Ota Fuchs - Academia.edu (original) (raw)
Papers by Ota Fuchs
International Journal of Molecular Sciences
Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and t... more Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).
Recent Developments in Myelodysplastic Syndromes [Working Title]
Acta Haematologica, 2011
Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We pr... more Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFβ/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.
Journal of Photochemistry and Photobiology B: Biology, 1998
The effect of 5aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and pro... more The effect of 5aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as nonnal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-( 4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM S-ALA, 4 h, 18 J cm ') reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT).
Acute Leukemia - The Scientist's Perspective and Challenge, 2011
RQ-PCR permits accurate quantification of PCR products during the exponential phase of the PCR am... more RQ-PCR permits accurate quantification of PCR products during the exponential phase of the PCR amplification process. Three main types of this analysis are used: (1) RQ-PCR using the hydrolysis probe format ("TaqMan probe"); (2) RQ-PCR using the hybridization probe format; and (3) RQ-PCR using SYBR Green Dye . Analysis with TaqMan probe uses 5´-3´ exonuclease activity of the Thermus aquaticus (Taq) polymerase to detect and quantify the PCR product. The hydrolysis probe is positioned within the target sequence and is conjugated with a reporter fluorochrome at the 5´ end and a quencher fluorochrome at the 3´ end. The quencher avoids the reporter from emission of a fluorescence signal as long as the probe is intact and both fluorochromes are in the close proximity. Upon amplification of the target sequence, the hydrolysis probe is displaced from the DNA strand by the Taq polymerase and subsequently hydrolysed by the 5´-3´ exonuclease activity of the Taq polymerase. This results in displacement of of the reporter from the quencher and the fluorescence of the reporter becomes detectable. Generally two quantification types (relative or absolute) in RQ-PCR are possible. A relative quantification based on relative expression of a target gene versus a reference gene is adequate for the most purposes. For absolute quantification, based either on an internal or an external calibration curve Ptaffl, 2001, Ptaffl et al. 2002, the methodology must be highly validated and the identical LightCycler PCR amplification efficiencies for standard material and target cDNA must be confirmed.
Current Signal Transduction Therapy, 2011
ABSTRACT
Current Signal Transduction Therapy, 2011
... survival was observed in patients with brain tumors (high-grade glioma and anaplastic astrocy... more ... survival was observed in patients with brain tumors (high-grade glioma and anaplastic astrocy-toma) who were intratumorally administered trabedersen, compared with patients receiving standard chemotherapy (Temozolomide or Procarbacine, Lomustine (CCNU), Vin-cristin). ...
The CCAAT/enhancer binding protein alpha (C/EBPα or CEBPA) is the founding member of a fam- ily o... more The CCAAT/enhancer binding protein alpha (C/EBPα or CEBPA) is the founding member of a fam- ily of related leucine zipper transcription factors that play important roles in myeloid differentiation. Target- ed inactivation of C/EBPα in mice demonstrates its im- portance in the proper development and function of liver, adipose tissue, lung and haematopoietic tissues. C/EBPα is highly expressed in these
Biochimica et Biophysica Acta
Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubat... more Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubation with isonicotinic acid hydrazide and transferrin-bound 59 Fe, were reincubated with various synthetic chelating agents and the amount of radioiron released ...
Ciba Foundation symposium
This paper reviews and reports the results of experiments on the mechanism by which iron is deliv... more This paper reviews and reports the results of experiments on the mechanism by which iron is delivered from extracellular transferrin to reticulocyte mitochondria in which haem is synthesized. It is suggested that transferrin donates the iron directly to mitochondria. Transferrin seems to be bound to mitochondria during the process of iron release. When the release of iron from transferrin is blocked by haem, the iron-transferrin complex remains bound to mitochondria so that the total amount of transferrin molecules associated with mitochondria increases in haem-treated reticulocytes. This also leads to an increase in the number of transferrin molecules in the cytosol. In haem-deficient reticulocytes, the rate of dissociation of iron from transferrin is accelerated and the uptake of iron by mitochondria is increased. When the synthesis of haem is inhibited, the non-haem iron in the cytosol (i.e. mainly low-molecular-weight and ferritin iron) comes from mitochondria. Greater amounts of non-haem iron can also be induced in reticulocytes incubated with highly saturated transferrin but, in this case, iron does not seem to be accumulated in mitochondria. These results represent an experimental basis for the elucidation of the excessive non-haem iron accumulation in erythroid cells observed in various clinical conditions.
