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R Gambari

Publications and source records attributed to R Gambari.

At least 145 records · Page 8Linked to original sources
PubMed1983

Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation.

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.

Acetamides↗
PubMed1982

[Expression of genes for human globin in the cell line K-562: an experimental model for the study of fetal erythropoiesis].

Human leukemia K-562(S) cells are a useful model system to study the relationship between cell proliferation and induced erythroid differentiation. In these studies K-562(S) cells were cultured in alpha -medium, 10% fetal calf serum and induced to express erythroid genes by 75 microM hemin, 1.2 mM butyric acid or 1.5 ng/ml actinomycin D. Cell number was determined using a ZF Coulter Counter and the increase in the proportion of hemoglobin-containing cells was detected by a specific colorimetric reaction with benzidine. The characterization of the synthesized hemoglobins was performed by cellulose-acetate gel electrophoresis of post-mitochondrial supernatants. By cloning K-562(S) cells in semi-solid medium (O,33% agar) containing 75 microM hemin a variant cell line, denominated K-562(S6), have been isolated which does not undergo terminal cell division but does express human globin genes and accumulates on the average 12 pg of Hb/cell. K-562(S6) cells accumulate, upon exposure to 75 microM hemin, mostly Hb Gower 1 (zeta 2 epsilon 2) and low amounts of Hb X (epsilon 2 gamma 2) and Hb Portland (zeta 2 gamma 2), being suitable for studies focused on the expression of embryonic globin genes and on the molecular mechanisms controlling the switching from embryonic-type to fetal-type hemoglobin accumulation, when in the human embryo zeta and epsilon globin genes become less active, being sharply increased accumulation of alpha and gamma globin chains.

Cell Count↗
PubMed1982

Tumor promoter-mediated effects on Friend leukemia cells induced by actinomycin D: flow microfluorometry analysis and erythroid differentiation.

Murine erythroleukemic cells can be induced to express globin genes and to differentiate by chemical compounds such as dimethylsulfoxide, hemin, butyric acid, hexamethylenebisacetamide. In addition, tumor promoters have been shown to inhibit this induced differentiation, thus suggesting that the conversion of a cell into a cancer cell can be thought as an alteration in its state of differentiation. We found that actinomycin D-induced erythroid differentiation is not inhibited by the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in two murine erythroleukemia cell lines. Thus we investigated the cellular distribution with respect to the cell cycle in synchronized cells cultured with TPA and actinomycin D in order to determine whether alteration of the cell cycle parameters can interfere to some extent with the process of differentiation. Cells synchronized in S phase progress through the cell cycle and differentiate when cultured with actinomycin D and TPA. On the contrary, some cells synchronized in G1 are prevented entry to S phase by the tumor promoter. Only in this cellular population was there found inhibition of intracytoplasmic hemoglobin accumulation. Therefore in the Friend leukemia cell system, the tumor promoter TPA does not inhibit DNA synthesis, but the progression of the cells through G1. In addition our results suggest that this progression through G1 and/or early S is a cellular event which is critical for the organization of the expression of globin and related genes.

Animals↗
PubMed1981

The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA.

We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.

Amino Acid Sequence↗
PubMed1981

Embryonic hemoglobins in man and other mammals.

This report attempts to review the present state of research on embryonic hemoglobins in humans and other mammals from a cytologic, molecular, and clinical point of view. In all mammals, the yolk sac is the site responsible for the "primitive" erythropoiesis, which produces an erythroid population characterized by peculiar cytologic features and globin gene expression. Morphological and molecular events that underlie prenatal erythropoiesis are described herein giving rise to questions regarding biology at large (i.e., the differential activity of genes capable of similar functions); molecular biology of eukaryotic genes (i.e., globin gene organization and structural subtleties); and clinical hematology (i.e., syndromes associated with the appearance of embryonic hemoglobins.

Aneuploidy↗
PubMed1981

Control of globin gene expression in mouse yolk sac erythroid cells.

In the present paper are reported results from our laboratory and data of the literature on the structure and the expression of embryonic globin genes in mouse fetuses. The following aspects are presented: 1. Erythropoiesis and synthesis of globin chains in fetal mice. 2. The transcriptional and post-transcriptional control of embryonic globin gene expression. 3. The chromosomal location and organization of mouse embryonic globin genes.

Animals↗
PubMed1981

Expression of erythroid differentiation and terminal cell division in induced Friend leukemia cells.

Hexamethylenebisacetamide and hemin are both inducers of Friend leukemia cells to express globin genes. In Friend leukemia cells treated with hexamethylenebisacetamide the accumulation and translation of globin mRNA and the accumulation of hemoglobin are associated with terminal cell division. As hemin does not induce the expression of the set of genes responsible for terminal cell division, the relationship between cell cycle and globin gene expression was investigated during the late differentiation of Friend leukemia cells treated with hemin. Our results demonstrate that terminal cell division is not necessarily performed in cells which accumulate both globin mRNA and hemoglobin, suggesting that these features are not necessarily coupled in leukemia erythroid differentiation.

Acetamides↗
PubMed1981

Translational discrimination between embryonic and adult mouse globin mRNAs in the rabbit reticulocyte lysate system.

