The long term goal of the research is to better understand the molecular biology of normal cell development, malignancy and its suppression. Towards this goal, cDNA clones of differentiation primary response (MyD) genes, activated in the absence of protein synthesis following induction of myeloid cell terminal differentiation and growth arrest, have been isolated. The overall aim of the proposed studies is to gain a better understanding of the events in the signal transduction from the cell membrane to the nucleus which induce these differentiation primary response genes at the onset of terminal differentiation and the role these genes play in the regulated ordered expression of specific genes, resulting in the conversion of proliferating, nondifferentiated cells into nonproliferating, highly specialized cells. In these studies. In these studies the research exploits the attractive biological features of the myeloid cell system, where in addition to being able to manipulate and analyze the growth and differentiation of normal myeloid precursor cells in vitro, using physiological myelopoietic factors, several leukemic and nonleukemic myeloblast cells lines are available. These include: D+ leukemic precursors, where growth has been uncoupled from differentiation, yet can be induced to differentiate, thereby suppressing the leukemic phenotype, and nondifferentiating (D-) leukemic and nonleukemic factor dependent cell lines. Thus, one can study at the molecular-genetic level early events associated with normal myeloid cell differentiation, differentiation of D+ leukemic cells associated with growth arrest and suppression of the leukemic phenotype, and possible genetic lesions associated with nondifferentiating myeloid leukemic and factor dependent cell lines. cDNA clones of three differentiation primary response genes, MyD1,2, & 3, which appear novel (based on hybridization studies and homology searches of sequence databanks), have been selected for further structural and functional analysis. Specifically: 1) The full length sequence of the MyD cDNA clones will be determined to search for homologies with various functional domains, to be able to deduce the sequence of the encoded protein, and to be able to construct expression vectors. 2) The expression of the MyD genes will be analyzed during normal myelopoiesis, compared to in D+ leukemic myeloblasts following induction of terminal differentiation and suppression of their leukemic phenotype, and further compared to in nondifferentiating (D-) leukemic and factor dependent blasts. 3) The role of the MyD genes in myeloid differentiation, growth inhibition, and suppression of certain leukemic phenotypes will be determined, via: a. The use of MyD antisense oligonucleotides in the culture media to block MyD gene expression in normal and D+ leukemic myeloblasts to determine its effects on normal myeloid differentiation as well as differentiation and leukemia suppression of D+ leukemic phenotypes. b. Introduction of MyD genes, either expressed constitutively or under the control of an inducible promoter into D+ & D- leukemic blasts to determine whether any of the MyD genes may abrogate the need for an exogenous source of differentiation factor in D+ leukemic blast &/or may functionally complement genetic lesions of nondifferentiating (D)- leukemic and factor dependent phenotypes. 4) Ascertain the mode of activation of the MyD genes, which occurs in the absence of protein synthesis, by defining cis and trans acting control elements. First, the 5' regulatory region of a specific MyD gene will be isolated from available genomic libraries. Functional assays with a reporter gene whose expression is dependent on the MyD regulatory region will be done to identify, dissect and map positive and negative cis acting functional domains, whereas a combination of gel mobility shift assays and in-vitro footprinting will be used to define MyD gene trans acting transcription factors and their cognate binding sites.
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