ABSTRACT
This corrects the article DOI: 10.1038/onc.2015.483.
ABSTRACT
Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.
Subject(s)
GTP Phosphohydrolases/genetics , Gene Expression Regulation, Neoplastic , Leukemia, Experimental/pathology , Leukemia, Myeloid, Acute/pathology , Membrane Proteins/genetics , Mutation/genetics , ral GTP-Binding Proteins/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , Female , Humans , Leukemia, Experimental/genetics , Leukemia, Experimental/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Mice, SCID , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Neoplasm Invasiveness , Oncogenes , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , ral GTP-Binding Proteins/geneticsABSTRACT
CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt ß-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.
Subject(s)
Colorectal Neoplasms/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Genes, Tumor Suppressor , Animals , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/pathology , Disease-Free Survival , Gene Deletion , Gene Expression Regulation, Neoplastic , Humans , Mice , Mutation , Signal TransductionABSTRACT
Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.
Subject(s)
B-Lymphocytes/metabolism , DNA Transposable Elements/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , STAT5 Transcription Factor/genetics , Animals , Blotting, Western , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression Regulation, Leukemic , Ikaros Transcription Factor/genetics , Ikaros Transcription Factor/metabolism , Janus Kinase 1/genetics , Janus Kinase 1/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Kaplan-Meier Estimate , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Insertional , Mutation , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SOS1 Protein/genetics , SOS1 Protein/metabolism , STAT5 Transcription Factor/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Kcnq1, which encodes for the pore-forming α-subunit of a voltage-gated potassium channel, was identified as a gastrointestinal (GI) tract cancer susceptibility gene in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. To confirm that Kcnq1 has a functional role in GI tract cancer, we created Apc(Min) mice that carried a targeted deletion mutation in Kcnq1. Results demonstrated that Kcnq1 is a tumor suppressor gene as Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, and some of these tumors progressed to become aggressive adenocarcinomas. Gross tissue abnormalities were also observed in the rectum, pancreas and stomach. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant mice compared with wild-type littermate controls, suggesting a role for Kcnq1 in the regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1, we carried out microarray studies in the colon and proximal small intestine. We identified altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, epidermal growth factor receptor and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2 and CFTR, two important regulators of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC), we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival.
Subject(s)
Gastrointestinal Neoplasms/genetics , Gastrointestinal Neoplasms/metabolism , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Disease Models, Animal , Disease Progression , Female , Gastrointestinal Neoplasms/mortality , Gastrointestinal Neoplasms/pathology , Gene Expression Profiling , Humans , Intestine, Small/metabolism , Intestine, Small/pathology , Male , Mice , Mice, Knockout , Mucin-2/genetics , Mucin-2/metabolism , Neoplasm Staging , Phenotype , Prognosis , Signal TransductionABSTRACT
Recognizing and avoiding aversive situations are central aspects of mammalian cognition. These abilities are essential for health and survival and are expected to have a prominent genetic basis. We modeled these abilities in eight common mouse inbred strains covering â¼75% of the species' natural variation and in gene-trap mice (>2000 mice), using an unsupervised, automated assay with an instrumented home cage (PhenoTyper) containing a shelter with two entrances. Mice visited this shelter for 20-1200 times/24 h and 71% of all mice developed a significant and often strong preference for one entrance. Subsequently, a mild aversive stimulus (shelter illumination) was automatically delivered when mice used their preferred entrance. Different genotypes developed different coping strategies. Firstly, the number of entries via the preferred entrance decreased in DBA/2J, C57BL/6J and 129S1/SvImJ, indicating that these genotypes associated one specific entrance with the aversive stimulus. Secondly, mice started sleeping outside (C57BL/6J, DBA/2J), indicating they associated the shelter, in general, with the aversive stimulus. Some mice showed no evidence for an association between the entrance and the aversive light, but did show markedly shorter shelter residence times in response to illumination, indicating they did perceive illumination as aversive. Finally, using this assay, we screened 43 different mutants, which yielded a novel gene, specc1/cytospinB. This mutant showed profound and specific delay in avoidance learning. Together, these data suggest that different genotypes express distinct learning and/or memory of associations between shelter entrance and aversive stimuli, and that specc1/cytospinB is involved in this aspect of cognition.
