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.

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

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.

Autocrine loop of tumor necrosis factor induced by interferon-alpha in tumor cells from hairy cell leukemia.

Interferon-alpha (IFN-alpha) is successfully used in the therapy of hairy cell leukemia (HCL). We previously reported that IFN-alpha administered to HCL patients stimulated the levels of tumor necrosis factor (TNF) receptors on hairy cells. Here, we investigated the interactions between IFN-alpha and TNF, using the HCL cell line Eskol as a model. We showed that increased levels of TNF receptors (TNFR) induced by IFN-alpha treatment were associated with stimulation of high M(r) TNFR. Low M(r) TNFR were not detected on Eskol cells or hairy cells, either before or after IFN-alpha treatment. Furthermore, the expression of TNF mRNA increased in Eskol cells treated with IFN-alpha for 1-4 h as compared to control cells. In vivo experiments showed that IFN-alpha given to HCL patients induced both TNF mRNA expression in hairy cells and secretion of active TNF protein in patients' serum. These results provide evidence that IFN-alpha may induce an autocrine loop of TNF in HCL.

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.

Up-regulation of aFGF expression in quiescent cells is related to cell survival.

Exogenously administrated acidic FGF modulates the proliferation of several cell types, controls cell differentiation, and promotes cell survival. Most cells that are sensitive to exogenous aFGF are also capable of expressing it at very low levels. Thus in order to establish the role of endogenous aFGF as a mitogenic, differentiation, or survival factor, we studied the regulation of aFGF expression by evaluating the level of mRNA by PCR amplification and the concentration of protein by Enzyme Immuno Assay (EIA). In the lens, the amount of aFGF transcripts in nondividing cells of the central epithelium and in the differentiated fiber cells located at the periphery of the lens is similar, suggesting that endogenous aFGF is not involved with lens differentiation. In cultures, depending on the growth conditions, the endogenous aFGF expressed by Bovine Epithelial Lens (BEL) cells is subject to modulation. Cells arrested either by contact inhibition or by serum deprivation express more aFGF transcripts and protein than in exponentially growing cells, implying that endogenous aFGF has no mitogenic role under these conditions. In serum-deprived cells, the addition of specific aFGF antisense primers inhibits endogenous aFGF expression and leads to the death of these cells. These results associated with the higher expression of aFGF in nondividing BEL cells, suggesting that, contrary to exogenous aFGF, endogenous aFGF is not a mitogenic factor but a survival factor.

Interferon-gamma modulates retinoblastoma gene mRNA in monocytoid cells.

To study the effect of interferon gamma (IFN-gamma) on the expression of the retinoblastoma (RB) susceptibility gene, we performed Northern-blot analysis on RNA extracted from Wish, HEL and monocytoid cell lines U-937 and THP-1 treated with 1,000 IU/ml of recombinant IFN-gamma. In U-937 and THP-1 cells, IFN-gamma increased the abundance of RB mRNA. In Wish and HEL cells, co-treatment with cycloheximide was required for IFN-gamma to increase the level of RB mRNA. Pre-treatment of THP-1 cells with cycloheximide prior to IFN-gamma treatment augmented the effects of IFN-gamma on RB gene expression. The effect of IFN-gamma in THP-1 cells was observed after 3 hr of treatment, being more pronounced after 6 hr and persisting until at least 18 hr, although at a lower level. These results suggest that IFN-gamma regulates the level of RB mRNA by different mechanisms in the different cell types. This cytokine increases the abundance of RB mRNA in monocytoid cell lines, reinforced by prior treatment with cycloheximide. Inhibition of protein synthesis is required in Wish and HEL cell lines before IFN-gamma has an effect on RB gene expression.

Collagen synthesis by long-lived mRNA in embryonic chicken lens.

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.

Human and bovine vascular endothelial cells. Comparative effect on cell growth and longevity of an eye-derived growth factor (EDGF) and of extracellular matrix.

Human and bovine vascular endothelial cells from the umbilical vein and the aorta, respectively, were cultured in the presence of EDGF (a growth factor prepared from bovine retina) on plastic or on extracellular matrix (ECM). Both EDGF and ECM are required to allow the maximal proliferation of human cells and their organization in a typical monolayer. Conversely, bovine aortic endothelial cells grow perfectly in the absence of both factors in 6% fetal calf serum. However, a requirement for EDGF can also be demonstrated in low serum conditions, or in cells at high passage number. ECM had no growth promoting activity by itself. Thrombin acts similarly to EDGF on bovine serum-starved cells. EDGF prolongs the in vitro lifespan of both types of cells. Cells at all stages still synthesize factor VIII antigen as revealed by immunofluorescence. Thus EDGF, like other growth factors from brain, FGF or ECGF, may have an important role in angiogenesis, a critical problem in pathological retinas.

Modification of human platelet behaviour in diabetes.

Among the different factors which could be responsible for the retinal vascular disturbances in diabetic retinopathy, we have investigated platelet populations, sialic acid content of platelets and collagen-induced platelet aggregation. The following results were obtained: a) there was no modification of platelet population distribution except for population A; b) there was a modification of collagen-induced platelet aggregation; the lag time was increased in diabetics.