MB-02 Cells undergo fetal to adult globin gene switching in response to erythropoietin (original) (raw)
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American Journal of Hematology, 1990
Human theta (01)-globin gene represents a member of the d i k e globin gene family residing on chromosome 16. el-Specific transcripts have been detected so far only in erythroid tlssues and in erythroleukemia K562 cells. To investigate systematically its inducible expression and developmental specificity, we analyzed at the RNA level five additional human erythroleukemia cell lines with diverse developmental globin programs, two somatic cell hybrids between K562 and mouse erythroleukemia (MEL) cells, a human fetal liver x MEL somatic cell hybrid, and reticulocytes and bone marrow cells from normal adults. el-Globin gene was expressed in all cell types. Inducible expression (twoto sixfold) was documented both in HEL and K562 erythroleukemia cells after 5azacytldine treatment. Like K562 cells, HEL cells also displayed hemin-Inducible el-globin gene expression. Following transfer of human chromosome 16 from embryonicifeta1 K562 to the adult MEL cells, el-globin gene remained active but lost its potential for Inducibility, suggesting probably a trans regulation mechanism. Higher levels of 81 mRNA were found in fetal liver cells compared with trace amounts in reticulocytes and normal adult bone marrow cells. These data clearly show that in contrast to the embryonic and adult patterns of expression of 5 and a-globin genes, respectively, el-globln gene displays a different profile, being active predominantly during the early stages of ontogeny, switching to lower levels of expression in adulthood.
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.
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...
Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia
Blood, 1991
Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst-forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticul...
Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin
Proceedings of the National Academy of Sciences of the United States of America, 1979
When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me(2)SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me(2)SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me(2)SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altere...
Developmental Biology, 1991
Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (y) to adult (0) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in tram by preparing hybrid cells containing marked fragments of the y and fl genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequcnccs prcscnt in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.
Determination of the hemoglobin F program in human progenitor-derived erythroid cells
Journal of Clinical Investigation, 1985
The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content of HbF was indistinguishable in the wellhemoglobinized progeny of marrow erythroid colony-forming units, marrow or blood BFU-E, or of mixed colony-forming units. The term HbF program refers to this inherent capacity to produce fetal hemoglobin (HbF) in the erythroid cells derived from these progenitors in vitro. The HbF content of marrow erythroblasts as determined by the same radioligand immunoassay was similar to that found in the peripheral blood, suggesting that the switch off of 'v-chain production occurs after the erythroid colony-forming unit stage of maturation. Increasing concentrations of a crude erythropoietin-containing preparation induced higher numbers of erythroid colonies, which were larger in size, but the HbF program was unaffected. In contrast to the hemoglobin accumulation in human progenitorderived colonies, simian progenitor-derived colonies produced considerably more HbF, and the amount of HbF was strongly influenced by progenitor maturity. Assays of the HbF content of erythroblasts derived from culture of the peripheral blood BFU-E of patients with nondeletion hemoglobinopathies and their parents showed that the HbF program in the progenitors of such patients is highly variable. Some produce only a slight excess of HbF in progenitor-derived erythroblasts, whereas others have extraordinarily high HbF programs. The molecular basis of this variability is presently unknown. Hemoglobin F in Human Progenitor-derived Erythroid Colonies 1359 J. Clin. Invest.
1992
HE HUMAN (Y and p globin gene clusters are well- T characterized gene families that have been extensively used in studies of tissue-specific and developmental gene expression.' In both complexes the genes are arranged in the order in which they are expressed during development (5'-52-(~2-a1-3', 5'-~-~y-*y-6-P-3') and both are under the control of sequences lying several kilobases upstream of the