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J Troppmair

Publications and source records attributed to J Troppmair.

At least 37 records · Page 2Linked to original sources
PubMed1996

The effect of c-raf antisense oligonucleotides on growth factor-induced proliferation of hematopoietic cells.

While it is well established that Raf-1 kinase is activated by phosphorylation in growth factor-dependent hematopoietic cell lines stimulated with a variety of hematopoietic growth factors, little is known about the biological effects of Raf-1 activation on normal hematopoietic cells. Therefore, we examined the requirement for Raf-1 in growth factor-regulated proliferation and differentiation of hematopoietic cells using c-faf antisense oligonucleotide. Raf-1 required for the proliferation of growth factor dependent cell lines stimulated by IL-2, IL-3, G-CSF, GM-CSF and EPO that bind to the hematopoietin class of receptors. Raf-1 is also required for the proliferation of cell lines stimulated by growth factors that use the tyrosine kinase containing receptor class, including SLF and CSF-1. In addition, Raf-1 is also required for IL-6, LIF- and OSM-induced proliferation whose receptors share the gp 130 subunit. In contrast to previous results which demonstrated that IL-4 could not activate Raf-1 kinase, c-raf antisense oligonucleotides also inhibited IL-4-induced proliferation of T cell and myeloid cell lines. Using normal hematopoietic cells, c-raf antisense oligonucleotides completely suppressed the colony formation of murine hematopoietic progenitors in response to single growth factors, such as IL-3, CSF-1 or GM-CSF. Further, c-raf antisense oligonucleotides inhibited the growth of murine progenitors stimulated with synergistic combinations of growth factors (required for primitive progenitor growth) including two, three and four factor combinations. In comparison to murine hematopoietic cells, c-raf antisense oligonucleotides also inhibited both IL-3 and GM-CSF-induced colony formation of CD 34+ purified human progenitors. In addition, Raf-1 is required for the synergistic response of CD 34+ human bone marrow progenitors to multiple cytokines; however, this effect was only observed when additional antisense oligonucleotides were added to the cultures at day 7 of a 14 day assay. Finally, Raf-1 is required for the synergistic response of human Mo-7e cells and of normal human fetal liver cells to five factor combinations. Thus, Raf-1 is required to transduce growth factor-induced proliferative signals in factor-dependent progenitor cells lines for all known classes of hematopoietic growth factor receptors, and is required for the growth of normal murine and human bone marrow-derived progenitors.

Animals↗
PubMed1995

Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells.

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.

Animals↗
PubMed1995

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.

Animals↗
PubMed1994

Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway.

We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.

3T3 Cells↗
PubMed1994

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation.

The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.

3T3 Cells↗
PubMed1994

The ins and outs of Raf kinases.

Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

Animals↗
PubMed1994

Lytic susceptibility of target cells to cytotoxic T cells is determined by their constitutive major histocompatibility complex class I antigen expression and cytokine-induced activation status.

Cytotoxic T-cell lines (TCL) were raised in vitro using stimulator cells with a defined major histocompatibility complex (MHC) mismatch and tested in a cytotoxic chromium-release assay against haemopoietic and non-haemopoietic target cells from the original stimulator. Monoclonal antibody (mAb)-blocking experiments and simultaneous determination of MHC class I, class II, lymphocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) density by quantitative radioimmunometric methods and flow cytometry on target cells demonstrated that lysis was restricted by MHC class I and dependent upon the constitutive MHC class I antigen expression. Measurements showed a high constitutive expression of class I MHC antigens on peripheral blood mononuclear cells (PBMC), but a low one on keratinocytes (K). Also, PBMC were more susceptible to lysis by TCL than K. Interferon-gamma (IFN-gamma) treatment of K resulted in increased MHC class I antigen expression and enhanced lytic susceptibility to TCL. IFN-alpha and tumour necrosis factor-alpha (TNF-alpha) treatment, which did not modulate MHC class I antigen expression on K, did not influence the amount of K lysis either. None of the cytokines tested in this analysis, however, increased the expression of MHC class I, class II, ICAM-1 and LFA-1 on PBMC. Only IFN-gamma pretreatment showed a minimal, statistically significant increase in MHC class I antigen expression. In spite of the minimal effect of IFN-gamma and no effect of IFN-alpha on class I MHC expression, pretreatment of target cells with both cytokines considerably increased their lytic susceptibility. The mechanism of cytokine-induced enhanced lytic susceptibility to TCL was not explained by increased MHC class I, LFA-1 or ICAM-1 expression, since no correlation was found between surface expression of these molecules and lytic susceptibility to TCL. These data demonstrate that: (1) the constitutive density of MHC class I antigens determines the extent of TCL lysis; (2) IFN-gamma, and not IFN-alpha or TNF-alpha controls the amount of K target cell lysis by increasing their MHC class I antigen expression; and (3) IFN-gamma and IFN-alpha control the amount of PBMC target cell lysis by a mechanism independent of MHC class I, ICAM-1 or LFA-1 expression.

