ABSTRACT
Alternative splicing of the oncogene murine double minute 2 (MDM2) is induced in response to genotoxic stress. MDM2-ALT1, the major splice variant generated, is known to activate the p53 pathway and impede full-length MDM2's negative regulation of p53. Despite this perceptible tumor-suppressive role, MDM2-ALT1 is also associated with several cancers. Furthermore, expression of MDM2-ALT1 has been observed in aggressive metastatic disease in pediatric rhabdomyosarcoma (RMS), irrespective of histological subtype. Therefore, we generated a transgenic MDM2-ALT1 mouse model that would allow us to investigate the effects of this splice variant on the progression of tumorigenesis. Here we show that when MDM2-ALT1 is ubiquitously expressed in p53 null mice it leads to increased incidence of spindle cell sarcomas, including RMS. Our data provide evidence that constitutive MDM2-ALT1 expression is itself an oncogenic lesion that aggravates the tumorigenesis induced by p53 loss. On the contrary, when MDM2-ALT1 is expressed solely in B-cells in the presence of homozygous wild-type p53 it leads to significantly increased lymphomagenesis (56%) when compared with control mice (27%). However, this phenotype is observable only at later stages in life (⩾18 months). Moreover, flow cytometric analyses for B-cell markers revealed an MDM2-ALT1-associated decrease in the B-cell population of the spleens of these animals. Our data suggest that the B-cell loss is p53 dependent and is a response mounted to persistent MDM2-ALT1 expression in a wild-type p53 background. Overall, our findings highlight the importance of an MDM2 splice variant as a critical modifier of both p53-dependent and -independent tumorigenesis, underscoring the complexity of MDM2 posttranscriptional regulation in cancer. Furthermore, MDM2-ALT1-expressing p53 null mice represent a novel mouse model of fusion-negative RMS.
Subject(s)
Carcinogenesis/genetics , Gene Expression Regulation, Neoplastic , Mice , Proto-Oncogene Proteins c-mdm2/genetics , Rhabdomyosarcoma/genetics , Alternative Splicing , Animals , B-Lymphocytes/metabolism , Cell Proliferation/genetics , Female , Humans , MCF-7 Cells , Male , Mice, Transgenic , NIH 3T3 Cells , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Oncogenes , Proto-Oncogene Proteins c-mdm2/metabolism , Rhabdomyosarcoma/pathology , Signal Transduction/genetics , Spleen/cytology , Spleen/pathology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Barbiturate euthanasia solutions are a humane and approved means of euthanasia. Overdosing causes significant tissue damage in a variety of laboratory animals. METHODS: One hundred seventeen non-human primates (NHP) representing 7 species including 12 fetuses euthanized for humane and research reasons by various vascular routes with Euthasol, Sodium Pentobarbital, Fatal Plus, Beuthanasia D, or Euthanasia 5 were evaluated for euthanasia-induced tissue damage. Lungs and livers were histologically graded for hemolysis, vascular damage, edema, and necrosis. Severity of tissue damage was analyzed for differences on the basis of agent, age, sex, dose, and injection route. RESULTS: Severity of tissue damage was directly related to dose and the intracardiac injection route, but did not differ by species, sex, and agent used. CONCLUSIONS: When the recommended dose of agent was used, tissue damage was generally reduced, minimal, or undetectable. Barbiturate-induced artifacts in NHPs are essentially the same as in other laboratory species.
Subject(s)
Callithrix , Cercopithecinae , Euthanasia , Pentobarbital/administration & dosage , Pentobarbital/pharmacology , Saguinus , Animals , Dose-Response Relationship, Drug , Drug Combinations , Female , Hypnotics and Sedatives/administration & dosage , Hypnotics and Sedatives/pharmacology , Liver/pathology , Lung/pathology , MaleABSTRACT
A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.
Subject(s)
Endothelium/enzymology , Peptidyl-Dipeptidase A/immunology , Animals , Antibodies, Monoclonal , Cattle , Cells, Cultured , Humans , MiceABSTRACT
This report describes the preparation of a highly specific reagent (monoclonal antibody) that reacts selectively with large ganglion cells of bovine retina. Other cells populations of bovine retina react weakly or not at all, but neurons in brain regions of ox other than retina also bind the immunoglobulins specifically. Species specificity was indicated by a weak reaction with cat and dog retinal neurons, and organ specificity was shown by lack of reaction with bovine liver. This highly specific reagent can be used to classify the subsets of large retinal ganglion cells on the basis of their antigenic properties and to identify neurons in different brains regions that share antigenic determinants.