Reactivating gamma gene expression which increases fetal hemoglobin (HbF) levels can improve the clinical symptoms of sickle cell disease. Recent, hydroxyurea was shown to be beneficial for reducing the number of painful episodes in sickle cell patients. Not all patients respond to therapy, therefore, novel approaches to induce fetal hemoglobin production are needed. The experiments proposed in this project will define the role of Interleukin-6 (IL-6) and IL-11 in gamma gene silencing during megakaryocyte maturation. Patients treated with these cytokines have a rapid onset reversible anemia. The investigators have demonstrated decreased HbF production in IL-6 treated K562 cells, which was abrogated by anti-IL-6 monoclonal antibodies. The investigators also observed inhibition of erythroid colony growth when this cytokine was added to cultures on day seven. The inhibitory role of IL-6 on globin gene expression may be a mechanism for the clinical anemia observed with IL-6 treatment. In vitro protein binding studies to elucidate the molecular mechanism for globin gene silencing by IL-6 showed significant increased binding of the proto-oncogene Ap-1/c-jun to the tandem NF-E2/AP=1 site, located in 5' hypersensitive site 2 from the beta globin locus control region. Ap-1/c-jun may act as an inhibitor of transcription. Therefore, these data suggest a role for Ap-1/c-jun in the mechanism for gamma gene silencing by IL-6. The hypothesis is: Proto- oncogene activation by cytokines contributes a modulatory effect on globin gene expression during lineage commitment. This hypothesis will be tested by the following Specific Aims:
Specific Aim 1 : To compare and contrast the effects of IL-6 with IL-11 on globin gene silencing during megakaryocytic lineages commitment.
Specific Aim 2 : To identify the nuclear trans-actin factor(s) which mediate the inhibition of gamma gene expression by IL-6 and/or IL-11.
Specific Aim 3 : To develop antisense oligodeoxynucleotide delivery strategies to block the inhibitory effects of nuclear trans-acting factor(s) on globin gene expression.
Specific Aim 4 : To evaluate the therapeutic effects of antisense oligodeoxynucleotide therapy in vivo, in a transgenic mouse model for globin gene regulation. Achieving these Specific Aims will establish a model system for modulating gamma gene expression and a basis for strategies to induce fetal hemoglobin production to ameliorate the symptoms of sickle cell disease.
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