scholarly journals Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line.

1991 ◽  
Vol 11 (7) ◽  
pp. 3528-3536 ◽  
Author(s):  
N L Lumelsky ◽  
B G Forget
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Regulation ◽  
Myristate Acetate ◽  
Globin Mrna ◽  
Megakaryocytic Differentiation ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line

1991 ◽  
Vol 11 (7) ◽  
pp. 3528-3536
Author(s):  
N L Lumelsky ◽  
B G Forget
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Regulation ◽  
Myristate Acetate ◽  
Globin Mrna ◽  
Megakaryocytic Differentiation ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

LIN28A-Mediated Expression Of Fetal Hemoglobin Ameliorates Erythrocyte Sickling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 313-313
Author(s):  
Jaira F. de Vasconcellos ◽  
Ross M. Fasano ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
Colleen Byrnes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Transgenic Expression ◽  
Sickle Hemoglobin ◽  
Gamma Globin ◽  
Alpha Globin ◽  
And Control

Abstract Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes. After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009). These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Ethanol Extract ofFructus trichosanthisPromotes Fetal Hemoglobin Production via p38 MAPK Activation and ERK Inactivation in K562 Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Hui Li ◽  
Chun Hay Ko ◽  
Suk Ying Tsang ◽  
Ping Chung Leung ◽  
Ming Chui Fung ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Fetal Hemoglobin ◽  
Ethanol Extract ◽  
K562 Cells ◽  
Globin Mrna ◽  
Mapk Activation ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Stimulation Of

Pharmacological stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of beta-thalassemia. In this study, the effects ofFructus trichosanthis(FT) were investigated in human erythroleukemic K562 cells for their gamma-globin mRNA and HbF-induction activities. The role of signaling pathways, including extracellular regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), was also investigated. It was found that the ethanol extract of FT significantly increased gamma-globin mRNA and HbF levels, determined by real-time reverse transcription polymerase chain reaction and enzyme linked immunosorbent assay, respectively, in dose- and time-dependent manner. Total Hb (THb) levels were also elevated in the concentrations without cytotoxicity (<80 μg mL−1). Pre-treatment with p38 MAPK inhibitor SB203580 blocked the stimulatory effects of FT extract in total and HbF induction. In contrast, no change in HbF was observed when treated with ERK inhibitor PD98059. Furthermore, FT ethanol extract activated p38 MAPK and inhibited ERK signaling pathways in K562 cells, as revealed in western blotting analysis. In addition, SB203580 significantly abolished p38 MAPK activation when the cells were treated with FT. In summary, the ethanol extract of FT was found to be a potent inducer of HbF synthesis in K562 cells. The present data delineated the role of ERK and p38 MAPK signaling as molecular targets for pharmacologic stimulation of HbF production upon FT treatment.

scholarly journals The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1078-1078 ◽  
Author(s):  
Joy Miyashiro ◽  
Asha Pant ◽  
Boris Tchernychev ◽  
Todd G Milne ◽  
Mark G Currie ◽  
...  
Keyword(s):  
Cell Line ◽  
Guanylyl Cyclase ◽  
Mode Of Action ◽  
Globin Gene ◽  
Soluble Guanylyl Cyclase ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Globin Mrna ◽  
No Signaling

Abstract Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178). The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively. In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). Disclosures Miyashiro: Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

scholarly journals Fetal hemoglobin silencing in humans

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2081-2086 ◽  
Author(s):  
Patricia A. Oneal ◽  
Nicole M. Gantt ◽  
Joseph D. Schwartz ◽  
Natarajan V. Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Postnatal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

scholarly journals Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
JL Miller ◽  
CE Walsh ◽  
PA Ney ◽  
RJ Samulski ◽  
AW Nienhuis
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Single Copy ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Adeno Associated Virus ◽  
Binding Motifs ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
New Strategy

Abstract The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

scholarly journals High-resolution analysis of the human gamma-globin gene promoter in K562 erythroleukemia cell chromatin

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 606-612 ◽  
Author(s):  
JM Gimble ◽  
EE Max ◽  
TJ Ley
Keyword(s):  
High Resolution ◽  
Transcription Initiation ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Initiation Site ◽  
Dnase I ◽  
Globin Genes ◽  
Hypersensitive Site ◽  
Gamma Globin

Abstract We performed high-resolution mapping studies of the DNAse I- hypersensitive sites located just 5′ to the human G gamma- and A gamma- globin genes of K562 erythroleukemia cells, in which these genes are constitutively expressed at low levels. This analysis revealed that the hypersensitive site extends from approximately -210 +/- 5 to -25 +/- 5 base pairs (bp) upstream from the transcription initiation site. Within this region, a GC-rich region located between the proximal CCAAT box and the TATA box is particularly accessible to nuclease digestion; however, the 5′ end of the hypersensitive site is less accessible to nucleases. The pattern of DNAse I cleavage does not change on either strand with hemin induction of K562 cells, which increases the rate of gamma-globin gene transcription about threefold. The region within the hypersensitive site includes all the consensus promoter elements of the gamma-globin genes as well as an octamer sequence located between -182 and -175, and a region associated with a variety of mutations that may cause hereditary persistence of fetal hemoglobin (HPFH).

scholarly journals Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
JL Miller ◽  
CE Walsh ◽  
PA Ney ◽  
RJ Samulski ◽  
AW Nienhuis
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Single Copy ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Adeno Associated Virus ◽  
Binding Motifs ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
New Strategy

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

scholarly journals High-resolution analysis of the human gamma-globin gene promoter in K562 erythroleukemia cell chromatin

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 606-612
Author(s):  
JM Gimble ◽  
EE Max ◽  
TJ Ley
Keyword(s):  
High Resolution ◽  
Transcription Initiation ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Initiation Site ◽  
Dnase I ◽  
Globin Genes ◽  
Hypersensitive Site ◽  
Gamma Globin

We performed high-resolution mapping studies of the DNAse I- hypersensitive sites located just 5′ to the human G gamma- and A gamma- globin genes of K562 erythroleukemia cells, in which these genes are constitutively expressed at low levels. This analysis revealed that the hypersensitive site extends from approximately -210 +/- 5 to -25 +/- 5 base pairs (bp) upstream from the transcription initiation site. Within this region, a GC-rich region located between the proximal CCAAT box and the TATA box is particularly accessible to nuclease digestion; however, the 5′ end of the hypersensitive site is less accessible to nucleases. The pattern of DNAse I cleavage does not change on either strand with hemin induction of K562 cells, which increases the rate of gamma-globin gene transcription about threefold. The region within the hypersensitive site includes all the consensus promoter elements of the gamma-globin genes as well as an octamer sequence located between -182 and -175, and a region associated with a variety of mutations that may cause hereditary persistence of fetal hemoglobin (HPFH).

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