Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases.

v-raf confers CSF-1 independent growth to a macrophage cell line and leads to immediate early gene expression without MAP-kinase activation.

The BAC-1.2F5 macrophage cell line depends on CSF-1 for proliferation and survival. Phosphorylation and activation of the RAF-1 kinase are among the early events in CSF-1 signal transduction. To characterize the role of RAF-1 in CSF-1-induced proliferation, we overexpressed oncogenically activated RAF-1, cellular RAF-1 and RAF-1 kinase-defective mutant proteins in BAC-1.2F5 cells. We were unable to establish stable cell lines expressing either kinase-negative or full length RAF-1 proteins, implying that expression of these molecules is not tolerated in BAC-1.2F5 cells. Oncogenically activated RAF-1 induces CSF-1-independent growth in the absence of autocrine growth factor production. Autonomous growth is not associated with dedifferentiation, since v-raf-expressing macrophages perform the same immunological functions as control cells. Intriguingly, autonomous growth correlates with the suppression of CSF-1-mediated MAP-Kinase activation and with the low constitutive expression of a number of CSF-1-inducible genes, including fos, jun, ets2, and myc, but also the genes for the inflammatory cytokines TNF alpha and IL-1 beta. Many of these genes have AP-1 binding sites in their promoters, and the v-raf-expressing cells contain constitutive AP-1 binding activity. These data indicate that RAF-1, but not MAP-Kinase, is a key component in CSF-1 mitogenic signal transduction, and are consistent with a working hypothesis in which RAF-1 mediates transcriptional activation of genes via AP-1.

The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability.

In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors' expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRAcell progenitors and 69% for MRACFU progenitors. Thus, the repopulation of hypoxic cultures with cells or CFU-GM closely reflected the presence of progenitors capable of repopulating, with cells or CFU-GM, the bone marrow of lethally irradiated syngeneic animals. Progenitors repopulating hypoxic cultures were, like MRA progenitors, significantly resistant to 5-fluorouracil, progenitors repopulating cultures with CFU-GM being two-fold more resistant than those repopulating cultures with cells. We concluded that the repopulation of day-14 hypoxic cultures occurring after their transfer to air is to be considered an indicator of the maintenance of MRA progenitors in hypoxia. The relevance of these results to stem cell biology and their potential practical applications are discussed.

Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages.

We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.

Interleukin-4 rapidly down-modulates the macrophage colony-stimulating factor receptor in murine macrophages.

The activation of macrophages interferes with their response to macrophage colony-stimulating factor (M-CSF), the main growth and differentiation factor for mononuclear phagocytes. We tested the rapid effects of interleukin-4 (IL-4), the alternative macrophage activator produced by Th2 helper lymphocytes, on the receptor for M-CSF (M-CSFR) expressed on the cell surface of murine macrophages. IL4 rapidly down-modulated M-CSFR in a dose-dependent fashion. This effect was unique to IL-4 among a number of Th2-produced cytokines, none of which, with the exception of IL4 itself, is able to activate macrophages. The down-modulation of M-CSFR by IL4 was partially prevented by the inhibition of the activity of phospholipase C or protein kinase C. The data are consistent with the hypothesis that the down-modulation of M-CSFR is a property common to, and exclusive of, macrophage activators, and is driven by different activators via a common mechanism.

Hemopoietic progenitor cells are sensitive to the cytostatic effect of pyruvate.

The addition of certain oxidizable substrates (such as pyruvate and oxalacetate) produced a marked diminution of the number of colonies formed in vitro by mouse bone marrow cells (BMC) stimulated by spleen cell-conditioned medium (SCM). Pyruvate apparently exerted an all-or-none inhibition on colony forming cells (CFCs), affecting neither the size nor morphology of detectable colonies, which were essentially composed of immature cells, neutrophils, and monocytes-macrophages. Pyruvate furthermore reduced BMC proliferation in SCM-stimulated liquid cultures, apparently without modifying the cell population's morphological profile. The effects of pyruvate on hemopoietic progenitor cells were further studied by replating aliquots of BMC liquid cultures with or without pyruvate into agar medium devoid of this substrate. Pyruvate did not interfere with the increase in CFC number observed in the controls during the first few days of incubation, indicating that CFC generation is not inhibited by this substrate. However, the plating efficiency of CFCs recovered from liquid cultures with or without pyruvate was strongly inhibited when these cells were seeded into pyruvate-containing agar plates, thus supporting the theory that CFCs generated in vitro in both conditions have the same sensitivity to pyruvate as those originally present in bone marrow. On the whole, our results indicated that the pyruvate cytostatic effect is a metabolic feature distinguishing CFCs from their progenitors. These differences are discussed in the light of the explanation advanced for the pyruvate cytostatic effect in other cell systems.

Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro.

Incubation in severe hypoxia (1% oxygen) increased the number of erythroid bursts generated from full-term CD34+, or premature mononucleated, human cord blood (CB) cells, in semisolid cultures containing stem cell factor (SCF), interleukin (IL)-3 and erythropoietin (EPO). Severe hypoxia also enhanced the maintenance of erythroid burst-forming units (BFU-E) in CB cell liquid cultures. These positive effects of hypoxia on the maintenance and cloning efficiency of BFU-E did not extend to the other progenitors assayed. Hypoxia, on the other hand, markedly reduced the size and level of hemoglobinization of bursts and, in liquid cultures, suppressed the growth factor-stimulated numerical increase in BFU-E and inhibited the expression of CD36, a marker of erythroid colony-forming units and maturing erythroid precursors. However, when transferred to clonal assays incubated in air, cells from liquid cultures incubated in hypoxia or in air generated fully expanded and hemoglobinized bursts, suggesting that in hypoxia the clonogenic potential of BFU-E was maintained and the development of erythroid clones reversibly inhibited. These results indicate that hypoxia inversely regulates two subsequent phases of erythropoiesis, i.e., it enhances the maintenance of BFU-E and the early development of erythroid clones but inhibits the terminal expansion and maturation of these clones. The cloning of CB cells selected for CD34 positivity, when compared with that of the total population of mononucleated CB cells, revealed that the early development of erythroid bursts was either hypoxia-enhanced or hypoxia-insensitive, reflecting the existence of two different types of BFU-E. Hypoxia-enhanced BFU-E are relatively immature, are maintained in hypoxia but not in air, and account for a large part of CD34+ BFU-E and for a high percentage of the BFU-E in premature CB. Hypoxia-insensitive BFU-E are mostly CD34- and are largely predominant in full-term CB, and most probably correspond to a more mature type of BFU-E.

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells.

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen. Salarin C induced mitotic cycle arrest, apoptosis and DNA damage. Salarin C also concentration-dependently inhibited the maintenance of stem cell potential in cultures in low oxygen of either CML cell lines or primary cells. Surprisingly, the drug also concentration-dependently enforced the maintenance of BCR/Abl signaling in low oxygen, an effect which was paralleled by the rescue of sensitivity of stem cell potential to IM. These results suggest a potential use of salarin C for the suppression of CML cells refractory to tyrosine kinase inhibitors.

Low M(r) phosphotyrosine protein phosphatase activity on fibroblast growth factor receptor is not associated with enzyme translocation.

Fibroblast growth factor receptor (class IV) shares a certain degree of similarity with class III members like platelet-derived growth factor and macrophage-colony-stimulating factor receptors, which, once activated, are substrates of low M(r) phosphotyrosine protein phosphatase. Up until now no phosphotyrosine phosphatase has been shown to act on this receptor in vivo. Here we demonstrate that low M(r) phosphotyrosine protein phosphatase is able to reduce receptor tyrosine phosphorylation and cell proliferation in response to basic fibroblast growth factor. Contrary to what was previously observed for platelet-derived growth factor, during cell stimulation with basic fibroblast growth factor, no enzyme redistribution among cellular compartments is observed.

Apoptosis : molecular regulation of cell death and hematologic malignancies.

Apoptosis, or programmed cell death, represents in cell biology a functional program as important as cell growth or differentiation. Programmed cell death is of basic importance for the development of multicellular organisms and its basic mechanisms are conserved during the evolution of metazoa. Mammalian cells exhibit several different apoptotic pathways that converge to a common endpoint. Each pathway is triggered by a different stimulus: growth factor default, irradiation, induction of the p53 oncosuppressor protein, glucocorticoid hormones (in lymphocytes), ligand binding to Fas/APO (CD95), or tumor necrosis factor receptor (TNF-R), perforin secreted by cytotoxic T cells (reviewed by Hale et al. [1]). As opposed to necrosis, apoptosis is a "clean" process: as the cell shrinks, the cell membrane turns into the "apoptotic shell," the nucleus is condensed and reduced in volume, and eventually the cell disappears from the tissue, due to phagocytosis by neighboring cells or professional phagocytes, such as macrophages.

Cytokine receptor signal transduction mechanisms in immuno-hematopoietic cells.

Novel aspects of cytokine receptor signal transduction are discussed and cytokine receptors classified based on ligand-dependent signalling. An introductory section presents an overview of the role of cytokines in hematopoiesis. A brief explanation of basic concepts, such as redundancy, pleiotropy, synergism, etc., important for the understanding of cell response to cytokines, is also included. Three of five classes of receptors show the involvement of tyrosine kinase activity as the key step in signal transduction. The importance of tyrosine phosphorylation in cellular response to cytokines is pointed out.

