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. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: Int J Biochem Cell Biol. 2008 May 29;40(11):2341–2347. doi: 10.1016/j.biocel.2008.04.024

Histone deacetylase inhibitors and hemoglobin F induction in β-thalassemia

Anna Rita Migliaccio a,b,*, Dante Rotili c, Angela Nebbioso d, George Atweh a, Antonello Mai c
PMCID: PMC2581454  NIHMSID: NIHMS66944  PMID: 18617435

Abstract

Epigenomic modifiers, such as histone deacetylase inhibitors, are compounds that regulate gene expression by interfering with the enzymatic machinery that maintains the proper chromatin structure of the nucleus. These compounds are at the forefront of novel therapeutic agents for the treatment of several diseases including cancer and genetic disorders such as β-thalassemia and sickle cell disease. Here we review the current understanding of the mechanism of action of epigenomic modifiers in the treatment of β-thalassemia and sickle cell anemia. We also discuss how the lessons learned from the study of the effects of these compounds on the β-globin locus, one of the best characterized regions of the human genome, might contribute to the understanding of the mechanism of action of these same compounds in cancer, where the specific regions of the genome that are responsible for the pathophysiology of the disease are often poorly defined.

Keywords: Histone deacetylase inhibitors, Hemoglobin F induction, β-Thalassemia, β-Globin locus

Introduction

β-Thalassemia and sickle cell anemia are two of the most common single gene disorders of humans. Both diseases result from different mutations of the β-globin gene that encodes two of the tetrameric globin chains that make up the major hemoglobin present in adult red cells (adult hemoglobin, HbA). β-Thalassemia may be traced to numerous genetic mutations that result in either loss or reduced expression of β-globin. Sickle cell anemia results from a missense mutation (glutamine to valine substitution) at the 6th amino acid of the β-globin chain. The resulting sickle Hb (HbS) forms insoluble polymers within the cytosol upon deoxygenation, with subsequent deformation of the red blood cells and vaso-occlusion. Patients with β-thalassemia and sickle cell disease do not have clinical complications of their disease at birth when their red cells contain the fetal form of Hb (HbF) (for a review see Stamatoyannopoulos & Grosveld, 2001).

One to three percent of normal adult red blood cells express HbF. Several mutations in regulatory regions of the β-globin gene cluster that lead to increased HbF synthesis in adult life have been described. These conditions are referred to collectively as hereditary persistence of fetal hemoglobin (HPFH) (Figure 1). Patients with sickle cell disease and β-thalassemia who co-inherit these mutations typically have a much milder clinical condition. In sickle cell disease, the presence of HbF reduces the effective concentration of HbS, thus decreasing the propensity for intracellular polymerization. The fetal γ-globin chains also interfere with the ability of HbS to polymerize by heterohybrid formation. In β-thalassemia, the presence of γ-chains compensates for the deficiency of β-chains. Furthermore, clinical studies have shown that induction of HbF to approximately 10–20% of total hemoglobin (or a 3- to 5-fold increase from baseline) could ameliorate the complications of sickle cell disease (see also Noguchi, Rodgers, Serjeant, & Schechter, 1988; Olivieri, 1999). These observations have led to considerable efforts to develop new therapies for sickle cell disease and β-thalassemia whose aim is to re-activate γ-globin expression in red blood cells during adult life.

Figure 1. Models of the mechanisms of reactivation of HbF expression in adult red cells.

Figure 1

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The first model is based on the observation that during the normal progression of erythroid maturation, activation of γ-globin gene expression precedes that of β-globin. This model predicts that the increased level of HbF observed in erythroid cells produced during the recovery from anemia, in which the maturation process is accelerated, is due to the fact that the cells are forced to mature earlier, by skipping division, when their cytoplasm contains relatively higher level of γ-globin mRNA. This model has lead to the identification of the HbF inducing ability of cytotoxic drugs such as hydroxyurea. The second model is based on the observation that red cells from individuals lacking the EKLF binding site of the β-globin promoter are normal despite the fact that they do not contain HbA but HbF (HPFH syndrome). This observation has inspired the search for chemical agents that, by interacting with the transcription machinery, would reactivate HbF expression by activating γ-globin and/or suppressing β-globin expression, such as the HDAC inhibitor butyrate (for further details on mechanism 2 see Figure 2) (see also Cao, 2004; Atweh & Schechter, 2001).

