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Biomedical subjects

R Gambari

Publications and source records attributed to R Gambari.

At least 127 records · Page 7Linked to original sources
PubMed1986

Lack of correlation between hypomethylation and expression of the HLA-DR alpha gene.

DNA methylation at the 5'-CCGG-3' sites of the HLA-DR alpha gene and relative flanking regions has been analyzed by Msp I and Hpa II enzymatic digestion in order to determine whether a correlation exists between DNA methylation and transcription of the HLA-DR alpha gene. Unexpectedly, and in contrast to the behavior of most eukaryotic genes, no positive correlation was found between hypomethylation and expression. In fact, HLA-DR alpha appears to be fully unmethylated at Msp I/Hpa II sites in K562 cells, not expressing DR molecules, and to exhibit a high degree of methylation in Colo 38 cells, which actively transcribe the gene. The unorthodox behavior of HLA-DR alpha is not unique to melanoma or erythroid cells since a similar, positive correlation between methylation and expression also exists when this analysis is extended to cell lines belonging to other histotypes.

Cell Line↗
PubMed1985

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

Long-term cultures of K562(S) cells in 50-75 microM 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 (zeta 2 epsilon 2) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly epsilon-globin mRNA (epsilon-globin mRNA/gamma-globin mRNA = 2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.

Cell Division↗
PubMed1985

Cytologic evidence for increased rRNA gene activity in hemin-induced K562(S) cells.

Hemin-induced K562(S) cells have been studied for the following parameters: cell proliferation, erythroid induction, hemoglobin accumulation, and activation of ribosomal gene clusters 48 hr after hemin induction. Increased transcriptional activity of rRNA genes has been demonstrated by cytochemical methods at both the cell population and single cell level. The following results have been obtained: (a) The vast majority of induced cells shows a highly significant increase in the number of active rRNA gene clusters per cell. At this time, the number of benzidine-positive cells and the quantity of hemoglobin per cell are almost doubled. (b) Specific rRNA gene clusters are activated within single cells. Activation can be visualized at the single gene cluster level. (c) The increase in the average number of active ribosomal gene clusters per cell is not due to clonal selection, but rather to diffuse activation of several gene clusters. (d) The transcriptional activity of rRNA genes has been shown to be regulated at the cellular level by an agent known to specifically induce derepression of genes responsible for erythroid differentiation.

Cell Differentiation↗
PubMed1985

Gene deletion in an Italian haemophilia B subject.

DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal.

Chromosome Deletion↗
PubMed1984

Human leukemia K562 cells: relationship between hemin-mediated erythroid induction, cell proliferation and expression of c-abl and c-myc oncogenes.

We have studied the expression of the c-myc and c-abl oncogenes in two human leukemic K562 cell lines which do express hemoglobin genes retaining a differential rate of cell proliferation. Our data indicate that in hemin-induced K562(S) cells the expression of c-abl oncogene decreases and appears to be related to a decrease in the proliferation capacity rather than to the activation of differentiated functions. The K562(hC) cell line, which produces large amounts of Hb Gower 1 retaining an efficient rate of cell proliferation, expresses indeed the c-abl oncogene at high level.

Cell Division↗
PubMed1984

Human leukemic K562 cells: differential effects of 5-azacytidine on DNA methylation of epsilon-, gamma-globin and 7SL RNA genes.

5 Azacytidine ribonucleoside (5 Aza CR), greatly enhances erythroid differentiation of the K562(h) cell line, with a sharp increase of embryonic and fetal globin gene expression. This phenomenon is correlated with the undermethylation of gamma-globin but not of epsilon-globin, as the epsilon-globin gene is already extensively undermethylated before 5AzaCR induction. By contrast no variations in both DNA methylation and expression are observed in 7SL RNA genes.

Azacitidine↗
PubMed1984

A monoclonal antibody to human transferrin receptor which inhibits erythroid differentiation of human leukemic K-562 cells.

In this paper we demonstrate that the 42/6 monoclonal antibody to human transferrin receptor (1) inhibits erythroid differentiation of human leukemic K-562 cells without affecting cell proliferation. Erythroid induction was monitored by benzidine-staining of K-562 cell suspensions and hemoglobin accumulation by cellulose acetate gel electrophoresis of post-mitochondrial cell lysates (4,5). Our results suggest that erythroid differentiation and cell growth require a different number of transferrin receptors.

Animals↗
PubMed1984

The mouse beta h1 gene codes for the z chain of embryonic hemoglobin.

