The long term objectives of this proposal include defining specific molecular changes related to both good and poor prognostic groups in AML and to understand the mechanisms by which these factors produce their effects. Focus will be placed on the RB, p53 and WAF 1 genes, since they are important components of cell cycle regulation (particularly at the G1/S boundary, and have been implicated by individuals in our program as potential independent prognostic markers in AML for remission duration, overall survival, and chemoresistance. Further studies to confirm the initial results will be undertaken. In addition, the percentage of cases within the low/no RB protein expression cohort which can be induced by growth factors to produce normal RB protein will be studied both in cell culture and the patient. Whether leukemias which remain RB negative contain RB mutations, and as those that subsequently produce RB protein after growth factor stimulation do not will be examined. The interaction among these genes will also be studied by transferring each gene separately into the appropriate cells using a tetracycline inducible system to allow RB, p53 and WAF 1 to be switched on and off in a reproducible manner. This system should also enable careful documentation of their role in the response to chemotherapeutic agents used or proposed for the treatment of AML and permit the determination of the mechanism(s) by which a specific gene may produce its effect. This in turn could be related to the chemosensitivity or resistance of a given AML patient to a particular agent and/or to overall survival. The relationship between changes in protein expression of these genes to differences in cell cycle and production of apoptosis before or after exposure to chemotherapeutic agents will also be determined. Once the tetracycline inducible cell lines have been isolated and studied in cell culture they will be examined in the SCID mouse model in a similar manner. Standard molecular biology techniques, plasmid construction, immunochemical staining and chemosensitivity assays will be utilized and cell cycle changes or production of apoptosis will be done in the FACS Core Facility within the Program Project. It is the goal of this project is to identify key molecular changes in AML involved in response to therapy and ultimately survival as well as understanding their interactions. This information will also be utilized to devise new therapies and strategies for the very poor prognostic group.
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