Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia.

Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (HbF) is well-recognized as a critical laboratory parameter: lower HbF is associated with a higher risk of vaso-occlusive complications, organ damage, and early death. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long-term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia.

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the (G)γ-globin promoter and miRNA expression within primary CD71(+) erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated (G)γ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Microarray analysis of liver gene expression in iron overloaded patients with sickle cell anemia and beta-thalassemia.

Chronic transfusion therapy is used clinically to supply healthy erythrocytes for patients with sickle cell anemia (SCA) or beta-thalassemia major (TM). Despite the benefits of red blood cell transfusions, chronic transfusions lead to iron accumulation in key tissues such as the heart, liver, and endocrine glands. Transfusion-acquired iron overload is recognized as a cause of morbidity and mortality among patients receiving chronic transfusions. At present, there is little understanding of molecular events that occur during transfusional iron loading and the reasons for the large inter-individual variation observed clinically in transfusion-acquired iron accumulation. To address these issues, we examined whether any liver-expressed genes in SCA or TM patients with transfusional iron overload were associated with the degree of iron accumulation. Specifically, we performed microarray analysis on liver biopsy specimens comparing SCA patients with mild or severe iron overload and also compared SCA with TM patients. Fifteen candidate genes were identified with significantly differential expression between the high and low liver iron concentrations. SCA patients and 20 candidate genes were detected between the SCA and TM patient comparison. Subsequent quantitative PCR experiments validated 12 candidate genes; with GSTM1, eIF5a, SULF2, NTS, and HO-1 being particularly good prospects as genes that might affect the degree of iron accumulation. Future work will determine the baseline expression of these genes prior to transfusional iron overload and elucidate the full impact of these genes on the inter-individual variation observed clinically in transfusion-acquired iron accumulation.

The roles of alpha IIb beta 3-mediated outside-in signal transduction, thromboxane A2, and adenosine diphosphate in collagen-induced platelet aggregation.

Collagen-induced activation of platelets in suspension leads to alpha(IIb)beta(3)-mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and alpha(IIb)beta(3)-mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that alpha(IIb)beta(3)-mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion. A high level of collagen can activate platelets deficient in PLC gamma 2, G alpha q, or TxA2 receptors, as well as platelets treated with a protein kinase C inhibitor, Ro31-8220. Thus, activation of alpha(IIb)beta(3) in response to a high level of collagen does not require these signaling proteins. Furthermore, a high level of collagen can cause weak TxA2 and ADP-independent aggregation, but maximal aggregation induced by a high level of collagen requires TxA2 or secretion.

The roles of LAT in platelet signaling induced by collagen, TxA2, or ADP.

The work presented here demonstrates that platelets from mice lacking LAT (linker for the activation of T cells) show reversible aggregation in response to concentrations of collagen that cause TxA2/ADP-dependent irreversible aggregation of control platelets. The aggregation defect of the LAT-deficient platelets was shown to be the result of almost no TxA2 production and significantly diminished ADP secretion. In contrast, the LAT deficiency does not affect aggregation induced by high concentrations of collagen because that aggregation is not dependent on TxA2 and/or ADP. Even though ADP and TxA2 provide amplification signals for platelet activation in response to low concentrations of collagen, LAT-deficient platelets hyperaggregate to low levels of U46619, a TxA2 analog, or ADP. Though the mechanism(s) of costimulatory signals by collagen, ADP, and TxA2 remains unidentified, it is clear that LAT plays a positive role in collagen-induced, TxA2/ADP-dependent aggregation, and a negative role in TxA2 or ADP-induced platelet aggregation.

Role of the Src family kinase Lyn in TxA2 production, adenosine diphosphate secretion, Akt phosphorylation, and irreversible aggregation in platelets stimulated with gamma-thrombin.

Members of the Src family of kinases are abundant in platelets. Although their localization is known, their role(s) in platelet function are not well understood. Lyn is a Src-family kinase that participates in signal transduction pathways elicited by collagen-related peptide; it has also been implicated through biochemical studies in the regulation of von Willebrand factor signaling. Here, we provide evidence that Lyn plays a role in gamma-thrombin activation of platelets. Unlike the wild-type platelets, platelets from Lyn-deficient mice do not undergo irreversible aggregation, produce thromboxane A2, or secrete adenosine diphosphate in response to submaximal gamma-thrombin concentrations that cause secretion-dependent irreversible aggregation. Phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase, also requires a higher concentration of gamma-thrombin in Lyn-deficient platelets than in wild-type platelets. These findings demonstrate that Lyn signaling is required for thrombin induction of secretion-dependent platelet aggregation. Specifically, Lyn is required under these conditions to enable thrombin-induced TxA2 production and adenosine diphosphate secretion, necessary steps in secretion-dependent platelet aggregation.