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N Weich

Publications and source records attributed to N Weich.

13 recordsLinked to original sources

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

Animals↗

RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes.

Myelopoiesis and lymphopoiesis are controlled by haematopoietic growth factors, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for G(alphai)- and G(alphaq)-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS18. RGS18 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS18 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS18 mRNA was not detected in cells of the lymphoid lineage. RGS18 was also highly expressed in mouse embryonic 15-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS1, RGS2 and RGS16, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS18 was shown to interact specifically with the G(alphai-3) subunit in membranes from K562 cells. Furthermore, overexpression of RGS18 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-1. In yeast cells, RGS18 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS18 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS18 might have an important role in the regulation of megakaryocyte differentiation and chemotaxis.

Amino Acid Sequence↗

Suppressive effects of TNF-alpha, TGF-beta1, and chemokines on megakaryocytic colony formation in CD34+ cells derived from umbilical cord blood compared with mobilized peripheral blood and bone marrow.

CD34+ cells from human umbilical cord blood (CB) were isolated and investigated for megakaryocytic (MK) colony formation in response to recombinant human (rh) stimulatory and suppressive cytokines and compared with their counterparts in normal BM and G-CSF-mobilized peripheral blood (mPBL). First, we observed that IL-11 by itself at any dosage had no stimulator activity on MK colony formation derived from CD34+ cells in CB, mPBL, and BM. IL-3, steel factor (SLF), or thrombopoietin (Tpo) alone stimulated numbers of colony-forming unit-megakaryocyte (CFU-MK) in a dose-dependent fashion. Maximum growth of MK progenitor cells was noted in the presence of a combination of cytokines: IL-11, IL-3, SLF, and Tpo. The frequency of CFU-MK in CB and mPBL was significantly greater than that in BM, and the size of colonies in CB and mPBL was significantly greater than that in BM, and the size of colonies was larger as well. In addition, an increased number of big mixed colonies containing MK were observed in CB and mPBL. In the presence of IL-11, IL-3, SLF, and Tpo, CFU-MK derived from CB, mPBL, and BM was suppressed by tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). CFU-MK derived from normal BM was inhibited by some chemokines evaluated, whereas CFU-MK derived from CB was suppressed only by platelet factor-4 (PF-4), IFN-inducible protein-10 (IP-10), Exodus-1, Exodus-2, and Exodus-3, but to a lesser degree. In CB, unlike granulocyte-macrophage (CFU-GM), erythroid (BFU-E), high-proliferative potential (HPP-CFC), or multipotential (CFU-GEMM) progenitors, at least a subpopulation of MK progenitors are in S-phase. Therefore, CB MK progenitors respond to the suppressive effects of some members of the chemokine family. Similar results were noted for burst-forming unit-MK (BFU-MK). Our results indicate that CB and mPBL are rich sources of MK progenitors and that MK progenitors in CB are responsive to the suppressive effects of TNF-alpha and TGF-beta1 and some members of the chemokine family.

Antigens, CD34↗

The role of recombinant interleukin 11 in megakaryocytopoiesis.

Recombinant human interleukin 11 (rHuIL-11) is a multifunctional cytokine with activities on a broad range of hematopoietic cells including primitive stem cells and mature progenitor cells. Analysis of rHuIL-11 in vitro has revealed that its hematopoietic activities are predominantly a result of synergistic interactions with other early-acting factors such as IL-3 and Steel factor. Studies indicate that rHuIL-11 acts directly on purified stem and progenitor cell populations and can support the growth of colony forming units-megakaryocyte in these cultures. In normal animals, rHuIL-11 has a potent effect on cells of the megakaryocyte (MK) lineage. Administration of rHuIL-11 results in a two- to threefold increase in circulating platelets, stimulation of bone marrow (BM) and spleen progenitor numbers, and enhanced MK maturation as measured by a shift to higher ploidy values. rHuIL-11 administration in preclinical models of myelosuppression induced by chemotherapy and/or irradiation has shown a reproducible acceleration of platelet recovery and, in some models, enhanced neutrophil and red blood cell recovery. rHuIL-11 has been tested in a non-human primate myelosuppression model using carboplatin. Administration of rHuIL-11 following carboplatin treatment was found to eliminate the period of severe thrombocytopenia (<20,000 platelets/ml) and enhance the recovery of platelets to normal levels (>100,000/ml). Recently, human clinical trials conducted with rHuIL-11 in patients treated with chemotherapy have demonstrated its potent thrombopoietic activity, including improved platelet nadirs, enhanced platelet recovery and a significant decrease in the number of patients who require platelet transfusions. Combined with the preclinical results, these studies confirm that this cytokine will be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and BM transplantation.

