Efficient cell proliferation and predominant accumulation of ɛ-globin mRNA in human leukemic K562 cells which produce mostly Hb Gower 1

Summary Long-term cultures of K562(S) cells in 50–75 M hemin allow the selection of hemin-resistant K562 cells together with cells which proliferate efficiently while fully induced to express the human embryonic globin genes, as the hemoglobin Gower 1 (₂₂) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly -globin mRNA (-globin mRNA/-globin mRNA=2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.We thank Dr Irene Bozzoni (Centro degli Acidi Nucleici, Università di Roma) for the pXCR7 probe. Address for reprint request: R.G. Centro Studi Biochimici sul Morbo di Cooley, Via Borsari 46, I-44100 Ferrara.     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Natural killer-like activity in human cultured lymphoid cells propagated in the presence of interleukin-2: acquired resistance to prostaglandin E2- or dexamethasone-mediated suppression

The cytotoxic activity of human peripheral blood lymphocytes against the natural killer-sensitive target K562 was suppressed both by prostaglandin E2 and dexamethasone. On the other hand, cultured lymphoid cells propagated in the presence of interleukin-2 showed strong cytotoxic reactivity against K562 targets, and were resistant to prostaglandin E2- or dexamethasone-mediated suppression.     

Natural killer-like activity in human cultured lymphoid cells propagated in the presence of interleukin-2: acquired resistance to prostaglandin E2- or dexamethasone-mediated suppression

Summary The cytotoxic activity of human peripheral blood lymphocytes against the natural killer-sensitive target K562 was suppressed both by prostaglandin E₂ and dexamethasone. On the other hand, cultured lymphoid cells propagated in the presence of interleukin-2 showed strong cytotoxic reactivity against K562 targets, and were resistant to prostaglandin E₂- or dexamethasone-mediated suppression.     

Cell-mediated cytotoxicity by natural killer and killer cells, lipid peroxidation and glutathione

In the course of spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral lymphocytes as effector cells, no lipid peroxidation occurred as measured by the production of ethane and thiobarbituric acid-reactive material. Furthermore, impairment of major cellular defense systems of target cells (K562 cells for SCMC, Chang liver cells for ADCC), by decreasing their glutathione content, had no effect on either lipid peroxidation or the cytotoxic response. These findings indicate that peroxidative damage is not a mechanism of NK and K cell-mediated cytotoxicity.     

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the ^A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.     

Staurosporine aglycone (K252-c) and arcyriaflavin A from the marine ascidian,Eudistoma sp

Staurosporine aglycone (K252-c) (compound1) and arcyriaflavin A (2) were isolated from a specimen of the marine ascidian,Eudistoma sp., collected off the coast of West Africa. In addition to expressing micromolar and submicromolar inhibition of enzyme activity against seven protein kinase C isoenzymes and inhibition of proliferation of the human lung cancer A549 and P388 murine leukemia cell lines,1 also inhibited cell adhesion of the EL-4.IL-2 cell line and expressed activity in the K562 bleb and neutrophil assays.     

Chromosomal territory segmentation in apoptotic cells

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.     

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE₂ increased [³H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE₂ increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE₂ obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE₂-induced cell cycle regulatory protein expression and thymidine incorporation. PGE₂ caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE₂-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells. Received 30 January 2009; received after revision 03 March 2009; accepted 10 March 2009     

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.     

Hemoglobin catabolism and the killing of intraerythrocyticPlasmodium falciparum by chloroquine

To evaluate how chloroquine kills malaria parasites, hemoglobin catabolism was studied at the various stages of intraerythrocytic parasite development. We found that hemoglobin catabolism is switched off whenPlasmodium falciparum parasites mature to the late trophozoite or early schizont stages and is switched on again during the ring stage. When hemoglobin catabolism is switched off, the parasites are resistant to the morphologic effects of chloroquine. Although the ring stage parasites failed to mature in the presence of chloroquine, some of them switched on hemoglobin ingestion and became stuffed with hemoglobin-filled vesicles, indicating a distal block in catabolism. In fact, we demonstrated a high-grade block in hemozoin production during a 22 h incubation of synchronized ring forms; ferriprotoporphyrin IX (FP) incorporation into the -hematin of hemozoin decreased from 900 to 50 pmol/10⁶ parasitized erythrocytes. We propose that the primary effect of chloroquine on hemoglobin catabolism is to block FP polymerization to -hematin. Secondarily, toxic FP and FP-chloroquine complexes accumulate and are available to exert their several toxicities, which include inhibition of hemoglobindegrading proteases and membrane damage. As a consequence, maturation is arrested and eventually the parasites die and lyse.     