Acta biologica et medica Germanica
Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid d... more Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.
Casopís lékar̆ů c̆eských
The transforming growth factor-beta (TGF-beta) family of cytokines consists of more than 30 secre... more The transforming growth factor-beta (TGF-beta) family of cytokines consists of more than 30 secreted structurally related polypeptides including TGF-beta s, activins and bone morphogenetic proteins (BMP). This family regulates a broad spectrum of biological functions, including cell proliferation, differentiation, apoptosis, migration, and extracellular matrix production. Signaling by these cytokines occurs via binding to specific receptors with serine/threonine kinase activity and activation of specific downstream intracellular effectors (the receptor-regulated Smad transcription factors). TGF-beta is a regulator of all stages of hematopoiesis. Depending on the differentiation stage of the target cell, the local environment and the concentration of TGF-beta, TGF-beta can be positive or negative regulator of proliferation, apoptosis or differentiation of hematopoietic cells. TGF-beta inhibits the growth of primitive hemopoietic cells with a stem cell immunophenotype but has no effect or in some cases stimulates the growth of committed progenitors. Persistent loss of TGF-beta signaling in the hemopoietic stem cells and the primitive hemopoietic progenitor cells may possibly be involved in the development of malignant transformation.
Ceskoslovenska fysiologie / Ustredni ustav biologicky
Casopís lékar̆ů c̆eských
The mRNA level for ferrochelatase was assessed in the total cytoplasmic RNA isolated from mouse e... more The mRNA level for ferrochelatase was assessed in the total cytoplasmic RNA isolated from mouse erythroleukaemic cells, line 707, in the course of induction of erythroid differentiation with hexamethylenebisacetamide and from spleen cells of mice in different stages of erythroid differentiation after phenylhydrazine administration. The level of this mRNA increased after five days of induction of erythroleukaemic cells about six times. An even more marked increase of the mRNA level for ferrochelatase (13.5X) was found in the total cytoplasmic RNA isolated from erythropoietic spleen cells of mice after induction of haemolytic anaemia by phenylhydrazine. Haem synthesis inhibitors (succinylacetone, isonicotinic acid hydrazide and penicillamine) caused a reduced gene expression for ferrochelatase in erythroleukaemic cells of mice induced with hexamethylenebisacetamide. Conversely addition of precursors of haem synthesis (5-aminolaevulinic acid or protoporphyrin IX) led to an increased mRNA synthesis for ferrochelatase. In addition to protoporphyrin synthesis the supply of another substrate, iron, is decisive for the gene expression ferrochelatase. Iron chelators (desferrioxamine or pyridoxal isonicotinoylhydrazone) reduced and iron donors (iron bound to transferrin or pyridoxal isonicotinoylhydrazone) enhanced mRNA synthesis for ferrochelatase. Added haemin reduces mRNA synthesis for ferrochelatase in fully induced erythroleukaemic cells but increases the gene expression ferrochelatase in the course of induction of erythroid differentiation because it acts as an inducing agent.
Neoplasma
The levels of iron-responsive element-binding protein (IRE-BP I) mRNA throughout the course of er... more The levels of iron-responsive element-binding protein (IRE-BP I) mRNA throughout the course of erythroid differentiation were investigated in several lines of murine erythroleukemia (MEL) cells (Friend 745, 707 and Fw cells). Fw cells are not inducible for ferrochelatase activity and heme synthesis. Cytoplasmic ferrochelatase mRNA and transferrin receptor (TfR) mRNA levels are only insignificantly increased in Fw cells after induction. We have found increased levels of (IRE-BP 1) mRNA during erythroid differentiation of MEL cells of all lines investigated. Run-on transcription reactions using isolated nuclei from Friend 707 cells showed increased (IRE-BP 1) gene transcription following induction of erythroid differentiation with 5 mmol hexamethylenebisacetamide (HMBA). The increase in (IRE-BP 1) gene transcription is only about 2-fold in comparison with 8-fold increase in the level of (IRE-BP 1) mRNA during 96 hours of Friend 707 cells induction. These findings indicate that the stability of (IRE-BP 1) mRNA might also play a role in the increase of (IRE-BP 1) mRNA levels after Friend 707 cells induction. The possible role of increased (IRE-BP) mRNA levels in the elevation of TfR numbers during erythroid differentiation is discussed.
