Mouse model systems to study multistep tumorigenesis.
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Animals↗
Biomedical subjects
J Domen
Publications and source records attributed to J Domen.
Pim-1 levels determine the size of early B lymphoid compartments in bone marrow.
The mouse proto-oncogene Pim-1, which encodes two cytoplasmic serine-threonine-specific protein kinases, is frequently activated by proviral insertion in murine leukemia virus-induced hematopoietic tumors. Transgenic mice overexpressing Pim-1 show a low incidence of spontaneous T cell lymphomas, whereas null mutant mice lack an obvious phenotype. We have analyzed the early B lymphoid compartment from both null mutant and E mu-Pim-1 transgenic mice. The level of Pim-1 expression appears to be a determining factor in the ability of these cells to respond to the growth factors interleukin 7 (IL-7) and SF (steel factor). The impaired response in null mutant mice could be rescued by introduction of a functional Pim-1 transgene. Moreover, overexpression of Pim-1 facilitates the derivation of primitive lymphoid cell lines that are dependent on combined stimulation with IL-7 and SF or insulin-like growth factor 1. These results for the first time identify the involvement of Pim-1 in a normal cellular function, as an important regulator of early B lymphopoiesis in mice.
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In vivo analysis of Pim-1 deficiency.
The Pim-1 proto-oncogene encodes a highly conserved serine/threonine phosphokinase which is predominantly expressed in hematopoietic organs and gonads in mammals. Overexpression of Pim-1 predisposes to lymphomagenesis in mice. To develop a further understanding of Pim-1 in molecular terms, as well as in terms of its potential role in hematopoietic development, we have generated mice deficient in Pim-1 function. Pim-1-deficient mice are ostensibly normal, healthy and fertile. Detailed comparative analyses of the hematopoietic systems of the mutant mice and their wild-type littermates showed that they are indistinguishable for most of the parameters studied. Our analyses revealed one unexpected phenotype that correlated with the level of Pim-1 expression: Pim-1 deficiency correlated with a erythrocyte microcytosis, whereas overexpression of Pim-1 in E mu-Pim-1-transgenic mice resulted in erythrocyte macrocytosis. In order to confirm that the observed decrease in erythrocyte Mean Cell Volume (MCV) was attributable to the Pim-1 deficiency, we developed mice transgenic for a Pim-1 gene construct with its own promoter and showed that this transgene could restore the low erythrocyte Mean Cell Volume observed in the Pim-1-deficient mice to near wild-type levels. These results might be relevant to the observed involvement of the Pim-1 gene in mouse erythroleukemogenesis. The surprising lack of a readily observed phenotype in the lymphoid compartment of the Pim-1-deficient mice, suggests a heretofore unrecognized degree of in vivo functional redundancy of this highly conserved proto-oncogene.
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Impaired interleukin-3 response in Pim-1-deficient bone marrow-derived mast cells.
The mouse Pim-1 gene encodes two cytoplasmic serine-threonine-specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (E mu-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-nitrosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow-derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.
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Analysis of Pim-1 function in mutant mice.
The Pim-1 gene has frequently been found activated by proviral insertion in haematopoietic tumors in mice. The fact that overexpression of Pim-1 can contribute to lymphomagenesis was formally proven by overexpressing a Pim-1 transgene in lymphoid cells. The transgene induces a low incidence of T cell lymphomas and an increased susceptibility to chemically (ENU) and virally (MoMuLV) induced lymphomas. The mouse Pim-1 gene encodes two cytoplasmic protein-serine/threonine kinases. Northern analysis shows the highest expression to be in haematopoietic tissues, especially early in development. High expression has also been noted in testis and ES cells. Expression can be induced by growth factors and mitogens. The gene is evolutionarily highly conserved. Inactivation of both Pim-1 alleles in ES cells or mice did not reveal any obvious abnormalities. In order to look more closely for possible haematopoietic abnormalities specific growth factor response were studied in vitro. The IL-3 response of bone marrow-derived mast-cell cultures (BMMC) was found to be severely impaired in mast cells derived from Pim-1 deficient mice.
Alleles↗
Collaboration of pim-1 and bcl-2 in lymphomagenesis.
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Animals↗
Herpesviruses encode an unusual protein-serine/threonine kinase which is nonessential for growth in cultured cells.
