Simvastatin and tBHQ Act Synergistically to Increase γ-Globin Gene Expression Through the Transcription Factors KLF2 and NRF2

Abstract Abstract 2149 While increased fetal hemoglobin (HbF) levels have proven therapeutic benefit for people with sickle cell disease and β-thalassemia, none of the current HbF inducing agents have the optimal combination of safety, efficacy and ease of use that would make them applicable to most hemoglobinopathy patients. In an effort to develop new strategies for HbF induction, we have recently shown that drugs that activate the NF-E2 related factor 2 (NRF2)/antioxidant response signaling pathway stimulate HbF production in primary human erythroid cells. This discovery prompted us to investigate ways to further enhance HbF levels achieved with NRF2 activators alone. Recent reports from the cardiovascular literature have uncovered a synergy between Kruppel-like factor 2 (KLF2) and NRF2. In vascular endothelial cells, shear stress induces a battery of genes that protect against atherosclerotic cardiovascular disease and this induction is mediated by the transcription factors KLF2 and NRF2 and includes synergistic activation of NRF2 target genes by the two factors. Interestingly, HMG-CoA reductase inhibitors (statins), are strong activators of the transcription factor, KLF2. These findings suggested to us that combining statins with drugs that activate NRF2 signaling might synergistically activate γ-globin gene expression and HbF production. An additional rationale for this approach is that several NRF2 activating drugs are either already approved or undergoing clinical testing and that statins are among the most widely used drugs. To test this hypothesis, we first treated K562 cells with various concentrations of simvastatin and observed a dose dependent increase in KLF2 mRNA and protein expression, with 5μM statin resulting in more than 200-fold increase in steady state mRNA levels but no change in γ-globin mRNA. When combined with tBHQ, 5μM statin synergistically increased γ-globin levels compared to either drug alone at 24 and 48hrs. To investigate the specificity of this synergy, we created a stable K562 cell line that overexpressed murine klf2. Treating these cells with tBHQ enhanced γ-globin expression compared to tBHQ treated WT K562 cells, reproducing the effect we saw with statin and tBHQ combination treatment in WT K562 cells. This suggests that KLF2 is responsible for the synergistic effects of statin when combined with tBHQ. To further investigate the mechanism of statin action we performed KLF2 and NRF2 ChIP studies. Statin treatment strongly increased KLF2 binding to HS2 of the β-globin LCR (30-fold over IgG) while tBHQ induced NRF2 binding to the NF-E2 region of LCR HS2 30-fold and 10-fold over IgG in K562 and in primary human erythroid cells, respectively. Binding at HS1, HS3 or HS4 was not increased for either factor. In a single experiment performed so far, combined tBHQ and statin treatment of differentiating primary human erythroid cells increased KLF2 and NRF2 target gene NQO1 mRNA. γ-globin mRNA was induced to levels equivalent to those seen with 5-azacytidine. These data provide preliminary evidence suggesting that combining NRF2 activators with widely used statins may be a safe and effective way to achieve therapeutic HbF levels in β-thalassmia and sickle cell disease patients. Disclosures: No relevant conflicts of interest to declare.

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Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Cell Disease ◽

Globin Mrna ◽

Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

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X-Ray and UV Irradiation and Heat Shock Induce γ-Globin Gene Expression in Erythroid Cells Via P38 MAPK Signaling.

Blood ◽

10.1182/blood.v114.22.459.459 ◽

2009 ◽

Vol 114 (22) ◽

pp. 459-459

Author(s):

Rachel West ◽

Rodwell Mabaera ◽

Sarah Conine ◽

Elizabeth Macari ◽

William J. Lowrey ◽

...

