Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia (original) (raw)
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Blood, 1985
In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or de...
Acceleration of the hemoglobin switch in cultures in neonate erythroid precursors by adult cells
Blood, 1980
Erythroid colonies from cord blood in which both late (CFU-E) and early (BFU-E) erythroid precursors are present, were grown by the plasma clot technique. Hemoglobin (Hb) synthesis was studied and compared in fresh reticulocytes. 7-day-old colonies. and 14-day-old colonies. In the 8 cases studied, the proportion of HbA synthesis progresssively increased from circulating reticulocytes to 7-day-old colonies and finally in 14-day-old colonies. This result brings evidence that Hb switch is programmed at least at the level of early erythroid precursors. In order to modify the cellular environment of the culture and to examine their influence on globin genes expression. neonate and adult irradiated light density blood cells were added. Irradiated cells from adults. in contrast to those from neonates. were able to increase HbA
Quantitative analysis of globin gene induction in single human erythroleukemic cells
Nucleic Acids Research, 2000
The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.
Proceedings of the National Academy of Sciences, 1988
To test directly whether the control of fetal hemoglobin (HbF) in the adult takes place at the level of erythroid progenitors or at the level of erythroblasts, we treated animals with high doses oferythropoietin and examined the effects of this manipulation on the globin gene programs of erythroid progenitors. We found that administration of erythropoietin produced a rapid expansion of all classes of erythroid progenitors. Almost all the expansion of colonyforming units-erythroid and 46-56% of erythroid clusters was due to the increase of HbF-programmed erythroid progenitors. The expansion of HbF-programmed erythroid progenitors was followed, 2-3 days later, by a wave of reticulocytes containing HbF in the peripheral blood. These results provide direct in vivo evidence that fetal-globin expression in the adult is controlled at the level of erythroid progenitors.
Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line
Blood, 1979
The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.
Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals
Proceedings of the National Academy of Sciences, 1976
The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hem...