Chromatin-associated CSF-1R binds to the promoter of proliferation-related genes in breast cancer cells.

The colony-stimulating factor-1 (CSF-1) and its receptor CSF-1R physiologically regulate the monocyte/macrophage system, trophoblast implantation and breast development. An abnormal CSF-1R expression has been documented in several human epithelial tumors, including breast carcinomas. We recently demonstrated that CSF-1/CSF-1R signaling drives proliferation of breast cancer cells via 'classical' receptor tyrosine kinase signaling, including activation of the extracellular signal-regulated kinase 1/2. In this paper, we show that CSF-1R can also localize within the nucleus of breast cancer cells, either cell lines or tissue specimens, irrespectively of their intrinsic molecular subtype. We found that the majority of nuclear CSF-1R is located in the chromatin-bound subcellular compartment. Chromatin immunoprecipitation revealed that CSF-1R, once in the nucleus, binds to the promoters of the proliferation-related genes CCND1, c-JUN and c-MYC. CSF-1R also binds the promoter of its ligand CSF-1 and positively regulates CSF-1 expression. The existence of such a receptor/ligand regulatory loop is a novel aspect of CSF-1R signaling. Moreover, our results provided the first evidence of a novel localization site of CSF-1R in breast cancer cells, suggesting that CSF-1R could act as a transcriptional regulator on proliferation-related genes.

AML1/ETO sensitizes via TRAIL acute myeloid leukemia cells to the pro-apoptotic effects of hypoxia.

We determined the effects of severe hypoxia (∼0.1% O2) on acute myeloid leukemia cells expressing the AML1/ETO oncogene. Incubation of Kasumi-1 cells in hypoxia induced growth arrest, apoptosis and reduction of AML1/ETO protein expression. The conditional expression of AML1/ETO in U937-A/E cells showed that hypoxia induces marked apoptosis in AML1/ETO-expressing cells only, pointing to AML1/ETO as a factor predisposing cells to hypoxia-induced apoptosis. In AML1/ETO-expressing cells, hypoxia enhanced TRAIL expression and its proapoptotic effects. AML1/ETO was found to bind TRAIL promoter and induce TRAIL transcription, although TRAIL expression was restrained by a concomitant relative transcription block. In hypoxia, such a TRAIL repression was removed and an increase of TRAIL expression was induced. Finally, blocking anti-TRAIL antibodies markedly reduced (Kasumi-1 cells) or completely inhibited (U937-A/E cells) hypoxia-induced apoptosis. Taken together, these results indicated that hypoxia induces apoptosis in AML1/ETO-expressing cells via a TRAIL/caspase 8-dependent autocrine loop and that TRAIL is a key regulator of hypoxia-induced apoptosis in these cells.

Selective anti-leukaemic activity of low-dose histone deacetylase inhibitor ITF2357 on AML1/ETO-positive cells.

We analysed the in vitro effects of a new hydroxamate derivative, ITF2357, on AML cells. ITF2357 potently induced histone acetylation. ITF2357 0.1 microM blocked proliferation and induced apoptosis in AML1/ETO-positive Kasumi-1 cells, while AML1/ETO-negative HL60, THP1 and NB4 cell lines were sensitive only to 1 microM ITF2357. Apoptosis was induced by 0.1 microM ITF2357 in AML1/ETO-positive primary blasts and U937-A/E cells induced to express AML1/ETO, but not in U937-A/E cells non-expressing AML1/ETO. In Kasumi-1 cells 0.1 microM ITF2357 induced AML1/ETO degradation through a caspase-dependent mechanism. ITF2357 0.1 microM also determined DNMT1 efflux from, and p300 influx to, the nucleus. Moreover, 0.1 microM ITF2357 determined local H4 acetylation and release of DNMT1, HDAC1 and AML1/ETO, paralleled by recruitment of p300 to the IL-3 gene promoter. ITF2357 treatment, however, did not induce re-expression of IL-3 gene. Accordingly, the methylation level of IL-3 promoter, as well as of several other genes, was unmodified. In conclusion, ITF2357 emerged as an anti-leukaemic agent very potent on AML cells, and on AML1/ETO-positive cells in particular. More relevantly, clearly emerged from our results that ITF2357 could be an ideal agent to treat AML subtypes presenting AML1/ETO fusion protein which determine HDAC involvement in leukaemogenesis.

CXCR3 and alphaEbeta7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes.

