The applicant wishes to test the hypotheses that hemopoietic cytokines exert their anti-apoptotic effects by inactivating p53 activity and thus lowering Bax levels in leukemic cells. Apoptosis will be induced in myeloid cell lines M1 and M07e by expressing the temperature sensitive (ts) mutant of p53 and by deprivation of serum in the culture medium in primary AML cells. Various cytokines (GM-CSF, G-CSF, IL6) and flt-3 ligand will either be used alone or in different combinations. Expression of Bcl-2, Bax, Bcl-X, and X will be measured in leukemic cells subjected to apoptotic stress. Changes in expression of these molecules will be correlated with anti-apoptotic effects. Since apoptosis is asynchronous in nature, the activity of the Bcl-2 family members will be separately measured in apoptotic and viable cells that have been sorted by FACS. To prove that a given molecule is important for apoptosis in leukemic cells, antisense oligonucleotides will be used to block their activity. Inactivation of p53 will be measured by testing p53 DNA binding activity with gel shift assay and the ability to activate transcription from Bax promoter driven reporter gene constructs.