Cell Adhesion Molecules↗
PubMed1994

Transformation by Raf and other oncogenes renders cells differentially sensitive to growth inhibition by a dominant negative c-jun mutant.

In NIH3T3 cells expressing active Raf-1 protein serine/threonine kinase (PSK) c-jun expression is constitutive while c-fos expression is attenuated. This alteration prompted us to determine whether oncogene transformation would render cells differentially sensitive to growth inhibition by a dominant negative mutant of c-jun, TAM 67. Growth inhibition was observed in three types of assays: (1) transfection of TAM 67 into cells stably transformed by a variety of oncogenes, (2) cotransfection of TAM 67 with oncogene expression plasmids into NIH3T3 cells and (3) titration of oncogene-expressing retroviruses on cells stably expressing TAM 67. The results clearly demonstrate that Raf-1 dependent oncogenes, which include receptor protein tyrosine kinases (PTKs)-, intracellular PTKs- and Ras-derived genes share the Raf phenotype of constitutive c-jun expression, attenuated c-fos induction, and high sensitivity to growth suppression by TAM 67. Additionally, the intracellular PSK oncogene, mos and the nuclear oncogenes c-myc, c-fos, and SV40 T antigen were TAM 67-sensitive for transformation. This universal pattern of altered growth regulation in oncogene transformed fibroblast cell lines highlights the potential usefulness of c-jun based inhibitors for control of tumor cell growth.

3T3 Cells↗
PubMed1994

v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.

Interleukin-3 (IL-3) is required for the proliferation, survival and differentiation of myeloid progenitors. In the absence of IL-3, murine myeloid 32D.3 cells accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death, or apoptosis. Here we demonstrate that enforced expression of the v-raf oncogene suppresses apoptosis of myeloid 32D.3 cells following the withdrawal of IL-3. Surprisingly, steady state levels of Bcl-2, an oncogene known to suppress apoptosis, were not dependent upon IL-3 in 32D.3 cells and its levels were not augmented in v-raf clones. This suggests that ability of v-raf to suppress apoptosis in the absence of ligand is either Bcl-2 independent or that v-raf kinase promotes Bcl-2 function. v-raf also promoted growth of these cells in the presence of IL-3. v-raf clones proliferated at an increased rate due to a shortened G1 phase and had decreased requirements for IL-3 for growth. Therefore, transformation of myeloid cells by v-raf involves signaling pathways which promote both cell cycle progression and cell survival.

Animals↗
PubMed1994

Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase.

The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.

Animals↗
PubMed1993

Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.

3T3 Cells↗
PubMed1993

Susceptibility and resistance to J3V1 retrovirus-induced murine plasmacytomagenesis in reconstituted severe combined immunodeficient mice.

To date much is known about the genetics of susceptibility and resistance to plasmacytoma induction in mice, however little is known about the cellular aspects of these phenotypes. The complexity of plasmacytomagenesis allows for susceptibility and resistance to reflect differences in B cells, T cells, accessory cells and/or stromal elements contributing to the disease process. Alternatively, these phenotypes may result from differential abilities to affect events critical to plasmacytomagenesis, such as myc deregulation. To address these possibilities, the v-myc-raf-containing retrovirus, J3V1, was used to induce plasmacytomas (PCTs) in severe combined immunodeficient (SCID) mice reconstituted with susceptible (Balb/c) and/or resistant (DBA/2) cells. The results demonstrate that Balb/c bone marrow (BM)-reconstituted SCID mice yielded PCTs of donor origin, while DBA/2 BM-reconstituted mice did not. Mice reconstituted with both DBA/2 BM and Balb/c peripheral lymphocytes, as well as those reconstituted with Balb/c peripheral lymphocytes alone, also yielded only Balb/c PCTs. These results indicate that: (1) a microenvironment supportive of plasmacytomagenesis is insufficient to allow PCT development among resistant cells; (2) DBA/2 BM-derived cells do not suppress plasmacytomagenesis by target cell elimination or microenvironment destruction; (3) resistance is not solely attributable to the inability of DBA/2 B cells to deregulate myc; and (4) potential PCT targets reside in a number of lymphoid tissues. Taken together, these results demonstrate that a major aspect of resistance/susceptibility to plasmacytomagenesis is dictated by the genotype of the target B cell.

