The long-term goals of this proposal are to define the molecular events that control the promyelocyte-specific """"""""program"""""""" of normal and leukemic cells, and to define the functions of the promyelocyte-specific enzyme cathepsin G (CG). Our laboratory has determined that the murine and human CG genes are expressed only in promyelocytes; cis-acting sequences within or near the human CG gene contain the promyelocyte-targeting signals. We intend to use this information to further characterize the biology of promyelocytes, via the following Specific Aims: 1. We will define the regulatory DNA sequences that target expression of CG to promyelocytes in transgenic mice. We have shown that a 6.0 kb fragment containing the human CG gene is targeted specifically to myeloid precursors in transgenic mice. We will dissect the sequences within this fragment to define the promyelocyte targeting elements, and identify proteins that interact specifically with these elements. 2. We will study the biology of acute promyelocytic leukemia (APL) by targeting PML-RARalpha and RARalpha-PML cDNAs to murine promyelocytes with CG-derived targeting sequences. We have created a novel targeting vector containing all of the sequences from the 6.0 kb CG transgene. We will use an E2A-PBX-1 cDNA to confirm the accuracy of the targeting vector, an to determine whether promyelocytes can be transformed in vivo. We will then target cDNAs created by the t(15;17) translocation associated with APL. A PML-RARalpha cDNA, and a reciprocal RARalpha-PML cDNA will be individually targeted to promyelocytes in transgenic mice to determine whether either of these cDNAs is capable of causing a block in promyelocyte differentiation or overt APL. Expressing transgenic lines will be bred to one another to determine whether the two transgenes synergize to produce APL. The transgenic animals will be carefully characterized to create models for the study of this disease. 3. We will create a loss of function model for murine CG. We will use standard strategies to create a targeted mutation in the murine CG gene in embryonic stem cells, and crate CG-/-mice. These mice will be examined for defects in neutrophil and macrophage functions in vitro and in vivo. CG may play a role in causing the DIC syndrome associated with human APL; if the """"""""APL"""""""" mice defined above develop DIC, we will breed them into the CG-/- background to further define this role.
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