Spontaneous tumor development in bone marrow-rescued DNA-PKcs(3A/3A) mice due to dysfunction of telomere leading strand deprotection.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at the Thr2609 cluster is essential for its complete function in DNA repair and tissue stem cell homeostasis. This phenomenon is demonstrated by congenital bone marrow failure occurring in DNA-PKcs(3A/3A) mutant mice, which require bone marrow transplantation (BMT) to prevent early mortality. Surprisingly, an increased incidence of spontaneous tumors, especially skin cancer, was observed in adult BMT-rescued DNA-PKcs(3A/3A) mice. Upon further investigation, we found that spontaneous γH2AX foci occurred in DNA-PKcs(3A/3A) skin biopsies and primary keratinocytes and that these foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation. Consistently, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs(3A/3A) cells as compared with wild-type and DNA-PKcs-knockout cells. In addition, a previously identified DNA-PKcs Thr2609Pro mutation, found in breast cancer, also induces a similar impairment of telomere leading-end maturation. Taken together, our current analyses indicate that the functional DNA-PKcs T2609 cluster is required to facilitate telomere leading strand maturation and prevention of genomic instability and cancer development.

Primary neoplasms of bones in mice: retrospective study and review of literature.

To compare and summarize the mechanisms, frequencies of occurrence, and classification schemes of spontaneous, experimental, and genetically engineered mouse skeletal neoplasms, the literature was reviewed, and archived case material at The Jackson Laboratory was examined. The frequency of occurrence of spontaneous bone neoplasms was less than 1% for most strains, with the exceptions of osteomas in CF-1 (5.5% and 10% in two studies) and OF-1 outbred strains (35%), and osteosarcomas in NOD/ShiLtJ (11.5%) and NOD-derived (7.1%) mice. The frequency was 100% for osteochondromas induced by conditional inactivation of exostoses (multiple) 1 (Ext1) in chondrocytes, osteosarcomas induced by tibial intramedullary inoculation of Moloney murine sarcoma virus, and osteosarcomas induced by conditional inactivation of Trp53-with or without inactivation of Rb1-in osteoblast precursors. Spontaneous osteogenic neoplasms were more frequent than spontaneous cartilaginous and vascular types. Malignant neoplasms were more frequent than benign ones. The age of occurrence for spontaneous neoplasms ranged from 37 to 720 days (M = 316.35) for benign neoplasms and 35 to 990 (M = 299.28) days for malignant. In genetically engineered mice, the average age of occurrence ranged from 28 to 70 days for benign and from 35 to 690 days for malignant. Histologically, nonosteogenic neoplasms were similar across strains and mutant stocks; osteogenic neoplasms exhibited greater diversity. This comparison and summarization of mouse bone neoplasms provides valuable information for the selection of strains to create, compare, and validate models of bone neoplasms.

Opportunistic bacterial infections in breeding colonies of the NSG mouse strain.

Spontaneous morbidity primarily affecting female breeders in 3 independent breeding colonies of NSG (NOD.Cg-Prkdc(scid) I12rg(tm1Wjl) /SzJ) mice prompted an investigation to uncover the cause of disease. Necropsies were performed on 264 (157 female and 107 male) spontaneously sick, experimentally unmanipulated NSG mice. In sum, 42 mice (15.9%) had acute or chronic renal inflammatory lesions, of which 12 had concurrent histologic evidence of an ascending urinary tract infection. From 94 kidneys cultured for bacterial organisms, 23 (24.5%) grew Enterococcus sp and 19 (20.2%) grew Klebsiella oxytoca. Female mice were twice more likely than males to present with nephritis. These findings indicate that bacterial nephritis is a major contributor to morbidity in the NSG strain.

The de-ubiquitinase UCH-L1 is an oncogene that drives the development of lymphoma in vivo by deregulating PHLPP1 and Akt signaling.

