The long term goal of the research is to better understand the molecular biology of normal blood cell development, leukemogenicity & its suppression. Towards this end clones of myeloid differentiation primary response (MyD) genes, activated in the absence of protein synthesis upon induction of myeloid terminal differentiation, were isolated. Both normal myeloid precursor cells, & M1 myeloid leukemic myeloblasts are used. M1 cells proliferate autonomously and undergo terminal differentiation which culminates in programmed cell death, & loss of leukemogenicity when treated with the physiological inducers Interleukin- 6 (IL-6), leukemia inhibitory factor (LIF), or conditioned media of mouse lungs (containing both factors). Also available are M1myc/M1myb cell lines, where the genetic program of myeloid maturation has been disrupted at distinct developmental stages by deregulated c-myc/c-myb transgenes. Along the lines of the previous proposal it was shown that LIF/IL-6, trigger the same immediate early MyD response, including protein phosphorylation steps essential for MyD gene activation. Also, it was found that two novel MyD genes, MyD116 & MyD118, encode proteins stikingly similar to proteins encoded by two novel genes, gadd34 & gadd45, coordinately activated by growth arrest/DNA-damage (gadd) stimuli. MyD116 & gadd34 were found to be homologues of the same gene, whereas MyD1118 and gadd45 represent two separate closely related genes, both induced in response to growth arrest/DNA-damage stimuli. Evidence has been accumulating to suggest that MyD116, MyD118 & gadd45 play pivotal roles in growth arrest & apoptosis of M1 myeloid precursor cells. This proposal is aimed towards: 1. Analysis of the roles MyD116, MyD118, gadd45 play in growth arrest and apoptosis of myeloid cells. M1 & normal myeloblasts will be genetically manipulated to alter the expression of MyD116, MyD118 & gadd45 in order to study the roles these genes play in the control of myeloid cell growth arrest & programmed cell death, including how cell maturation is linked to growth arrest and apoptosis. Interactions between MyD116, MyD118, gadd45 and other positive & negative regulators, possible mechanisms of action, & the role of other players that participate in the regulation of myeloid cell growth suppression and apoptosis also will be studied. 2. Analysis of the molecular mechanisms utilized by hematopoietic differentiation inducers to activate MyD gene expression. Advantage will be taken of MyD cis-acting elements localized within the promoter region of a prototype MyD gene to clone & characterize pre-existing MyD trans-acting-factor(s), & analyze the molecular mechanisms employed by hematopoietic differentiation inducers to convert it to an active form that plays a role in promoting MyD gene transcription. Information should result which leads to increased understanding of terminal differentiation, including transduction of differentiation signals, control of growth arrest & programmed cell death, & how perturbing normal controls can block cell maturation & contribute to leukemogenesis, ultimately aiding in diagnosis, prognosis & eventual therapy.
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