Subject(s)
Avoidance Learning , Genotype , Nuclear Proteins/genetics , Phenotype , Phosphoproteins/genetics , Animals , Cell Cycle Proteins , Cytoskeletal Proteins , High-Throughput Screening Assays/methods , Male , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Phosphoproteins/metabolismSubject(s)
Antimetabolites, Antineoplastic/therapeutic use , Cytarabine/therapeutic use , Gene Expression Profiling , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Oligonucleotide Array Sequence Analysis/methods , Amyloid beta-Protein Precursor/genetics , DNA Mismatch Repair , Drug Resistance, Neoplasm , Humans , Proto-Oncogene Proteins c-bcl-2/geneticsSubject(s)
Cytarabine/therapeutic use , Leukemia, Myeloid/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurofibromin 1/genetics , Protein Kinases/metabolism , Acute Disease , Animals , Cell Death/drug effects , Cell Line, Tumor , Cytarabine/pharmacology , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid/drug therapy , Mice , Neurofibromin 1/deficiency , Neurofibromin 1/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine KinasesABSTRACT
Micronucleus assay and dual color-fluorescence in situ hybridization (DC-FISH), using centromere-specific and whole chromosome-specific painting probes, are considered a useful screening test to determine the incidence of micronucleus, their origin and contents. The patients with acute lymphoblastic leukemia (ALL), who had undergone chemotherapy, were analysed before and after treatment with vincristine, methotrexate, daunomycin, prednisone, and asparaginase. The incidence of micronuclei after the antileukemic agent treatment was significantly higher than before the treatment. Application of DC-FISH using a combination of whole chromosome-specific painting probes and the same chromosome-specific alpha-satellite centromeric probe showed that there were no significant differences in the micronucleus incidence for any specific chromosome (chromosomes 7, 8, 11, 17, X, and Y). There were no significant differences between the incidence of centromere-positive micronuclei and the incidence of centromere-negative micronucleus. We concluded that antileukemic agents induced the somatic genetic damage but this damage is not related to any specific chromosome studied.
Subject(s)
Antineoplastic Agents/adverse effects , In Situ Hybridization, Fluorescence , Micronuclei, Chromosome-Defective/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Child, Preschool , Humans , Incidence , Infant , Micronucleus Tests , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
We present a rare case of a paediatric myelodysplastic syndrome (MDS) with congenital anomalies (frontal bossing and premature closure of anterior fontanelle). The case showed the clinical and biological features of a refractory anaemia excess blasts (RAEB). Bone marrow (BM) cytogenetics demonstrated a hyperdiploid karyotype, with several numerical abnormalities and unidentified rearrangements. Fluorescence in situ hybridization (FISH) using chromosome specific alpha-satellite and whole chromosome-specific painting probes verified the hyperdiploid karyotype, and confirmed the origin of the unknown markers and rearrangements more reliably than would be possible using conventional cytogenetic techniques.
Subject(s)
Myelodysplastic Syndromes/genetics , Polyploidy , Abnormalities, Multiple , Anemia, Refractory, with Excess of Blasts/genetics , Chromosome Aberrations , Cytogenetic Analysis , Humans , In Situ Hybridization, Fluorescence , Infant , Karyotyping , MaleABSTRACT
Juvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1 gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137-143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of the Myb gene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1 cooperate with loss of Nf1 to cause AML.
