Proteomic Studies for the Investigation of γ-Globin Induction by Decitabine in Human Primary Erythroid Progenitor Cultures.

Reactivation of γ-globin is considered a promising approach for the treatment of ß-thalassemia and sickle cell disease. Therapeutic induction of γ-globin expression, however, is fraught with lack of suitable therapeutic targets. The aim of this study was to investigate the effects that treatment with decitabine has on the proteome of human primary erythroid cells from healthy and thalassemic volunteers, as a means of identifying new potential pharmacological targets. Decitabine is a known γ-globin inducer, which is not, however, safe enough for clinical use. A proteomic approach utilizing isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in combination with high-pH reverse phase peptide fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to investigate the effects of decitabine treatment. Bioinformatics analysis making use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) was employed for functional annotation of the 192 differentially expressed proteins identified. The data are available via ProteomeXchange with identifier PXD006889. The proteins fall into various biological pathways, such as the NF-κB signaling pathway, and into many functional categories including regulation of cell proliferation, transcription factor and DNA binding, protein stabilization, chromatin modification and organization, and oxidative stress proteins.

ASH1L (a histone methyltransferase protein) is a novel candidate globin gene regulator revealed by genetic study of an English family with beta-thalassaemia unlinked to the beta-globin locus.

In 1993, we described an English family with beta-thalassaemia that was not linked to the beta-globin locus. Whole genome sequence analyses revealed potential causative mutations in 15 different genes, of which 4 were consistently and uniquely associated with the phenotype in all 7 affected family members, also confirmed by genetic linkage analysis. Of the 4 genes, which are present in a centromeric region of chromosome 1, ASH1L was proposed as causative through functional mRNA knock-down and chromatin-immunoprecipitation studies in human erythroid progenitor cells. Our data suggest a putative role for ASH1L (Trithorax protein) in the regulation of globin genes.

The investigation of resveratrol and analogs as potential inducers of fetal hemoglobin.

Βeta-thalassemia, is a hemoglobinopathy characterized by reduced beta-globin chain synthesis, leading to imbalanced globin chain production, ineffective erythropoiesis and anemia. Increasing gamma-globin gene expression is a promising therapeutic approach as it reduces this imbalance by combining with the excess alpha globin chains and producing fetal hemoglobin (HbF). Furthermore, increased iron absorption and repeated blood transfusions lead to iron overload and tissue damage secondary to reactive oxygen species. Compounds exhibiting both antioxidant and HbF inducing activities are, therefore, highly desirable therapeutic agents. Resveratrol, a natural phytoalexin, combines these two activities but is also cytotoxic. Nine hydroxystilbenic resveratrol derivatives were synthesized in an attempt to identify compounds that retain the HbF-inducing and antioxidant activities of resveratrol but exhibit reduced cytotoxicity. Three derivatives (P1, P4 and P11) exhibited similar hemoglobin-inducing properties to resveratrol in K562 cells, however, only P11 showed reduced cytotoxicity. All three derivatives demonstrated variable HbF-inducing activity in primary erythroid progenitor cells from healthy donors. Resveratrol and P11 increased HbF induction significantly, with P11 having the highest activity. Additionally, P4 significantly increased progenitor numbers. A combinatorial treatment in K562 cells using resveratrol and decitabine resulted in a statistically significant increase in hemoglobin-inducing activity only above the level shown by resveratrol alone.

Origins, actions and dynamic expression patterns of the neuropeptide VGF in rat peripheral and central sensory neurones following peripheral nerve injury.

BACKGROUND: The role of the neurotrophin regulated polypeptide, VGF, has been investigated in a rat spared injury model of neuropathic pain. This peptide has been shown to be associated with synaptic strengthening and learning in the hippocampus and while it is known that VGFmRNA is upregulated in dorsal root ganglia following peripheral nerve injury, the role of this VGF peptide in neuropathic pain has yet to be investigated. RESULTS: Prolonged upregulation of VGF mRNA and protein was observed in injured dorsal root ganglion neurons, central terminals and their target dorsal horn neurons. Intrathecal application of TLQP-62, the C-terminal active portion of VGF (5-50 nmol) to naïve rats caused a long-lasting mechanical and cold behavioral allodynia. Direct actions of 50 nM TLQP-62 upon dorsal horn neuron excitability was demonstrated in whole cell patch recordings in spinal cord slices and in receptive field analysis in intact, anesthetized rats where significant actions of VGF were upon spontaneous activity and cold evoked responses. CONCLUSION: VGF expression is therefore highly modulated in nociceptive pathways following peripheral nerve injury and can cause dorsal horn cell excitation and behavioral hypersensitivity in naïve animals. Together the results point to a novel and powerful role for VGF in neuropathic pain.