Human papillomavirus 16 E5 up-regulates the expression of vascular endothelial growth factor through the activation of epidermal growth factor receptor, MEK/ ERK1,2 and PI3K/Akt

The E5 oncoprotein of human papillomavirus (HPV) 16 plays an important role in early cervical carcinogenesis. Vascular endothelial growth factor (VEGF) plays a central role in switching on the angiogenic phenotype during early cervical carcinogenesis. However, the relationship between E5 and VEGF has not previously been examined. To clarify the regulatory role of E5 in VEGF expression, we transferred the E5 gene into various cell types. E5 increased VEGF expression. The addition of epidermal growth factor receptor (EGFR) inhibitor significantly suppressed VEGF expression, demonstrating that E5 stimulates VEGF expression through the activation of EGFR. E5-mediated EGFR activation was accompanied by phosphorylation of Akt and ERK1/2, which are also involved in VEGF expression. Furthermore, the mRNA stability of VEGF was not affected by E5, but VEGF promoter activity could be modulated by inhibitors of the EGFR, MEK-ERK1/2 and PI3K/Akt pathways in E5-expressing cells. Collectively, these novel results suggest that HPV 16 E5 increases VEGF expression by activating EGFR, MEK/ERK1/2 and PI3K/Akt. Received 23 November 2005; received after revision 10 January 2006; accepted 9 February 2006     

Glutamine stimulates translation of uncoupling protein 2mRNA

Uncoupling protein 2 (UCP2) belongs to a family of transporters/exchangers of the mitochondrial inner membrane. Using cell lines representing natural sites of UCP2 expression (macrophages, colonocytes, pancreatic beta cells), we show that UCP2 expression is stimulated by glutamine at physiological concentrations. This control is exerted at the translational level. We demonstrate that the upstream open reading frame (ORF1) in the 5’ untranslated region (5’UTR) of the UCP2 mRNA is required for this stimulation to take place. Cloning of the 5’ UTR of the UCP2 mRNA in front of a GFP cDNA resulted in a reporter gene with which GFP expression could be induced by glutamine. An effect of glutamine on translation of a given mRNA has not been identified before, and this is the first evidence for a link between UCP2 and glutamine, an amino acid oxidized by immune cells or intestinal epithelium and playing a role in the control of insulin secretion. Received 26 January 2007; received after revision 16 April 2007; accepted 8 May 2007 C. Hurtaud, C. Gelly: These authors contributed equally to this work.     

Differential effects of prostaglandins and isoproterenol on cAMP content and Na, K pump activity in rat submandibular acini

The beta-adrenergic agonist isoproterenol and prostaglandins E1 and E2 (but not F2 alpha) increased the cAMP content of rat submandibular acini in vitro, but only isoproterenol enhanced ouabain-sensitive 86Rb (K) uptake. These findings suggest that cAMP is not involved in the activation of the Na, K pump in salivary cells.     

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K _D of CA52?CDARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs?? potentialities to target, to purify, and to modulate the function of cellular markers.     

Postnatal decline of (Ca2+ + Mg2+)-activated membrane ATPase in cattle red cells.

Acitivity of membrane bound (Ca2+ + Mg2+)-stimulated ATPase, associated with Ca2+ outward transport, in calf red cells is high at birth and declines with a rate constant of 0.041 d-1 after the 3rd week. The decline parallels the disappearance of fetal hemoglobin.     

Immunological properties of oxygen-transport proteins: hemoglobin,hemocyanin and hemerythrin

It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte–pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.     

Malate dehydrogenase isoenzymes in the pancreatic islets of obese-hyperglycemic mice

Zusammenfassung Mit der Polyacrylamidgel-Elektrophorese lassen sich aus Langerhansschen Inseln fettsüchtig-hyperglykämischer Mäuse 2 Fraktionen von Malatdehydrogenase isolieren. Die rascher wandernde Franktion scheint eine grössere Aktivität gegen Malat zu besitzen, während in der Aktivität gegen Oxalacetat kein Unterschied besteht.

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This work was supported by grants from the U.S. Public Health Service (No. AM-05759-06), the Medical Faculty in Umea, Therese and Johan Anderssons Stiftelse, the Swedish Medical Research Council (No. K67-12x-562-03) and the Swedish Diabetes Association.     

Predominant expression of σ and ε globin genes in human leukemia K-562(S6) variant cell line

Summary In the human leukemia K-562(S6) cell line (a) the accumulation ofa-globin chains is low or absent, (b) -globin gene expression is correlated with expression of -chains and (c) the genes responsible for the terminal cell division are not operated within 8–12 cell cycles, while K-562 (S6) cells are fully induced to erythroid differentiation.This work was supported by grants from the Italian National Research Council (C.N.R.) G. R. is a recipient of a fellowship from the National Committee for Nuclear Energy, Rome. Address for reprint requests: R. G., Centro di Studi Biochimici, Via L. Borsari 46, I-44100 Ferrara.