[Biochemical characterization of the optic nerve in mice overexpressing the P53 gen. Oxidative stress assays]. / Caracterización bioquímica del nervio óptico en el ratón que sobreexpresa el gen P53. Análisis de estrés oxidativo.

PURPOSE: The tumour inhibitor p53 gene has the ability of triggering proliferation arrest and cellular death by apoptosis subsequent to several factors, among them oxidative stress. The p53 protein is a major regulator of gene expression. Using genetically manipulated mice carrying an extra copy of gene p53 (transgenic mice super p53) versus control mice, we have investigated the generation of reactive oxygen species and antioxidant activity in the optic nerve of mice in relation to p53 availability. METHODS: We studied two groups of 12-month-old mice of the strain C57BL/6: 1) super p53 group (Sp53) and 2) wild-type control group (CG). Mice were anesthetized in ether atmosphere and the eyeball and retrobulbar optic nerves were excised, washed, soaked in PBS, and stored in liquid nitrogen at -85 degrees C until processing. Three-four optic nerves from the same group were placed in an eppendorf tube, homogenized and enzymatic-colorimetric methods used to determine oxidative and antioxidant activities and the nitric oxide synthesis. RESULTS: A significant increase in free radical formation (via lipid peroxidation; p<0.001), antioxidant activity (p<0.001) and nitric oxide synthesis (p<0.001) was found in the optic nerves from transgenic super p53 mice compared to respective controls. CONCLUSION: The presence of an extra copy of the p53 gene correlated with redox status in the mouse optic nerve. This transgenic mouse could be useful as an experimental model to study cell resistance to neurodegenerative processes in relation to oxidative stress and to apoptosis induction, such as glaucomatous optic neuropathy or age-related macular degeneration.

Caracterización bioquímica del nervio óptico en el ratón que sobreexpresa el gen p53. Análisis de estrés oxidativo / Biochemical characterization of the optic nerve in mice overexpressing the P53 gen. Oxidative stress assays

Objetivos: El gen supresor tumoral p53 detiene la proliferación y la muerte celular por apoptosis subsecuente a la acción de diversos factores, entre ellos el estrés oxidativo. La proteína p53 es fundamentalmente un regulador de la expresión génica. Utilizando ratones genéticamente manipulados para presentar una copia extra del gen p53 (transgénicos super p53) frente a ratones controles, hemos investigado el estado oxidativo y antioxidante en los nervios ópticos, en relación a p53. Método: Se han utilizado ratones de la cepa C57BL/6 de 12 meses de edad en dos grupos: 1) grupo super p53 (Sp53) y 2) grupo de controles wild-type (GC). Los ratones fueron anestesiados en atmósfera de éter, extrayendo los globos oculares y nervios ópticos que se lavaron en PBS, manteniendo las muestras en nitrógeno líquido y en congelador de –85ºC hasta su procesamiento. Se homogeneizaron 3-4 nervios ópticos por cada eppendorf, clasificando por grupos y determinando mediante métodos enzimático-colorimétricos la actividad traperoxidativa y actividad antioxidante total y la concentración de oxido nítrico. Resultados: Existe aumento significativo en la formación de radicales libres via peroxidación lipídica (p<0,001), de la actividad antioxidante (p<0,001) y síntesis de óxido nítrico (p<0,05) en los nervios ópticos de los ratones transgénicos super p53, frente a los ratones controles. Conclusiones: La presencia de una copia extra del gen p53 está ligada a modificaciones de la actividad redox en el nervio óptico del ratón, sugiriendo que p53 otorga una mayor resistencia a la agresión oxidativa. Valoramos la utilización de este modelo de ratón transgénico en procesos neurodegenerativos relacionados con el estrés oxidativo y la inducción de la apoptosis, como la neuropatía óptica glaucomatosa o la degeneración macular asociada a la edad

Purpose: The tumour inhibitor p53 gene has the ability of triggering proliferation arrest and cellular death by apoptosis subsequent to several factors, among them oxidative stress. The p53 protein is a major regulator of gene expression. Using genetically manipulated mice carrying an extra copy of gene p53 (transgenic mice super p53) versus control mice, we have investigated the generation of reactive oxygen species and antioxidant activity in the optic nerve of mice in relation to p53 availability. Methods: We studied two groups of 12-month-old mice of the strain C57BL/6: 1) super p53 group (Sp53) and 2) wild-type control group (CG). Mice were anesthetized in ether atmosphere and the eyeball and retrobulbar optic nerves were excised, washed, soaked in PBS, and stored in liquid nitrogen at –85ºC until processing. Three-four optic nerves from the same group were placed in an eppendorf tube, homogenized and enzymatic-colorimetric methods used to determine oxidative and antioxidant activities and the nitric oxide synthesis. Results: A significant increase in free radical formation (via lipid peroxidation; p<0.001), antioxidant activity (p<0.001) and nitric oxide synthesis (p<0.001) was found in the optic nerves from transgenic super p53 mice compared to respective controls. Conclusion: The presence of an extra copy of the p53 gene correlated with redox status in the mouse optic nerve. This transgenic mouse could be useful as an experimental model to study cell resistance to neurodegenerative processes in relation to oxidative stress and to apoptosis induction, such as glaucomatous optic neuropathy or age-related macular degeneration

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.

Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1.

A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27(Kip1) but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15(INK4b), p16(INK4a), p19( INK4d), and p21(Cip1) as well as other negative cell cycle regulators such as p53 and p19(ARF), implies that this senescence-related growth arrest is independent of the activity of p53, p19(ARF), p16(INK4a), and p21(Cip1), which are associated with replicative senescence. The p27(Kip1) binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27(Kip1) can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27(Kip1)expression. In summary, these observations taken together suggest that p27(Kip1) is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27(Kip1) is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived from p27(Kip1-/-) mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27(Kip1). This is the first description that p130 may have a role in inhibiting CDK activity during senescence.