Inactivation of aldophosphamide by human aldehyde dehydrogenase isozyme 3.
Tumors resistant to chemotherapeutic oxazaphosphorines such as cyclophosphamide often overexpress aldehyde dehydrogenase (ALDH), some isozymes of which catalyze the oxidization of aldophosphamide, an intermediate of cyclophosphamide activation, with formation of inert carboxyphosphamide. Since resistance to oxazaphosphorines can be produced in mammalian cells by transfecting them with the gene for human ALDH isozyme 3 (hALDH3), it seems possible that patients receiving therapy for solid tumors with cyclophosphamide might be protected from myelosuppression by their prior transplantation with autologous bone marrow that has been transduced with a retroviral vector causing overexpression of hALDH3. We investigated whether retroviral introduction of hALDH3 into a human leukemia cell line confers resistance to oxazaphosphorines. This was examined in the polyclonal transduced population, that is, without selecting out high expression clones. hALDH3 activity was 0.016 IU/mg protein in the transduced cells (compared with 2x10(-5) IU/mg in untransduced cells), but there was no detectable resistance to aldophosphamide-generating compounds (mafosfamide or 4-hydroperoxycyclophosphamide). The lack of protection was due, in part, to low catalytic activity of hALDH3 towards aldophosphamide, since, with NAD as cofactor, the catalytic efficiency of homogeneous, recombinant hALDH3 for aldophosphamide oxidation was shown to be about seven times lower than that of recombinant hALDH1. The two polymorphic forms of hALDH3 had identical kinetics with either benzaldehyde or aldophosphamide as substrate. Results of initial velocity measurements were consistent with an ordered sequential mechanism for ALDH1 but not for hALDH3; a kinetic mechanism for the latter is proposed, and the corresponding rate equation is presented.
Giorgianni
F
F
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 38105, Memphis, TN, USA.
Bridson
P K
PK
Sorrentino
B P
BP
Pohl
J
J
Blakley
R L
RL
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000080924:Aldehyde Dehydrogenase 1 Family; D000447:Aldehydes / Q000378:metabolism; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001547:Benzaldehydes / Q000378:metabolism; D003001:Cloning, Molecular; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000494:pharmacology; D018076:DNA, Complementary / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010752:Phosphoramide Mustards / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D050697:Retinal Dehydrogenase; D017421:Sequence Analysis; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
C033253:5-chlorocyclophosphamide; D000447:Aldehydes; D000970:Antineoplastic Agents; D001547:Benzaldehydes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010752:Phosphoramide Mustards; D011994:Recombinant Proteins; C030863:corneal protein 54, bovine; C006446:aldophosphamide; D003520:Cyclophosphamide; C005556:propionaldehyde; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; C032175:benzaldehyde