MAFOSFAMIDE INDUCES DNA FRAGMENTATION AND APOPTOSIS IN HUMAN T-LYMPHOCYTES - A POSSIBLE MECHANISM OF ITS IMMUNOSUPPRESSIVE ACTION

Cyclophosphamide, an alkylating agent belonging to the family of nitro gen mustards, is commonly used to treat progressive autoimmune disease s in humans. At the molecular level, its cytotoxicity results from DNA double strand crosslinks and, at higher concentrations, from DNA stra nd breaks. At the cellular level, cyclophosphamide may selectively aff ect mature lymphocytes with relative sparing of the respective precurs or cells. In this study, we show that 4-hydroxycyclophosphamide (4-OH- CP), the active metabolite of cyclophosphamide, induces apoptosis in m ature human lymphocytes at concentrations that are achieved in vivo. S ince cyclophosphamide requires enzymatic conversion in the liver to yi eld its active metabolite, 4-OH-CP was generated in vitro by non-enzym atic hydrolysis of mafosfamide. Apoptotic cell death of lymphocytes wa s characterized by typical morphological changes, nucleosomal DNA frag mentation, and quantified by 3'-OH end labeling of fragmented DNA. The percentage of apoptotic cells both depended on drug concentration and time of exposure. Cycloheximide or ZnSO4 did not suppress 4-OH-CP ind uced apoptosis. Etoposide, a topoisomerase II inhibitor known to induc e apoptosis in human tumor cell lines like 4-OH-CP, did induce detecta ble DNA fragmentation in only a minor proportion of T-lymphocytes but suppressed T-cell proliferation.