Altered transcription of the stem cell leukemia gene in myelofibrosis with myeloid metaplasia.

An increased number of circulating CD34+ hematopoietic progenitors with a prominent proliferation of the megakaryocytic (MK) population are the hallmarks of the myeloproliferation in myelofibrosis with myeloid metaplasia (MMM). Analyzing the potential contribution of the stem cell leukemia (SCL) gene in MMM myeloproliferation was doubly interesting for SCL is expressed both in primitive-uncommitted progenitor cells and erythroid/MK cells, its transcription differentially initiating from promoter 1b and 1a, respectively. Our results show that: (i) the expression of SCL transcript is increased in peripheral blood mononuclear cells (PBMCs) from patients; (ii) SCL gene transcription is altered in MMM CD34+ progenitor cells sorted into CD34+CD41+ and CD34+CD41- subpopulations. Actually, in patients, SCL transcription initiated at promoter 1b is restricted to primitive CD34+CD41- progenitor cells, while it is detectable in both cell subsets from healthy subjects; (iii) the full-length isoform of SCL protein is present in patients' CD34+ cells and in PBMC; in the latter the SCL-expressing cells mainly belong to the MK lineage in which its sublocalization is both nuclear and cytoplasmic, which contrasts with the sole nuclear staining observed in normal MK cells. Our demonstration of altered expression and transcription of SCL in patients' hematopoietic cells emphasizes the possible contribution of this regulatory nuclear factor to the hematopoietic dysregulation, which is a feature of myelofibrosis with myeloid metaplasia.

Involvement of the fibrogenic cytokines, TGF-beta and bFGF, in the pathogenesis of idiopathic myelofibrosis.

Idiopathic Myelofibrosis (IMF), is a chronic myeloproliferative disorder characterized by the association of myeloproliferation and myelofibrosis. The pathophysiological mechanisms resulting in this disease remain still unclear. The myeloproliferation appeared to result from the clonal amplification of hematopoietic progenitors. In contrast, fibroblasts participating in myelofibrosis were shown to be polyclonal, thus suggesting that myelofibrosis was a reactive process. We studied the role of two growth factors TGF-beta and bFGF, which display potent fibrogenic properties and are major regulators of primitive hematopoiesis, in IMF pathogenesis. We demonstrated an increase of TGF-beta and bFGF expression in circulating megakaryocytic cells and platelets, together with alterations of the expression of these cytokines and their receptors in hematopoietic CD34+ progenitor cells from IMF patients. Our results suggested that TGF-beta and bFGF are involved both in myelofibrosis and myeloproliferation which characterize IMF.

Lack of interferon-alpha-induced tyrosine phosphorylation of Vav proto-oncogene in patients with myelofibrosis with myeloid metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is an uncommon chronic myeloproliferative disorder characterized by clonal stem cell proliferation and reactive non-clonal proliferation of bone marrow fibroblasts with fibrosis. In the absence of curative therapy, the current management for the majority of patients is directed towards alleviation of symptoms and improvement in quality of life. A number of experimental therapies have been investigated, among which is the use of type I interferon (IFN)-alpha, whose overall results are disappointing. We recently showed that the Vav proto-oncogene product, p95Vav, which is phosphorylated under IFN-alpha treatment, associates with both chains that constitute the functional type I IFN receptor and contributes to the antiproliferative effects of IFN-alpha. The involvement of p95Vav in IFN-alpha signalling and the frequent non-responsiveness of patients to IFN-alpha led us to investigate for any functional defect(s) of Vav in response to IFN in MMM patients. Our results showed that in the majority of patients Vav is constitutively hyperphosphorylated and that IFN-alpha failed to increase substantially such a tyrosine phosphorylation of p95Vav.

p95(vav) associates with the type I interferon (IFN) receptor and contributes to the antiproliferative effect of IFN-alpha in megakaryocytic cell lines.

The vav proto-oncogene product is a 95 kDa protein predominantly expressed in hematopoietic cells. Vav presents a wide range of functional domains, including structural domains known to be involved in signal transduction. Triggering of various cytokine receptors among which type I interferon receptor induces a rapid and transient tyrosine phosphorylation of p95(vav). Nevertheless, the biological functions of p95(vav) are still unclear. This report is the first documentation on the physical association of p95(vav) with both alpha and beta type I interferon receptor chains, as demonstrated by co-immunoprecipitation and Western blot analysis in megakaryocytic cells (Dami and UT7). This interaction is increased by interferon-alpha/beta stimulation. Moreover, p95(vav) phosphorylated subsequently to type I interferon treatment, is translocated in the nucleus; a concomitant increase of its association with the regulatory subunit of the nuclear DNA-dependent protein kinase, KU-70 is observed in the nucleus. To determine whether p95(vav) participates in the biological response to type I interferons, we studied the effects of non modified Vav oligodeoxynucleotides on the antiproliferative effect of interferon-alpha on megakaryocytic cells. By this oligodeoxynucleotide strategy, we show that p95(vav) contributes greatly to the cell proliferation inhibition induced by type I IFN.

