Marion A. Brach

Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells

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.

Interleukin 6: presence and future

SummaryThis review article deals with the basic biological characteristics of the multifunctional cytokine interleukin-6 (IL-6) in man. Three central issues will be addressed more closely: the pathophysiological role of unbalanced IL-6 production in various disease states, the diagnostic usefulness of measurements of IL-6 in biological fluids, and the possible role of IL-6, IL-6 antagonists, and IL-6 derivatives as therapeutic tools in clinical medicine.

Regulation of M-CSF Expression by M-CSF: Role of Protein Kinase C and Transcription Factor NFkB

Macrophage-colony-stimulating factor (M-CSF), also referred to as CSF-1, regulates the survival, growth, differentiation and functional activity of monocytes by binding to a single class of high-affin

Mechanisms of differential regulation of interleukin-6 mRNA accumulation by tumor necrosis factor alpha and lymphotoxin during monocytic differentiation

In the present report we compare the capacity of two related cytokines, tumor necrosis factor (TNF) alpha and lymphotoxin (LT), to modulate mRNA levels of interleukin‐6 (IL‐6) in cells representing different stages of monocytic differentiation including the human leukemia cell lines HL 60, U 937, THP‐1, MonoMac 1 and peripheral blood monocytes. We show that the capacity of TNF alpha and LT to induce IL‐6 mRNA accumulation increases as monocytic differentiation proceeds with TNF alpha being more potent than LT, suggesting that alternate pathways may be used by differentiating cells to control expression of IL‐6. In contrast, in monocytes which constitutively synthesize IL‐6 transcripts, TNF alpha and LT treatment had opposite effects on levels of IL‐6 mRNA accumulation. In these cells TNF alpha enhanced steady state levels of IL‐6 transcripts due to mRNA stabilization, whereas LT shortened IL‐6 mRNA half‐life, most likely due to induction of a RNA destabilizer since LT‐mediated downregulation of levels of IL‐6 mRNA in monocytes could be prevented by inhibition of protein synthesis. Neither TNF alpha nor LT altered IL‐6 mRNA accumulation by interfering with preexisting transcription factors since both TNF alpha and LT required de novo protein synthesis to exert their effects.

Regulation of gene expression of macrophage-colony stimulating factor in human fibroblasts by the acute phase response mediators interleukin (IL)-1β, tumor necrosis factor-α and IL-6

Fibroblasts constitute a major element of the bone marrow stroma. They play a pivotal role in blood cell development by providing the scaffolding required for cellular organization and tissue cohesion and by producing soluble molecules including colony stimulating factors (CSFs) and various interleukins regulating hematopoiesis. Our data demonstrate that the acute phase response mediators interleukin (IL)‐1 β, tumor necrosis factor (TNF)‐α and IL‐6 which are abundantly produced by activated monocytes, enhance levels of macrophage‐colony stimulating factor (M‐CSF) in fibroblasts by both transcriptional and post‐transcriptional mechanisms. The action of these proteins to induce M‐CSF transcript levels was dependent on synthesis of new proteins and was not mediated by protein kinase C (PKC) stimulation as depletion of cellular PKC pools by prolonged exposure of fibroblasts to phorbolester TPA did not prevent factor induced synthesis of M‐CSF transcripts. However, blockade of PKC by the isoquinoline sulfonamide derivative H7 and thus inhibition of phosphorylation was associated with augmentation of the fibroblasts response to TNF‐α and IL‐6.

Differential regulation of interleukin-6 expression in human fibroblasts by tumor necrosis factor-α and lymphotoxin

The treatment of human diploid fibroblasts with tumor necrosis factor (TNP)‐α and with lymphotoxin (LT) is associated with induction of interleuk‐in‐6 (IL‐6) transcripts with TNF‐α being 10‐fold more potent than LT. Here we report on the TNF‐α/LT‐induced signaling mechanisms responsible for the regulation of IL‐6 gene expression in these cells. Run‐on assays demonstrated that both TNF‐α and LT increase IL‐6 mRNA levels by transcriptional activation of this gene. Stability studies of IL‐6 transcripts in fibroblasts showed that TNF‐α delayed IL‐6 mRNA decay but not LT. The induction of IL‐6 transcripts by TNF‐α and LT was not inhibited by the isoquinoline sulfonamide derivative H7. Similarly, depletion of protein kinase C (PKC) by 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) did not change the ability of TNF‐α and LT to induce IL‐6 transcripts, demonstrating that stimulation by these agents may not be mediated by activation of PKC. Stimulation of IL‐6 transcripts in fibroblasts did also not require new protein synthesis as exposure to the protein synthesis inhibitor cycloheximide (CHX) enhanced accumulation of IL‐6 mRNA in the presence or absence of TNF‐α or LT.

Induction of monocytic differentiation and modulation of the expression of c-fos, c-fms and c-myc protooncogenes in human monoblasts by cytokines and phorbolester.