Neoplasma
Mouse erythroleukemia (MEL) cells transformed by Friend virus and induced to undergo erythroid di... more Mouse erythroleukemia (MEL) cells transformed by Friend virus and induced to undergo erythroid differentiation by treatment with hexamethylenebisacetamide (HMBA) increase erythroid specific 5-aminolevulinate synthase (ALAS-E) mRNA levels by 4-15-fold and decrease "housekeeping" 5-aminolevulinate synthase (ALAS-N) mRNA levels by 1.2-1.4-fold. Iron affects translation of (ALAS-E) mRNA but nothing is known about its effect at the pretranslational level of the expression of (ALAS-E) mRNA. The aim of this study was to examine the effect of iron on the synthesis of (ALAS-E) mRNA and (ALAS-N) mRNA. This effect was compared with the effect of iron on the iron on the synthesis of H-ferritin and transferrin receptor mRNAs. Incubation of uninduced or induced MEL cells with iron chelator pyridoxal isonicotinoyl hydrazone (PIH) or desferrioxamine (Desferal) and 3H-uridine decreased the level of the 3H-labeled (ALAS-E) mRNA. The treatment with either diferric transferrin or Fe-PIH increased the level of the 3H-labeled (ALAS-E) mRNA. The opposite effect was observed on the level of the 3H-labeled (ALAS-N) mRNA. These findings indicate that iron might play its role also at the pretranslational level of the expression of ALAS-E or in the stability of (ALAS-E) mRNA.
Casopís lékar̆ů c̆eských
Haem is a constituent of various haemoproteins which are essential for the function of all living... more Haem is a constituent of various haemoproteins which are essential for the function of all living cells. The haem biosynthetic pathway is now well understood and the molecular biology of its function and dysfunction in sideroblastic anemias and porphyrias is currently intensively investigated. Each type of these disorders is the result of a specific reduction in the activity of one of the enzymes of the heme biosynthetic pathway. In this paper a review of the recent progress using molecular probes in the study of sideroblastic anemias and porphyrias is presented. Until now different mutations in genes for haem biosynthesis pathway have been described and other mutations will be identified in the near future and this will provide new tools for the diagnosis of mutated genes carriers. This is particularly important in porphyrias with acute manifestations (acute intermittent porphyria, coproporphyria and variegate porphyria) since the prevention of acute attacks rests on the detection of asymptomatic carriers among members of affected families.
Biomedica biochimica acta
The effect of changes of iron availability in the culture medium on the expression of TfR (transf... more The effect of changes of iron availability in the culture medium on the expression of TfR (transferrin receptor) and glutathione peroxidase (GSHPx) genes was investigated in uninduced or induced murine erythroleukemia (Friend) cells of lines 707 and Fw labeled with (3H)uridine for 3 h. The level of the labeled cytoplasmic TfR mRNA exhibited about 2-3-fold increase and the level of the labeled GSHPx mRNA about 2-fold increase in induced Friend 707 cells in comparison with uninduced cells. Raising the levels of intracellular iron by treatment of Friend 707 cells with either hemin, Fe-pyridoxal isonicotinoyl hydrazone (PIH) or diferric transferrin (Tf) resulted in decreased levels of the labeled TfR mRNA. On the other hand, hemin and Fe-PIH caused an increase in the labeled cytoplasmic GSHPx mRNA. Conversely, treatment with PIH or desferrioxamine stimulated synthesis of TfR mRNA and decreased the levels of the labeled GSHPx mRNA. In Fw cells we did not find any difference between the levels of the labeled cytoplasmic TfR mRNA in induced and uninduced cells and the levels of labeled cytoplasmic GSHPx mRNA were only slightly increased by induction. Changes of the intracellular iron pool caused the same effect as in Friend 707 cells.
