INDUCTION OF APOPTOSIS AND CATHEPSIN-D IN LIMBS EXPOSED IN-VITRO TO AN ACTIVATED ANALOG OF CYCLOPHOSPHAMIDE

Apoptosis, a form of active cell death, plays a role during normal lim b development. The present study was done to test the hypothesis that the teratogen cyclophosphamide, an alkylating agent and commonly used anticancer drug, produces malformations by disturbing the regulation o f apoptosis in the limb. The effects of a preactivated analog of cyclo phosphamide, 4-hydroperoxycyclophosphamide, on limb development and on apoptosis in the limb were determined in vitro. Cathepsin D is a lyso somal protease which is induced in tissues undergoing destruction by a poptosis. To further examine the process of apoptosis in the limb, the effects of 4-hydroperoxycyclophosphamide exposure on cathepsin D prot ein concentration and on the immunolocalization of cathepsin D in limb buds were assessed. limb buds from gestational day 12 mice were excis ed and cultured in roller bottles in a chemically defined medium for u p to 6 days. The addition of 4-hydroperoxycyclophosphamide (1 or 10 mu g/ml) to the culture medium produced time- and concentration-dependen t limb malformations. Electrophoresis of the DNA extracted from both c ontrol and treated limbs revealed a DNA fragmentation pattern characte ristic of apoptosis. Limbs cultured in the control medium showed a ''D NA ladder'' only after 72 hours in vitro; however, those in the drug-t reated groups showed fragmentation within 12 hours of drug exposure. A cridine orange staining and ex amination of cell ultrastructure with t he electron microscope further confirmed that apoptotic cell death in the interdigital areas was accelerated in drug-exposed limbs. The rela tive abundance of cathepsin D in limbs exposed to 4-hydroperoxycycloph osphamide for 24 hours was increased compared to control limbs. Using immunohistochemical staining at the light microscope level, the cathep sin D protein in control limbs was localized mainly to the interdigita l and apical ectodermal ridge areas; staining in these areas was incre ased in limbs exposed to 4-hydroperoxycyclophosphamide. Using immunogo ld electron microscopy, cathepsin D immunoreactivity was found to be l ocalized in phagocytosed apoptotic bodies; this observation suggests t hat it is the process of phagocytosis which induces cathepsin D. Thus, exposure to 4-hydroperoxycyclophosphamide increased apoptosis in the interdigital areas and apical ectodermal ridge of mouse limb buds in v itro. Moreover, cathepsin D may play an important role in mediating th e phagocytosis of apoptotic bodies in the teratogen-exposed limbs. (C) 1995 Wiley-Liss, Inc.