The following hypothesis is to be tested. """"""""Transient activation ofapoptotic nucleuses Initiates MIL translocations with leukemogenicpotentiar. It is proposed that apoptotic execution may be arrested, after targeted DNA damage has occurred, in cells that survive. Preliminary data shows that apoptotictriggers initiate cleavage of MLL, which is subsequently translocated ioAF9, creating the leukemogenicMLL-AF9 fusion gene that is transcribed in cells capable of division. The relevance to health care is that this process would represent a novel pathwayfor the generation of fusion genes implicated in leukemogencsisthat would be open to therapeutic intervention.
The Aims sequentially address the mechanism controllingcleavage within MLL, the growth of cells that survive and validationof these in-vitrodata with a clinical model of early stage leukemogenesis.
Aim 1 Mechanism of MIX cleavage and translocation in response to apoptotic triggers. It is proposed that apoptotic cleavage of MLL is regulated by adjacent sequence motifs, including ATC tracts or topoisomerase LI binding sites. These possibilities will be tested by modifying each motif using site directed mutagenesis within an MLL containingepisome (pCEP4) that reproduces genomic MLL cleavage in cells undergoing apoptosis.
Aim 2. Selection and analysis of cells that survive apoptotic activation. Cell lines will be selected using a pCEP4 episome containing a 367 bp MLL apoptotic cleavage motif adjacent to an out offrame neomycin resistance gene. Apoptotic cleavage and mis-rejoining ofthe MLL motif mayplace the neomycin back into its correct readingframe, allowing selection ofapoptotic survivors.
Aim 3. Detection of. MLL translocations in patients at risk of therapy related leukemia. Blood taken from patients both before and during treatment for non-Hodgkins lymphoma or breast cancer will be examined for cells containing MLL-AF9 message or MLLrearrangements at the apoptotic cleavage site using inverse PCR. The ability of such cells to divide will be determined by the presence of duplicated genomic breakpoint junctions within each patients blood.
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