International Journal of Molecular Sciences
Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and t... more Compared to solid tumors, the role of PD-L1 in hematological malignancies is less explored, and the knowledge in this area is mostly limited to lymphomas. However, several studies indicated that PD-L1 is also overexpressed in myeloid malignancies. Successful treatment of the acute myeloid leukemia (AML) is likely associated with elimination of the residual disease by the immune system, and possible involvement of PD-L1 in this process remains to be elucidated. We analyzed PD-L1 expression on AML primary cells by flow cytometry and, in parallel, transcript levels were determined for the transcription variants v1 and v2. The ratio of v1/v2 cDNA correlated with the surface protein amount, and high v1/v2 levels were associated with worse overall survival (p = 0.0045). The prognostic impact of PD-L1 was limited to AML with mutated nucleophosmin and concomitant internal tandem duplications in the FLT3 gene (p less than 0.0001 for this particular AML subgroup).
Recent Developments in Myelodysplastic Syndromes [Working Title]
Acta Haematologica, 2011
Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We pr... more Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFβ/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.
Journal of Photochemistry and Photobiology B: Biology, 1998
The effect of 5aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and pro... more The effect of 5aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as nonnal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-( 4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM S-ALA, 4 h, 18 J cm ') reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT).
Acute Leukemia - The Scientist's Perspective and Challenge, 2011
RQ-PCR permits accurate quantification of PCR products during the exponential phase of the PCR am... more RQ-PCR permits accurate quantification of PCR products during the exponential phase of the PCR amplification process. Three main types of this analysis are used: (1) RQ-PCR using the hydrolysis probe format ("TaqMan probe"); (2) RQ-PCR using the hybridization probe format; and (3) RQ-PCR using SYBR Green Dye . Analysis with TaqMan probe uses 5´-3´ exonuclease activity of the Thermus aquaticus (Taq) polymerase to detect and quantify the PCR product. The hydrolysis probe is positioned within the target sequence and is conjugated with a reporter fluorochrome at the 5´ end and a quencher fluorochrome at the 3´ end. The quencher avoids the reporter from emission of a fluorescence signal as long as the probe is intact and both fluorochromes are in the close proximity. Upon amplification of the target sequence, the hydrolysis probe is displaced from the DNA strand by the Taq polymerase and subsequently hydrolysed by the 5´-3´ exonuclease activity of the Taq polymerase. This results in displacement of of the reporter from the quencher and the fluorescence of the reporter becomes detectable. Generally two quantification types (relative or absolute) in RQ-PCR are possible. A relative quantification based on relative expression of a target gene versus a reference gene is adequate for the most purposes. For absolute quantification, based either on an internal or an external calibration curve Ptaffl, 2001, Ptaffl et al. 2002, the methodology must be highly validated and the identical LightCycler PCR amplification efficiencies for standard material and target cDNA must be confirmed.
Current Signal Transduction Therapy, 2011
ABSTRACT
Current Signal Transduction Therapy, 2011
... survival was observed in patients with brain tumors (high-grade glioma and anaplastic astrocy... more ... survival was observed in patients with brain tumors (high-grade glioma and anaplastic astrocy-toma) who were intratumorally administered trabedersen, compared with patients receiving standard chemotherapy (Temozolomide or Procarbacine, Lomustine (CCNU), Vin-cristin). ...
The CCAAT/enhancer binding protein alpha (C/EBPα or CEBPA) is the founding member of a fam- ily o... more The CCAAT/enhancer binding protein alpha (C/EBPα or CEBPA) is the founding member of a fam- ily of related leucine zipper transcription factors that play important roles in myeloid differentiation. Target- ed inactivation of C/EBPα in mice demonstrates its im- portance in the proper development and function of liver, adipose tissue, lung and haematopoietic tissues. C/EBPα is highly expressed in these
Biochimica et Biophysica Acta
Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubat... more Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubation with isonicotinic acid hydrazide and transferrin-bound 59 Fe, were reincubated with various synthetic chelating agents and the amount of radioiron released ...
Ciba Foundation symposium
This paper reviews and reports the results of experiments on the mechanism by which iron is deliv... more This paper reviews and reports the results of experiments on the mechanism by which iron is delivered from extracellular transferrin to reticulocyte mitochondria in which haem is synthesized. It is suggested that transferrin donates the iron directly to mitochondria. Transferrin seems to be bound to mitochondria during the process of iron release. When the release of iron from transferrin is blocked by haem, the iron-transferrin complex remains bound to mitochondria so that the total amount of transferrin molecules associated with mitochondria increases in haem-treated reticulocytes. This also leads to an increase in the number of transferrin molecules in the cytosol. In haem-deficient reticulocytes, the rate of dissociation of iron from transferrin is accelerated and the uptake of iron by mitochondria is increased. When the synthesis of haem is inhibited, the non-haem iron in the cytosol (i.e. mainly low-molecular-weight and ferritin iron) comes from mitochondria. Greater amounts of non-haem iron can also be induced in reticulocytes incubated with highly saturated transferrin but, in this case, iron does not seem to be accumulated in mitochondria. These results represent an experimental basis for the elucidation of the excessive non-haem iron accumulation in erythroid cells observed in various clinical conditions.
