Giorgianni F, Bridson P K, Sorrentino B P, Pohl J, Blakley R L
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 38105, Memphis, TN, USA.
Biochem Pharmacol. 2000 Aug 1;60(3):325-38. doi: 10.1016/s0006-2952(00)00344-0.
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.
对环磷酰胺等化疗用氮杂磷类药物耐药的肿瘤通常会过度表达醛脱氢酶（ALDH），其中一些同工酶催化环磷酰胺活化中间体醛磷酰胺的氧化，形成惰性的羧基磷酰胺。由于通过用人类ALDH同工酶3（hALDH3）基因转染哺乳动物细胞可使其对氮杂磷类药物产生耐药性，因此接受环磷酰胺治疗实体瘤的患者，有可能通过预先移植用逆转录病毒载体转导导致hALDH3过表达的自体骨髓，从而免受骨髓抑制。我们研究了将hALDH3通过逆转录病毒导入人白血病细胞系是否能赋予其对氮杂磷类药物的耐药性。这在多克隆转导群体中进行了检测，即未筛选出高表达克隆。转导细胞中hALDH3活性为0.016 IU/mg蛋白（未转导细胞中为2×10⁻⁵ IU/mg），但对产生醛磷酰胺的化合物（马法兰或4-氢过氧环磷酰胺）未检测到耐药性。缺乏保护作用部分归因于hALDH3对醛磷酰胺的催化活性较低，因为以NAD为辅因子时，均一的重组hALDH3对醛磷酰胺氧化的催化效率比重组hALDH1低约7倍。hALDH3的两种多态形式以苯甲醛或醛磷酰胺为底物时具有相同的动力学。初始速度测量结果与ALDH1的有序顺序机制一致，但与hALDH3不一致；提出了后者的动力学机制，并给出了相应的速率方程。