In the present study mouse adult (alpha, beta) and embryonic globin (x, y, z) mRNas are compared for their capacity of being translated in a micrococcus nuclease treated rabbit reticulocyte lysate. The results of these experiments indicate that: 1) alpha and beta adult RNAs are translated with a higher efficiency as compared to the alpha-like and beta-like embryonic mRNAs; 2) adult beta and embryonic beta-like messengers are translated more efficiently than their corresponding adult alpha and embryonic alpha-like messengers; 3) the alpha/beta synthetic ratio, both for adult and embryonic globins is highly dependent upon the concentration of globin mRNAs. For embryonic globins the ratio decreases from 0.21 at low mRNA concentration to 0.11 at high mRNA concentration. For adult globin mRNAs the alpha/beta chain synthetic ratio decreases from 1.4 to 0.9 within the same range of concentration.

Animals↗
PubMed1979

Tumor promoter-mediated inhibition of cell differentiation: suppression of the expression of erythroid functions in murine erythroleukemia cells.

Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cell division cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (delta-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.

Cell Differentiation↗
PubMed1979

Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Murine erythroleukemia cells (MELC) are induced to erythroid differentiation by culture with 4 mM hexamethylenebisacetamide. The relationship between onset of accumulation of globin mRNA and cell cycle phase has been examined in MELC fractionated with respect to cell cycle (G1, early S, mid S, and late S/G2) by the technique of centrifugal elutriation. This technique provides synchronized populations of cells without use of chemicals that block cell function (e.g., DNA synthesis). It is shown that the initial onset of accumulation of globin mRNA occurs during the first G1 phase after a complete S traversed in culture with inducer. Once globin mRNA synthesis is initiated, it continues throughout the cell cycle. Studies of synchronized MELC populations exposed to hexamethylenebisacetamide starting in G1, mid S, or late S/G2, provide evidence that an effect of the inducer during G1 or early S is critical to initiation of accumulation of newly synthesized globin mRNA and to the prolongation of G1, both of which are associated with terminal erythroid differentiation.

Acetamides↗
PubMed1979

Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells.

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.

Acetamides↗
PubMed1978

Isolation and characterization of the messenger RNAs for mouse embryonic globin chains.

Messenger RNAs for mouse embryonic globins were purified from yolk sac erythroid cells by oligodeoxythymidilate-cellulose chromatography and sucrose density centrifugation. Full-sized complementary DNA copies of embryonic globin mRNAs were synthesized. Acrylamide gel electrophoresis of these RNAs indicate an average molecular weight of 220 000, including a polyadenylated sequence of about 35 residues, as determined by hybridization to [3H]polyuridylate. The wheat-germ translation products of mRNAs have the size and the ionic characteristics of the four embryonic globin chains alpha, x, y and z. Hybridization kinetics in vast RNA excess were performed and compared to standard r0t curves of adult globin messengers, demonstrating a total base sequence complexity of about 880 000 daltons, that is four different RNA sequences of 220 000 molecular weight. The titration of embryonic globin cDNAs with increasing amounts of their complementary RNA templates indicates that the embryonic globin messengers were isolated at a high degree of purity.

Animals↗
PubMed1978

Presence of spectrin in untreated Friend erythroleukemic cells. Its accumulation upon treatment of the cells with dimethyl sulfoxide.

Friend leukemia cells (FLC) are nucleated erythroid precursors, and are markedly stimulated towards more advanced stages of differentiation by treatment with dimethyl sulfoxide (DMSO). The presence of spectrin, an erythrocyte membrane protein, has been investigated in untreated and in DMSO-treated FLC by indirect immunofluorescence and by analysis in SDS-polyacrylamide gel electrophoresis of low-ionic-strength cell extracts immunoprecipitated with a monospecific anti-spectrin serum. Spectrin is detectable in significant amounts in the "inducible" clones prior to DMSO stimulation, and accumulates 4- to 5-fold upon addition of this compound to the cultures. Spectrin accumulation occurs rather early (24 hours after cell seeding) and reaches its peak on the third day to decline thereafter. Semiquantitative determinations of spectrin amounts present in DMSO-stimulated 745A and A degree 1 cells on the third day after treatment were 2.4 X 10(5) and 3.0 X 10(5) molecules/cell, respectively. Spectrin is also detectable in very low amounts in an "uninducible" line of FLC, and is not accumulated upon DMSO treatment thereof, whereas treatment with hemin does cause a significant increase of spectrin-positive cells. These data indicate that spectrin is a convenient "early" marker for in vitro studies of erythropoiesis.

Animals↗
PubMed1978

Synthesis of globin mRNA in relation to the cell cycle during induced murine erythroleukemia differentiation.

The relationship between the synthesis of globin mRNA and the phase of cell cycle was examined in synchronized murine erythroleukemia cells. Cells were synchronized with respect to the cell division cycle either by culture with 2 mM thymidine or 2 mM thymidine followed by 0.5 mM hydroxyurea, which caused cells to accumulate in late G1 or early S (referred to as G1/S boundary). Cells were induced to erythroid differentiation by culture with 280 mM dimethyl sulfoxide or 4 mM hexamethylene bisacetamide. These inducers do not alter the progression of cells from the G1/S boundary through S, G2, and M, but do cause prolongation of the subsequent G1 phase. Accumulation of newly synthesized globin mRNA is first detected when cells are in this G1 phase.

Acetamides↗