Hematopoietic growth factor mimetics.

Hematopoietic growth factors are glycoproteins of 15-70 kDa. Although much clinical success has been obtained using recombinant proteins produced in mammalian cell lines and in microbial fermentation processes, the full-length polypeptides necessarily are expensive to produce, require parenteral administration, and in some cases have provoked detrimental immune responses. With the availability of high throughput biological function and receptor binding assays it has become possible to screen millions, if not billions, of randomly produced organic compounds and relatively short peptides to identify lead compounds for the development of small molecular mimetics of hematopoietic growth factors. Herein the strategies used to screen libraries of small molecules and peptides and the successes in finding mimetics and antagonists for/to erythropoietin, granulocyte colony-stimulating factor, and thrombopoietin are reviewed. Finally, the structural study of mimetic-receptor complexes has provided us with many molecular details of growth factor-induced receptor activation and is likely to yield new insights into the molecular basis of hematopoietic signal transduction.

Use of thrombopoietic growth factors in acute leukemia.

Several hematopoietic growth factors have been shown to affect megakaryocyte development, and two, interleukin (IL)-11 and thrombopoietin (TPO) are presently being evaluated for use in patients with thrombocytopenia. In two studies patients who required one or more platelet transfusions during their first course of chemotherapy were found to require fewer platelet transfusions if their second cycle was augmented with IL-11. The drug was generally safe, with cardiovascular compromise the only significant complication occurring in a minority of patients. Although these reports included patients with various malignancies, studies of IL-11 in patients with myeloproliferative disorders have not been presented. In several clinical trials in cancer patients treatment with TPO was safe, and when administered early following a moderately aggressive cytotoxic insult was effective in accelerating platelet recovery. In addition, in both pre-clinical and clinical trials, TPO given to stem cell donors during mobilization lead to accelerated hematopoietic recovery. Finally, TPO appears safe when administered to patients with acute myelogenous leukemia (AML), both with respect to acute toxicity and long-term outcome of the leukemia. However, when used following a 7-day course of standard chemotherapy, the agent does not appear to accelerate platelet recovery. As such, additional clinical trials to test different growth factor regimens are ongoing. A number of studies have suggested that megakaryocytic growth factors may play a role in the biology of myeloproliferative disorders. Given the potential for adversely affecting patients with these disorders, the affects of IL-11 or TPO in patients with AML must continue to be carefully studied.

The thrombocytopenia of cancer. Prospects for effective cytokine therapy.

The previous 10 years have witnessed the development of increasing needs for platelet transfusion in support of aggressive therapies of malignancy. Despite gains in our understanding of platelet preparation, storage, and transfusion, alternative therapies are clearly desirable. During the late 1980s at least six distinct cytokines that display effects on megakaryocyte growth and differentiation-IL-3, KL, GM-CSF, IL-6, IL-11, and LIF- and a synthetic growth factor, PIXY 321, were cloned and characterized. Although none of these cytokines fulfill all of the physiologic roles of thrombopoietin, in its absence several have undergone extensive preclinical and preliminary clinical testing. Of these, IL-11 and PIXY 321 hold promise for clinical amelioration of thrombocytopenia in cancer patients. With the recent cloning of thrombopoietin and its promise in preclinical trials, the role of each of these recombinant proteins in clinical medicine is undergoing careful evaluation. As with erythropoietin and G-CSF before it, given its normal role in the regulation of platelet production, Tpo would appear to provide the greatest physiologic stimulus to platelet production in states of natural and iatrogenic marrow failure. Careful clinical trials of the agent are needed to determine whether the hormone will fulfill this promise. The following decade will most certainly see the resolution of many of the complications of thrombocytopenia and its transfusional support.

Thrombopoietin: the primary regulator of megakaryocyte and platelet production.

The development of blood cells requires the interplay of hematopoietic stem and progenitor cells, marrow stroma and polypeptide growth factors. Although many proteins support the expansion of megakaryocytic precursor cells, identification of the late acting, lineage specific growth factor for platelet production, termed thrombopoietin, has remained elusive. Recently, characterization of the proto-oncogene c-mpl revealed structural homology with the hematopoietic cytokine receptor family. Based on the cell of origin of its cDNA, we hypothesized that the ligand for c-Mpl might be identical with Tpo, and together with scientists at ZymoGenetics, Inc. recently cloned its cDNA. Using recombinant protein we have shown that the mpl-ligand displays all of the expected biological properties of the major regulator of megakaryocyte development, and proposed that it be termed thrombopoietin.

Physiology and preclinical studies of thrombopoietin.

Until recently, the molecular basis for the control of platelet production was largely unknown. In the past year, several groups have obtained complementary DNA for thrombopoietin, the substance first theorized nearly 40 years ago to regulate this process. Cellular and molecular studies have confirmed many of the properties previously attributed to this molecule, and have revealed some surprises. It is now clear that thrombopoietin is the critical regulator of platelet production. Detailed study of the molecule will likely yield important physiologic insights into megakaryocyte biology, and its application to states of iatrogenic and natural marrow failure will almost certainly provide therapeutic advances.

Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.

Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.

Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.