Acta biologica et medica Germanica
Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid d... more Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.
Casopís lékar̆ů c̆eských
The transforming growth factor-beta (TGF-beta) family of cytokines consists of more than 30 secre... more The transforming growth factor-beta (TGF-beta) family of cytokines consists of more than 30 secreted structurally related polypeptides including TGF-beta s, activins and bone morphogenetic proteins (BMP). This family regulates a broad spectrum of biological functions, including cell proliferation, differentiation, apoptosis, migration, and extracellular matrix production. Signaling by these cytokines occurs via binding to specific receptors with serine/threonine kinase activity and activation of specific downstream intracellular effectors (the receptor-regulated Smad transcription factors). TGF-beta is a regulator of all stages of hematopoiesis. Depending on the differentiation stage of the target cell, the local environment and the concentration of TGF-beta, TGF-beta can be positive or negative regulator of proliferation, apoptosis or differentiation of hematopoietic cells. TGF-beta inhibits the growth of primitive hemopoietic cells with a stem cell immunophenotype but has no effect or in some cases stimulates the growth of committed progenitors. Persistent loss of TGF-beta signaling in the hemopoietic stem cells and the primitive hemopoietic progenitor cells may possibly be involved in the development of malignant transformation.
Ceskoslovenska fysiologie / Ustredni ustav biologicky
Casopís lékar̆ů c̆eských
The mRNA level for ferrochelatase was assessed in the total cytoplasmic RNA isolated from mouse e... more The mRNA level for ferrochelatase was assessed in the total cytoplasmic RNA isolated from mouse erythroleukaemic cells, line 707, in the course of induction of erythroid differentiation with hexamethylenebisacetamide and from spleen cells of mice in different stages of erythroid differentiation after phenylhydrazine administration. The level of this mRNA increased after five days of induction of erythroleukaemic cells about six times. An even more marked increase of the mRNA level for ferrochelatase (13.5X) was found in the total cytoplasmic RNA isolated from erythropoietic spleen cells of mice after induction of haemolytic anaemia by phenylhydrazine. Haem synthesis inhibitors (succinylacetone, isonicotinic acid hydrazide and penicillamine) caused a reduced gene expression for ferrochelatase in erythroleukaemic cells of mice induced with hexamethylenebisacetamide. Conversely addition of precursors of haem synthesis (5-aminolaevulinic acid or protoporphyrin IX) led to an increased mRNA synthesis for ferrochelatase. In addition to protoporphyrin synthesis the supply of another substrate, iron, is decisive for the gene expression ferrochelatase. Iron chelators (desferrioxamine or pyridoxal isonicotinoylhydrazone) reduced and iron donors (iron bound to transferrin or pyridoxal isonicotinoylhydrazone) enhanced mRNA synthesis for ferrochelatase. Added haemin reduces mRNA synthesis for ferrochelatase in fully induced erythroleukaemic cells but increases the gene expression ferrochelatase in the course of induction of erythroid differentiation because it acts as an inducing agent.
Neoplasma
The levels of iron-responsive element-binding protein (IRE-BP I) mRNA throughout the course of er... more The levels of iron-responsive element-binding protein (IRE-BP I) mRNA throughout the course of erythroid differentiation were investigated in several lines of murine erythroleukemia (MEL) cells (Friend 745, 707 and Fw cells). Fw cells are not inducible for ferrochelatase activity and heme synthesis. Cytoplasmic ferrochelatase mRNA and transferrin receptor (TfR) mRNA levels are only insignificantly increased in Fw cells after induction. We have found increased levels of (IRE-BP 1) mRNA during erythroid differentiation of MEL cells of all lines investigated. Run-on transcription reactions using isolated nuclei from Friend 707 cells showed increased (IRE-BP 1) gene transcription following induction of erythroid differentiation with 5 mmol hexamethylenebisacetamide (HMBA). The increase in (IRE-BP 1) gene transcription is only about 2-fold in comparison with 8-fold increase in the level of (IRE-BP 1) mRNA during 96 hours of Friend 707 cells induction. These findings indicate that the stability of (IRE-BP 1) mRNA might also play a role in the increase of (IRE-BP 1) mRNA levels after Friend 707 cells induction. The possible role of increased (IRE-BP) mRNA levels in the elevation of TfR numbers during erythroid differentiation is discussed.