BACKGROUND: We previously demonstrated the existence of two distinct subsets of T cell receptor (TCR)alphabeta+CD8alphabeta+ single positive (SP) cells in human postnatal thymus which express the chemokine receptor CCR7 or CXCR3 and migrate in vitro in response to their specific ligands. AIM: To investigate whether these two CD8+ thymocyte subsets had distinct peripheral colonisation. METHODS: TCRalphabeta+CD8+ SP cells were obtained from normal postnatal thymus, mesenteric lymph node (LNs), small bowel, and peripheral blood (PB) specimens. Cells were then evaluated for expression of surface molecules, cytolytic potential, telomere length, and profile of cytokine production. RESULTS: CD8+CCR7+CXCR3- thymocytes exhibited CD62L, in common with those which localise to LNs. In contrast, CD8+CCR7-CXCR3+ thymocytes lacked CD62L but exhibited CD103, similar to intraepithelial lymphocytes (IELs) present in the gut mucosa where the CXCR3 ligand, CXCL10, and the CD103 ligand, E-cadherin, are highly and consistently expressed. In addition, thymocytes and gut CD8+CXCR3+CD103+ cells showed comparable telomere length, which was higher than that of PB CXCR3+CD8+ T cells. However, both of these populations contained perforin and granzyme A, and displayed the ability to produce interferon gamma and interleukin 2. Of note, CXCR3 deficient, in comparison with wild-type C57Black/6, mice showed decreased proportions of CD3+CD8alphabeta+ and increased proportions of CD3+CD8alphaalpha+ lymphocytes at gut level. Moreover, adoptive transfer of CD3+CD8alphabeta+ thymocytes from wild-type into CXCR3 deficient mice resulted in a significant increase in CD3+CD8alphabeta+ T cells in the gut mucosa but not in other tissues. CONCLUSIONS: The results of this study demonstrate the existence of a previously unrecognised subset of TCRalphabeta+CD8alphabeta+ SP CXCR3+CD103+ thymocytes which share phenotypic and functional features with CD8+ IELs, thus suggesting the possibility of their direct colonisation of the gut mucosa.

The role of hypoxia in the maintenance of hematopoietic stem cells.

Bone marrow cell liquid cultures were incubated at various oxygen concentrations ranging from 0% to 18% (air). The total number of cells in culture (CT) at the end of a 6-day incubation was found to be directly proportional to the oxygen concentration. As compared with air-incubated controls, cells recovered from severely hypoxic (1% oxygen) day-5 liquid cultures showed (1) the same day-7 colony-formation efficiency in semisolid culture (neutrophilic/monocytic colonies) or in spleen; (2) a higher day-14 spleen colony-formation efficiency; (3) an enhanced radio-protection ability; and (4) an increased marrow repopulation ability, as measured by determining either total cell number in recipient marrow MRAcell, or the capacity of the latter of generating day-7 neutrophilic/monocytic colonies in secondary in vitro assays (MRACFU-NM). Taking into account CT, the absolute numbers of progenitors in culture were also computed. The results showed that, with respect to time 0, incubation in air produced an increase in the number of day-7 CFUs and a decrease in the number of the other progenitors, whereas in hypoxic cultures all types of progenitors decreased. However, as compared with air-incubated controls, all progenitors, except cells sustaining MRACFU-NM, were reduced in hypoxic cultures. The degree of reduction paralleled the position of the progenitor in the hematopoietic hierarchy, being maximum for day-7 CFUs and null for cells sustaining MRACFU-NM, which, in fact, were better preserved in hypoxic cultures.

Alterations in CSF-1 receptor expression and protein tyrosine phosphorylation in autonomous mutants of a CSF-1 dependent macrophage cell line.

One approach to the problem of growth factor signal transduction is to identify alterations in growth factor-regulated events in mutants possessing an altered proliferative response. This approach was used in a study of the alterations in protein tyrosine phosphorylation in 22 independently-arising autonomous mutants of the CSF-1-dependent mouse macrophage cell line, BAC1.2F5. Only 4 of the mutants produced CSF-1 and/or factors that were capable of down-regulating the CSF-1 receptor (CSF-1R), suggesting that the majority were altered in the signal transduction pathway for proliferation. All of the mutants possessed lower numbers (13-89%) of cell surface CSF-1Rs than wild type cells. With two possible exceptions, the phosphorylation of the anti-phosphotyrosine-reactive cell surface CSF-1Rs from CSF-1 stimulated cells was directly proportional to cell surface CSF-1R number. In the absence of CSF-1, three of the 22 mutants exhibited tyrosine phosphorylation of proteins that was not observed in wild type cells under the same conditions. The results indicate that this approach will be useful in the analysis of the growth factor regulated pathway for cell proliferation.