Keyword(s):

Gene Expression ◽

Heat Shock ◽

P38 Mapk ◽

Globin Gene ◽

K562 Cells ◽

Mapk Signaling ◽

Erythroid Cells ◽

Globin Mrna ◽

X Ray ◽

Globin Gene Expression

Abstract Abstract 459 We have recently presented a cell stress signaling model to explain the ability of a wide variety of mechanistically distinct drugs to induce fetal hemoglobin (HbF) production (Mabaera et al, Exp Hematol 2008). This model proposes that HbF inducing agents, including DNMT and HDAC inhibitors, short-chain fatty acid derivatives and cytotoxic agents induce HbF production by activating intracellular cell stress signaling pathways and that the p38 MAPK (mitogen-activated protein kinase) pathway plays a central role in the induction process. The major alternative to this model is that each class of drugs works through a distinct mechanism, such as global DNA hypomethylation or histone hyperacetylation. If our model is correct, then non-pharmacologic inducers of p38 MAPK signaling, such as X-ray and UV irradiation, heat shock, and osmotic shock should also increase γ-globin gene expression. If these physical stresses do not up-regulate γ-globin gene expression, then our model is likely to be incorrect. To mimic our previous experiments in which we treated differentiating human primary erythroid cells with inducing agents over several days, we exposed K562 cells to physical stresses daily for 5 days at doses that did not cause cell death. To test whether X-irradiation induces the transcription of γ-globin mRNA, cells were irradiated at doses ranging from 0 to 1.0 cGy/day, in the absence and presence of SB203580 (a p38 MAPK inhibitor). Experiments were first performed on K562 cells, and then during in vitro erythroid differentiation of primary human CD34+ cells. These treatments stimulated a strong, dose-dependent increase of γ-globin mRNA in both K562 cells (up to 6-fold over untreated cells) and in primary erythroid cells (up to 5-fold) as measured by quantitative RT-PCR. SB203580 abolished this effect in both cell types. The inhibitor also caused a decrease in γ-globin mRNA expression in untreated control cells, suggesting that p38 MAPK signaling plays a role in basal γ-globin gene expression. The same X-ray doses also induced phosphorylation of the down-stream p38 MAPK target, HSP-27 and up-regulated expression of stress-induced transcription factor genes including GADD34, CHOP (GADD153), and ATF3. Expression of GADD34 and CHOP genes and HSP-27 phosphorylation were inhibited by SB203580. We next tested the ability of UV light (254 nm) administered daily for 5 days to induce γ-globin mRNA in K562 cells. A dose-dependent increase in γ-globin mRNA was observed following exposure to doses as low as 10 J/m2. Cells treated with 35 J/m2 had 2.7-fold higher levels of steady state γ-globin mRNA compared to untreated control cells. This induction was also inhibited by SB203580. Preliminary experiments with heat shock have yielded similar results. Following a single 180-minute exposure to 42°C, K562 cells showed a 2.2-fold increase in γ-globin mRNA compared to untreated cells. Shorter exposures to higher temperatures (e.g., 50°C for 15 minutes) caused an approximately 3-fold increase in γ-globin steady-state mRNA after 24 hours. These 2- to 3-fold increases in expression are similar to those we have previously observed in primary human erythroid cells with 5-azacytidine and butyrate. In none of our experiments did the K562 cells become benzidine positive, indicating that increased γ-globin expression was not the result of activating an erythroid differentiation program. Together with other published studies, these data support the hypothesis that p38 MAPK pathway signaling, whether caused by drugs or physical stress, is a key component of γ-globin gene induction. This in turn suggests that the components of the p38 MAPK pathway could serve as novel targets for the development of new HbF inducing agents. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v59.4.738.738 ◽

1982 ◽

Vol 59 (4) ◽

pp. 738-746 ◽

Cited By ~ 18

Author(s):

BL Tonkonow ◽

R Hoffman ◽

D Burger ◽

JT Elder ◽

EM Mazur ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Globin Gene ◽

Leukemia Cell ◽

K562 Cells ◽

Leukemia Cell Line ◽

Human Leukemia ◽

Globin Mrna ◽

Human Leukemia Cell Line ◽

Globin Gene Expression

Abstract 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.

