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Biomedical subjects

W Ostertag

Publications and source records attributed to W Ostertag.

At least 55 records · Page 3Linked to original sources
PubMed1996

A novel hematopoietic multilineage clone, Myl-D-7, is stromal cell-dependent and supported by an alternative mechanism(s) independent of stem cell factor/c-kit interaction.

A strictly stroma-dependent hematopoietic clone, Myl-D-7, with lympho-myeloid potential has been isolated. A subset of cells expresses myeloid-macrophage (Mac-1 and Gr-1), erythroid (TER119), and lymphoid (Thy-1 and B220) lineage markers. Spontaneous differentiation to the myeloid-macrophage, erythroid, or lymphoid pathway can be seen by morphologic criteria, detection of beta major globin synthesis, or expression of the early lymphoid specific transcription factor, Ikaros. By sorting lineage marker (Mac-1, Gr-1, B220, and TER119)-negative (LIN-) cells, we showed that the LIN- population actively self-renews on top of MS-5 stromal cells, and differentiates to LIN+ cells. Removal of stroma induces apoptosis and none of the growth factors tested can prevent apoptosis. Granulocyte-macrophage colony-stimulating factor accelerates the differentiation towards the myeloid-macrophage lineage. Using this clone, we show that (1) contact with stroma induces expression of bcl-2, (2) stromal cells derived from SI/SI homozygous fetuses can support long-term growth, and (3) conditioned media of specific stromal cells contains an activity that supports proliferation and self-renewal of the clone. Myl-D-7 can thus be used as an indicator cell for unknown factors that may provide stromal cell support.

Animals↗
PubMed1996

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase.

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony-forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.

Animals↗
PubMed1996

Transcriptional regulation of the c-fms proto-oncogene mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) in murine cell lines.

Differentiation of blood cells is paralleled by a timely ordered expression of cytokine receptor genes. We show here that the expression of the c-fms gene which encodes the lineage-specific receptor for macrophage colony-stimulating factor (M-CSF or CSF-1) is directly linked to ligand-mediated activation of the receptor for the granulocyte/macrophage colony-stimulating factor (GM-CSF). In interleukin-3 (IL-3) dependent multipotent progenitor cells, FDC-Pmix GMV#2 cells, GM-CSF treatment results in the rapid formation of full-length c-fms transcripts. Surprisingly, this upregulation of c-fms transcripts is also observed in mouse NIH3T3 fibroblasts stably transfected with genes coding for the alpha- and beta-subunits of the GM-CSF receptor. These results indicate a direct control by the GM-CSF receptor that takes place regardless of cell differentiation. Furthermore, a 2.1 kb genomic fragment containing the c-fms proximal promoter directs GM-CSF-inducible expression of a reporter gene, suggesting a regulation of c-fms gene expression on the transcriptional level.

3T3 Cells↗
PubMed1996

Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection.

Therapeutic gene transfer into hematopoietic cells is critically dependent on the evolution of methods that allow ex vivo expansion, high-frequency transduction, and selection of gene-modified long-term repopulating cells. Progress in this area needs elaboration of defined culture and transduction conditions for long-term repopulating cells and improvement of gene transfer systems. We have optimized retroviral vector constructions based on murine leukemia viruses (MuLV) to overcome the transcriptional repression encountered with the use of conventional Moloney MuLV (MoMuLV) vectors in early hematopoietic progenitor cells (HPC). Novel retroviral vectors, termed FMEV (for Friend-MCF/MESV hybrid vectors), were cloned that mediate greatly improved gene expression in the myeloerythroid compartment. Transfer of the selectable marker multidrug resistance 1 (mdr1), FMEV, in contrast to conventional MoMuLV-related vectors currently in use for clinical protocols, mediated background-free selectability of transduced human HPC in the presence of myeloablative doses of the cytostatic agent paclitaxel in vitro. Furthermore, FMEV also greatly improved chemo-protection of hematopoietic progenitor cells in a murine model system in vivo. Finally, when a second gene was transferred along with mdr1 in an FMEV-backbone, close to 100% coexpression was observed in multidrug-resistant colonies. These observations have significant consequences for a number of ongoing and planned gene therapy trials, for example, stem cell protection to reduce the myelotoxic side effects of anticancer chemotherapy, correction of inherited disorders involving hematopoietic cells, and antagonism of HIV infection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Activity of Friend mink cell focus-forming retrovirus during myelo-erythroid hematopoiesis.