Induction of HbF by cytotoxic drugs

Red blood cells from individuals recovering from anemia or from patients recovering from stem cell ablation in preparation for bone marrow transplantation were shown to have higher levels of HbF than normal (Papayannopoulou, Abkowitz, D’Andrea, & Migliaccio, 2005). These observations lead to the development of strategies for the reactivation of HbF in β-thalassemia and sickle cell disease that mimick the accelerated erythropoiesis that occurs in response to the anemic stress (summarized in model 1 of Figure 1). Indeed, HbF was shown to be induced in primates by cell cycle–specific cytotoxic drugs such as 5-azacytidine, cytosine arabinoside (AraC), hydroxyurea, and vinblastin. These drugs trigger erythroid regeneration secondary to their cytotoxic effects on cycling cells (Atweh & Schechter, 2001). Similarly, AraC and hydroxyurea induced HbF in patients with sickle cell disease (Veith, Galanello, Papayannopoulou, & Stamatoyannopoulos, 1985). These observations provided the rational for the clinical trials that established the clinical efficacy of hydroxyurea for the treatment of sickle cell disease (Bradai, Abad, Pissard, Lamraoui, Skopinski, & De Montalembert, 2003; Charache, Terrin, & Moore, 1995). Hydroxyurea, however, is not effective in many patients with sickle cell disease, and it is only minimally effective in patients with β-thalassemia. It is also a drug that can induce DNA damage, and its long-term safety in patients with sickle cell disease is not yet established. The search for additional effective and less toxic agents is, therefore, still ongoing (Atweh & Schechter, 2001).

Epigenetic modifications and HbF synthesis

Two enzyme superfamilies, histone acetyltransferases (HATs) and histone deacetylases (HDACs) exert antagonistic epigenetic control on gene expression through the regulation of chromatin structure (Figure 2). HATs, by inducing histone acetylation, favour chromatin relaxation, exposing gene regulatory regions to the transcription machinery. In contrast, HDAC catalyze histone deacetylation that condenses the chromatin into tightly compact, and therefore transcriptionally silent “heterochromatin” regions (Figure 2).

Figure 2. Diagrammatic schemes of the enzyme complexes that control gene expression by regulating the chromatin configuration, the histone acetylation state and the RNA polymerase II recruitment of the β-globin locus.

Figure 2

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A) Loci of active chromatin configuration contain acetylated forms of histone H3 and H4. Histone acetylation is catalyzed by complexes containing the catalytic and regulatory elements of histone acetyltransferases (HATs). These complexes contain additional proteins and transcription factors that, by binding to specific DNA sequences, ensure specificity of DNA binding to the complex. This relaxed configuration of the chromatin structure allows binding of the RNA polymerase (pol II) to the promoter region of the gene and its subsequent transcription (see also Hassig & Schreiber, 1997; Felsenfeld & Groundine, 2003).

B) Loci of silent chromatin configuration contain, instead, de-acetylated forms of H3 and H4. These histones are kept in a de-acetylated state by HDAC. To date, eighteen distinct mammalian HDACs have been reported, grouped into four classes (I, II, III, and IV) depending on their primary homology to the Saccharomyces cerevisiae deacetylases. Class I (HDAC1-3, 8), class II (HDAC4-7, 9, 10), and class IV (HDAC11) HDACs, homologues of yeast RPD3 (class I/IV) and HDA1 (class II), are Zn2+-dependent deacetylases, whereas class III HDACs (SIRT1-7, sirtuins) are homolog to the yeast Sir2, use NAD+ as co-factor for their enzymatic activities. Class I, II and IV HDACs and sirtuins are sensitive to very different classes of inhibitors. It is debated whether each HDAC might be involved in the control of a specific cell functions (see also Figure 3). HDACs perform their biological function by forming complexes with other proteins. These complexes include specific combinations of HDAC isoforms, transcription factors, protein kinases and other proteins still to be identified. The function of each specific element in the complex remains to be established. Transcription factors may facilitate the recognition of specific DNA binding sites while protein kinases might regulate HDAC activity by phosphorylation of specific protein residues. The reason why the complex usually contain more than one HDAC isoform is also unknown. The observation that the recombinant forms of some HDAC isoforms, such as HDAC3, retain enzymatic activity while those of others, as an example HDAC4, do not has suggested that one of the HDAC present in the complex may represent the functional enzyme, while the other isoform exerts regulatory functions. To date, only few of the HDAC complexes have been characterized. As an example, HDAC1 and 2 are part of the same complexes (Sin3, NuRD and CoREST), while HDAC3, the HDAC isoform, is associated with HDAC4 and 5 in complexes whose precise composition is still poorly defined. The understanding of the lineage-specific spectrum of action of the different HDAC complexes is still in its early stages. It is conceivable that the identification of which complex is expressed and regulated in which cell lineage will greatly facilitate the design of HDACi that would target specific cell functions (for further details see also De Ruijter, van Gennip, Caron, Kemp, & van Kuilenburg, 2003; Mai et al., 2005 b; Minucci & Pelicci, 2006).

