Asterios Tsiftsoglou - Academia.edu (original) (raw)

Papers by Asterios Tsiftsoglou

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2003

Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow a... more Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G 1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype.

Pharmaceutics

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in s... more Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistri...

Journal of Biological Research-Thessaloniki, 2021

Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or re... more Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Results Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-XaSITE-α-globin-HA to interact in vitro with the previously produced 10xHis-XaSITE-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and conf...

Molecular Pharmaceutics, 2018

Protein Replacement Therapy (PRT) has been applied to treat severe monogenetic/metabolic disorder... more Protein Replacement Therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders, characterized by a protein deficiency. In disorders, where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. β-thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult β-globin chain. The resulting imbalance of α-/β-globin chains of adult hemoglobin (α 2 β 2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD)-technology has emerged as a promising therapeutic approach, we produced a human recombinant β-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature β-globin chain. Notably, the produced human recombinant β-globin chain without the TAT peptide, used as internal negative control, failed to be transduced

Molecular Genetics and Metabolism, 2004

The human Sco2 protein is a cytochrome c oxidase assembly protein that participates in mitochondr... more The human Sco2 protein is a cytochrome c oxidase assembly protein that participates in mitochondrial copper pathway, acting downstream of Cox17 protein. In a previous work, we detected mutations in the human SCO2 gene in three unrelated infants with fatal cardioencephalomyopathy and COX deficiency. In this study, full-length processed recombinant wild-type and two mutated forms of hSco2p (w/t-rhSco2p, E140K-rhSco2p, and S225F-rhSco2p) were produced in bacteria as soluble recombinant peptides for the first time and evaluated for differences in their physical state and ability to bind copper. Our data indicate the following: (a) w/t-rhSco2p and S225F-rhSco2p were found to be in a monomeric form in contrast to E140K-rhSco2p that was in a major non-reducible dimer and a minor monomer form; (b) wild-type and mutated rhSco2p exhibited clear differences in their physical conformational state, as shown by circular dichroism and thermal denaturation analyses; (c) copper binding studies showed that E140K-rhSco2p bound markedly less copper while S225F-rhSco2p more than expected as compared to amount of the copper bound with w/t-rhSco2p. rhCox17p served as positive control experiment. These data indicate that S225F and E140K mutations found in the SCO2 gene derived from patients alter the physical conformational state of encoded hSco2p that may disturb the normal copper transport pathway in mitochondria. These findings are valuable for understanding the molecular basis of fatal cardioencephalomyopathy and COX deficiency and for designing appropriate pharmacological interventions.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2010

Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been f... more Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with L-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) L-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time-and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [ 35 S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders.

Springer eBooks, 1996

Providing both basic aspects and recent findings in the molecular and cellular biology of maligna... more Providing both basic aspects and recent findings in the molecular and cellular biology of malignant tumours, this work focuses on the mechanisms by which normal and neoplastic cells proliferate, differentiate and undergo apoptosis. The differentiation of hemopoietic, epithelial and neuronal cells is treated with respect to growth factors, signal transduction, transcription factors and genes regulating the cell cycle and the commitment to maturation. The role of oncogenes in neoplastic cell growth and cell death is also covered. The book deals with the regulation of globin gene expression by the LCR locus and the mechanisms of RNA stability for iron-binding and other proteins. The induction of differentiation of neoplastic cells as an alternative approach to cancer therapy is also discussed.

Biochemical Pharmacology, Nov 1, 1995

We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block com... more We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block commitment of murine erythroleukemia (MEL) cells to terminal erythroid maturation. In this study, we further investigated the mechanism of this blockade. Treatment of MEL cells with N6mAdo inhibited cell growth, prevented accumulation of committed cells, suppressed methylation of total cytoplasmic RNA, and erased the expression of "memory" response, an event that precedes initiation of commitment. Furthermore, N6mAdo increased the level of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and altered the SAH/SAM ratio that influences methylation of ribonucleic acid (RNA). Moreover, analysis of the intracellular extracts revealed that N6-mAdo is converted into S-(N6-methyl)-adenosylhomocysteine (N6-SAH) in MEL cells, an active intermediate that affects methylation of RNA. Therefore, we conclude that N6-mAdo prevents induction of MEL cell differentiation by affecting methylation of critical RNA transcripts involved in expression of "memory" and initiation of commitment. It is likely that this inhibition occurs via conversion of N6mAdo into N6-SAH.