Neoplasma
Mouse erythroleukemia (MEL) cells transformed by Friend virus and induced to undergo erythroid di... more Mouse erythroleukemia (MEL) cells transformed by Friend virus and induced to undergo erythroid differentiation by treatment with hexamethylenebisacetamide (HMBA) increase erythroid specific 5-aminolevulinate synthase (ALAS-E) mRNA levels by 4-15-fold and decrease "housekeeping" 5-aminolevulinate synthase (ALAS-N) mRNA levels by 1.2-1.4-fold. Iron affects translation of (ALAS-E) mRNA but nothing is known about its effect at the pretranslational level of the expression of (ALAS-E) mRNA. The aim of this study was to examine the effect of iron on the synthesis of (ALAS-E) mRNA and (ALAS-N) mRNA. This effect was compared with the effect of iron on the iron on the synthesis of H-ferritin and transferrin receptor mRNAs. Incubation of uninduced or induced MEL cells with iron chelator pyridoxal isonicotinoyl hydrazone (PIH) or desferrioxamine (Desferal) and 3H-uridine decreased the level of the 3H-labeled (ALAS-E) mRNA. The treatment with either diferric transferrin or Fe-PIH increased the level of the 3H-labeled (ALAS-E) mRNA. The opposite effect was observed on the level of the 3H-labeled (ALAS-N) mRNA. These findings indicate that iron might play its role also at the pretranslational level of the expression of ALAS-E or in the stability of (ALAS-E) mRNA.
Casopís lékar̆ů c̆eských
Haem is a constituent of various haemoproteins which are essential for the function of all living... more Haem is a constituent of various haemoproteins which are essential for the function of all living cells. The haem biosynthetic pathway is now well understood and the molecular biology of its function and dysfunction in sideroblastic anemias and porphyrias is currently intensively investigated. Each type of these disorders is the result of a specific reduction in the activity of one of the enzymes of the heme biosynthetic pathway. In this paper a review of the recent progress using molecular probes in the study of sideroblastic anemias and porphyrias is presented. Until now different mutations in genes for haem biosynthesis pathway have been described and other mutations will be identified in the near future and this will provide new tools for the diagnosis of mutated genes carriers. This is particularly important in porphyrias with acute manifestations (acute intermittent porphyria, coproporphyria and variegate porphyria) since the prevention of acute attacks rests on the detection of asymptomatic carriers among members of affected families.
Biomedica biochimica acta
The effect of changes of iron availability in the culture medium on the expression of TfR (transf... more The effect of changes of iron availability in the culture medium on the expression of TfR (transferrin receptor) and glutathione peroxidase (GSHPx) genes was investigated in uninduced or induced murine erythroleukemia (Friend) cells of lines 707 and Fw labeled with (3H)uridine for 3 h. The level of the labeled cytoplasmic TfR mRNA exhibited about 2-3-fold increase and the level of the labeled GSHPx mRNA about 2-fold increase in induced Friend 707 cells in comparison with uninduced cells. Raising the levels of intracellular iron by treatment of Friend 707 cells with either hemin, Fe-pyridoxal isonicotinoyl hydrazone (PIH) or diferric transferrin (Tf) resulted in decreased levels of the labeled TfR mRNA. On the other hand, hemin and Fe-PIH caused an increase in the labeled cytoplasmic GSHPx mRNA. Conversely, treatment with PIH or desferrioxamine stimulated synthesis of TfR mRNA and decreased the levels of the labeled GSHPx mRNA. In Fw cells we did not find any difference between the levels of the labeled cytoplasmic TfR mRNA in induced and uninduced cells and the levels of labeled cytoplasmic GSHPx mRNA were only slightly increased by induction. Changes of the intracellular iron pool caused the same effect as in Friend 707 cells.