Subject(s)
Leukemia, Experimental/genetics , Leukemia, Myeloid/genetics , Acute Disease , Animals , Gene Deletion , Genes, Neurofibromatosis 1 , Genes, myb , Genetic Predisposition to Disease , Leukemia, Experimental/etiology , Leukemia, Experimental/virology , Leukemia, Myeloid/etiology , Leukemia, Myeloid/virology , Mice , Retroviridae/genetics , Virus Integration/geneticsABSTRACT
OBJECTIVE: The homeobox gene Hoxb8 is activated in the murine myelomonocytic cell line WEHI-3B as a result of intracisternal A particle integration. Cooperative activation between Hoxa9 and Meis1 is induced by retroviral integration in BXH2 murine myeloid leukemias and the myeloid leukemia cell line M1. The present study was conducted to examine possible Meis gene activation and cooperative DNA binding of homeobox proteins in WEHI-3B and to reveal the specific role of Hox and Meis genes in myeloid differentiation. MATERIALS AND METHODS: Northern blot analysis and reverse transcriptase polymerase chain reaction were performed to examine homeobox genes expression. Electrophoretic mobility shift assay was performed to evaluate DNA binding of homeobox proteins. Myeloid differentiation of 32Dcl3 was induced by granulocyte colony-stimulating factor. RESULTS: Meis2 was coactivated with Hoxb8 in WEHI-3B cells. DNA-protein complexes including Hox, Meis, and Pbx were observed in WEHI-3B and 32Dcl3. Expression and the DNA-binding complex of Hoxa9, Hoxb8, Meis1, and Meis2 were down-regulated during myeloid differentiation of 32Dcl3 cells. Enforced expression of Hox or Meis genes inhibited myeloid differentiation of 32Dcl3. CONCLUSION: The results indicate that Meis2 is an important Meis gene for myeloid leukemogenesis and that Hox and Meis are important genes for myeloid leukemogenesis through differentiation block.
Subject(s)
Homeodomain Proteins/genetics , Leukopoiesis/genetics , Animals , Cell Differentiation/genetics , Cell Line , Gene Expression Regulation , Mice , Transcriptional ActivationABSTRACT
We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation-site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)-trap transposon vectors, were outcrossed to wild-type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics.
Subject(s)
DNA Transposable Elements/genetics , Gene Transfer Techniques , Mutagenesis, Insertional , Spermatozoa , Transposases/genetics , Animals , Base Sequence , Crosses, Genetic , DNA/genetics , DNA/metabolism , Female , Gene Expression , Genes, Synthetic , Male , Mice , Mice, Transgenic , Models, Animal , Molecular Sequence Data , Transformation, GeneticABSTRACT
Introducing dominant oncogenic alterations uncovered in human myeloid malignancies into the mouse germline provides a powerful approach for studying leukemogenesis. However, little is known about how gene inactivation contributes to the development of myeloid malignancies. We describe how Nf1 mutant mice provide one example in which disrupting a tumor suppressor gene has been used to generate an informative murine leukemia model. We also discuss how chromosome engineering technologies are being harnessed to model the segmental deletions found in myeloid malignancies, and how these approaches can be combined with retrovirally medicated insertional mutagenesis to generate new models and for gene discovery.
Subject(s)
Disease Models, Animal , Genes, Tumor Suppressor , Leukemia, Myeloid/genetics , Animals , Chromosome Deletion , Humans , Mice , Mice, Mutant StrainsABSTRACT
Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factor CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show that CalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification of CalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.
Subject(s)
Guanine Nucleotide Exchange Factors/genetics , Leukemia, Myeloid/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA , Fibroblasts/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Humans , Leukemia, Myeloid/pathology , Mice , Molecular Sequence Data , Proto-Oncogene Mas , Rats , Signal Transduction , Tumor Cells, CulturedSubject(s)
Cell Transformation, Neoplastic/genetics , Gene Dosage , Genes, Tumor Suppressor , Leukemia, Promyelocytic, Acute/etiology , Neoplasm Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis/genetics , Leukemia, Promyelocytic, Acute/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , TransgenesABSTRACT
The cellular receptor for murine thymic stromal lymphopoietin (TSLP) was detected in a variety of murine, but not human myelomonocytic cell lines by radioligand binding. cDNA clones encoding the receptor were isolated from a murine T helper cell cDNA library. TSLP receptor (TSLPR) is a member of the hematopoietin receptor family. Transfection of TSLPR cDNA resulted in only low affinity binding. Cotransfection of the interleukin 7 (IL-7)Ralpha chain cDNA resulted in conversion to high affinity binding. TSLP did not activate cells from IL-7Ralpha(-/)- mice, but did activate cells from gammac(-/)- mice. Thus, the functional TSLPR requires the IL-7Ralpha chain, but not the gammac chain for signaling.
