The basic theme of this application is to establish the functional role of site specific hypophosphorylation of topoisomerase II in DNA cleavable complex formation. The applicant hypothesizes that phosphorylation of the topoisomerase II alpha (170 kDa) and/or topoisomerase II beta protein is dependent on alterations in intracellular free calcium and this affects etoposide stabilized DNA cleavable complex formation and cytotoxicity. Experiments will be carried out in sensitive or progressively adriamycin resistant HL-60 tumor model systems. To test the effect of intracellular calcium transients on phosphorylation of topoisomerase II alpha or topoisomerase II beta, sensitive or resistant HL-60 human leukemia cells will be treated without or with the intracellular calcium buffer 1,2-bis (o- aminophenoxy) ethane-N,N,N',N', -tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) and labeled with [(32)P]-orthophosphoric acid. Using immunoprecipitates of topoisomerase II alpha or topoisomerase II beta the applicant will determine selectivity of etoposide stabilized DNA cleavable complex by band depletion and site specific alterations in the phosphorylation of topoisomerase II beta protein by two dimensional tryptic mapping of phosphopeptides. The phosphorylation sites in the topoisomerase II alpha and topoisomerase II beta protein which are affected by treatment with BAPTA-AM will be determined by two approaches: (a) Edman sequencing of (32)P labeled peptides separated by high performance liquid chromatography (HPLC); and (b) phosphopeptide mapping and sequencing by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). Topoisomerase II alpha and topoisomerase II beta protein with mutations in phosphoacceptor sites will be expressed in an inhibitor sensitive (ISE2) mutant strain of yeast or by transient transfection of cells to evaluate the functional role of site specific phosphorylation by characterizing wild-type or mutant stabilized DNA cleavable complex formation and drug sensitivity. Studies proposed with topoisomerase II alpha and topoisomerase II beta will provide novel information on the functional role of calcium and phosphorylation in cleavable complex formation, and in the long term an understanding of mechanisms in the evolution of drug resistance to topoisomerase II inhibitors.
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