Animals↗
PubMed1993

Raf revertant cells resist transformation by non-nuclear oncogenes and are deficient in the induction of early response genes by TPA and serum.

A revertant cell line was generated from v-raf transformed NIH/3T3 fibroblasts. These cells, termed CHP25, express a functional v-raf oncogene. However, they are non-tumorigenic, do not form colonies in soft agar and possess a flat morphology. CHP25 cells are resistant to re-transformation by sis, ras, tyrosine kinase- as well as serine/threonine kinase-encoding oncogenes suggesting that Raf functions downstream of most membrane associated signal transducers. In contrast to v-raf transformed cells, in which the endogenous Raf-1 protein kinase is constitutively activated, v-Raf in CHP25 cells does not activate endogenous Raf-1 kinase. Since mitogen regulation of Raf-1 kinase in CHP25 cells is intact, we conclude that CHP25 cells are blocked at the level of Raf-1 substrate phosphorylation. Consistent with this interpretation CHP25 cells show specific alterations of early gene induction. The serum induction of c-fos and junD as well as the serum and TPA (12-O-tetradecanoylphorbol-13-acetate) induction of junB and egr-1 are almost completely abolished. Only v-fos can transform CHP25, whereas c-fos, v-myc, c-jun and junB are ineffective. These data suggest that the lesion responsible for the revertant phenotype of CHP25 cells is the inability to activate the AP-1 complex. We conclude that Raf-1 signaling is essential for transformation of NIH/3T3 cells by peripheral oncogenes and for regulation of a subset of early response genes by TPA and serum growth factors.

3T3 Cells↗
PubMed1992

Ras controls coupling of growth factor receptors and protein kinase C in the membrane to Raf-1 and B-Raf protein serine kinases in the cytosol.

A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.

3T3 Cells↗
PubMed1990

Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.

Amino Acid Sequence↗
PubMed1990

v-myc and v-raf act synergistically to induce B-cell tumors in pristane-primed adult BALBC mice.

In a variety of systems, evidence is accumulating which suggests that neoplastic transformation requires the action of two or more genes such as mutated or over-expressed proto-oncogenes. To determine whether the cytoplasmic serine/threonine kinase oncogene raf could complement a deregulated myc gene and induce tumors in adult mice, BALBC mice were primed with an intraperitoneal (ip.) injection of mineral oil (pristane) and then given an ip. injection of a retroviral construct, J1, J2 or J5, which expresses either v-raf (J1), v-myc (J5) or both (J2). The J1 virus induced no tumors in 150 days in 38 mice, except for 5 helper virus-associated T-cell lymphomas. Under identical conditions the J5 virus, which expresses only v-myc, induced exclusively monocytic neoplasms in 93% of 15 mice. The J2 virus expresses both v-myc and v-raf and caused equal numbers of monocytic and B cell tumors in 66% of 30 mice. Under these conditions, it appears that v-raf expression acts synergistically with v-myc to induce the transformation of B cells, which neither oncogene could do alone. The J3 virus, which originally contained a complete v-myc and an inactivated v-raf, can induce tumors of later stage B cells (plasmacytomas, Potter et al., 1987). Recent studies of virus recovered from these plasmacytomas (called the J3V1 virus, Troppmair et al., 1989) show that the J3 virus has undergone deletions which have reactivated v-raf in a mutated form. Only J3V1, not J3, induced tumors in vivo. Our data presented here corroborate Troppmair et al. and extend Potter et al. (1987) which reported that J3 (presumably J3V1) induced 10% myeloid tumors and 90% plasmacytomas. In light of the discovery, our J2 and J3 data indicate that in combination with the same form of v-myc, different forms of v-raf induce different spectra of tumors.

Animals↗