Construction of a versatile set of retroviral vectors conferring hygromycin resistance.
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Animals↗
Biomedical subjects
W Ostertag
Publications and source records attributed to W Ostertag.
CSF-1 deficiency in the op/op mouse has differential effects on macrophage populations and differentiation stages.
Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.
Animals↗
Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation.
Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.
Animals↗
Retroviral vectors related to the myeloproliferative sarcoma virus allow efficient expression in hematopoietic stem and precursor cell lines, but retroviral infection is reduced in more primitive cells.
Retroviral vectors are considered to be the most suited vehicles for somatic gene therapy with hematopoietic stem cells as targets. Retrovirus-mediated gene transfer into differentiation-restricted hematopoietic precursor (FDC-P1, FDC-P2) and multipotent progenitor (stem) cell lines (FDC-Pmix) is inefficient. Two cellular restrictions are involved. One is specific for stem but not precursor cells and is at the level of transcription. Due to a unique property of the transcriptional control region of the myeloproliferative sarcoma virus (MPSV), vectors derived from MPSV are not affected by this block. The second restriction occurs before proviral DNA synthesis and integration. This inhibition of effective viral infection depends on the state of differentiation, being more pronounced in multipotent clonogenic blast cells. This block to retroviral infection affects all retroviral vectors tested.
Animals↗
Abrogation of the requirement for feeder cell interaction and T cell receptor stimulation of lymphocytes infected with retroviral vectors.
Infection of sensitive adult mice with myeloproliferative sarcoma virus (MPSV) results in a myeloproliferative syndrome. Two components of the viral genome are required to induce this unique pathology: the mos oncogene and sequences within the U3 region of the long terminal repeat (LTR). In studies designed to identify the target cell of MPSV and thus better understand the mechanism by which a myeloproliferative syndrome is induced, we have infected a series of T cell lines with MPSV-based vectors. The results presented here show that infection with neoR MPSV abrogates the requirement for an antigen-specific or feeder cell-dependent stimulation, without altering the requirement for interleukin 2. Significantly, this response is not dependent on the mos oncogene, but requires sequences within the U3 region of the MPSV LTR. No alteration in the constitutive or induced levels of lymphokines released by these cells was observed. These results suggest a model in which T cells acquire a proliferative advantage by uncoupling the proliferative response from the lymphokine synthesis that is induced by activation of the T cell receptor. These cells are thus poised for antigen stimulation and secretion of cytokines that stimulate myelopoiesis.
Cell Line↗
Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.
The expression of Moloney murine leukemia virus and vectors derived from it is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have developed a retroviral vector, the murine embryonic stem cell virus (MESV), that is active in embryonal carcinoma and ES cells. MESV was derived from a retroviral mutant [PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV)] expressed in embryonal carcinoma cells but not in ES cells. The enhancer region of PCMV was shown to be functional in both cell types, but sequences within the 5' untranslated region of PCMV were found to restrict viral expression in ES cells. Replacement of this region by related sequences obtained from the dl-587rev retrovirus results in MESV, a modified PCMV virus that confers G418 resistance to fibroblasts and ES cells with similar efficiencies. Expression of MESV in ES cells is mediated by transcriptional regulatory elements within the 5' long terminal repeat of the viral genome.
Animals↗
The malignant histiocytosis sarcoma virus, a recombinant of Harvey murine sarcoma virus and Friend mink cell focus-forming virus, has acquired myeloid transformation specificity by alterations in the long terminal repeat.
The malignant histiocytosis sarcoma virus (MHSV), in contrast to other viruses with the ras oncogene, induces acute histiocytosis in newborn and adult mice. Molecular structure and function studies were initiated to determine the basis of its unique macrophage-transforming potential. Characterization of the genomic structure showed that the virus evolved by recombination of the Harvey murine sarcoma virus (Ha-MuSV) and a virus of the Friend-mink cell focus-forming virus family. Structural analysis of MHSV showed two regions of the genome that are basically different from the Ha-MuSV: (i) the ras gene, which is altered by a point mutation in codon 181 leading to a Cys----Ser substitution of the p21 protein, and (ii) the U3 region of the long terminal repeat, which is largely derived from F-MCFV and contains a deletion of one direct repeat as well as a duplication of an altered enhancer-like region. Biological studies of Ha-MuSV, MHSV, and recombinants between the two viruses show that the U3 region of the MHSV long terminal repeat is essential for the malignancy and specificity of the disease. A contributing role of the ras point mutation in determining macrophage specificity, however, cannot be excluded.
