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Leukaemic cells from a patient in the blast crisis of chronic myeloid leukaemia were subjected to a surface marker analysis using a panel of monoclonal antibodies recognizing differentiation antigens of myeloid (MY7, MY906, VIM D5, M phi P9), erythroid (VIE G4), megakaryocyte (AN51), T-lymphoid (WT1, 10.2, OKT3, OKT4, OKT6, OKT8, OKT11A) and B-lymphoid cells (B1, B2, Y29/55), common ALL-antigen (VILA1), non-lineage-restricted antigens (OKT9, OKT10), monomorphic HLA-DR determinants (7.2) as well as TdT. When the patient entered his first blast crisis, his blasts expressed a phenotype corresponding to an immature myeloid cell (7.2+, MY7+, My906+, VIM D5-). Ph1-chromosome-positive blasts from this patient's first relapse had completely changed their surface marker characteristics: they had become TdT-positive and exhibited surface features characteristic of early T blasts (WT1+, 10.2+, OKT9+, OKT10+, 7.2-, OKT6-). Together, these features provide evidence that myeloid cells may share a common precursor with T cells.

The activation of MAPKAP kinase 2 was investigated under heat-shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor-alpha (TNF-alpha). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10-15 min after stimulation either by heat shock or TNF-alpha. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF-alpha treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.

Transforming growth factor (TGF)-alpha is a pleiotropic polypeptide which mediates a variety of tissue-specific cellular responses such as induction of proliferation, cell migration, vascularization, and formation of extracellular matrix. TGF-alpha is produced by certain tumor cells and embryogenic tissues, as well as by normal cells of different origin. Within the granulocytic lineage, TGF-alpha production has been shown in promyelocytic leukemia cells induced to differentiate, as well as in blood eosinophils of patients with the idiopathic hypereosinophilic syndrome. The present study was carried out in order to examine expression of the TGF-alpha gene in polymorphonuclear (PMN) and mononuclear (MN) blood cells of normal healthy donors. While MN and neutrophilic PMN failed to synthesize TGF-alpha transcripts and protein, eosinophils constitutively exhibited TGF-alpha transcripts accompanied by the release of immunoreactive TGF-alpha protein. Exposure of PMN and MN cells to the leukocyte-activating cytokines interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor resulted in a several-fold increase of TGF-alpha mRNA expression and protein release by eosinophils, but not by neutrophils and MN cells. PMN and MN were insensitive to induction of TGF-alpha release by IL-8 and granulocyte colony-stimulating factor. These results point to a functional role of eosinophils in disorders characterized by unbalanced TGF-alpha production such as disease states associated with abnormal matrix formation and neovascularization which may be explained by the present demonstration of TGF-alpha production in these cells.

The product of the c-raf-1 proto-oncogene, Raf-1, is known to encode a 74-kDa ubiquitously expressed cytoplasmic serine/threonine kinase. Various growth factors such as epidermal growth factor, acidic fibroblast growth factor, platelet-derived growth factor, insulin, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2, IL-3 and erythropoietin have been shown to induce phosphorylation of Raf-1, thereby activating Raf-1 kinase. Raf-1 is, thus, believed to play a role in coupling growth factor receptors to proliferation. We have examined the role of Raf-1 in the mitogenic response of human peripheral blood-derived IL-2 receptor expressing T cells to human recombinant IL-2 employing c-raf antisense (AS) oligodeoxyribonucleotide. Uptake studies of oligonucleotides indicated that incorporation of oligomers was maximal at 4 h and oligodeoxynucleotides remained stable in these cells for up to 24 h. Treatment of T cells with the AS oligodeoxyribonucleotide in intracellular duplex formation followed by efficient translation blockade of c-raf-1. In contrast, sense (S) and nonsense (NS) oligodeoxynucleotides failed to form intracellular duplexes and did not interfere with translation of c-raf-1, suggesting specific elimination of c-raf-1 by the AS oligomer. Proliferation of T cells ([3H]thymidine incorporation) following exposure to IL-2 was substantially reduced when the c-raf-1 AS oligodeoxyribonucleotide was added to cultures, while the mitogenic response to this factor remained almost unaffected in the presence of S and NS oligodeoxyribonucleotides.

In this study we report the identification of a novel transcription factor, termed Nuclear Factor-jun (NF-jun). This factor contributes to inducible transcription of the c-jun gene in human myeloid leukemia cells. NF-jun was, however, undetectable in nuclear proteins from human monocytes, granulocytes, resting T lymphocytes and lung fibroblasts. NF-jun shares several features with the well characterized NF-kappa B in that binding activity can be generated in cytosolic extracts by treatment with dissociating agents. In addition, binding of NF-jun to its recognition site is enhanced by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, tumor necrosis factor alpha or the protein synthesis inhibitor cycloheximide (CHX). However, as revealed by competition assays and electrophoretic mobility shift assays, purified NF-kappa B fails to bind to the c-jun fragment which contains the NF-jun site, and this fragment fails to compete with NF-kappa B for binding. UV crosslinking showed that NF-jun contains a 55 and a 125 kDa protein species. These findings demonstrate that the c-jun gene can be regulated by a transcription factor distinct from AP-1. Our findings also indicate that while NF-jun has several features in common with the NF-kappa B binding protein including its subcellular localization and its ability to translocate from the cytoplasm to the nucleus, this factor recognizes a unique DNA sequence. Moreover, the activity of this protein is differentially regulated in various cell types. NF-jun might function as a signal transducing molecule in order to mediate rapid induction of the early response gene c-jun in a cell type- and stimulus-specific manner.