Friend mink cell focus-forming (FMCF) viruses are recombinants between the Friend murine leukemia virus (F-MuLV) and endogenous polytropic retroviruses involved in a number of retrovirus-induced malignancies of the myelo-erythroid compartment. To analyze the contribution of the viral cis regulatory elements to the host range determinants within the hematopoietic system, we performed a series of marker gene experiments using both transient transfection and retroviral-mediated stable transduction of indicator cell lines representing distinct developmental stages. According to our data, the U3 region in the long terminal repeat (LTR) of FMCF viruses possesses an enhancer assembly that allows efficient transcription in both early and late myelo-erythroid stem and progenitor cells. Retroviral gene expression, however, is subjected to stage-dependent transcriptional controls during blood cell maturation. We obtained evidence that a repressor element overlapping with the primer binding site in the viral leader region compromises U3-mediated gene expression in a stage-dependent manner, with the strongest restriction observed in the most primitive cells analyzed, FDCP-mix. In addition, our data indicate a second hurdle for retroviral gene expression in early hematopoietic cells that is independent of the primer binding site and most likely related to inefficient utilization of U3-located enhancers. These data shed light on the mechanisms of host range restriction within the hematopoietic system and define a basis for the design of retroviral vectors aimed to overcome transcriptional inefficiency in early hematopoietic cells. Thus, we developed novel retroviral vectors combining FMCF-type U3 regions with a permissive leader from the murine embryonic stem cell virus. These vectors are highly efficient for gene transfer and expression in both early and late myelo-erythroid cells, indicating that they will be of great use for a variety of experimental and therapeutic applications.

3T3 Cells↗
PubMed1995

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.

Animals↗
PubMed1995

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event.

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT-PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.

Adult↗
PubMed1995

Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.

We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells. Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner. Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells. In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells. In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus. On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus. When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

3T3 Cells↗
PubMed1995

Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

Amino Acid Sequence↗
PubMed1994

Selectable retrovirus vectors encoding Friend virus gp55 or erythropoietin induce polycythemia with different phenotypic expression and disease progression.

The Friend spleen focus-forming virus induces a massive expansion of erythroid progenitor cells resulting in polycythemia and splenomegaly. The pathogenic agent is the membrane glycoprotein gp55, encoded by the env gene. Recent evidence indicates that gp55 binds to and activates the erythropoietin (Epo) receptor. It is not clear, however, whether gp55 completely mimics the natural receptor ligand (Epo). To directly compare both effectors, we constructed selectable retroviral vectors which carry either the env or the Epo gene. The selection marker allowed for clonal analysis of infected cells. After infection of DBA/2J mice, the spleen weight, hematological indices, and Epo titer of peripheral blood were monitored. Although both viruses induced an acute erythrocytosis, there were significant differences in disease phenotype and progression. The Epo virus caused an enhanced increase of hematocrit and erythrocytes, whereas with the env virus the pool of late progenitors (CFU-erythroid) was dramatically expanded, resulting in a more severe splenomegaly. The distribution of cytologically recognizable erythroid precursors was shifted towards immature cell types by the env vector compared with Epo. These data suggest that Epo and gp55 differentially affect proliferation and differentiation. Gp55 appears to promote proliferation over differentiation, whereas Epo preferentially drives differentiation.

Animals↗
PubMed1994

Coordinate expression of the lineage-specific growth factor colony-stimulating factor (CSF)-1 and its receptor selectively promotes macrophage maturation during differentiation of multipotential progenitor cells.

The multipotent hematopoietic precursor line A4GMV#2, derived by infection of FDCP-mix cells with a retroviral vector expressing the granulocyte-macrophage colony-stimulating factor (CSF) gene, proliferates continuously in interleukin 3 and presents the unique advantage of synchronous granulocyte and macrophage differentiation upon interleukin 3 withdrawal. Using this system, we showed previously that the mRNAs for lineage-specific receptors (granulocyte-CSF receptors, CSF-1 receptors, and Erythropoietin receptors) and ligands (granulocyte-CSF and CSF-1) are up-regulated during myeloid maturation. Here we address the specific question of the regulation of the expression of CSF-1 and its receptor and of their relevance to macrophage differentiation. Both genes were transcribed with equal efficiency in undifferentiated and differentiating cells. CSF-1 mRNA was detected in undifferentiated cells and increased slightly in the early phases of differentiation. CSF-1 receptor mRNA, absent in undifferentiated cells, accumulated early in differentiation (24 h) and remained constant thereafter. The production of both proteins, detected later during the differentiation of A4GMV#2 cells and of bone marrow-derived myeloid precursors, was therefore controlled at the posttranscriptional level. CSF-1 was produced by cells of the macrophage lineage and accumulated in mature phagocytes. A neutralizing anti-CSF-1 serum selectively impaired macrophage differentiation of A4GMV#2 cells and, most significantly, of primary myeloid precursors. These data indicate that CSF-1 and its receptor interact productively during differentiation and that the resulting autocrine stimulation selectively promotes macrophage maturation.

Amino Acid Sequence↗
PubMed1994

Mutants of a multipotent hematopoietic cell line blocked in GM-CSF-induced differentiation are leukemogenic in vivo.

FDCP-Mix cells infected with a retroviral vector expressing the GM-CSF gene (GMV-FDCP-Mix) self-renew in the presence of interleukin-3 (IL-3), are multipotent, and undergo differentiation into granulocytes and macrophages coupled with clonal extinction after removal of IL-3. Mutants of GMV-FDCP-Mix were isolated that escape clonal extinction after differentiation induction by the autocrine secreted GM-CSF. Some of these mutant clones have a blast cell morphology and are blocked in differentiation, whereas others exhibit all stages of granulocyte and macrophage differentiation without clonal extinction. In contrast to the parental GMV-FDCP-Mix cells, all the mutants tested were leukemogenic when injected into syngeneic mice. Depending on the in vitro differentiation capacity of the transplanted mutant cell lines, the animals developed undifferentiated blast cell leukemias or CML-like syndromes. Thus, these mutant cell lines can be used to define the cooperating steps in autocrine myeloid leukemia.