Amino Acid Sequence↗
The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.
Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.
Animals↗
Retrotransposons as mutagens in the induction of growth autonomy in hematopoietic cells.
Factor-independent mutants of hematopoietic cells, especially of multipotent cells, are valuable tools to identify genes that regulate stem cell proliferation and differentiation and thus may be important in leukemogenesis. Factor-independent mutants from both myeloid precursor and hematopoietic stem cell lines were isolated. The frequency of such mutants in a given cell population was one to two orders of magnitude lower for the multipotent cell line FDC-Pmix (3.6 x 10(-9)) than for the myeloid precursors, FDC-P1-M (1.7 x 10(-8)) and D35 (2.2 x 10(-7)). Analysis of these mutants revealed several mechanisms by which growth autonomy was obtained, either with or without direct contribution of growth factor gene activation. The molecular basis of spontaneous activation of the Multi-CSF (Interleukin3) gene was determined and compared to activation of the GM-CSF gene in a previous study. Multi-CSF gene activation in both precursor and stem cells was caused by the insertion of an intracisternal A particle (IAP) provirus. In two independent mutants of the D35 cell line, activation of the Multi-CSF or the GM-CSF gene was caused by almost identical IAPs with a 99% homology in the U3 and R region of the long terminal repeat. This result demonstrates that only one class of IAPs, or perhaps a single provirus, is involved in transposition and gene activation in a particular cell line. A unique example of anti-sense promotion from an IAP provirus in one Multi-CSF mutant underlines the versatility of these elements as natural insertional mutagens.
Amino Acid Sequence↗
Mechanisms of growth factor expression in acute myeloid leukemia (AML).
To investigate possible mechanisms of growth factor expression in acute myeloid leukemia, genes for granulocyte macrophage colony-stimulating factor (GM-CSF) were analyzed by Southern blots in 20 patients, for M-CSF in 13, for interleukin-6 (IL-6) in 14, for IL-6 receptor in 14 and for G-CSF in five patients. Only in one patient a complex rearrangement of the G-CSF gene with possible amplification was noted indicating rarity of direct alterations of growth factor genes in acute myelogenous leukemia (AML). Spontaneous m-RNA expression for GM-CSF was found in only one of 20 patients, and for IL-6 in eight of 11 patients. In vitro incubation of AML cells of eight patients with recombinant tumor necrosis factor for 24 hr revealed induction of GM-CSF m-RNA expression in three cases and GM-CSF protein expression in two of them. These data suggest that spontaneous GM-CSF production occurs rarely in AML and that monokines, such as tumor necrosis factor, may induce GM-CSF in AML cells. Therefore, interactions of AML cells with normal or malignant accessory cells may be important for autocrine stimulation in AML. Our data suggest that ectopic growth factor secretion is not the primary cause of generating AML but may contribute to progression of the disease. Alternatively, AML may represent a heterogenous group of leukemias with different etiology but similar phenotype.
Blotting, Northern↗
Virus-transformed macrophage-like cell line as tool for eicosanoid research.
The ability of a virus-transformed murine macrophage like cell line HA 38 to produce different eicosanoid metabolites was examined. HA 38 cells release similar amounts of prostaglandins and leukotrienes as did murine peritoneal macrophages in response to both physiological and non-physiological stimuli. Enzyme systems known to be involved in the regulation of eicosanoid synthesis are expressed. HA 38 cells thus are a well defined macrophage model system and are well suited to study eicosanoid synthesis in macrophages and effects of drugs on the prostaglandin and leukotriene synthesis pathways.
Animals↗
Aberrant expression of the multi-CSF gene in hematopoietic precursor and stem cell lines initiates leukemogenic progression.
