The objectives of this proposal are to examine the mechanism of glucocorticoid action during the cell cycle in synchronized HeLa S3 cells. We will test the unique hypothesis that physiological modifications in the glucocorticoid receptor proteins can result in steroid responsive and resistant phases of the human cell cycle. Previous investigations have shown the presence of a """"""""charge"""""""" modified unproteolyzed glucocorticoid receptor during cell cycle stages when dexamethasone does not induce alkaline phosphatase (AP) in HeLa S3 cells. This receptor does not nuclear translocate at 37C in whole cells or activate by warming at 25C in vitro. We propose to characterize further the physical properties of dexamethasone-mesylate affinity laveled glucocorticoid receptors during the cell cycle and determine the molecular basis for modification in receptor as well as study the regulation of this modification. Based on the literaturwe and our own studies the modification may likely involve phosphorylation/dephosphorylation of receptor. This possibility will be explored in vitro with highly purified affinity labeled receptors and in whole cells during the cell cycle. Furthermore monoclonal antibodies will be raised to the various modified forms of affinity labeled glucocrticoid receptors and used as unique probes to establish receptor structure/function relationships. This goal will be achieved by 1) selecting responsive and nonresponsive cells with monoclonal antibodies to alkaline phosphatase on a cell sorter and physically characterizing their receptors, 2) microinjecting monoclonal antibodies to the modified receptor and normal receptor forms into HeLa S3 cells and assessing alkaline phosphatase production in response to a dexamethasone challenge. These studies should provide needed biochemical information on whether the glucocorticoid receptor is a modifiable protein and whether such epigenetic mechanisms can account for steroid resistance during the cell cycle. A thorough testing of this hypothesis and an understanding of the concepts developed should provide an adequate biochemical basis to accurately predict the state of steroid responsiveness in human neoplasia and other diseases involving the adrenal axis.
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