SummaryGrowing evidence suggests that proto-oncogenes regulate central aspects of cellular physiology such as cell proliferation and differentiation. The proto-oncogenes c-fos, c-fms and c-myc are thought to be involved in these processes. In this study the human myelomonoblast line THP-1 has been used to study monocytic differentiation in response to various cytokines and the phorbolester TPA. After treatment of THP-1 cells with Tumor Necrosis Factor (TNF)-alpha, Interleukin (IL-6) and TPA the cells became adherent, lost their division potential and expressed new surface structures associated with monocytic differentiation. The expression of cfos and c-fms transcripts was rapidly induced within 45 min by these agents and declined to undectable levels within 24 h. Exposure of THP-1 to TNF-alpha, IL-6 and TPA was associated with a rapid downregulation of c-myc expression, that returned to starting levels within 36 h. However, treatment of THP-1 with other cytokines including Granulocyte (G)-, Macrophage (M)-Granulocyte/Macrophage (GM)-Colony Stimulating Factor (CSF), Interleukin (IL)-3 and Interleukin (IL)-4 failed to result in monocytic differentiation. These data suggest that changes in c-fms, c-myc and c-fos expression may be related to induction of monocytic differentiation and that their appearance or downregulation can be induced by certain cytokines.

Expression of functional receptors for interleukin‐6 by human polymorphonuclear, leukocytes Downregulation by granulocyte‐macrophage colony‐stimulating factor

Surface interleukin‐6 receptors were identified on human polymorphonuclear leukocytes (PMNL) by monoclonal anti p80‐chain antibody MT 18 Cytoplasmic RNA harvested from PMNL also contained IL‐6‐R transcripts. Binding of recombinant human (rh) interleukin‐6 (IL‐6) to IL‐6‐R bearing PMNL was identified by flow cytometry using phycocrythrin (PE)‐conjugated ligand. Treatment of PMNL with rh granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) led to the inability of PMNL to bind MT 18 monoclonal antibody (moAb) and to display binding sites for PE‐conjugated rh IL‐6. Levels of IL‐6‐R transcripts in PMNL exposed to GM‐CSF were about 5‐fold below those of PMNL cultured in medium only. Though a definitive role for IL‐6 to modulate the function of PMNL was not found, treatment of PMNL with rh IL‐6 clearly resulted in an enhancement of transcript levels of the early response genes c‐fox and c‐jun in these cells, thus indicating that IL‐6 binding is followed by signal transduction.

MODULATION OF CYTOTOXICITY AND DIFFERENTIATION-INDUCING POTENTIAL OF ARABINOFURANOSYLCYTOSINE IN MYELOID LEUKEMIA CELLS BY HEMATOPOIETIC CYTOKINES

Hematopoietic growth factors may be useful in improving the clinical effectiveness of arabinofuranosylcytosine (ara-C). In vitro studies have indicated that interleukin 3(IL-3) and, to a lesser extent, granulocyte-macrophage colony-stimulating factor (GM-CSF), but not G-CSF or M-CSF, may be capable of specifically augmenting the ability of ara-C to kill leukemic myeloid cells by pharmacological and cytokinetic mechanisms including increase of intracellular ara-CTP/dCTP pool ratios and enhanced ara-C DNA incorporation in leukemic blast cells, decrease of IC 90 of ara-C for leukemic colony-forming cells (CFC) as compared with normal CFC growth, and recruitment of quiescent leukemic cells into the cell cycle. In contrast, the combination of ara-C with M-CSF or with the leukemia inhibitory factor (LIF) appears to be useful in overcoming the block in differentiation of leukemic blast, while the effects of GM-CSF and IL-3 on ara-C-induced differentiation appear limited. The combined treatment of human myeloid leukemia cells by ara-C and LIF is associated with down-regulation of c-myc gene expression, transcriptional activation of jun/fos gene expression, and features of functional differentiation (e.g., the capability to reduce nitroblue tetrazolium, to express lysozyme, or to display differentiation-related surface receptors including C3bi and the c-fms protein). On the basis of these in vitro studies first clinical trials are underway that are examining the efficacy of ara-C combinations with these molecules for the treatment of myeloid disorders.

Modulation of Cytotoxicity and Differentiation-Inducing Potential of Cytosine Arabinoside in Myeloid Leukemia Cells by Hematopoietic Cytokines

Cytosine arabinoside (ara-C) has been used extensively in the treatment of acute myelogenous leukemia (AML) for many years (see [4] for review). Besides the conventional low-dose regimen worked out by Frei et al. [16], high dose regimens [29] as well as combinations of ara-C with anthracycline derivatives [30] have further expanded and improved today’s therapeutic repertoire for remission induction of AML. Despite considerable success in improving complete remission (CR) rates using new ara-C-based treatment modalities, we still face appaIlingly high relapse rates. Further improvement of AML induction therapy is thus mandatory.