C) Diagram of the β-globin locus on chromosome 11 and acetylation status of H3 (top panel) and H4 (middle panel) and polymerase II (pol II, bottom panel) recruitment at the promoter region of the ε, γ and β globin genes in cells expressing high (fetal erythroblasts or adult erythroblasts treated with butyrric acid, white columns) or low (adult cells, grey column) levels of HbF. Acetylation of both H3 and H4 and binding of the pol II complex is relatively higher at the γ or β locus in cells expressing high or low levels of HbF, respectively (modified from Aerbajinai, Zhu, Gao, Chin, & Rodgers, 2007; Yin et al., 2007; Fathallah, Weinberg, Galperin, Sutton, & Atweh, 2007).

The study of epigenetic modifications and HbF synthesis was inspired by the discovery that the genes in the β-globin locus are differentially methylated, a landmark of chromosome inactive regions, during human ontogenesis (Mavilio et al., 1983) and that treatment of adult erythroid cells in vitro with bromo-deoxyuridine, a nucleotide analog that inhibits ex-novo DNA methylation, reactivates HbF expression in these cells (Comi et al., 1986). An indication on the molecular mechanisms with which epigenomic modifiers might act was provided by the demonstration that, red blood cells from individuals who harbor a mutation in the promoter of the β-globin gene that disrupts a binding site for the transcription factor EKLF contain high levels of HbF (HPFH syndrome). This suggested that agents that would mimic the HPFH mutation by preventing binding of “transcription factors” to the β-globin promoter would favor expression of the γ-globin gene and ameliorate the severity of β-thalassemia and sickle cell disease (Atweh & Schechter, 2001). Another seminal observation in this field was the demonstration that specific histone acetylation patterns play a role in the developmental switch of the murine β-globin genes, suggesting that HDACs might also play a role in silencing γ-globin gene expression in human adult red cells (Forsberg, Downs, Christensen, Im, Nuzzi, & Bresnick, 2000). Hence, HDAC inhibitors (HDACi), by favoring the transition from a transcriptional silent to an active chromatin state (model 2 of Figure 1), are promising candidates for pharmacological reactivation of HbF (Cao, 2004).

The proof-of-concept for the use of HDACi as pharmacologic inducers of HbF was provided by the demonstration that pregnant sheep who were continuously infused with sodium butyrate during pregnancy displayed a delayed HbF to HbA switch (Perrine, Rudolph, & Faller, 1988). Butyrate is a well known HDACi, although it is relatively weak and requires millimolar concentrations for its full activity. Subsequent studies showed that butyrate induces HbF synthesis in human erythroid cultures in adult baboons (Constantoulakis, Knitter, & Stamatoyannopoulos, 1989), in certain patients with β-thalassemia (Perrine, Ginder, Faller, Dover, Ikuta, Witkowska, Cai, Vichinsky, & Olivieri, 1993; Sher, Ginder, Little, Yang, Dover, & Olivieri, 1995), and in a majority of patients with sickle cell disease (Atweh, Sutton, & Nassif, 1999). Other butyrate analogues (e.g. phenylbutyrate, isobutyramide and other short chain fatty acids) also induce HbF expression in human erythroid cells. These compounds have been used in small clinical trials in β-thalassemia (Cao, 2004). The chemical structure of butyrate has little similarity with the theoretical pharmacophoric model for HDACi (see Table 1).

TABLE 1.

