Our group has focused on the pharmacological augmentation of fetal hemoglobin levels in patients with sickle cell disease and beta- thalassemia. Our previous observations that hydroxyurea (HU) is capable of increasing fetal hemoglobin (HbF) levels in about 75-80% of patients with sickle cell disease by 2-10 fold, culminated in a controlled clinical trial in adult patients which substantiated unambiguously HU?s clinical efficacy. An updated analysis of 52 consecutive NIH patients entered on the HU trial, consisting of an initial in-hospital phase, and more prolonged outpatient observation periods has allowed us to draw important conclusions about the response rate, the kinetic aspects of the response to therapy, and the optimal dosage schedules for patients. In addition, in on-going collaborative studies in China and Thailand we have found that some patients with beta-thalassemia intermedia, who are not transfusion-dependent may benefit from HU treatment mainly by improving the quality of the newly formed RBC, and thereby increase their blood counts. In related laboratory studies, we have found that HU treatment at high concentrations (i.e. greater than 100 micro-molar) of a human adult erythroid cell system, is accompanied by a 2 to 4-fold induction of HbF levels and is associated with the loss of binding of three specific transcription factors or factor complexes in the proximal region of the gamma globin gene promoter. This observation required the modification of the standard in vivo footprinting methodology, termed ENU in vivo footprinting, to permit the identification of footprints extending into ?G? motifs. Subsequent analysis has in part implicated an inhibition in the expression of the erythroid specific transcription factor, GATA-1, to parallel the increase gamma globin expression and apoptosis of erythroid cells at these HU concentrations. Studies are underway to confirm the specificity of these observations as well as to further characterize, purify, and eventually clone the upstream targets and downstream signals. We have also coupled this liquid erythroid culture methodology with the technique of differential display has been applied to examine for novel mRNA species induced or repressed following HU treatment of human erythroid cell in a liquid medium system. We have identified two interesting candidate genes ? clone 39-13 shares 91% homology with the mouse ras-like GTP binding protein and is ubiquitously expressed; clone 52-12 is 85% homologous to a high-throughput X-linked gene of unknown function. We believe that these lab-based studies may clarify the molecular mechanism of pharmacological-induced fetal hemoglobin increases, and may shed light on other promising compounds. - Human Subjects