Subject(s)
Hematopoiesis/drug effects , Lymphocytes/drug effects , Receptors, Cytokine/physiology , Receptors, Interleukin-7/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chromosome Mapping , Cloning, Molecular , Cytokines/pharmacology , Humans , Interleukin-7/pharmacology , Lymphocytes/physiology , Male , Mice , Molecular Sequence Data , RNA, Messenger/analysis , Receptors, Cytokine/chemistry , Receptors, Cytokine/genetics , Receptors, Interleukin-7/chemistry , Recombinant Proteins/chemistry , Signal TransductionABSTRACT
This study was undertaken to develop a reliable method to enumerate and map somatically acquired, clonal, murine leukemia virus (MuLV) proviral insertions in acute myeloid leukemia (AML) cells from the BXH-2 mouse strain. This was achieved by using fluorescence in situ hybridization combined with tyramide signal amplification (FISH-TSA) and an 8.8 kilobase pair (kb) full-length ecotropic MuLV or 2.0 kb MuLV envelope (env) gene probe. Two-color FISH was utilized combining chromosome-specific probes for regions near the telomere and/or centromere and the MuLV probes. The technique reliably detected germline and somatically acquired, tumor-specific, MuLV proviruses in BXH-2 AML cell lines. It was possible to readily verify homozygous insertions at endogenous ecotropic MuLV loci, Emv1 (chromosome 5), Emv2 (chromosome 8) and a BXH-2 strain-specific locus (chromosome 11). This strategy also verified the presence of molecularly cloned proviral insertions within the mouse Nf1 gene and another locus on distal chromosome 11, as well as on chromosome 7 and chromosome 9 in BXH-2 AML cell line B117. The technique was also used to detect several new tumor-specific, proviral insertions in BXH-2 AML cell lines.
Subject(s)
DNA Transposable Elements/genetics , Leukemia Virus, Murine/genetics , Leukemia, Myeloid/genetics , Leukemia, Myeloid/virology , Proviruses/genetics , Acute Disease , Animals , Chromosome Mapping/methods , Crosses, Genetic , DNA Probes , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Disease Models, Animal , Female , In Situ Hybridization, Fluorescence/methods , Male , Mice , Mice, Inbred C57BL , Retroviridae Infections/genetics , Retroviridae Infections/virology , Tumor Virus Infections/genetics , Tumor Virus Infections/virology , Tyramine , Virus Integration/geneticsABSTRACT
The study of myeloid leukemia induced by slow transforming murine leukemia viruses (MuLV) in the laboratory mouse has led to discovery of many important genes with critical roles in regulating the growth, death, lineage determination and development of hematopoietic precursor cells. This review provides an overview of the susceptible strains and virus isolates that cause acute myeloid leukemia (AML) in mice. In addition, newer methodologies, involving the use of the polymerase chain reaction, that have been used to identify cancer genes mutated by proviral insertion in mouse models, will be discussed. As cancer is a multi-gene disease, a system in which pairs of oncogenic mutations are classified as redundant, neutral or synergistic is described. The potential to combine MuLV mutagenesis with recent advances in mouse transgenesis in order to model specific forms of myeloid leukemia or genetic pathways common in human AML will be discussed. Finally, a general strategy for maximizing these genetically rich models to foster a better understanding of AML physiology and developing therapies is proposed.