We have performed large-scale random oligonucleotide insertion mutagenesis on a 41-kbp genomic segment derived from the unique long (UL) region of the alphaherpesvirus pseudorabies virus (PRV). This procedure has resulted in the generation of a series of PRV strains, each carrying a single gene whose termination of translation is induced by the inserted oligonucleotide. To relate the genes that were involved in the mutagenization to genes previously identified in herpes simplex virus type 1, the prototype alphaherpesvirus, we have performed cross-hybridization studies. In this way, we have mapped the location of the homolog of a gene which was described to have sequence characteristics of a eukaryotic phosphotransferase. We characterized the phenotype of a mutant PRV strain lacking this putative phosphotransferase also the phenotype of a PRV strain lacking, in addition to the UL-encoded putative phosphotransferase, the protein kinase encoded within the unique short region of the virus. To assess the enzymatic activity of the UL region-encoded phosphotransferase, we expressed the gene transiently in a eukaryotic expression system. Immunoprecipitation of the protein followed by kinase assays and phosphoamino acid analyses revealed protein-serine/threonine kinase activity. Implications of sequence divergence of this protein from classical protein-serine/threonine kinases for kinase structure and function are discussed in view of the recent resolution of the structure of the catalytic domain of cyclic AMP-dependent protein kinase.
Amino Acid Sequence↗
The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.
The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.
Amino Acid Sequence↗
Mice bearing the E mu-myc and E mu-pim-1 transgenes develop pre-B-cell leukemia prenatally.
Previously, it has been shown that E mu-pim-1 transgenic mice are predisposed to T-cell lymphomas, whereas E mu-myc transgenic mice are predisposed to pre-B-cell lymphomas. Here we show that double-transgenic E mu-myc E mu-pim-1 mice exhibit pre-B-cell leukemia in utero. Upon transplantation into recipient mice, embryo-derived double-transgenic leukemic cells frequently progressed to highly malignant monoclonal tumors, indicating that additional (epi)genetic events had occurred during the progression of the disease.
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Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors.
Transgenic mice bearing the pim-1 gene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukemia virus long terminal repeat express pim-1 mRNA at high levels in both B and T cells. Between 5% and 10% of the pim-1 transgenic mice develop clonal T cell lymphomas before 7 months of age, whereas none of the age-matched control mice do, providing direct evidence for the oncogenic potential of pim-1. Histological examination and FACS analysis revealed no abnormalities in hematopoietic tissues of disease-free pim-1 transgenic mice. When newborn pim-1 transgenic mice are infected with MuLV, T cell lymphomas develop much faster (latency 7-8 weeks) than in nontransgenic mice (latency 22 weeks). In all these T cell lymphomas either c-myc or N-myc was activated by proviral insertion, suggesting strong cooperation between pim-1 and myc in lymphomagenesis.
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Comparison of the human and mouse PIM-1 cDNAs: nucleotide sequence and immunological identification of the in vitro synthesized PIM-1 protein.
Pim-1 is a putative oncogene which was discovered as a target for transcriptionally activating proviral insertions in T-cell lymphomas in mice. Its genomic and cDNA sequence have been published (Selten et al., 1986, Cell 46, 603-611). We have isolated and sequenced pim-1 cDNA clones from the human leukemia cell line K562 (h-pim-1). Comparison of human and mouse cDNA sequences reveals that both contain: (i) a GC rich leader sequence, (ii) an open reading frame encoding a 313 amino acid protein (94% conserved) showing obvious homology to protein kinases and (iii) a 1.3 kb 3' untranslated region with two polyadenylation signals and five copies of the mRNA destabilizing motif ATTTA. "In vitro" translation experiments show that both mouse and human cDNA derived RNA can translate into a protein of the expected size. Their identity was confirmed by immuno-precipitation with antisera raised against synthetic pim-1 oligopeptides.
Amino Acid Sequence↗
The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases.
We have shown previously that the putative oncogene pim-1 is frequently activated by provirus insertion in murine leukemia virus-induced T cell lymphomas. Here we describe the structure of the pim-1 gene as determined by sequencing genomic and cDNA clones. The gene has an open reading frame, encoding a protein of 313 amino acids, extending over six exons and preceded and followed by stop codons in all reading frames. Proviruses always integrate outside the protein-encoding domain, showing a high preference for a small region in the 3'-terminal exon; integration in the 3' exon results in relatively high levels of pim-1 mRNA. Computer search reveals homology between pim-1 and protein kinases: all the domains characteristic of protein kinases are conserved in the pim-1 amino acid sequence. The highest homologies were observed with the protein-serine kinases.
Amino Acid Sequence↗
A vital staining method for measuring the efficiency of transfection of eucaryotic cells.
The xylE gene encodes catechol 2,3-dioxygenase, which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. The expression of this gene in eucaryotic cells can be detected simply by addition of catechol to the growth medium of the cells: cells that have a sufficient level of expression of the xylE gene stain yellow because of the accumulation of 2-hydroxymuconic semialdehyde. The number of stained cells is thus dependent upon the transfection efficiency as well as the level of expression of the xylE gene and is a measure of the combined transfection/expression efficiency in a particular cell type. Since the staining procedure does not affect the viability of the culture, the cells can be harvested afterward and analyzed for the expression of other, cotransfected, genes. This system for measuring transfection efficiency is especially useful when only small amounts of tissue are available.
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