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5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

Blood ◽

10.1182/blood.v108.11.1581.1581 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1581-1581

Author(s):

Rodwell Mabaera ◽

Christine Richardson ◽

Sarah Conine ◽

Christopher H. Lowrey

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Erythroid Differentiation ◽

Maximal Response ◽

Mrna Levels ◽

Erythroid Cells ◽

Globin Mrna ◽

Potent Inducer ◽

Cellular Dna ◽

Globin Gene Expression

Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

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Talen-Mediated Knock Outs Of Cis and Trans Elements Potentially Involved In Globin Gene Switching

Blood ◽

10.1182/blood.v122.21.436.436 ◽

2013 ◽

Vol 122 (21) ◽

pp. 436-436

Author(s):

Patrick A Navas ◽

Yongqi Yan ◽

Minerva E Sanchez ◽

Ericka M Johnson ◽

George Stamatoyannopoulos

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Globin Gene ◽

K562 Cells ◽

Globin Mrna ◽

Core Elements ◽

Gene Knockouts ◽

Gene Switching ◽

Cis And Trans ◽

Globin Gene Expression

Abstract Transcription activator-like effector nucleases (TALEN) are engineered proteins used for precise genome editing by generating specific DNA double strand that are repaired by homologous recombination and by non-homologous end joining. TALENs can be used to study gene regulation by deleting putative regulatory elements in the context of the native chromosome and measuring mRNA synthesis. We designed TALENs to delete individual DNAse I-hypersensitive sites (HS) of the β-globin locus control region (LCR) followed by an assessment of globin gene expression and assessment of epigenetic effects in K562 erythroleukemia cells. The β-globin LCR is composed of five HSs and functions as a powerful regulatory element responsible for appropriate levels of the five β-like globin genes during development. Introduction of plasmid DNA encoding a pair of TALENs and targeting individually the flanking region of the HS2, HS3 and HS4 core elements along with a donor 100 base single-stranded oligonucleotide resulted in the successful deletions of each of the three core elements in K562 cells. Individual K562 cells were seeded to produce clones and the mutations were screened by PCR to identify both heterozygous and homozygous clones. The TALEN-mediated 288 bp HS2 core deletion resulted 32 heterozygous (48.5%) and 6 homozygous clones (9.1%) in a total of 66 clones screened. K562 carries three copies of chromosome 11 emphasizing the robustness of TALEN technology to target each of the alleles. In the 199 bp HS3 core deletion, from 113 clones we identified 28 heterozygous (24.8%) and 3 (2.7%) homozygous clones. Lastly, the 301 bp HS4 core deletion yielded 9 homozygous (5.9%) and 12 heterozygous (7.9%) clones from 151 clones screened. Total RNA was isolated from wild-type K562 cells, and from both the heterozygous and homozygous mutant clones and subjected to RNase Protection analysis to quantitate the levels of globin mRNA. Deletion if the HS3 core in K562 cells in a ∼30% reduction in ε-globin mRNA and 2-fold reduction in γ-globin mRNA. A more dramatic effect on globin expression is observed in the HS2 core deletion, as ε- and γ-globin expression is reduced by 2- and 5-fold, respectively. These results suggest that HS2 contributes the majority of the LCR enhancer function in K562 cells. The HS4 core deletion resulted in a modest ∼20% reduction in both ε- and γ-globin expression. TALENs were designed to knockout trans-acting factors implicated to be involved in globin gene regulation and/or globin switching. TALENs bracketing the gene promoters and the first exon of 25 genes encoding either a transcription factor or histone-modifying enzyme were synthesized and post-transfection PCR screens of the transfected pool of K562 cells resulted in the successful identification of 17 gene knockouts. The 17 target genes are PRMT5, LDB1, EIF2AK3, BCL11A, HBSIL, MYB, SOX6, NFE4, NR2F2, NR2C1, NR2C2, CHTOP, NFE2, DNMT3A, RBBP4, MTA2 and MBD2. Single cell clones have been generated by limited dilution of transfected K562 pools and thus far we have identified heterozygous and homozygous clones of 8 of 17 gene knockouts, importantly all clones were identified without selection. The frequency of identifying the knockout clones, represented by the number of clones screened/ number of heterozygous clones/ number of homozygous clones, are as follows: HBS1L (63/3/0), SOX6 (68/13/2), NFE4 (56/13/7), LBD1 (300/2/0), MBD2 (301/0/1), CHTOP (288/66/6), NFE2 (712/44/5) and NR2C1 (96/40/11). The remaining nine gene knockouts and globin gene expression data will be presented at the meetings. These studies highlight a powerful TALEN-mutagenesis platform for target deletions of both cis- and trans-elements to study globin gene switching. TALENs can be synthesized in several days and the screening of the individual clones for the desired knockouts is completed within two weeks. This highly efficient mutagenesis platform will further our understanding of the molecular basis of globin switching. Disclosures: No relevant conflicts of interest to declare.