Dual implication of fibrogenic cytokines in the pathogenesis of fibrosis and myeloproliferation in myeloid metaplasia with myelofibrosis.

Though the diagnostic criteria of myeloid metaplasia with myelofibrosis (MMM) are now well established, the origin and pathophysiological mechanisms of this myeloproliferative disorder remain unclear. Concerning its pathophysiology, myeloproliferation and myelofibrosis are the intrinsic characteristics of the disease. Whereas the myeloproliferation was shown to result from a clonal amplification of primitive progenitor cells, fibroblast proliferation appeared to be polyclonal, thus suggesting that myelofibrosis was a reactive process. The myeloproliferation observed in MMM patients is characterized by an increased number of circulating CD34(+) hematopoietic progenitors. When cultured at high concentration without added exogenous growth factors, unpurified progenitors from MMM patients gave rise to spontaneous colonies of all myeloid lineages. Such an autonomous growth disappeared when purified CD34(+) progenitors were plated. These results suggested that growth factors are involved in the dysregulation of proliferation and/or differentiation of MMM hematopoietic progenitors. Cytokines such as PDGF, TGF-beta, and bFGF, produced mainly by megakaryocytes, have been proposed to be involved in the abnormal activation of fibroblasts, resulting in fibrosis. Recently the role of the fibrogenic cytokines, TGF-beta and bFGF, in the regulation of primitive hematopoiesis has been reported. The aim of this review is to address the question of the potential dual implication of TGF-beta and bFGF in the pathogenesis of both myelofibrosis and myeloproliferation in MMM patients.

Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis.

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented. We previously reported increased expression of TGF-beta in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium. consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.

Differential expression of transforming growth factor-beta, basic fibroblast growth factor, and their receptors in CD34+ hematopoietic progenitor cells from patients with myelofibrosis and myeloid metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is a myeloproliferative disorder characterized by clonal expansion of hematopoiesis and marrow fibrosis. Previous results from our group have shown an increased production of two potent fibrogenic factors also involved in the regulation of primitive hematopoietic cells, namely transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), in patients with MMM. It is likely to assume that the myeloproliferation characteristic of this disease may result from an abnormal proliferation of CD34+ hematopoietic progenitors. Thus, we were particularly concerned in studying the gene and protein expression of these cytokines and their receptors in CD34+ progenitors purified from the peripheral blood of MMM patients by using semiquantitative reverse transcriptase-polymerase chain reaction and immunolabeling methods. Our data showed that the expression of TGF-beta1 is not altered in patients CD34+ cells; in contrast, the expression of TGF-beta type II receptor is significantly decreased in such cells, as compared with CD34+ cells from healthy subjects. Regarding bFGF, the very low expression of the cytokine and its type I and II receptors detected in normal CD34+ cells contrasts with that observed in patients' CD34+ cells, which is significantly higher. Our results might be a clue for a better understanding of the mechanism(s) involved in the dysregulation of hematopoiesis in MMM. Actually, the increased expression of bFGF and its receptors associated with the reduction of the TGF-beta binding receptor in CD34+ progenitors from MMM patients might facilitate the expansion of hematopoietic progenitors, not only by stimulating their growth and/or survival, but also by overcoming negative regulatory signals.

TGF-beta and megakaryocytes in the pathogenesis of myelofibrosis in myeloproliferative disorders.

Myeloproliferative disorders are clonal disorders of the hematopoietic stem cell and comprise a spectrum of more or less well-defined clinical entities: polycythaemia vera, chronic myeloid leukemia, essential thrombocythaemia, and agnogenic myeloid metaplasia. Myelofibrosis, which contributes substantially to the impaired hematopoiesis, is commonly observed in myeloproliferative disorders but it represents the criterion of agnogenic myeloid metaplasia also termed idiopathic myelofibrosis. Although progress has been made in the elucidation of the pathogenesis of myelofibrosis, it still remains unclear. The aim of this review is to address the new insights that outline the potential role of TGF-beta in the promotion of myelofibrosis, through its release from megakaryocytes/platelets, particularly in agnogenic myeloid metaplasia.

