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.

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 >/=50 mumol/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.

Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca(2+) receptor.

UNLABELLED: 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. INTRODUCTION: The human Ca(2+) receptor (CaR), which plays a central role in the regulation of [Ca(2+)](0) homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca(2+) stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca(2+) and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. RESULTS: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca(2+) was also increased by binding of 5C8 and decreased by binding of 1A8. CONCLUSIONS: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.