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Exploiting the NRF2/Antioxidant Response Element Signaling Pathway to Induce γ-Globin Gene Expression and HbF Production

Blood ◽

10.1182/blood.v116.21.2074.2074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2074-2074

Author(s):

Elizabeth R. Macari ◽

Rachel J. West ◽

Christopher H. Lowrey

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Globin Gene ◽

K562 Cells ◽

Antioxidant Response ◽

Mrna Levels ◽

Antioxidant Response Element ◽

Globin Mrna ◽

Nrf2 Pathway ◽

Globin Gene Expression

Abstract Abstract 2074 Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including hydroxyurea, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoiesis and the possibility of long-term side effects. Two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemopreventative agents where these and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor, NF-E2 related factor 2 (NRF2), which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between the two CAAT boxes, suggesting that it too may be activated by NRF2. This led us to hypothesize that drugs that activate the ARE/NRF2 pathway may provide a less toxic approach to HbF induction. To test this hypothesis, we treated K562 cells with various NRF2 pathway activators. We initially tested six compounds that are known to induce antioxidant response genes at doses that did not inhibit proliferation and found the most pronounced γ-globin induction with tert-butylhydroquinone (tBHQ) (2.8 fold). We next tested tBHQ in two different primary cell culture models: erythroid precursors isolated from normal human bone marrow and in vitro erythroid differentiation of primary human CD34+ cells. In both of these models, tBHQ treatment increased γ-globin steady state mRNA levels and induced expression of NRF2 target genes. Treatment of differentiating erythroid cells caused a dose dependent increase in γ/(γ+β) mRNA and % HbF. HPLC analysis revealed the highest non-toxic concentration of tBHQ, 5μM, produced 10% HbF while the positive control, 0.5μM of 5-Azacytidine, resulted in 12% HbF compared to the untreated control at 3%. Since similar mRNA induction was seen in K562 cells, we used these cells to characterize the mechanism of tBHQ induced γ-globin expression. First, we used siRNA to decrease NRF2 mRNA levels. This resulted in a greater than 75% knockdown of NRF2 mRNA and protein and reduced tBHQ induction of γ-globin and NQO1 gene expression by 90% and 75%, respectively, compared to samples transfected with scrambled siRNA (p < 0.01). Subsequent experiments showed that tBHQ treatment resulted in NRF2 translocation to the nucleus and binding to the NQO1 and γ-globin promoters but not at negative control sites. In addition, inducing NRF2 translocation by transiently suppressing levels of its inhibitor, KEAP1, did not result in full induction of γ-globin mRNA expression, suggesting that NRF2 translocation alone is not sufficient for γ-globin induction. However, when combined with tBHQ, suppression of KEAP1 did enhance γ-globin induction. Taken together, these results suggest that NRF2/ARE pathway activation induces γ-globin mRNA expression and HbF production in primary human erythroid cells and that this is a promising strategy for further pre-clinical development. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v59.4.738.bloodjournal594738 ◽

1982 ◽

Vol 59 (4) ◽

pp. 738-746

Author(s):

BL Tonkonow ◽

R Hoffman ◽

D Burger ◽

JT Elder ◽

EM Mazur ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Globin Gene ◽

Leukemia Cell ◽

K562 Cells ◽

Leukemia Cell Line ◽

Human Leukemia ◽

Globin Mrna ◽

Human Leukemia Cell Line ◽

Globin Gene Expression

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.