HDACi Pharmacophoric Model and HDAC Inhibitory Activity of Compounds 9, 11, and 24

Pharmacophoric modela Compd. Maize HDACsb Human HDACsc
IC50 (µM) (% of inhibition at 2 µM)
graphic file with name nihms66944t1.jpg no R HD1-B
(Class I)		 HD1-A
(Class II)		 Class II
selectivity ratio		 HDAC1
(Class I)		 HDAC4
(Class II)
graphic file with name nihms66944t2.jpg 9 3-Cl 31.4 0.44 71.4 0 10.2
11 3-F 38.8 0.22 176.4 0 54.9
graphic file with name nihms66944t3.jpg 24 3-Cl 0.012 0.006 2 85.8 69.2
graphic file with name nihms66944t4.jpg 0.028 0.178 - 84.5 66.2
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a

Crystallography data on the interaction between the catalytic domain of a bacterial HDAC and TSA and SAHA have allowed to define the pharmacophoric model for compounds that by mimicking the substrate inhibit the catalytic activity of class I/II/IV HDACs, as described on the top of the Table. This model predicts that substrate mimetic compounds should have 4 domains: a zinc binding group (ZBG), a hydrophobic spacer (HS), a connection unit (CU) and an interaction domain with the rim of the catalytic pocket (CAP).

b

HD1-B and HD1-A are considered homologues of mammalian class I and class II HDACs, respectively. Class II selectivity ratio was calculated as IC50 HD1-B/ IC50 HD1-A.

c

Purified by immunoprecipitation with specific antibodies from U937 cells (HDAC1) and from the breast cancer line ZR75.1 (HDAC4), respectively.

d APHA (Aroyl-Pyrrolyl Hydroxy-Amide) and UBHA (Uracil-Based Hydroxy-Amide) derivatives are indicated in yellow and blue, respectively. R indicates the chemical substituent on the benzene ring.

(Modified by permission from Mai et al., 2007, 2005 a and 2005 b)

Recent studies have started to elucidate the molecular mechanism by which butyrate re-activates γ-globin expression in erythroid cells. Butyrate was shown to displace HDAC3 (but not HDAC1 or HDAC2) and its adaptor protein NCoR from the γ-globin promoter. Knockdown of HDAC3 by siRNA was shown to induce the expression of the γ-globin gene. These results suggest that from all the HDACs expressed in erythroid cells, HDAC3 might be the isoform that is primarily responsible for γ-globin silencing (Mankidy et al., 2006). In addition to their effects on HDAC activity and histone acetylation status, HDACi exert additional biological functions. For example, exposure to butyrate was shown to alter the DNA methylation status of the β-globin locus (Fathallah, Weinberg, Galperin, Sutton, & Atweh, 2007). It is not clear whether this is a direct or indirect effect of butyrate exposure. The HDACi effects on DNA methylation might be a reflection of the fact that HDAC complexes that dock to a specific DNA sequence contain methyl-binding proteins. Therefore, HDACs may compete with methyl-transferases for binding to sites of DNA methylation (Mai et al, 2005 b). Furthermore, butyrate was shown to increase the efficiency of the translation of the globin mRNA (Weinberg et al., 2005). It is also not clear whether this effect on mRNA translation is related to a protein deacetylase activity of butyrate.

Considerable variability has been observed in the individual responsiveness of patients with sickle cell disease and β-thalassemia to butyrate. This variability of response can be replicated in vitro when erythroid progenitors from the same patients are cultured in the presence of butyrate (Fathallah, Weinberg, Galperin, Sutton, & Atweh, 2007). These observations suggest that the responsiveness of a patient to butyrate may be determined by the epigenetic configuration of the β-globin gene cluster. . The elucidation of the role of this epigenetic variability is a major challenge for the effective use of these compounds in the treatment of patients with hemoglobin disorders. It should be remembered that the chromatin structure of the locus is determined by the acetylation state of the histones and the state of its DNA methylation. In this regard, it has been debated whether the HbF inducing effects of inhibitors of DNA methyltransferase like 5-azacytidine and decitabine are a reflection of their cytotoxic effects or their ability to inhibit de novo DNA methylation and, hence, reactivation of silenced genes. Thus, therapeutic strategies that combine a demethylating agent like 5-azacytidine with an HDACi like butyrate might represent a very effective strategy for epigenomic modifier-based therapies for sickle-cell disease and β-thalassemia.