Animals↗

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors. The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells. The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

Amino Acid Sequence↗

Influence in vitro of IL-3/Epo fusion proteins compared with the combination of IL-3 plus Epo in enhancing the proliferation of single isolated erythroid and multipotential progenitor cells from human umbilical cord blood and adult bone marrow.

Human interleukin-3/erythropoietin (IL-3/Epo) fusion protein have been constructed, expressed, and tested for biological activity. These fusion proteins were previously shown to be active on erythroid progenitors (BFU-E) from unseparated human bone marrow. We evaluated if these fusion proteins could stimulate erythroid and multipotential progenitor cells directly at the single-cell level. Two IL-3/Epo fusion proteins containing short (SL-3E, two amino acids) and long (LL-3E, 23 amino acids) linker sequences as well as a short linker Epo/IL-3 sequence (SL-E3, three amino acids) were tested. Highly enriched CD34 or BFU-E enriched CD34 CD33- cells from human umbilical cord blood or CD34 HLA-DR+CD33- cells from normal adult bone marrow were sorted as single cells into single wells. The combination of Epo plus IL-3 synergized to enhance the proliferation of BFU-E and multipotential progenitors (CFU-GEMM) in comparison to the individual effects of these cytokines. The three fusion proteins also enhanced proliferation of BFU-E and CFU-GEMM at the single-cell level and were at least as active as the combination of Epo and IL-3, demonstrating that IL-3/Epo fusion proteins directly stimulate proliferation of BFU-E and CFU-GEMM and that biological activity of IL-3 and Epo in vitro can be maintained when these proteins are fused. The activity of the combination of Epo and IL-3 or the fusion proteins was partially neutralized by preincubation with monoclonal antibodies to either Epo or IL-3 and was neutralized by greater than 90% by the combination of both antibodies, suggesting that the Epo and IL-3 components of the fusion proteins were both involved in the enhancing activity of these proteins. Additionally, use of monoclonal antibody to the human Epo receptor completely blocked the stimulating/enhancing activity of Epo alone, Epo plus IL-3, or the fusion proteins for stimulation of colony formation by BFU-E and CFU-GEMM but not for granulocyte-macrophage progenitors (CFU-GM), suggesting that the enhancing effects of the fusion proteins are most likely mediated, at least in part, by the Epo receptor.

Bone Marrow Cells↗

Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system.

The development of blood cells from hematopoietic stem cells is controlled by multiple cytokines. These growth factors influence survival, cell cycle status, differentiation into lineage-committed progenitors, final maturation into blood cells, and perhaps self-renewal of stem cells. The specific contribution of IL-6 to these processes in vivo was evaluated in mice with a targeted disruption of the IL-6 gene. Decreases in the absolute numbers of CFU-Sd12 and preCFU-S, as well as in the functionality of LTRSC in these mutant mice, suggests a role for IL-6 in the survival, self-renewal, or both of hematopoietic stem cells and early progenitors. In addition, as a result of the IL-6 deficiency, the control between proliferation and differentiation of the progenitor cells of the granulocytic-monocytic, megakaryocytic, and erythroid lineages into mature blood cells is altered, leading to abnormal levels of committed progenitors of these lineages and to a slow recovery from hematopoietic ablation.

Animals↗

Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation.

Erythropoietin mediates the rapid phosphorylation of Raf-1 in the murine cell lines HCD-57 and FDC-P1/ER, which proliferate in response to this cytokine. Phosphorylation occurs at both serine and tyrosine residues and as such is similar to the Raf-1 phosphorylation seen after interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and interleukin-2 stimulation in other murine cell lines. Such data suggest that these growth factors may share a common mechanism(s) of Raf-1 phosphorylation. Furthermore, in association with Raf-1 phosphorylation, erythropoietin induces a 2-3-fold increase in Raf-1 kinase activity as measured in immune complex kinase assays in vitro. Finally, a c-raf antisense oligodeoxyribonucleotide, which specifically decreases intracellular Raf-1 levels, also substantially inhibits both erythropoietin and IL-3-directed DNA synthesis. Together, these results provide evidence that activated Raf-1 is a necessary component of erythropoietin and IL-3 growth signaling pathways.