Animals↗
PubMed1993

Induction of monocytic differentiation and tumorigenicity by v-Ha-ras in differentiation arrested hematopoietic cells.

Activated ras genes are often associated with human myeloid leukemias with a monocytic differentiated phenotype. To investigate the role of the activated ras gene in leukemogenesis, a myeloid nontumorigenic cell line (FDC-P1) was infected with a selectable retroviral vector carrying the v-Ha-ras gene (H1neo). Infected FDC-P1 cells were not only tumorigenic, but also showed increased monocytic differentiation in vitro. Monocytic differentiation and tumorigenicity in vivo were correlated with several-fold increased levels of activated ras gene expression. Tumorigenic cells were arrested with respect to monocytic marker expression at a much later stage of macrophage differentiation than the parental noninfected FDC-P1 cells. These data thus suggest a model of how activated ras genes could be involved in the preferential induction of hematopoietic malignancies with a myelomonocytic phenotype.

Animals↗
PubMed1993

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

Animals↗
PubMed1993

Upregulation of lineage specific receptors and ligands in multipotential progenitor cells is part of an endogenous program of differentiation.

Multipotent hematopoietic progenitor cell lines (FDCP-Mix) infected with a retroviral vector expressing the GM-CSF gene show functional downregulation of the GM-CSF receptor when maintained in IL-3 and activation of the receptor resulting in synchronous differentiation into mature granulocytes and macrophages on withdrawal of IL-3. This system has now been used to investigate whether or not receptors for some of the other growth factors are also influenced as a consequence of differentiation. We show here the lineage specific receptors for M-CSF, G-CSF and erythropoietin are all upregulated, regardless of whether or not differentiation is induced by GM-CSF or by other conditions. Concomitant induction of the mRNA coding for the ligands M-CSF and G-CSF, but not for erythropoietin, suggests that M-CSF and possibly G-CSF facilitate macrophage or granulocyte differentiation by an autocrine stimulation of the lineage specific receptors. FDCP-Mix mutants that are blocked in their ability to differentiate on exposure to GM-CSF, but that still require GM-CSF for proliferation, do not express increased levels of M-CSF receptor nor M-CSF. Based on these data, we suggest that expression of these lineage specific receptors is part of the intrinsic endogenous program of myeloid differentiation.

Animals↗
PubMed1993

Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line.

Retroviral insertion mutagenesis has been used extensively in vivo but not in vitro to induce and identify critical mutations during oncogenic progression and differentiation. We have developed a tissue culture system using the human, growth factor-dependent, hematopoietic precursor cell line TF-1 that permits the use of retroviral vectors to induce a large (up to 28-fold) increase in the mutation frequency to growth factor independence and thus the isolation of many mutants. The mutation frequency, as expected, is directly proportional to the number of retroviral insertions (2.2 x 10(-7) mutants per insertion). The mutant phenotypes can be subdivided into mutants that release growth factors and those that do not ("autonomous" mutants). The majority of growth factor-producing mutants release an unidentified ligand. A subset of the autonomous mutants shows alterations in expression of the alpha subunit of either the GM-CSF or the IL-3 receptor. One mutant expresses neither GM-CSF nor IL-3 alpha receptor chains, thus showing coordinate regulation of the alpha receptor subunits.

Cell Line↗
PubMed1992

v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant.

The myeloproliferative sarcoma virus (MPSV) v-mos protein was predicted to be identical in size to p39c-mos because of an observed one-base deletion in the seventh codon of the env-mos open reading frame, which would allow translation to initiate at the methionine equivalent to codon 32 of the env-mos gene. On the basis of published results, p39c-mos is known to have greatly reduced in vitro protein kinase activity compared with p37env-mos encoded by Moloney murine sarcoma virus. Unexpectedly, the relative activity of the MPSV v-mos protein kinase was comparable to that of p37env-mos. Consistent with this finding, the size of MPSV v-mos protein was found to be similar to the size of p37env-mos. Moreover, the pattern and sizes of phosphorylated bands produced by autophosphorylation of the MPSV v-mos protein were similar to those of p37env-mos. These results were confirmed by in vitro transcription-translation of the MPSV v-mos gene. Resequencing portions of the MPSV mos gene failed to show the deletion within codon 7. Except for the codon 262 deletion, other mutations characteristic of MPSV and temperature-sensitive MPSV v-mos genes were confirmed. A glycine-to-arginine mutation at residue 338 of the MPSV env-mos sequence, previously shown to cause thermosensitivity of the mutant virus (termed ts159) transforming function, yielded a v-mos protein that had significantly reduced protein kinase activity in vitro. These findings indicate that MPSV, like other Moloney murine sarcoma virus strains, also encodes a functional env-mos protein.

Amino Acid Sequence↗