Here we seek to understand the relevance of the mitochondrial Hsp70 protein, mortalin, in acute myelologenous leukemia (AML) cancers. In these studies, we will address two overlapping specific aims by asking the following questions: a) is extra-mitochondrial, overexpressed mortalin protein responsible for tethering the p53 transcription factor protein in the cytoplasm of cells from the KG1 cell line and from a subset of cancer patients, all of which demonstrate cytoplasmic sequestration and inactivation of the apoptotic functions of p53? and b) will mortalin targeted agents promote nuclear translocation of p53 and result in apoptosis of these cells? These areas of study are significant because: p53 in these cells is likely to be wild type, should induce apoptosis transcriptionally and thus eliminate these cancer cells. Here we will use observations from our previous published studies on the clam hemocyte leukemia model to inform our interrogation of a repository of AML cells available from our collaborator, Dr. Claxton, Penn State Hershey Cancer Institute (see his letter of collaboration). ______________________________________________________________________ The two overlapping areas to be investigated and the methods to be used are: a) to determine the role of mortalin by employing co-immunolocalization and also co- immunoprecipitation of p53 and mortalin to determine if p53 is bound to mortalin and also QPCR and western blotting to determine if mortalin is overexpressed in the cytoplasm of AML cells positive for cytoplasmic sequestration of p53. b) to determine if mortalin targeted agents will reduce mortalin binding to p53 or mortalin concentration and lead to nuclear translocation of p53 and apoptosis of AML cells positive for cytoplasmic sequestration of p53 by treatment with agents that competitively bind to the p53 binding site in mortalin and also with mortalin RNAi. These are initial steps in determining the relevance of overexpressed mortalin in the inactivation of the apoptotic functions of p53 in a subclass of human AML that have recently been identified and have not been investigated before in studies of this kind.
This proposal is relevant to public health by studying the role of mortalin (the mitochondrial Hsp70 protein) in inactivating the p53 transcription factor protein in cancer cells. The specific models used in this study are human acute myelogenous leukemia cells from both a cell line (KG1) and from a subset cancer patients, all of which demonstrate cytoplasmic sequestration and inactivation of p53 protein. Results and broad conclusions from this study will be directly relevant to developing targeted therapies that reactivate p53 in cancer cells where extra- mitochondrial, over-expressed mortalin tethers p53 in the cytoplasm and inactivates its transcriptional apoptotic functions.
