Autologous bone marrow transplantation (ABMT) in human acute leukemia is limited by the presence of occult viable tumor cells in the marrow graft. The proposed preclinical research will develop strategies to eliminate potentially drug-resistant leukemic cells from marrow suspensions before ABMT by ex vivo incubation with antineoplastic agents (""""""""chemopurging""""""""), using the model of human acute myeloid leukemia (AML) in the Lewis x Brown Norway hybrid (LPN) rat. Suspensions of LBN AML and/or normal marrow cells will be incubated with graded concentrations of selected chemotherapeutic agents (4-hydroperoxy-cyclophosphamide [4HC], bleomycin, etoposide, m-AMSA, or vincristine). The dose-dependent effects of incubation with each drug on the growth of leukemic and normal hematopoietic cells will be compared with complementary in vitro clonogenic assays for leukemic (CFU-Leuk) and normal hematopoietic progenitors (CFU-GM), and in vivo assessment of engraftment, prolongation of median survival time (MST), and/or leukemia- free survival (LFS) after transplantation of chemopurged normal LBN marrow, AML cells, or mixtures thereof (to approximate residual leukemia in remission bone marrow) into syngeneic recipients. Agents that demonstrate synergistic antitumor effects without additive stem cell toxicity will thus be identified and further evaluated in sequential drug-incubation studies. To determine the efficacy of these chemopurging regimens in a model of relapsed drug-resistant human AML, a cyclophosphamide (CY)-resistant subline of LBN AML will be developed by chronic exposure to CY in vivo and to 4HC in vitro. Potential mechanisms of CY resistance such as elevated aldehyde dehydrogenase (ALDH) activity, and strategies to reverse resistance by ex vivo incubation with ALDH inhibitors, an approach that is relevant to chemopurging of autografts in human AML, will be evaluated in this subline. Finally, methods that may enhance differential sensitivity of leukemic vs. normal marrow stem cells in ex vivo chemopurging regimens will be studied. The effects of incubation with bryostatin (BRYO), a novel antitumor agent that activates protein kinase C and stimulates normal human hematopoiesis, on the growth of leukemic and normal rat marrow cells will be examined by in vitro clonogenic assays, by observation of engraftment, increases in MST, and/or LFS after transplantation of BRYO-treated normal marrow and/or AML cells into LBN rats, and by in vivo administration of BRYO to leukemic animals. The ability of coincubation with BRYO to potentiate the antileukemic effects and minimize the stem-cell toxicity of chemotherapeutic agents will also be determined.
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