The objective of the proposed studies is to investigate potentially new molecular and biochemical mechanisms of the resistance to intercalating agent mAMSA, specifically the role of a protein with an apparent molecular weight of 76,000 (p76), found to be consistantly associated with primary, in vitro induced resistance to mAMSA in numerous independently derived human acute myelogenous leukemia (AML) cell lines. We propose to use recombinant DNA clones and antibodies to p76 as detection probes to relate the function of p76 to mAMSA resistance in AML cell lines. After further purification of the p76 to homogeneity we will determine amino acid sequence of any internal peptides and also produce antibodies to p76. Based on the amino acid sequence, a unique oligonucleotide probe will be constructed and used to obtain full length cDNA corresponding to p76. The isolated p76 cDNA will be transfected using a suitable vector into mAMSA resistant mutants. The phenotype of transfected cells will be screened for p76 expression using p76 antibodies; for drug sensitivity and for drug induced changes in the Topoisomerase II activity. With the advent of the described molecular probes and p76 antibodies we will screen leukemic samples from AML patients in various stages of their disease i.e. prior treatment and during the course of chemotherapy with mAMSA and emergence of clinical resistance. The ultimate goal of the study is to clarify the role of p76 in cellular response of leukemic cells to mAMSA and in the development of mAMSA resistance, both in experimental models and clinical situations.
