ID: mdl-33948568

ABSTRACT

Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies.


Subject(s)
Hypoxia/metabolism , Immunotherapy, Adoptive , Receptors, Chimeric Antigen/genetics , T-Lymphocytes/metabolism , Tumor Microenvironment/immunology , Animals , Cell Line, Tumor/metabolism , Disease Models, Animal , Genes, erbB/genetics , Humans , Hypoxia/genetics , Immunotherapy, Adoptive/methods , Mice, Transgenic , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays/methods
10.
ILAR J ; 62(1-2): 133-168, 2021 12 31.
Article in English | MEDLINE | ID: mdl-33712827

ABSTRACT

Animal models provide a valuable tool and resource for biomedical researchers as they investigate biological processes, disease pathogenesis, novel therapies, and toxicologic studies. Interpretation of animal model data requires knowledge not only of the processes/diseases being studied but also awareness of spontaneous conditions and background lesions in the model that can influence or even confound the study results. Species, breed/stock, sex, age, anatomy, physiology, diseases (noninfectious and infectious), and neoplastic processes are model features that can impact the results as well as study interpretation. Here, we review these features in several common laboratory animal species, including ferret, dog (beagle), pig, sheep, and goats.


Subject(s)
Goats , Swine Diseases , Animals , Animals, Laboratory , Disease Models, Animal , Dogs , Ferrets , Sheep , Swine
11.
PLoS One ; 15(6): e0230162, 2020.
Article in English | MEDLINE | ID: mdl-32542000

ABSTRACT

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.


Subject(s)
Animal Husbandry/instrumentation , Internationality , Tarsal Bones/injuries , Animals , Male , Mice , Mice, Inbred C57BL , Tarsal Bones/diagnostic imaging , Tomography, X-Ray Computed
12.
J Immunol ; 205(1): 78-89, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32414808

ABSTRACT

Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the TCR and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T (Treg) cell function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation, including chronic inflammatory conditions, cancer, and autoimmune diseases. Idelalisib (PI3Kδ), alpelisib (PI3Kα), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and, in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis is a mouse model of T cell-driven CNS inflammation, in which Treg cells play a key protective role. In this study, we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signaling resulted in increased experimental autoimmune encephalitis disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signaling in Treg cell-mediated protection against CNS inflammation.


Subject(s)
Class I Phosphatidylinositol 3-Kinases/metabolism , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Peripheral Nerves/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Autoimmunity/genetics , Class I Phosphatidylinositol 3-Kinases/genetics , Class Ib Phosphatidylinositol 3-Kinase/genetics , Encephalomyelitis, Autoimmune, Experimental/blood , Encephalomyelitis, Autoimmune, Experimental/diagnosis , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Humans , Male , Mice , Mice, Transgenic , Myelin-Oligodendrocyte Glycoprotein/administration & dosage , Myelin-Oligodendrocyte Glycoprotein/immunology , Peptide Fragments/administration & dosage , Peptide Fragments/immunology , Peripheral Nerves/pathology , Severity of Illness Index , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes, Regulatory/metabolism
13.
J Clin Invest ; 129(9): 3640-3656, 2019 06 04.
Article in English | MEDLINE | ID: mdl-31162141

ABSTRACT

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.


Subject(s)
Hypoxia-Inducible Factor-Proline Dioxygenases , RNA Interference/immunology , Signal Transduction , T-Lymphocytes, Regulatory , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/immunology , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/immunology , Mice , Mice, Transgenic , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism
14.
Sci Rep ; 9(1): 20398, 2019 12 31.
Article in English | MEDLINE | ID: mdl-31892712

ABSTRACT

The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modified genetically. We have identified a point mutation in Col4a4 in mice where disease is modified by strain background, providing further evidence of the genetic modification of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identified early differences in disease progression, including expression of podocyte-specific genes and podocyte morphology. In C57BL/6J mice podocyte effacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We find that there is evidence of differential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an inflammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function.