We demonstrate that macrophage-colony stimulating factor (M-CSF) is expressed in human fibroblasts at the mRNA and protein level. Following activation with both interleukin (IL)-1 beta and IL-4, fibroblasts synthesized M-CSF transcripts detectable by Northern blot analysis with peak expression occurring at 8 h and 12 h, respectively. Exposure of fibroblasts to both cytokines resulted in M-CSF protein release at 60 h of c. 500 U/ml (for IL-1 beta) and 1000 U/ml (for IL-4), relative to a control preparation of recombinant human M-CSF in a murine bone marrow colony assay. Both interleukins synergized to enhance M-CSF mRNA accumulation and their ability to induce M-CSF transcripts could be abolished by treatment with specific neutralizing antibodies. These observations provide support for the idea that fibroblasts may control monocyte/macrophage development and function, and that IL-1 beta and IL-4 are involved in the regulation of this process.

How cancer cells become resistant to chemotherapy is not completely understood, but it is believed that resistance is usually associated with overexpression of drug resistance genes. Drug resistance mediated by the MDR-1 gene is the first well characterized form of drug resistance in human cancer. MDR-1 encodes a phosphoglycoprotein, P-GP, that serves as an energy-dependent drug efflux pump, reducing intracellular drug accumulation and thereby cytotoxicity. We have used ribozymes to reverse the multiple drug resistance phenotype. A hammerhead ribozyme recognizing the GUC sequence at position -6 to -4 close to the translation start site of the 4.5 kb MDR-1 mRNA was prepared by in vitro transcription (MDR-1-RZiv) or chemical synthesis (MDR-1-RZs). Both MDR-1-RZiv and MDR-1-RZs specifically cleaved the MDR-1 mRNA into two parts of the expected size under physiological conditions in an extracellular system with MDR-1-RZiv being more effective. Site-specific cleavage was dependent on time, temperature and [MgCl2]. To examine the in vivo potential of MDR-1-RZ, MDR-1-RZiv and MDR-1-RZs were transfected into a human pleural mesothelioma cell line and into one adriamycin-resistant and one vindesine-resistant subline thereof by liposome-mediated transfer. Incorporation of ribozymes resulted in significantly reduced expression of the MDR-1 gene, with MDR-1-RZs being more potent than MDR-1-RZiv in vitro. MDR-1-RZ reduces P-GP overexpression at the protein level. Liposome-mediated transfer of MDR-1-RZiv or MDR-1-RZs reversed the multiple drug resistance phenotype and restored sensitivity towards chemotherapeutic drugs.

Colony-stimulating factors are required for survival proliferation, differentiation and functional activation of granulocytes, macrophages and their precursor cells. In the present report, however, we demonstrate antiproliferative activity of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) on monoblast cell line U-937 and provide evidence for the involvement of tumor necrosis factor alpha TNF-alpha and interleukin 1 beta (IL 1 beta) in its growth inhibitory action. GM-CSF (but not granulocyte CSF, G-CSF or macrophage CSF, M-CSF) suppressed DNA synthesis and self renewal of U-937 cells. Similarly, medium conditioned by U-937 cells in response to GM-CSF (GM-CSF U-937-CM) was able to reduce clonogenicity and [3H]thymidine uptake by U-937 cells. Since neutralization of GM-CSF present in GM-CSF U-937-CM by monoclonal antibody to GM-CSF did not abrogate the autoinhibitory activity present in GM-CSF U-937-CM, we considered the possibility that other soluble molecules are released by U-937 cells upon GM-CSF stimulation. Neutralization by antibodies to IL 1 beta and TNF-alpha suggested that both monokines could be the antiproliferative principle operating in GM-CSF U-937-CM. Moreover, employing IL 1 beta-specific enzyme-linked immunosorbent assay, TNF-alpha specific radioimmunoassay, Northern analysis using a cloned TNF-alpha-specific cDNA and an oligonucleotide probe for IL 1 beta, we demonstrate GM-CSF-inducible IL 1 beta and TNF-alpha gene expression by U-937 cells at the mRNA and protein level. Although M-CSF expression was induced under similar conditions, M-CSF failed to inhibit growth of U-937 cells.

Clinical and prognostic relevance of the Kiel classification of non-Hodgkin lymphomas (NHL) was investigated in 1127 patients entering a prospective multicenter observation study. Survival of the 782 (69.4 per cent) patients with low-grade malignant NHL (lymphocytic lymphomas, predominantly B-CLL, LP immunocytoma, centrocytic lymphoma, centroblastic-centrocytic lymphoma) exceeded that of the 341 patients (30.2 per cent) with high-grade malignant NHL (centroblastic, immunoblastic, lymphoblastic lymphomas). Prognosis was best in centroblastic-centrocytic lymphoma and in B-CLL and least favorable in immunoblastic and lymphoblastic lymphomas. Survival of LP immunocytoma and centrocytic lymphoma patients was intermediate after 2 to 2.5 years of follow-up. Corresponding to histopathology, pattern of survival curves of low-grade malignant NHL (slow decline, no plateauing) differed from that of high-grade malignant NHL (rapid decline, subsequent plateauing). Prognosis of B-CLL was superior to that of LP immunocytoma. Stages I and II were more frequent in centroblastic-centrocytic lymphoma (21 per cent) than in LP immunocytoma (2.5 per cent) and centrocytic lymphoma (11 per cent). Ability of radiotherapy to induce stable complete remissions in stage III of centroblastic-centrocytic lymphoma indicates prolonged restriction of lymphoma to the lymphatic system. In immunoblastic and centroblastic lymphomas, stages I and II were diagnosed in 34 and 38 per cent of cases, respectively, but only in stage I/IE of centroblastic lymphoma prolonged remissions were achieved by radiotherapy. In advanced high-grade malignant NHL marked improvement of prognosis was solely possible by induction of complete remissions whereas in corresponding low-grade malignant lymphomas also partial remissions were prognostically relevant.