Evaluation of the foetal time to death in mice after application of direct and indirect euthanasia methods.

Directive 2010/63/EU on the protection of animals used for scientific purposes requires that the killing of mammal foetuses during the last third of their gestational period should be accomplished through effective and humane methods. The fact that murine foetuses are resistant to hypoxia-mediated euthanasia renders the current euthanasia methods ineffective or humane for the foetuses when these methods are applied to pregnant female mice. We have assessed the time to death of foetuses after performing either indirect (dam euthanasia) or direct (via intraplacental injection--a new approach to euthanasia) euthanasia methods in order to determine a euthanasia method that is appropriate, ethical and efficient for the killing of mouse foetuses. The respective times to death of foetuses after performing the three most commonly used euthanasia methods (namely cervical dislocation, CO2inhalation and intraperitoneal sodium pentobarbital administration) were recorded. Absence of foetal heartbeat was monitored via ultrasound. We consider that the most effective and humane method of foetal euthanasia was the one able to achieve foetal death within the shortest possible period of time. Among the indirect euthanasia methods assessed, the administration of a sodium pentobarbital overdose to pregnant female mice was found to be the fastest for foetuses, with an average post-treatment foetal death of approximately 29.8 min. As for the direct euthanasia method assessed, foetal time to death after intraplacental injection of sodium pentobarbital was approximately 14 min. Significant differences among the different mouse strains employed were found. Based on the results obtained in our study, we consider that the administration of a sodium pentobarbital overdose by intraplacental injection to be an effective euthanasia method for murine foetuses.

Tumorigenic activity of p21Waf1/Cip1 in thymic lymphoma.

The cell cycle inhibitor p21Waf1/Cip1 is among the most important mediators of the tumor suppressor p53. However, there is increasing evidence indicating that p21 could favor tumorigenesis in specific cell types. In particular, the absence of p21 delays the development of thymic lymphomas induced either by ataxia-telangiectasia mutated deficiency or by ionizing irradiation. Here, we extend these observations to the context of p53-deficient mice. The absence of p21 results in a significant extension of the lifespan of p53-null and p53-haploinsufficient mice, and this effect can be attributed exclusively to a decrease in the incidence of spontaneous thymic lymphomas. Specifically, despite the occurrence of a variety of tumor types in the context of p53 deficiency, the only tumors that were significantly impaired by the absence of p21 were thymic lymphomas. Moreover, the absence of p21 also delays the incidence of radiation-induced thymic lymphomas in p53-deficient mice. Interestingly, p21-deficient lymphomas have a higher apoptotic rate than p21-proficient lymphomas, and this could be on the basis of the delayed incidence of thymic lymphomas in the absence of p21. Together, our results indicate that p21 plays an oncogenic role restricted to thymic lymphomas that is mechanistically independent of p53 and associated to a lower tumor apoptotic rate.

Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.

We have introduced a point mutation in the first coding exon of the locus encoding the cyclin-dependent kinase 4 (Cdk4) by homologous recombination in embryonic stem cells. This mutation (replacement of Arg24 by Cys) was first found in patients with hereditary melanoma and renders Cdk4 insensitive to INK4 inhibitors. Here, we report that primary embryonic fibroblasts expressing the mutant Cdk4R24C kinase are immortal and susceptible to transformation by Ras oncogenes. Moreover, homozygous Cdk4(R24C/R24C) mutant mice develop multiple tumors with almost complete penetrance. The most common neoplasia (endocrine tumors and hemangiosarcomas) are similar to those found in pRb(+/-) and p53(-/-) mice. This Cdk4 mutation cooperates with p53 and p27(Kip1) deficiencies in decreasing tumor latency and favoring development of specific tumor types. These results provide experimental evidence for a central role of Cdk4 regulation in cancer and provide a valuable model for testing the potential anti-tumor effect of Cdk4 inhibitors in vivo.