Subject(s)
Collagen Type IV/genetics , Genetic Background , Mutation , Nephritis, Hereditary/genetics , Animals , Disease Models, Animal , Disease Progression , Genetic Predisposition to Disease , Kidney/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Nephritis, Hereditary/metabolism , Podocytes/metabolism
15.
Cell Rep ; 25(12): 3315-3328.e6, 2018 12 18.
Article in English | MEDLINE | ID: mdl-30566859

ABSTRACT

Mutations in genes essential for mitochondrial function have pleiotropic effects. The mechanisms underlying these traits yield insights into metabolic homeostasis and potential therapies. Here we report the characterization of a mouse model harboring a mutation in the tryptophanyl-tRNA synthetase 2 (Wars2) gene, encoding the mitochondrial-localized WARS2 protein. This hypomorphic allele causes progressive tissue-specific pathologies, including hearing loss, reduced adiposity, adipose tissue dysfunction, and hypertrophic cardiomyopathy. We demonstrate the tissue heterogeneity arises as a result of variable activation of the integrated stress response (ISR) pathway and the ability of certain tissues to respond to impaired mitochondrial translation. Many of the systemic metabolic effects are likely mediated through elevated fibroblast growth factor 21 (FGF21) following activation of the ISR in certain tissues. These findings demonstrate the potential pleiotropy associated with Wars2 mutations in patients.


Subject(s)
Organ Specificity , Oxidative Phosphorylation , Stress, Physiological , Tryptophan-tRNA Ligase/genetics , Adipose Tissue, Brown/pathology , Adipose Tissue, White/pathology , Adiposity , Alleles , Alternative Splicing/genetics , Animals , Base Sequence , Body Weight , Brain/pathology , Cardiomyopathy, Hypertrophic/blood , Cardiomyopathy, Hypertrophic/complications , Cardiomyopathy, Hypertrophic/physiopathology , Disease Models, Animal , Electron Transport , Evoked Potentials, Auditory, Brain Stem , Exons/genetics , Fibroblast Growth Factors/blood , Fibroblasts/metabolism , Hearing Loss/blood , Hearing Loss/complications , Hearing Loss/genetics , Hearing Loss/physiopathology , Mice , Mice, Mutant Strains , Muscle, Skeletal/metabolism , Mutation/genetics , Organelle Biogenesis , Tryptophan-tRNA Ligase/metabolism , Up-Regulation
16.
Leukemia ; 32(6): 1435-1444, 2018 06.
Article in English | MEDLINE | ID: mdl-29556020

ABSTRACT

Epidemiological studies have demonstrated an increased leukemia incidence following ionizing radiation exposure, but to date, the target cells and underlying mechanisms of radiation leukemogenesis remain largely unidentified. We engineered a mouse model carrying a different fluorescent marker on each chromosome 2, located inside the minimum deleted region occurring after radiation exposure and recognized as the first leukemogenic event. Using this tailored model, we report that following radiation exposure, more than half of asymptomatic CBA Sfpi1 GFP/mCh mice presented with expanding clones of preleukemic hematopoietic cells harboring a hemizygous interstitial deletion of chromosome 2. Moreover, following isolation of preleukemic hematopoietic stem and progenitor cells irradiated in their native microenvironment, we identified the presence of Sfpi1 point mutations within a subpopulation of these preleukemic cells expanding rapidly (increasing from 6% to 55% in 21 days in peripheral blood in one case), hence identifying for the first time the presence of such cells within a living animal. Importantly, we also report a previously undescribed gender difference in the phenotype of the preleukemic cells and leukemia, suggesting a gender imbalance in the radiation-induced leukemic target cell. In conclusion, we provide novel insights into the sequence of molecular events occurring during the (radiation-induced) leukemic clonal evolution.


Subject(s)
Leukemia, Radiation-Induced/etiology , Preleukemia/etiology , Animals , Clonal Evolution , Disease Progression , Female , Hematopoietic Stem Cells/radiation effects , Male , Mice , Mice, Inbred CBA , Point Mutation , Proto-Oncogene Proteins/genetics , Sex Characteristics , Trans-Activators/genetics
17.
Mol Cancer Ther ; 17(2): 381-392, 2018 02.
Article in English | MEDLINE | ID: mdl-28839000