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A Comparison of Five Different Culture Protocols Shows That Growth of Human CD34+ Peripheral Blood Stem Cells in SCF, IL-3, Flt-3 Ligand and EPO Results in Exponential Erythroid Expansion and a Globin Gene Expression Pattern Which Is Similar to That Seen in Human Bone Marrow.

Blood ◽

10.1182/blood.v106.11.3649.3649 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3649-3649

Author(s):

Rodwell H. Mabaera ◽

Christopher H. Lowrey

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Globin Gene ◽

Gene Expression Pattern ◽

Erythroid Cells ◽

Globin Mrna ◽

Peripheral Blood Stem Cells ◽

Blood Stem Cells ◽

High Level ◽

Globin Gene Expression

Abstract Pharmacologic reactivation of γ-globin globin gene expression offers a potential strategy for ameliorating the consequences of β-thalassemia and sickle cell disease. While previous clinical and laboratory studies have established the effectiveness of inhibitors of DNA methylation in stimulating the expression of the fetal globin genes, the molecular mechanisms by which this effect is achieved are not well understood. In order to study the mechanisms and pharmacologic properties of these agents in a clinically relevant laboratory model, we have compared five different in vitro human erythroid differentiation protocols. In performing these experiments we sought a system which would yield a large number of erythroid cells exhibiting a pattern of globin gene expression which closely matched the pattern seen in adult bone marrow. FACS purified CD34+ peripheral blood stem cells (PBSC) from healthy donors were obtained from the NHLBI Programs of Excellence in Gene Therapy Hematopoietic Cell Processing Core (PEGT-HCPC) at the Fred Hutchinson Cancer Research Center. PBSC were cultured in the following different combinations of recombinant human cytokines: A) EPO alone for 12d; B) EPO, SCF and IL-3 for 14d; C) EPO, SCF and IL-3 for 7d followed by EPO alone for 7d; D) EPO, GM-CSF and IL-3 for 12d; and E) SCF, IL-3 and Flt-3 ligand for 7d followed by EPO alone for 11d. Cells were counted every day and differentiation assessed by light microscopy and flow cytometry using CD34, glycophorin A (GPA) and transferrin antibodies. Globin gene expression was measured by real time RT-PCR. Cultures B, C and E underwent exponential expansion from d4, while A and D showed no appreciable expansion. By day 6, all cultures that had EPO from d0 (A–D) consisted mainly of CD34−, GPA+ proerythroblasts. Basophilic erythroblasts, followed by polychromatophilic forms were evident at 8–10 days. By the end of each experiment more than 90% of cells in these cultures were erythroid. In contrast, condition E showed persistent expression of CD34 until removal of IL-3, SCF and flt-3 ligand. Proerythroblasts appeared on day 10 followed by basophilic and polychromatophilic forms at 13–15 days. At the end of the culture period 63% erythroid cells (by flow) were seen in a background of maturing monocytes and granulocytes. RT-PCR showed that induction of globin mRNA occured in all cultures at or just before appearance of basophilic erythroblasts (day 7–9 for cultures A–D and day 11–13 for E). While conditions C and E showed the highest levels of globin gene expression, peak expression under condition C for γ- and β-globins were equivalent and their expression overlapped. Condition E showed a much higher level of β- than γ-globin expression (β/γ ratio of 8:1), the rise in β-globin mRNA (d9–14) was accompanied by a fall γ-globin mRNA and β-globin expression persisted at a high level until the end of the experiment (d14–18). Of the 5 differentiation protocols tested, condition E appears to be the best choice for future studies of pharmacological reactivation of γ-globin gene expression as it produced a large number of erythroid cells which exhibited a gene expression pattern similar to that seen in normal human bone marrow and had a period of stable high-level β-globin gene expression which persisted over several days.

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Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.1224.1224 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1224-1224

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Histone Deacetylases ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Erythroid Cells ◽

Cell Disease ◽

Globin Mrna ◽

In Erythroid Cells

Abstract High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal. Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3

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