Induction of apoptosis and cathepsin D in limbs exposed in vitro to an activated analog of cyclophosphamide

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Abstract
Apoptosis, a form of active cell death, plays a role during normal limb 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 of apoptosis in the limb. The effects of a preactivated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide, on limb development and on apoptosis in the limb were determined in vitro. Cathepsin D is a lysosomal protease which is induced in tissues undergoing destruction by apoptosis. To further examine the process of apoptosis in the limb, the effects of 4-hydroperoxycylophosphamide exposure on cathepsin D protein concentration and on the immunolocalization of cathepsin D in limb buds were assessed. Limb buds from gestational day 12 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days. The addition of 4-hydroperoxycyclophosphamide (1 or 10 μg/ml) to the culture medium produced time- and concentration-dependent limb malformations. Electrophoresis of the DNA extracted from both control and treated limbs revealed a DNA fragmentation pattern characteristic of apoptosis. Limbs cultured in the control medium showed a “DNA ladder” only after 72 hours in vitro; however, those in the drug-treated groups showed fragmentation within 12 hours of drug exposure. Acridine orange staining and examination of cell ultrastructure with the electron microscope further confirmed that apoptotic cell death in the interdigital areas was accelerated in drug-exposed limbs. The relative abundance of cathepsin D in limbs exposed to 4-hydroperoxycyclophosphamide for 24 hours was increased compared to control limbs. Using immunohistochemical staining at the light microscope level, the cathepsin D protein in control limbs was localized mainly to the interdigital and apical ectodermal ridge areas; staining in these areas was increased in limbs exposed to 4-hydroperoxycyclophosphamide. Using immunogold electron microscopy, cathepsin D immunoreactivity was found to be localized in phagocytosed apoptotic bodies; this observation suggests that it is the process of phagocytosis which induces cathepsin D. Thus, exposure to 4-hydroperoxycylophosphamide increased apoptosis in the interdigital areas and apical ectodermal ridge of mouse limb buds in vitro. Moreover, cathepsin D may play an important role in mediating the phagocytosis of apoptotic bodies in the teratogen-exposed limbs. ©1995 Wiley-Liss, Inc.