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 were isolated. Both normal progenitor enriched bone- marrow (BM) cells, whose differentiation capabilities are studied in vitro & in animal model systems in vivo, & hematopoietic cell lines are used. M1 myeloblastic leukemia cells proliferate autonomously & undergo terminal differentiation which culminates in programmed cell death, & loss of leukemogenicity when treated with the physiological inducers IL-6/LIF. 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. The human myeloblastic leukemia HL60 cell line, which also proliferates autonomously, can be induced to differentiate to either macrophages or granulocytes, depending on the inducer, providing a good model to study cell lineage determination. Finally, the IL-3 dependent 32Dc13 myeloid cell line can be induced for granulocytic differentiation upon removal of IL-3 & addition of G-CSF. The research has shown that MyD genes which encode for transcription factors of the fos/jun family are positive regulators of terminal differentiation & apoptosis. Also, it was observed that the MyD transcription factor EGR-1 is essential for, restricts, & determines differentiation along the macrophage lineage. The proposed research plan entails deciphering how the transcription factors fos/jun and EGR-1 function as positive regulators of terminal differentiation, apoptosis & lineage specific development of hematopoietic cells, & what effect altering their function/expression may have in the progression of leukemias. The main thrust of the work involves genetic manipulation of leukemic & normal myeloid precursor cells to express either conditionally functional fos or EGR-1 transgenes (where function is dependent on beta-oestradiol in the culture medium and not on de novo synthesis of mRNA or protein), to clone fos/jun & EGR-1 target genes, & to assess their effect on terminal differentiation, apoptosis, lineage specific development & leukemogenicity in vitro & in vivo. How fos/jun interact with myc/myn & myb, negative regulators of myeloid differentiation which accelerate apoptosis, and the interactive effects of p53 and the Wilms tumor gene product (WT1) on the function of EGR-1, will be investigated at the level of normal & abnormal hematopoietic cell development. Finally, normal BM cells, as well as BM from fos-deficient or p53-deficient mice will be used to further assist in this research. These studies should lead to an increased understanding of the role of fos/jun & EGR-1 nuclear factors in terminal differentiation, apoptosis, and lineage specific differentiation, and how genetic lesions which effect their normal functions may contribute to leukemogenesis, ultimately aiding in diagnosis, prognosis and eventual therapy.