ABSTRACT

As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and bladder, for such patients. We developed methods to determine normal tissue toxicity severity in intestine and bladder in vivo, using novel radiotherapy techniques on a small animal radiation research platform (SARRP). The effects of panobinostat on in vivo tumor growth delay were evaluated using subcutaneous xenografts in athymic nude mice. Panobinostat concentration levels in xenografts, plasma, and normal tissues were measured in CD1-nude mice. CD1-nude mice were treated with drug/irradiation combinations to assess acute normal tissue effects in small intestine using the intestinal crypt assay, and later effects in small and large intestine at 11 weeks by stool assessment and at 12 weeks by histologic examination. In vitro effects of panobinostat were assessed by qPCR and of panobinostat, TMP195, and mocetinostat by clonogenic assay, and Western blot analysis. Panobinostat resulted in growth delay in RT112 bladder cancer xenografts but did not significantly increase acute (3.75 days) or 12 weeks' normal tissue radiation toxicity. Radiosensitization by panobinostat was effective in hypoxic bladder cancer cells and associated with class I HDAC inhibition, and protein downregulation of HDAC2 and MRE11. Pan-HDAC inhibition is a promising strategy for radiosensitization, but more selective agents may be more useful radiosensitizers clinically, resulting in fewer systemic side effects. Mol Cancer Ther; 17(2); 381-92. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."


Subject(s)
Histone Deacetylase Inhibitors/therapeutic use , Radiation-Sensitizing Agents/therapeutic use , Urinary Bladder Neoplasms/radiotherapy , Animals , Disease Models, Animal , Histone Deacetylase Inhibitors/pharmacology , Humans , Mice , Mice, Nude , Radiation-Sensitizing Agents/pharmacology , Transfection
18.
ILAR J ; 59(1): 4-12, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30624739

ABSTRACT

In translational research, animal models are an important tool to aid in decision-making when taking potential therapies into human clinical trials. Recently, there have been a number of papers that have suggested limited concordance of preclinical animal experiments with subsequent human clinical experience. Assessments of preclinical animal studies have led to concerns about the reproducibility of data and have highlighted the need for an emphasis on rigor and quality in the planning, conduct, analysis, and reporting of such studies. The incorporation of a wider role for the comparative pathologist using pathology best practices in the planning and conduct of animal model-based research is one way to increase the quality and reproducibility of data. The use of optimal design and planning of tissue collection, incorporation of pathology methods into written protocols, conduct of pathology procedures using accepted best practices, and the use of optimal pathology analysis and reporting methods enhance the quality of the data acquired from many types of preclinical animal models and studies. Many of these pathology practices are well established in the discipline of toxicologic pathology and have a proven and useful track record in enhancing the data from animal-based studies used in safety assessment of human therapeutics. Some of this experience can be adopted by the wider community of preclinical investigators to increase the reproducibility of animal study data.


Subject(s)
Pathology/methods , Translational Research, Biomedical/methods , Animals , Humans , Reproducibility of Results , Research Design
19.
Mol Metab ; 6(11): 1419-1428, 2017 11.
Article in English | MEDLINE | ID: mdl-29107289

ABSTRACT

OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.


Subject(s)
Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Animals , Anxiety/metabolism , Base Sequence , Brain/metabolism , Chromosome Mapping , Databases, Genetic , Female , Gene Expression , Gene Expression Regulation, Developmental/genetics , Genes, Homeobox , Homeodomain Proteins/physiology , Humans , Hypothalamus/metabolism , Male , Mice , Nerve Tissue Proteins/physiology , Neurosecretory Systems/metabolism , Obesity/metabolism , Transcription Factors/genetics , Transcriptome/genetics
20.
Nat Commun ; 8(1): 1804, 2017 11 27.
Article in English | MEDLINE | ID: mdl-29180704

ABSTRACT

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.


Subject(s)
Insulin Resistance , Mitochondria/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , AMP-Activated Protein Kinases/metabolism , Animals , Autophagy/physiology , Cell Line, Tumor , Class III Phosphatidylinositol 3-Kinases , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Gene Knock-In Techniques , Glucose/analysis , Glucose/metabolism , Glucose Tolerance Test , Glycolysis/physiology , Hepatocytes , Heterozygote , Humans , Insulin/metabolism , Liver/cytology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Myoblasts , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Primary Cell Culture
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