THE ROLE OF P53 AND CELL-DEATH BY APOPTOSIS AND NECROSIS IN 4-HYDROPEROXYCYCLOPHOSPHAMIDE-INDUCED LIMB MALFORMATIONS

The exposure of embryonic murine limbs in vitro to an activated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide (4OOH-CPA), induce d limb malformations and apoptosis, The purpose of this study was to i nvestigate the role of the tumor suppressor/cell cycle checkpoint gene , p53, and of cell cycle arrest in the response of the limbs to cyclop hosphamide. Limbs, excised on day 12 of gestation from wild-type, hete rozygous or homozygous p53-knockout transgenic murine embryos, were tr eated with vehicle (water) or 4OOH-CPA (0.3, 1.0 or 3.0 mu g/ml) and c ultured for 6 days. Exposure of wild-type (+/+) limbs to 400H-CPA resu lted in limb malformations, and reduced limb areas and developmental s tares. The homozygous (-/-) limbs mere dramatically more sensitive to the effects of 4OOH-CPA, as assessed by limb morphology, area and scor e. Heterozygous limbs exposed to the drug were intermediate for each p arameter, Apoptosis, as assessed by the formation of a DNA ladder, was increased in drug-exposed wild-type limbs, but not in the drug-expose d homozygous limbs. Light and electron microscopy examination of the l imbs revealed that drug treatment of mild-type limbs induced the morph ological changes typical of apoptosis, particularly in the interdigita l regions. In contrast, there was no evidence of apoptosis in homozygo us limbs exposed to 4-OOH-CPA; morphological characteristics of necros is such as cell membrane breakdown, mitochondrial swelling and cellula r disintegration were evident throughout these limbs, Heterozygous lim bs had cells dying with the characteristics of both apoptosis and necr osis, Fragments of poly(ADP-ribose) polymerase characteristic of necro sis predominated in the drug-treated heterozygous and homozygous limbs . 4OOH-CPA-treatment of limbs from wild-type embryos led to arrest of the cell cycle at the G(1)/S phase. No cell cycle arrest was observed after drug treatment of homozygous limbs, in which populations of cell s in S and G(2)/M phases, as well as a population of sub G(1) cells, w ere found. Thus, the presence of p53 and of p53-dependent apoptosis pr otect organogenesis-stage limbs from insult with a teratogen, The abse nce of p53 may decrease DNA repair capacity and contribute to the accu mulation of DNA damage in limb cells and their daughter cells; the fai lure of apoptosis to eliminate cells with DNA damage may result in inc reased cell death by necrosis and major limb malformations.