De-ubiquitinating enzymes (DUBs) can reverse the modifications catalyzed by ubiquitin ligases and as such are believed to be important regulators of a variety of cellular processes. Several members of this protein family have been associated with human cancers; however, there is little evidence for a direct link between deregulated de-ubiquitination and neoplastic transformation. Ubiquitin C-terminal hydrolase (UCH)-L1 is a DUB of unknown function that is overexpressed in several human cancers, but whether it has oncogenic properties has not been established. To address this issue, we generated mice that overexpress UCH-L1 under the control of a ubiquitous promoter. Here, we show that UCH-L1 transgenic mice are prone to malignancy, primarily lymphomas and lung tumors. Furthermore, UCH-L1 overexpression strongly accelerated lymphomagenesis in Emu-myc transgenic mice. Aberrantly expressed UCH-L1 boosts signaling through the Akt pathway by downregulating the antagonistic phosphatase PHLPP1, an event that requires its de-ubiquitinase activity. These data provide the first in vivo evidence for DUB-driven oncogenesis and suggest that UCH-L1 hyperactivity deregulates normal Akt signaling.

Retrospective study of spontaneous osteosarcomas in the nonobese diabetic strain and nonobese diabetic-derived substrains of mice.

In this retrospective study, spontaneous osteosarcomas were found in 85 of 1,202 (7.1%) nonobese diabetic (NOD) and NOD-derived mice. Gross tumors were evident at an average age of 155.8 days in male mice and 151.4 days in female mice. Compared with male mice, female mice had a statistically insignificant higher incidence: 56 cases (8.3% of 672) versus 28 cases (6.1% of 458). NOD/ShiLtJ mice had the highest incidence, with 39 cases among all the strains and substrains represented (3.2% of 1,202 necropsies), whereas NOD.SCID substrains had the highest incidence, with 16 cases among the various NOD-derived substrains (1.3% of 1,202 necropsies). There was a statistically significant difference in tumor incidence between NOD/ShiLtJ and NOD.SCID mice. Tumors were more frequent in the appendicular skeleton (55.7%) than in the axial skeleton (44.3%) and most often arose from the femurs. Histologically, osteoblastic osteosarcoma was the most common tumor type, with 79 cases (94%), followed by mixed osteosarcoma, with 5 cases (6%). Metastases were rare, with only 2 cases (2.3%).

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rgamma(null)) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rgamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

Non-obese diabetic-recombination activating gene-1 (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment.

Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non-obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene-1 (Rag1(null)) and interleukin (IL)-2 receptor common gamma chain (IL2rgamma(null)), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17-Prkdc(scid) (NOD-Prkdc(scid)) IL2rgamma(null) mice. We observed that immunodeficient NOD-Rag1(null) IL2rgamma(null) mice tolerated much higher levels of irradiation conditioning than did NOD-Prkdc(scid) IL2rgamma(null) mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation-conditioned adult mice, leading to multi-lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1(null) and scid stocks of immunodeficient NOD mice harbouring the IL2rgamma(null) mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD-Rag1(null) IL2rgamma(null) mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

A new immunodeficient hyperglycaemic mouse model based on the Ins2Akita mutation for analyses of human islet and beta stem and progenitor cell function.

AIMS/HYPOTHESIS: To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects. METHODS: We generated and characterised a new strain of immunodeficient spontaneously hyperglycaemic mice, the NOD-Rag1null Prf1null Ins2Akita strain and compared this strain with the NOD-scid Il2rgammanull (also known as Il2rg) immunodeficient strain rendered hyperglycaemic by administration of a single dose of streptozotocin. Hyperglycaemic mice were transplanted with human islets ranging from 1,000 to 4,000 islet equivalents (IEQ) and were monitored for normalisation of blood glucose levels. RESULTS: NOD-Rag1null Prf1null Ins2Akita mice developed spontaneous hyperglycaemia, similar to Ins2Akita-harbouring strains of immunocompetent mice. Histological examination of islets in the host pancreas validated the spontaneous loss of beta cell mass in the absence of mononuclear cell infiltration. Human islets transplanted into spontaneously diabetic NOD-Rag1null Prf1null Ins2Akita and chemically diabetic NOD-scid Il2rgammanull mice resulted in a return to euglycaemia that occurred with transplantation of similar beta cell masses. CONCLUSIONS/INTERPRETATION: The NOD-Rag1null Prf1null Ins2Akita mouse is the first immunodeficient, spontaneously hyperglycaemic mouse strain described that is based on the Ins2Akita mutation. This strain is suitable as hosts for human islet and human beta stem and progenitor cell transplantation in the absence of the need for pharmacological induction of diabetes. This strain of mice also has low levels of innate immunity and can be engrafted with a human immune system for the study of human islet allograft rejection.

