We have focused on pharmacological augmentation of fetal hemoglobin levels in patients with sickle cell disease and beta-thalassemia. In particular, our group has shown that hydroxyurea (HU), a cell-cycle specific agent that blocks DNA synthesis by inhibiting ribonucleotide reductase, is capable of increasing fetal hemoglobin (HbF) levels in about 75-80% of patients with sickle cell disease by 2-10 fold. These data have been substantiated by others and has been recently culminated by a controlled clinical trial in adult patients in which the HU treated group experienced a significant reduction in the frequency of vaso-occlusive crisis, hospitalization, transfusion requirements and the incidence of acute chest syndrome. An updated analysis of 21 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. Although HU treatment of transfusion-dependent b-thalassemia patients has been reported to be disappointing in contrast to the results in sickle cell patients, we have found that some patients with b- thalassemia intermedia, who are not transfusion-dependent may benefit from HU treatment mainly by improving the quality of the newly formed RBC, apparently due to increased HbA levels. In addition, patients double heterozygous for beta-thalassemia and HbE display a reciprocal increase in HbF levels with a decline in HbE in response to HU. Thus, not only is the effect of HU likely to be broader than previously appreciated (i.e., including stimulation of betaA-synthesis in some patients) but is also may be efficacious in other clinically significant hemoglobinopathies. Current studies are in progress to determine whether particular types of beta-thalassemia mutations are more susceptible to gamma- or beta- enhancing effects, to examine the effects of HU on the transcript levels of other globin genes, and to study the effects of HU on transcriptional and post-transcriptional events. Along these lines, we have found that HU treatment of a human adult erythroid cell system, which is accompanied by a 2 to 4-fold induction of HbF levels 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 to permit the identification of footprints extending into """"""""G"""""""" motifs. Studies are underway to confirm the specificity of this observation as well as to further characterize, purify, and eventually clone these factors responsible for fetal globin gene reactivation. Toward this end the technique of differential display has been applied to examine for novel mRNA species induced or repressed following HU treatment of human adult erythroid cell in a liquid medium system.