Cell Biology International, Nov 24, 2011

Repetitive sequences consist of .50% of mammalian genomic DNAs and among these SINEs (short inter... more Repetitive sequences consist of .50% of mammalian genomic DNAs and among these SINEs (short interspersed nuclear elements), e.g. B1 elements, account for 8% of the mouse genome. In an effort to delineate the molecular mechanism(s) involved in the blockade of the in vitro differentiation program of MEL (murine erythroleukaemia) cells by treatment with methylation inhibitors, we detected a DNA region of 559 bp in chromosome 7 located downstream of the 39-end of the b major globin gene (designated B1-559) with unique characteristics. We have fully characterized this B1-559 region that includes a B1 element, several repeats of ATG initiation codons and consensus DNA-binding sites for erythroid-specific transcription factors NF-E2 (nuclear factor-erythroid-derived 2), GATA-1 and EKLF (erythroid Krü ppel-like factor). Fragments derived from B1-559 incubated with nuclear extracts form protein complexes in both undifferentiated and differentiated MEL cells. Transient reporter-gene experiments in MEL and human erythroleukaemia K-562 cells with recombinant constructs containing B1-559 fragments linked to HS-2 (hypersensitive site-2) sequences of human b-globin gene LCR (locus control region) indicated potential cooperation upon erythropoiesis and globin gene expression. The possible interaction between the B1-559 region and b major globin gene transcriptional activation upon execution of erythroid MEL cell differentiation programme is discussed.

Several studies over the past years have established that a large variety of coordination compoun... more Several studies over the past years have established that a large variety of coordination compounds exert potent antineoplastic activity on a number of neoplasms (1). These findings have prompted us to search systematically for new and more potent metal containing antitumor agents. Our efforts have been directed towards the synthesis and biological study of copper(II) bi- and poly-nuclear complexes involving multiatom bridging units. Our rationale in this drug development has been as follows: i. copper is a natural metal ion constituting polynuclear reaction sites in biological processes and it is thought to be essential for normal animal metabolism (2). ii. copper(II) bi- and poly-nuclear complexes bridged by multiatom units exhibit a diverse range of interactions between the metal centers, which can be engineered by judiciously choosing the bridging and terminal ligands (3). Obviously, the establishment of any correlations between the nature and magnitude of these interactions and the biological activity of compounds, it is hoped to lead to new foundations for a molecular engineering of coordination compounds with biological properties forseable both in nature and magnitude.

Annals of the New York Academy of Sciences, Dec 1, 1982

Biochimica Et Biophysica Acta - Biomembranes, Nov 1, 1981

Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation ... more Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in 3H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24-96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development.

Archives of Oral Biology, Dec 1, 2018

Mesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair dam... more Mesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair damaged tissue, immunosuppression, ability to differentiate into several cell types and their secretome. Earlier studies have demonstrated their angiogenic potential in vitro and in vivo. However, little is known regarding proangiogenic inducers of stable endothelial transdifferentiation of MSCs. Here, we employed human MSCs from the Apical Papilla (SCAP) and investigated whether recombinant human erythropoietin-alpha (rhEPOa) could act as such inducer. Design: Cultured SCAP cells were exposed to rhEPOa and assessed for cell growth kinetics, viability and morphology, as well as their capacity to form capillary tubule structures in selected microenvironments. RT-PCR was used to monitor endothelial markers and activation of EPO/EPOR pathway signaling components; while gelatin zymographies to assess activation of MMP-2. Results: rhEPOa treatment initially (48 h) accelerated cell proliferation and allowed SCAP to sprout microtubular structures. Morphological and biochemical differentiation was accompanied by activation of MMP-2 and upregulation of PECAM-1, VEGFR2, vWF and VE-cadherin/CDH5. SCAP expressed the cognate EPO-R, while rhEPOa-treated SCAP exhibited higher expression of molecules involved in EPO/EPOR pathway (EPOR and JAK2). Conclusion: rhEPOa is capable of promoting endothelial transdifferentiation of SCAP which may be of clinical value in treating of ischemic disorders.