Animals↗

Characteristics of erythroleukemia cells selected for vincristine resistance that have accelerated inducer-mediated differentiation.

The induction of murine erythroleukemia cells (MELC; DS19/Sc9) to terminal differentiation by hexamethylenebisacetamide (HMBA) is characterized by a latent period of 10-12 hr before onset of commitment to terminal-cell division and increased transcription of globin genes. MELC variants, derived from this parental cell line, selected for resistance to vincristine (VC), can be induced to differentiate with little or no latent period. This study shows that accelerated HMBA-induced commitment is characteristic of MELC with a low level (2- to 5-fold) of VC resistance in four independently derived cell lines. Both resistance to VC and accelerated differentiation are stable phenotypes for at least 50 passages (approximately 5 months) in the absence of VC. Low-level VC-resistant MELC do not display increased levels of P-glycoprotein or mdr1, mdr2, and mdr3 mRNAs, nor do they exhibit cross-resistance to colchicine or doxorubicin. These cells do show (i) increased level of protein kinase C activity, (ii) reduced accumulation of [3H]VC, and (iii) restoration of VC sensitivity in the presence of verapamil. MELC selected for higher levels of VC resistance (approximately 500-fold) do express high levels of P-glycoprotein and the mdr3 gene. During HMBA-induced differentiation, DS19/Sc9 decrease [3H]VC accumulation, but P-glycoprotein content does not change. A VC-transport-associated protein, also critical for the process of induced differentiation, may be constitutively present in VC-resistant MELC, accounting for their enhanced sensitivity to inducer. This protein accumulates by exposure of VC-sensitive cells to HMBA, contributing to their differentiation and decreased level of VC accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Relationship between globin mRNA accumulation and commitment to terminal cell differentiation in inducer sensitive and resistant erythroleukemia variants.

The relationship between the kinetics of commitment to terminal cell differentiation and the rates of accumulation of globin mRNA has been examined during the induction of erythroid differentiation by polar/apolar chemical inducers in murine erythroleukemia cells (MELC), under conditions of more and less rapid commitment. Two differentiation inducers and three MELC variants have been studied. Hexamethylene bisacetamide (HMBA) initiates more rapid commitment than does dimethylsulfoxide (Me2SO). MELC variant DR10 is resistant to induction by Me2SO and responds sluggishly to HMBA, in comparison with the DS19-Sc9 variant. V3.17, an MELC variant resistant to low concentrations of vincristine, shows increased sensitivity to the inducers and an accelerated rate of commitment to terminal differentiation compared with DS19-Sc9. It is demonstrated that commitment and the actual expression of differentiation, as measured by the accumulation of alpha-, beta maj-, and beta min-globin mRNA, are temporally coordinated functions during induced differentiation of a transformed cell line by exposure to polar/apolar agents.

Acetamides↗

Regulation of murine alpha-, beta major-, and beta minor-globin gene expression.

Transcriptional properties of the mouse globin genes and their promoters were examined in COS cells using transient expression assays. Transfected intact mouse alpha 1-globin genes generate transcripts, whereas beta maj and beta min globin transcripts are detected only if the genes are associated with a strong exogenous (SV40) enhancer sequence. Under these conditions the ratio of accumulated beta maj and beta min mRNA sequences approximates the 4:1 ratio observed in reticulocytes and murine erythroleukemia cells (MELC) induced to differentiate by hexamethylene bisacetamide (HMBA). As determined using hybrid genes consisting of globin gene 5' regions fused to the chloramphenicol acetyltransferase (CAT) structural gene, this 4:1 ratio appears dependent upon the relative activities of the two 5' promoter regions.

Acetamides↗

Vincristine-resistant erythroleukemia cell line has marked increased sensitivity to hexamethylenebisacetamide-induced differentiation.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A.DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3.17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iii) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBA-mediated DS19 differentiation. We suggest that this V3.17 MEL cell line may express a factor that circumvents HMBA-mediated early events, which prepare the cells for commitment to terminal differentiation.

Acetamides↗

Hypomethylation of DNA during differentiation of Friend erythroleukemia cells.

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.

Animals↗