Transforming growth factor-beta and megakaryocytes in the pathogenesis of idiopathic myelofibrosis.

Although the disease is well described, the pathogenesis of bone marrow fibrosis in idiopathic myelofibrosis still remains unclear. We previously reported elevated intraplatelet transforming growth factor-beta (TGF-beta) levels in patients with this myeloproliferative disorder, compared with healthy subjects. Here, in a series of 16 patients, we show that TGF-beta expression is also increased in patients' peripheral blood mononuclear cells (PBMC): (i) at the mRNA level analysed by Northern blot hybridization and/or reverse transcription-polymerase chain reaction (RT-PCR); (ii) and/or at the secreted peptide level as evaluated in conditioned media from patients' mononuclear cells by a growth inhibition assay on CC164 cells. By immunostaining with a polyclonal anti-TGF-beta 1 antibody, TGF-beta was localized in morphologically heterogenous cells; these cells were characterized as megakaryocytes by labelling with a gpIIbIIIa monoclonal antibody. Thus we provide evidence that both TGF-beta and megakaryocytes are linked in the pathogenesis of idiopathic myelofibrosis.

Characterization of specific functional receptors for HuIFN-alpha on a human megakaryocytic cell line (Dami): expression related to differentiation.

Interferon-alpha (IFN-alpha) treatment has been shown to be highly effective in inhibiting human megakaryocytopoiesis and controlling thrombocytosis in patients with myeloproliferative disorders. These observations suggest that IFN-alpha might play some role in the biological feature of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN-alpha on human megakaryocytic cells, i.e. the Dami cell line, and to study the regulation of their expression. Our study demonstrates that [125I]-recombinant human IFN-alpha ([125I]rHu-IFN-alpha) binds to high-affinity specific receptor on these cells. Scatchard analysis of binding data indicates the presence of homogeneous binding sites estimated in the range of 3000-5000, with an apparent equilibrium dissociation constant, Kd, of 1-2 x 10(-9) M. Also, [125I]rHuIFN-alpha binding capacity decreased in Dami cells incubated with unlabelled rHuIFN-alpha. This down-regulation which was dose-dependent appeared to result from a reduction of IFN-alpha cell surface receptors and was observed at doses that elicited antiproliferative effects in Dami cells. Cross-linking of [125I]rHuIFN-alpha to Dami membrane proteins using a bifunctional reagent yielded to a radioactive complex of approximately 150,000 kD on SDS-PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cells as evaluated by surface marker and ploidy analysis, a 3-fold increase of the number of specific membrane receptors for IFN-alpha was observed, without any modification of either the affinity or the M(r) value of the cross-linked complex. Such an increase appeared to be restricted to IFN-alpha receptors; actually it was not observed in [125I]IFN-gamma binding experiments. Transcript analysis indicated that down-regulation and increased expression of the IFN-alpha receptor after PMA treatment are post-transcriptional events.

Interferon-gamma in vivo reverses the increased platelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia.

We previously reported high platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) levels in platelets from patients with myelofibrosis with myeloid metaplasia. We report here the effects of in vivo administration of recombinant human interferon-gamma on these growth factor levels. Of six patients who entered this study in our institution, four completed the 6-month therapy trial with interferon-gamma PDGF and TGF-beta levels in platelets were evaluated before and after treatment. PDGF levels were determined using a radioimmunoassay and TGF-beta activity was measured using an inhibition growth assay on CC164 cells. In these four patients, IFN-gamma therapy led to a reduction of the increased PDGF and TGF-beta levels in platelets towards the normal range. PDGF and TGF-beta values decreased by 25-45% and 32-46%, respectively. An acute deterioration was observed in one patient 5 months after the end of IFN-gamma therapy. In this patient, the deterioration of his clinical state correlated with an increase in intraplatelet PDGF and TGF-beta levels.

Increased intraplatelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia.