Cells, Aug 20, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Blood, Nov 18, 2011

Abstract 3860 Primary myelofibrosis (PMF) comprises a myeloproliferative neoplasia accompanied by... more Abstract 3860 Primary myelofibrosis (PMF) comprises a myeloproliferative neoplasia accompanied by imbalance of various tissues of the mesoderm, let alone the hematopoietic tissue. Involvement of multiple hematopoietic lineages during disease progression suggests the clonality of myelofibrosis that can be attributed to an initial stem cell defect at the very early stage of the stem cell hierarchy. Hematopoietic and endothelial phenotypes of circulating multipotent stem cells in patient peripheral blood, along with the increased microvascular density in the bone marrow, leads to the hypothesis that the critical event in PMF involves malignant transformation of a stem cell with hemangioblastic potential. Former studies have provided functional evidence that activated JAK2 signalling in primitive human hematopoietic cells is sufficient to drive key processes involved in the pathogenesis of the disease. In this study, the functionality and differentiation potential of circulating primitive JAK2V617F+ stem cells from primary myelofibrosis patients is assessed. Primitive stem cells were isolated from peripheral blood of 25 patients. All patients participating in the study were diagnosed with primary myelofibrosis, have been untreated, and were found positive for JAK2V617F mutation. Isolated stem cells were analysed for purity and assessed for the expression of markers characteristic for the hemangioblast phenotype (CD34, CD133, CD45, VEGFR2, VE-Cadherin, E-Cadherin, CD31) with flow cytometry. Genomic DNA was isolated from various stem cell populations to determine the mutational status by PCR. Our results indicate that long term repopulating stem cells circulating in peripheral blood bear the JAK2V617F mutation. Hemangioblast resembling populations within the isolated prime stem cells were also found positive for the mutation. Long term repopulating stem cells bearing different allele burden for JAK2V617F mutation from PMF patient peripheral blood were expanded for up to 4 months. Various colonies formed after seeding in semisolid media were characterised by morphological features (CFU-GEMM, CFU-GM, CFU-E, CFU-M, CFU-Endo) and expressing genes by quantitative PCR. Moreover, allele burden determination for various progenitors of both hematopoietic and endothelial lineages was performed. JAK2V617F allele burden varied within individual progeny phenotypes, indicating the acquisition of the mutation that boosts the outgrowth of the malignant clone within the hemangioblast compartment of the bone marrow. Endothelial and macrophage progenitors appear heterozygotic while all rest progenitors of various hematopoietic lineages can be either heterozygotic or homozygotic. This indicates high genomic instability of the JAK2V617F+ malignant clone as it is driven into hematopoietic differentiation. Our results indicate the existence of a malignant clone with hemangioblast phenotype in PMF which can differentiate into hematopoietic and/or endothelial progenitors in vitro. Our experiments shed light to the pathogenesis of PMF by characterising the potential of the defective stem cell subpopulation responsible for the disease. Disclosures: No relevant conflicts of interest to declare.

Journal of Cellular Physiology, 1982

The effects of dimethyl sulfoxide (DMSO)‐induced differentiation of Friend leukemia cells in vitr... more The effects of dimethyl sulfoxide (DMSO)‐induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [14C] glycerol or [3H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO‐diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.

Biochimica et biophysica acta, Apr 1, 1981

Advances in enzyme regulation, 1977

Chemischer Informationsdienst, May 1, 1979

ChemInform Abstract Ausgehend von D-Mannosamin-hydrochlorid (I) werden die Halogenacetamido-zucke... more ChemInform Abstract Ausgehend von D-Mannosamin-hydrochlorid (I) werden die Halogenacetamido-zucker (II) und ihre Tetraacetyl-Derivate (III) hergestellt. Die β-Konfiguration von (III) wird durch Vergleich der optischen Drehwerte und der 1H-und 13C-NMR-Daten bekannterähnlicher Verbindungen ermittelt. Die Wirkung auf Tumor-infizierte Mäuse wird untersucht und mit denen analoger Derivate in der Glucose-und Galaktose-Reihe verglichen.