Because of their very short half-life, their inconvenient mode of intravenous administration, their requirement of relatively high concentrations for activity and the heterogeneity of the individual patient responsiveness, short chain fatty acids like butyrate are clearly not optimal drugs for the treatment of patients with sickle cell disease and β-thalassemia. Thus, there is a considerable interest in identifying alternative and possibly more potent HDACi that will induce HbF. Additional HDACi that are being investigated for the treatment of cancer (Mai et al., 2005 b), can also induce HbF in vitro and/or in vivo. These agents are represented by hydroxamide derivatives of short chain fatty acids, valproic acid, and other products with a chemical structure similar to the pharmacophoric model illustrated in Table 1 [trichostatin A and analogues, trapoxin and derivatives, HC-toxin, suberoylanilide hydroxamic acid (SAHA), MS-275, apicidin, scriptaid and analogues] (Cao, 2004). However, because of their modest HbF-inducing activity and relatively high toxicity, a potential role for these compounds in the treatment of patients with β-hemoglobinopathies is uncertain.

New HDACi: the experience from a comparative search for new anticancer and γ-globin inducers in the same center

Based on the pharmacophoric model described in Table 1, we designed a library of potential new HDACi that consists of two classes of synthetic compounds defined as APHAs (Aroyl-Pyrrolyl Hydroxy-Amides) and UBHAs (Uracil-Based Hydroxy-Amides). The APHA compounds are selective both for maize and human class II HDAC enzymes while UBHA compounds are not (Table 1). This library was screened against the maize homologues of mammalian class I and class II HDACs. Twenty-four of the most active compounds against the maize enzymes were identified and independently tested for their ability to induce HbF in different models of erythroid differentiation (Mai et al., 2007). The same compounds were also tested for the ability to induce cytodifferentiation and/or apoptosis of a myeloid leukemic cell line. These last two assays are used as predictive tests for the efficacy of the compounds as anticancer agents (Mai et al., 2005 a). This strategy allowed the direct comparison of the efficacy of a compound in two different hematopoietic cell lineages, the myeloid and the erythroid lineage. Interestingly, the 24 compounds exerted very different biological effects in the two systems. In particular, two compounds, compound 9 and 24, were active as γ-globin mRNA inducers in normal erythroblasts (Figure 3). The two compounds increased the γ/(γ+β) ratio in normal erythroid cells by different mechanisms. Compound 9 increased the levels of γ-globin mRNA by two-fold, while compound 24 increased the level of γ-globin by 3-fold and decreased that of β-globin mRNA by 2-fold. As a result, the γ/(γ+β) ratio increased by 6-fold and HbF content increased to 50%. Compound 9 and 24 were also tested as γ-globin mRNA inducers in erythroblasts from patients with β0-thalassemic. Progenitor cells from 5 patients with β0-thalassemia generated morphologically normal pro-erythroblasts in vitro. However, unlike normal erythroid cells, β0-thalassemic cells fail to mature in vitro and express very low β-globin mRNA levels. Both compounds were effective in ameliorating the impaired in vitro maturation of β0-thalassemic erythroblasts. The improved maturation was associated with detectable increases in the γ/(γ+β) mRNA ratio in three of the five patients tested, again highlighting the individual genetic variability of the responsiveness to HDACi (Fathallah, Weinberg, Galperin, Sutton, & Atweh, 2007). In this context, it is interesting to note that one of the compounds (compound 11) whose chemical structure is very similar to that of compound 9 did not induce γ-globin expression and was cytotoxic for erythroid cells. The fact that compound 11 is more selective than compound 9 for class II HDACs illustrates the poor correlation between the chemical structure and the HDAC inhibitory activity of a compound with its biological effects. Interestingly, compound 11 did not have cytotoxic effects in the U937 myelomonocytic cells but induced their differentiation, as measured by CD11c expression. In contrast, compound 9 was not cytotoxic and compound 24 had modest cytotoxic activity in both myeloid and erythroid cells.

Figure 3. Comparison of the effects of three new HDACi, compound 9, 11, 24 and SAHA (as control) on the apoptotic state (A) and on the histone H4 acetylation levels (C) in a cell line model of myeloid leukaemia (the U937 cell lines) and in primary human erythroblasts obtained in vitro from normal donors. The effect of the compounds as inducers of myeloid differentiation in U937 cells and as inducer of γ-globin expression [γ/(γ+β) mRNA ratio] in primary erythroblasts is also compared (B). The chemical structure of the compounds is described in Table 1.

