Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of gamma-globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human gamma-globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including beta-thalassemia and sickle cell anemia.     

Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.     

Accumulation of γ-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of γ‐globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well‐known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two‐phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and γ‐globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of γ‐globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically‐mediated regulation of the expression of human γ‐globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including β‐thalassemia and sickle cell anemia.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors

OBJECTIVE: Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on gamma-globin gene activity was studied. METHOD: Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on gamma-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on gamma-globin mRNA synthesis was analyzed. RESULTS: NB and TSA activated p38 MAPK and increased gamma-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gamma-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased gamma-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. CONCLUSION: This study supports a novel role for p38 MAPK in gamma-globin regulation in human erythroid progenitors.     

Fetal hemoglobin silencing in humans

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.     

Isolation and characterization of a K562 cell line resistant to 1-beta-D-arabinofuranosylcytosine-mediated erythroid induction

The isolation of a K562 cell line, K562(S)R, resistant to 1-beta-D-arabinofuranosylcytosine (ara-C)-mediated erythroid induction, is described. Ara-C (10-50 microM) inhibits cell growth of K562(S)R cells but is not able to activate the program of erythroid induction. This failure is associated with the lack in the increase of accumulation of epsilon-globin and gamma-globin mRNA sequences in ara-C-treated K562(S)R cells. This cell line could be of interest for studies focused on molecular mechanisms of activation of globin genes in K562 cells.     

Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species

OBJECTIVE: Several compounds, including butyrate and trichostatin A, have been shown to activate gamma-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H(2)O(2)) in drug-mediated fetal hemoglobin (HbF) induction. METHODS: H(2)O(2) levels were measured using 2',7'-dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H(2)O(2) in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block gamma-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation. RESULTS: Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H(2)O(2)-dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated gamma-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF. CONCLUSION: The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A.     

Epigenetic analysis of the human alpha- and beta-globin gene clusters

K562 erythroleukemia cells have been widely used as a model for the study of globin gene regulation. A number of agents have been shown to activate or suppress globin gene expression in these cells. However, the molecular effects of these agents on the epigenetic configuration of the alpha- and gamma-globin genes that encode HbF are not known. In this report, we investigated the relationship between globin expression and histone acetylation of the human alpha- and beta-globin clusters in the fetal erythroid environment of K562 cells. Our studies suggest that acetylation of histone H3 may be important in regulating developmental stage-specific expression of the different beta-like globin genes while acetylation of both histones H3 and H4 may be important for the regulation of tissue-specific expression of these genes. In contrast, acetylation of both histones H3 and H4 at the alpha-like globin promoters appears to be important for both developmental stage- and tissue-specific expression. Interestingly, butyrate-induced activation of alpha-globin gene expression in K562 cells is associated with significant increase in histone acetylation levels while TPA-induced inhibition is associated with decreased histone acetylation at its promoters. In contrast, changes in histone acetylation and DNA methylation do not appear to be important in the regulation of gamma-globin gene expression by the same agents. These data suggest that the butyrate-mediated induction of the fetal gamma-globin genes in K562 cells is not a direct result of its histone deacetylase inhibitor activity of butyrate on the chromatin of the gamma-globin promoters, while the induction of the alpha-globin genes could be a result of a direct effect of butyrate on chromatin at its promoters. This is another example of the important differences in the molecular mechanisms of regulation of the genes of the alpha- and beta-like globin clusters.     

A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction

Gene therapy might fall short in achieving a complete reversion of the β-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of β-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the β-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from β(0)39-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human β-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of β-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from β-thalassemic patients with T9W, which expresses the human β-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired α-globin chains, combination of β-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of α-globin proteins in thalassemic erythroid progenitor cells.     

Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells

OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.     

Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3-4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3-5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.     

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.