Tumorigenesis of hemopoietic cells and acquisition of factor independence as a consequence of aberrant growth factor release are closely correlated. In previous work we were able to dissect two stages leading to growth factor autonomy of cells: the first step requires the secretion of the constitutively expressed CSF gene product and extracellular interaction with its cognate receptor. This requirement for external stimulation is abrogated by a second step. We were interested in characterizing the parameters that influence the conversion from nonautonomous to autonomous growth properties of hematopoietic precursor cells. The frequency with which this alteration occurs varies and correlates with the level of growth factor production. However, a significant increase of CSF production accompanying the progression to autonomy could not be detected. We thus conclude that there is no direct link between level of CSF production and acquisition of true autonomy but an indirect influence enhancing the frequency of genetic alteration(s) that lead to growth autonomy. Lang et al. have suggested that the acquisition of autonomous growth occurs due to internal receptor-ligand interaction. Indeed, Keating and Williams have claimed that PDGF may react with an intracellular PDGF receptor resulting in autocrine stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Infection of multipotent IL-3-dependent stem cells with a retroviral vector containing the IL-3 gene confers density-dependent growth autonomy without blocking differentiation.
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Animals↗
Biological effects of retroviral transfection of the murine interleukin-3 gene into FDCP-Mix cells.
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Animals↗
Conversion of factor-dependent myeloid cells to factor independence: autocrine stimulation is not coincident with tumorigenicity.
It has been postulated that the disruption of the normal hormonal regulation of blood cell formation and proliferation leads to the autonomous growth of hematopoietic progenitors or stem cells and thus to leukeamia. We have utilized established hematopoietic cell lines to establish the different mechanism by which growth autonomy is acquired. The analysis of thirteen spontaneous factor-independent mutants revealed that the majority (12/13) secreted a factor that stimulated growth of the parental cell line. Thus, autocrine stimulation may be a important mechanism by which normal growth control is disrupted. This is supported by the observation of Young and Griffin (1987) that some cells isolated from patients with acute myeloblastic leukemia (AML) autogenously produce growth factor. In the majority of Dind mutants more closely examined, growth factor gene activation was due to the juxtapostion of a retrotransposon. Although the exact nature of the involvement of human retroviruses in inducing leukemia has not been elucidated, one could envisage that altered growth factor regulation due to integration of the virus may play an important role. The existence of a second class of Dind mutants that have obtained factor-independence by a mechanism not involving factor production concurs with the acquisition of factor-independent growth in hematopoietic cells after introduction of some oncogenes. Several models have been proposed to explain how oncogenes may "short circuit" and thus activate the normal signal transduction pathway by mimicking the active receptor, transducer, or effector (Weinberg, 1985). To investigate more closely the role of autocrine stimulation in the induction of growth autonomy and tumorigenicity, retroviral vectors expressing either GM-CSF or IL3 were introduced into factor-dependent hematopoitic cell lines. Non-linear clonability of infected cell lines in the absence of exogenous growth factor and inhibition of proliferation by antiserum supported a model of autocrine stimulation. However, a secondary event, correlated with amount of factor released, often occurred that abrogated the requirement for secreted CSF. Growth of cells in which this alteration had occured was cell-density independent and could not be blocked by antibody. It has been postulated that autogenous factor may react with its receptor intracellularly (Lang et al., 1985). The results presented here cannot exclude that the secondary events may allow the internal interaction of receptor and factor.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.
At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.
Animals↗
Identification of genes involved in growth autonomy of hematopoietic cells by analysis of factor-independent mutants.
The factor-dependent myeloid precursor cell line D35 mutates spontaneously at a frequency greater than 2.4 x 10(-7) to growth factor autonomy. This frequency could be increased at least 20-fold by retrovirus insertional mutagenesis. The isolation and characterization of factor-independent mutants allowed the identification of genes involved in growth autonomy. Mutants could be subdivided into two sets: those that secreted a stimulating factor (10/11) and those that did not (1/11). In one case, the factor released was distinct from previously characterized growth factors. In most mutants (6/9), the activation of a growth factor gene was associated with rearrangement that could be attributed to the insertion of a transposable-like element either 5' or 3' of the factor coding region in all cases examined, excluding oncogene involvement. All factor-independent mutants were tumorigenic, consistent with the hypothesis that growth-factor independence initiated by aberrant growth factor gene activation is an important and early step in tumorigenesis.
Alleles↗