Platelet-derived growth factor (PDGF) is thought to play some role in the genesis of fibrosis associated with myeloproliferative disorders. In addition, transforming growth factor-beta (TGF-beta) has been confirmed to promote fibrotic process. Both PDGF and TGF-beta have been shown to cooperate with epidermal growth factor (EGF) in regulating the growth of human marrow fibroblasts. All three are contained in platelet alpha-granules. We report the results of a study in patients with myelofibrosis with myeloid metaplasia (MMM). We evaluated PDGF, TGF-beta and EGF-like activities in circulating platelets from patients compared to healthy subjects. In contrast to EGF-like intraplatelet levels which were similar in patients and in normal donors (1-4 ng/10(9) platelets), we found constantly higher values for both PDGF and TGF-beta in MMM patients. In both radioimmunoassay (RIA) and assay for mitogenic activity on human bone marrow fibroblasts, PDGF levels were increased on the average 2-3.5-fold over the levels found in normal donors (P less than 0.01 and P less than 0.001, respectively). PDGF serum levels in patients were consistent with those found in platelets. In platelet-poor plasma (PPP), PDGF concentrations were undetectable or congruent to 2 ng/ml in patients and in control donors as well. The total TGF-beta activity in platelet lysates, determined using a competitive radioreceptor binding assay on Swiss 3T3 mouse cells and an inhibition growth assay on CCL64 cells, was found 2-3-fold increased in patients with MMM as compared to control subjects (P less than 0.003). These results emphasize that, not only PDGF, but also TGF-beta are implicated in the myelofibrosis with myeloid metaplasia.

Platelet PDGF and TGF-ß Levels in Myeloproliferative Disorders.

Myeloproliferative disorders mainly including essential thrombocythemia, polycythemia vera, chronic myeloid leukemia and myelofibrosis with myeloid metaplasia are clonal myeloproliferative diseases in which myelofibrosis is commonly observed. The pathogenesis of myelofibrosis still remains unclear. However it was proposed that an inappropriate release of PDGF from either megakaryocytes in bone marrow or platelets in circulation might promote medullary fibrosis. Recently the role of another peptide growth factor, namely TGF-ß, in the fibrotic process was emphasized. This review will focus on the different studies aimed at the evaluation of intraplatelet PDGF and TGF-ß content in patients with the various MPD, and outline the salient results lending support to the potential role of PDGF and TGF-ß in the promotion of myelofibrosis.

Short- and long-term synergistic effects of human tumor necrosis factor and interferon-gamma on A549 human lung cancer cells maintained in three-dimensional culture.

We have studied the short- and long-term effects of human recombinant tumor necrosis factor (TNF) and TNF/recombinant human interferon-gamma (IFN-gamma) mixtures on A549 human lung carcinoma cells maintained in organotypic culture. Continuous treatments with 2 x 10; 2 x 10(2); 2 x 10(3) and 2 x 10(4) U/ml TNF or with mixtures of TNF/IFN-gamma at 2 x 10(2) and 10(3) U/ml, respectively, were administered. Nodule growth, cell proliferation and cell survival were studied. On the 2nd day of treatment with TNF, only the highest dose (2 x 10(4) U/ml) diminished cell proliferation significantly, as measured by tritiated thymidine uptake into DNA. On the 10th day, only the lowest TNF dose (2 x 10 U/ml) induced no significant growth inhibition. Necrosis and nodule disintegration were apparent in the 2 x 10(4) U/ml-treated nodules where DNA synthesis decreased. In this case, using agar cloning assays, no cell survival could be observed. Similar results could be obtained with TNF at low concentration (2 x 10(2) U/ml) in combination with INF-gamma (10(3) U/ml), showing a synergistic effect on inhibition of cell proliferation. In the long-term experiments, with the lower TNF doses, in situ evidence of regrowth was observed (outgrowing zones in the nodules) on about the 40th day of treatment, and nodule recovery was confirmed by the resumption of DNA synthesis measured on the 50th day of treatment. No regrowth, however, occurred when the IFN-gamma/TNF combination was used, and the nodules disintegrated completely on the 35th day of treatment without evidence of any cellular survival.

Increase of epidermal growth factor receptor expression associated with a lack of antiproliferative effect of IFN-beta in human lung cancer nodules in organotypic culture.

The possible relationship between the effects of alpha 2-, beta- and gamma-interferons (IFNs) on the growth of alveolar II pulmonary tumor cells (A549) maintained in tridimensional organotypic culture (nodules) and the modulation of epidermal growth factor receptor (EGF-R) expression was investigated. Treatment with rHu IFN-alpha 2 or IFN-gamma which results in the inhibition of the growth of A549 nodules had no effect on the binding of 125I-EGF to these cells. In contrast, treatment with rHu IFN-beta which exhibits no antiproliferative activity on A549 nodules resulted in a reproducible increase of the binding of 125I-EGF. Scatchard analysis of the EGF binding data indicated that a 48-hour exposure period resulted in an increase in the apparent number of cell surface EGF-R but did not significantly alter receptor affinity. Results from Northern blot analysis showed that the enhanced expression of EGF-R on the A549 nodule cells treated with IFN-beta correlates with an increase in mRNA for EGF-R. No modification of the EGF-R mRNA expression was observed in nodules treated with IFN-alpha 2 or IFN-gamma. In this model, our results suggest a relationship between resistance to antiproliferative effect of IFN-beta and modulation of EGF-R.