Comparative therapeutic effects of orally administered 1,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(3) on type-1 diabetes in non-obese diabetic mice fed a normal-calcaemic diet.

Frequent injections of the hormonal form of vitamin D(3), 1,25 dihydroxyvitamin D(3) (1,25D3) reportedly inhibits autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by correcting some of the abnormalities in antigen-presenting cells which contribute the development of pathogenic T cell responses. This route of administration greatly elevates the levels of these compounds in the bloodstream for hours after treatment, which requires mice to be fed diets formulated to contain much reduced levels of Ca to avoid the toxic effects of hypercalcaemia. In the current work, we demonstrate that feeding 1,25D3 or its synthetic precursor, 1alpha(OH) vitamin D(3) (1alphaD3), as part of a T1D supportive chow diet containing normal levels of Ca, is an effective means of reducing the incidence of disease in NOD mice, but the doses required for protection elicited hypercalcaemia. However, T1D protection elicited by D3 analogue feeding appears, at least partially, to have an immunological basis, as splenic T cells from treated mice had a decreased capacity to adoptively transfer disease. Protection is associated with an increased proportion of T cells with CD4+ forkhead box P3+ regulatory phenotype within the islet infiltrate of treated animals. The 1alphaD3 precursor is converted rapidly to the active 1,25D3 isoform in vivo. However, feeding the 1alphaD3 analogue elicited stronger T1D protection than the 1,25D3 compound, but also induced more severe hypercalcaemia. In future, the dietary supplementation of novel low-calcaemic D3 analogues may enable their continuous delivery at levels that inhibit T1D development in susceptible humans consuming normal levels of Ca.

The nonhomologous end joining factor Artemis suppresses multi-tissue tumor formation and prevents loss of heterozygosity.

Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ARTEMIS cause radiation-sensitive severe combined immunodeficiency in humans and may increase susceptibility to lymphoma in some settings. We now report that deficiency for Artemis (encoded by Dclre1c/Art in mouse) accelerates tumorigenesis in several tissues in a Trp53 heterozygous setting, revealing tumor suppression roles for NHEJ in lymphoid and non-lymphoid cells. We also show that B-lineage lymphomas in these mice undergo loss of Trp53 heterozygosity by allele replacement, but arise by mechanisms distinct from those in Art Trp53 double null mice. These findings demonstrate a general tumor suppression function for NHEJ, and reveal that interplay between NHEJ and Trp53 loss of heterozygosity influences the sequence of multi-hit oncogenesis. We present a model where p53 status at the time of tumor initiation is a key determinant of subsequent oncogenic mechanisms. Because Art deficient mice represent a model for radiation-sensitive severe combined immunodeficiency, our findings suggest that these patients may be at risk for both lymphoid and non-lymphoid cancers.

Development of a new canine osteosarcoma cell line.

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1-deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.

Disseminated infection with Balamuthia mandrillaris in a dog.

Bilateral chronic granulomatous nephritis and meningoencephalitis were diagnosed on necropsy of a 2-year-old male Great Dane dog. The causative agent was identified as Balamuthia mandrillaris, based on morphologic features, immunohistochemical staining, and deoxyribonucleic acid detection using the polymerase chain reaction with newly designed primer pairs. Trophozoite and cystic forms of the amoeba were evident within the kidneys and brain parenchyma. This is the first report on a B. mandrillaris infection in a dog.