Disruption of murine limb development in vitro by a teratogen : role of P53 dependent and independent cell death

Abstract

In this thesis, the effects of an active analog of cyclophosphamide (4-hydroperoxycyclophosphamide, 4OOH-CPA), an alkylating agent and teratogen, on mouse limb development, cell death and the role of the p53 gene in this process were studied. A mouse limb bud culture system was used for this purpose. Limbs developed normally in this system and showed physiological apoptosis in a spatio-temporal-controlled manner. Drug treatment caused limb reduction malformations, increased the amount of apoptosis and triggered its occurrence prematurely, primarily in areas with physiological apoptosis. This indicated that drug-treatment might have engaged the already available program of apoptosis in the developing limb, and suggested this might be the cause of limb malformations. Next we examined the induction pattern of three proteins, cathepsin D, transglutaminase and clusterin, known to be involved in the molecular mechanism of apoptosis. The pattern of their induction suggested that cathepsin D was involved in the digestion of apoptotic debris, transglutaminase was active in preserving the cellular integrity during apoptosis, and clusterin played a role in later phases of apoptosis. Since 4OOH-CPA causes DNA damage, we studied the role of the p53 gene, a DNA-damage response gene involved in regulating cell cycle and apoptosis, in a p53-transgenic mouse model. Absence of p53 led to more severe limb malformations in the treated limbs. To investigate the reason(s) for this higher sensitivity to drug insult, cell death and cell cycle patterns were studied. While wild-type limbs showed an induction of apoptosis by drug treatment, which might be crucial in eliminating damaged cells, the null limbs were resistant to this effect. Furthermore, in response to 4OOH-CPA-treatment, the cell cycle was not arrested in p53 -/- limbs, in contrast to wild-type limbs; this arrest is necessary for DNA repair. Further investigation revealed the occurrence of necrosis in p53 -/- limbs. Thus, w