Figure 3

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A) Induction of apoptosis in the myeloid leukaemia U937 cell line (white bars) and normal erythroid cells (grey bars) of compound 9, 11 and 24. The results are compared to the effects exerted in the same cells by SAHA. The three compounds have dramatic different effects as inducers of apoptosis in myeloid and erythroid cells. Compound 9 was less cytotoxic (<5%) than SAHA in both cell models. Compounds 11 had modest toxic effects on U937 cells, but, in spite of the similarity of its structure with compound 9, was a strong inducer (90%) of apoptosis in erythroid cells. Compound 24, in stead, induced, although at low levels (<20%), apoptosis in both cell systems. Compounds were tested at 2–5 µM concentration in all cases (modified from Mai et al., 2007, 2005 a, and unpublished results).

B) Effects of selected HDACi on the induction of myeloid differentiation (CD11c expression) and on the induction of γ-globin [γ/(γ+β) mRNA expression ratio] expression in the myeloid U937 cell line (white bars) and in primary normal erythroblasts (grey bars), respectively. Compounds 11 and 24 were equally potent as inducer of myeloid differentiation than SAHA. In contrast, compound 24, but not 11 was as potent as SAHA as γ-globin inducers in primary erythroblasts. On the other hand, compound 9 induced γ-globin expression as efficiently as SAHA (modified from Mai et al., 2007, 2005 a, and unpublished results).

C) Effects of compounds 9 and 24 on the level of histone H4 acetylation in U937 cells and in primary erythroblasts obtained from normal donors. U937 cells and human erythroblasts were cultured without HDACi (Control, top panels) or with DMSO (vehicle, negative control), or with compound 9 or 24, as indicated. After 4 days, the cells were harvested and the levels of H4 acetylation measured by flow cytometry. The white and grey area correspond to the fluorescence intensity expressed by cells labelled with an irrelevant antibody or with the anti-acetyl-histone H4, respectively. The average fluorescence intensity (AFU, in arbitrary units) expressed by cells incubated with the anti-acetyl-histone H4 is reported on the right, and is proportional to the acetylation state of histone H4 in the cell population analyzed. Compound 9 and 24 did not induce detectable levels of H4 acetylation in U937 cells. Compound 24 was also a weak inducer of H4 acetylation in primary human erythroblasts. By contrast, compound 9 was a strong inducer of the H4 acetylation in primary erythroblasts. This results reflect the overall potency of a compound as inducer of H4 acetylation in a certain cell system. They do not necessarily reflect the action of the compound on the status of H4 acetylation at a specific locus i.e. the globin locus. Specific ChIP analyses are necessary to clarify this important point. (modified from Mai et al., 2007, and unpublished results).

The differences in biological activity of the compounds in myeloid and erythroid cells led us to compare their effects on the acetylation status of histones in the two cell lineages. Both compounds 9 and 24 induced similar levels of H3 acetylation in myeloid and erythroid cells as assessed by western blotting (Mai et al., 2007 and results not shown). Both compounds, however, were not highly effective as inducers of H4 acetylation in U937 cells as assessed by flow cytometric analyses (Figure 3). In contrast, compound 9, and to a lesser extent compound 24, induced significant H4 acetylation in primary erythroblasts. The different effects of compound 9 and 24 on the acetylation of histone H4 in myeloid vs erythroid cells is consistent with a possible lineage-restricted composition of HDAC complexes. It is difficult, however, to draw any firm conclusions from this data on the mechanisms by which these HDACi exert their biological effects. Although the results of the H4 acetylation assays in the two cell lineages that were obtained by flow cytometry were confirmed by western blotting assays, findings from assays of the state of global histone acetylation in cells are poorly predictive of the effects of compounds on specific loci within the same cells. As a matter of fact, the different effects of compound 9 and 24 on H4 acetylation provide no information on why one of the compounds (compound 9) induced γ-globin expression while the other (compound 24) increased γ-globin and decreased β-globin expression. Furthermore, these results provide no clues why cells from different β0-patients responded to the HDACi so differently. These observations emphasize the importance of combining the identification of new HDACi with studies of the molecular organization of chromatin in the locus of the gene to be targeted and how this organization may vary in different individuals in order to fully understand the biological effects of these potent chemical effectors. For these reasons, the study of the biological effects of the different HDACi in patients with hemoglobin disorders, where the structure and the regulation of the relevant target gene locus is well characterized, may make it possible to optimize the use of these drugs in patients with cancer, where the targeted gene(s) and their epigenetic configuration(s) are poorly defined.

Acknowledgements

This work was partially supported by grants from AIRC 2007 (A.M.), PRIN 2006 (A.M.), NY-STAR grant (A.R.M.) and P01-CA108671 from the National Cancer Institute (USA) (A.R.M.).

Footnotes

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