Beta h0 and beta h1 are two beta-like globin genes in the mouse beta-globin gene cluster whose functions have not previously been established. Transcripts of both beta h0 and beta h1 are found in yolk sac-derived erythroid cells from mouse embryos and in the murine erythroleukemic cell line GM979. S1 nuclease analysis shows that beta h1 is the more abundant of the two transcripts in both embryonic erythroid cells and the cell line GM979. Hybridization of a beta h1-specific probe to RNA from erythroid cells of 10-, 11-, 12-, 13-, and 14-day-old mouse embryos indicates that levels of beta h1 mRNA are high at 10 and 11 days and then decline during further fetal development. The in vitro translation product of hybrid-selected beta h1 mRNA was analyzed by acid/urea polyacrylamide gel electrophoresis. The beta h1 translation product has the electrophoretic mobility expected for the z chain. We conclude that beta h1 is a fully functional gene which codes for the embryonic z chain.

Animals↗
PubMed1984

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine.

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.

Azacitidine↗
PubMed1984

Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x.

Cytoplasmic poly(A)+mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the alpha-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic alpha-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3' untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known alpha globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the alpha- and beta-globin gene complexes.

Amino Acid Sequence↗
PubMed1983

[Inhibition of heme biosynthesis in erythroid precursors (BFU-e) isolated from human peripheral blood: effects on globin synthesis].

We studied the relationship between heme accumulation and globin synthesis in human erythroid precursors which were stimulated by 2 I.U. of erythropoietin in semi-solid cultures (1% methyl-cellulose, 20% fetal calf serum) and treated with 6-9 micrograms/ml of desferrioxamina (DF), a potent inhibitor of heme synthesis (6). Heme accumulation was detected by specific reaction with benzidine (4), globin synthesis by CM-cellulose column chromatography. Our results demonstrate that globin gene expression occurs in DF-treated erythroid cells which do not accumulate heme molecules. As heme does affect translation and stability of globin mRNA (10) our system might be suitable for studies focused on pathological alterations of erythropoiesis associated with the presence of unstable globin mRNAs and/or unstable globins.

Chromatography, Ion Exchange↗
PubMed1983

[Commitment to erythroid differentiation and globin gene expression in the absence of heme accumulation in Friend leukemia cells].

Friend leukemia cells (FLC) induced to erythroid differentiation by 5 mM hexamethylenebisacetamide (HMBA) are suitable for studies focused on the expression of different genes involved in the same differentiative program. In our studies FL cells have been cultured in alpha-medium (Gibco), 10% fetal calf serum (FCS, Flow Laboratories). Erythroid differentiation has been analysed by benzidine staining of the cells and globin synthesis by pulse-labelling the cells with 3H-leucine (New England Nuclear) and electrophoresis of newly-synthesized proteins on polyacrylamide gels. Hemoglobin production was determined by cellulose acetate gel electrophoresis. Our results demonstrate that heme synthesis is suppressed by the iron-chelator desferrioxamine. In addition "commitment" to erythroid differentiation and transcription and translation of globin mRNAs occur in HMBA-treated FLC in the absence of heme accumulation.

Acetamides↗
PubMed1983

Desferrioxamine inhibits induced erythroid differentiation of human leukemic K-562 cells.

The iron chelator desferrioxamine reversibly inhibits heme accumulation and globin synthesis in human leukemic K-562 cells induced to express erythroid genes by butyric acid. These results suggest that iron metabolism can modulate globin gene expression. In addition we describe experimental conditions (6.25 micrograms/ml desferrioxamine) which do not suppress transcription of globin genes and translation of globin mRNA but prevent heme synthesis. Therefore expression of globin genes in butyric acid induced K-562 cells does not require accumulation of heme molecules. Human leukemic K-562 cells cultured with different inducers and treated with desferrioxamine should be used as a useful model system to further analyse the relationship between expression of erythroid genes, iron metabolism and heme accumulation.

Butyrates↗
PubMed1983

Desferrioxamine-mediated inhibition of heme and hemoglobin synthesis in murine erythroleukemia cells: expression of globin genes and commitment to erythroid differentiation.

The iron-chelator desferrioxamine inhibits heme accumulation and globin synthesis in induced murine erythroleukemia cells, suggesting that iron metabolism can modulate globin gene expression. However we found experimental conditions (12.5 micrograms/ml desferrioxamine) which do not suppress hexamethylenebisacetamide-mediated induction of globin synthesis, commitment of the cells to erythroid differentiation and competence for transcription of beta major globin genes, but prevent heme synthesis. Therefore expression of these erythroid functions might be independent from accumulation of heme molecules.

Acetamides↗