High-dose chemotherapy with or without total body irradiation, followed by allogeneic or autologous bone marrow transplantation, is of demonstrated clinical utility in the treatment of a number of hematological malignancies, e.g. acute lymphoblastic leukemia and non-Hodgkin's lymphoma, as well as a few solid tumors, e.g. breast cancer. However, while remissions are readily achieved, complete success is limited by a dismally high relapse rate. Oxazaphosphorines are often used as part of these therapeutic strategies either in vivo (cyclophosphamide or ifosfamide) as part of the high-dose chemotherapy regimen (autologous and allogeneic BMT) and/or ex vivo (4-hydroperoxycyclophosphamide or mafosfamide) to purged bone marrow of malignant cells (autologous BMT). Potentially, several enzymes, especially some (but not all) aldehyde dehydrogenases, may act to catalyze the detoxification of the oxazaphosphorines in target malignant cells thus protecting such cells from the cytotoxic action of the drug. The overall objectives of the investigations described herein are to test the notions that inherent and/or acquired insensitivity to the oxazaphosphorines is a contributing factor to the high relapse rates, and that the biological basis for this insensitivity is a higher than usual level of aldehyde dehydrogenase-catalyzed detoxification of the oxazaphosphorines. Leukemic bone marrow samples and bone marrow or surgical lymph node specimens will be obtained from the oxazaphosphorine-naive and oxazaphosphorine-treated relapsed ALL and NHL patients, respectively, and a clonogenic assay will be used to quantify the sensitivity of these samples to mafosfamide. Aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide to carboxyphosphamide by these samples will also be quantified. On-going studies designed to identify and characterize human aldehyde dehydrogenases that are relevant with regard to catalyzing the detoxification of oxazaphosphorines will be continued. Column chromatography and isoelectric focusing will be used to separate and semi-purify these enzymes. A spectrophotometric assay utilizing the substrate of interest, viz. aldophosphamide, will be used to kinetically characterize relevant enzymes. The possibility that other enzymes, e.g. aldehyde oxidase, aldehyde reductase and/or phosphoroamidases, mediate inherent and/or acquired insensitivity to the oxazaphosphorines will also be explored. Human tumor sublines that are highly resistant to the oxazaphosphorines and established in culture will be used for this purpose. The underlying molecular basis for any increased enzyme activity will be sought in all cases. Techniques common to molecular biology, vis, Western, Northern and Southern blots, will be used for this purpose. Ultimately, this information is expected to serve as the basis for the optimal therapeutic utilization of the oxazaphosphorines in BMT protocols.
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