Human lung cancer nodules in organotypic culture: no evidence of correlation between the antiproliferative effects of interferons and the induction of 2',5'-oligoadenylate synthetase.

Alveolar II pulmonary tumor cells (A549), maintained in continuous tridimensional organotypic culture, were used in an attempt to investigate whether there could be a relationship of the 2',5'-oligoadenylate (2,5A) synthetase pathway to the antiproliferative activity of interferons (IFNs) in this particular tumor cell model. IFN-alpha 2, -beta and -gamma were used separately and in combinations. IFN-alpha 2 and -gamma demonstrated an inhibitory effect on the nodule growth, whereas IFN-beta did not. Moreover, combinations of IFN-alpha 2 and -gamma resulted in a significant synergistic antiproliferative activity; IFN-beta only potentiated slightly the effect of IFN-gamma. All three IFNs induced an increase in the 2,5A synthetase activity, indicating a discrepancy with the pattern of anticellular activity. Furthermore, whereas the combination of IFN-alpha 2 and -gamma resulted in a synergistic antiproliferative effect, no synergism was observed in the induction of the enzyme. These results show that there is a lack of correlation between the sensitivity or the resistance to IFNs of A549 tumor nodules and the induction of the 2,5A synthetase activity.

Potentiation of antiproliferative activity by mixtures of human recombinant IFN-alpha 2 and -gamma on growth of human cancer nodules maintained in continuous organotypic culture.

Alveolar II pulmonary tumor cells (A549 cells) maintained in continuous tridimensional organotypic culture were used to evaluate the eventual potentiation effect of mixtures of recombinant human interferon-alpha 2 and -gamma on growth inhibition of the tumor nodules. A continuous 45 day treatment (interferon renewed three times a week) with 10, 10(2) or 10(3) U/ml of IFN-alpha 2 or -gamma combined with a fixed high dose (10(3) U/ml) of either IFN-alpha 2 or -gamma resulted in an additive or synergistic growth inhibition according to the doses used. There was a close dose-effect relation, the percentage of inhibition increasing proportionally to the variable IFN doses added to the fixed high dose; moreover, the growth inhibition effect occurred earlier with the mixtures than with IFNs used separately. Furthermore, the growth inhibition observed with 2000 U/ml of the mixture (1000 U/ml of each IFN) was greater than that induced by 2000 U/ml of IFN-alpha 2 or -gamma used alone. A 35-day treatment with IFN-alpha 2 1000 U/ml plus IFN-gamma 1000 U/ml led to a complete growth inhibition and necrosis of the nodules. These data demonstrate that IFN-alpha 2 and -gamma cooperate to potentiate the IFN antiproliferative activity.

Modulation of CALLA and HLA-DR on a non T non B leukemic cell line by lipid treatment.

The relationship between membrane lipid fluidity and expression of HLA-DR and cALL (CALLA) antigens was studied in a human non T non B acute lymphoblastic leukemia cell line (Reh). The membrane fluidity was modulated by treatment with cholesteryl hemisuccinate or phospholipids (e.g. egg lecithin) and monitored by fluorescence polarization. HLA-DR and CALLA expression was measured in an indirect immunofluorescence test with a Fluorescence Activated Cell Sorter (FACS 440), on 24, 48, 72 and 96 hour-cultured cells. Significant antigenic modulation was obtained with cholesteryl hemisuccinate treatment on 48 hour-cells where a slight increase in HLA-DR and a marked decrease in CALLA were observed. In contrast no antigenic modification was observed on lecithin-treated cells.

[Ultrastructural peroxidases and immunological markers in acute adult leukaemia: therapeutic applications (author's transl)]. / Peroxydases ultrastructurales et marqueurs immunologiques dans les leucémies aiguës de l'adulte: incidence thérapeutique.

In a retrospective study of 112 adult patients with acute leukaemia (AL), the percentage of undifferentiated leukaemia was reduced to almost 1% by an analysis of routine cytochemical reactions (PAS, peroxidase, esterases and esterase inhibition), B and T lymphocyte markers (IgS, E-rosettes, HuTLA) cAll antigen and ultrastructural detection of peroxidase activity (PO-ME). A simultaneous study of cALL antigen and PO-ME showed reciprocal exclusion of these markers, except in one case of mixed leukaemia. Therapeutically, the value of these tests lies in that patients with typical PAS reaction and cALL antigen consistently respond to vincristine- corticosteroid treatment, whereas patients without cALL and with PO-ME are frequently resistant to that combination of drugs.