Nicole M. Gantt

Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming

Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34(+) cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca2+ receptor

We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies.

Cl‐IB‐MECA inhibits human erythropoiesis

Candidate drugs are being sought for the suppression of human erythropoiesis. Cl‐IB‐MECA [2‐chloro‐N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide] is a derivative of adenosine that inhibits the growth of leukaemic cell lines. To determine the effects of Cl‐IB‐MECA upon erythropoiesis, studies were performed by using an ex vivo culture system of primary human CD34+ cells. Cl‐IB‐MECA suppressed erythroblast growth and maturation at doses ≥50u2003μmol/l through a mechanism of cell cycle inhibition and accumulation of cells in the G1/G0 phase. These findings demonstrate that Cl‐IB‐MECA inhibits human erythropoiesis, and suggest that further consideration of this drug is warranted for patients with erythrocytosis or polycythemia syndromes.

Inhibition of erythroblast growth and fetal hemoglobin production by ribofuranose-substituted adenosine derivatives.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5-deoxy adenosine and 2,3-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.