Nonrandom chromosomal translocation plays a major role in the pathogenesis of acute leukemia. However, the molecular mechanism of pathogenesis is relatively unknown. Studies on the molecular pathology of acute promyelocytic leukemia (APL) strongly support the importance of the fusion protein PML-RARalpha, encoded from the t(15;17), in the development of APL. Based on the finding by the investigators that PML is a growth suppressor and results reported from others, a model of APL pathogenesis was proposed. In this model, PML-RARalpha plays a central role as a dominant negative inhibitor against PML and RXR. Sequestration of these two proteins results in growth stimulation and differentiation block at the promyelocyte stage which leads to APL pathogenesis. This model represents the first to emphasize the importance of a dominant negative inhibitor in the development of acute leukemia. The investigator's finding that PML is a growth suppressor may contribute to a better understanding of APL pathogenesis. Studies on the biologic function of PML will provide critical information to further understand APL. The two major goals of this proposal are: (1) to elucidate the molecular mechanism of APL pathogenesis. Experiments are designed to support the proposed model using dominant negative inhibitors against PML, RARalpha (or RXR), and a mutant PML-RARalpha driven by an inducible promoter. Stable transfectants of these mutants will be established to investigate their effect on growth and differentiation of human leukemia cells and primary fibroblasts. The effect of over-expression of RXR and PML in the APL-derived NB4 cells on clonogenicity, differentiation and growth will be investigated. Dominant negative mutants of PML, RARalpha and cell lines are available in the laboratory for this study. (2) To study the biologic function of PML. Results demonstrated a highest number of PODs at the G1 phase; a nuclear diffused PML pattern at the S phase coincided with a decreased in PODs; a significant increased in PODs shortly after gamma-irradiation and that both tyrosine and serine residues of PML are phosphorylated. These findings suggest that modification of PML during cell cycle progression may be important for its biologic function. The participants propose to investigate the role of phosphorylation on the biologic function of PML. Site directed mutagenesis will be performed to identify and to alter the phosphoamino acid to a nonphosphorylated form. Their ability to form PODs in NIH/3T3 cells and to suppress transformation by neu will be investigated. It will be investigated as to whether PML is phosphorylated by a cell cycle related kinase. PML deletion mutants have been created, we found that the ability of PML to form POD is essential for its transformation suppressor function. The investigators will continue to study their effect on suppressing transcription activity of EGFR promoter. Stable transfectants of PML in NB4 cells will be used to investigate whether PML induces differentiation, apoptosis, or cell cycle arrest. Its effect on tumorigenicity and clonogenicity will also be investigated. The investigators have found that PML enhances cell survival after radiation exposure, and will investigate whether PML affect cell cycle distribution and inhibits apoptosis in these cells. Finally, identification and characterization of the PML associated proteins by 32P-labelled PML probe and by the yeast two-hybrid system will carried out.
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