RESUMO
Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl-N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/genética , Opacidade da Córnea/genética , Pálpebras/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Queratinócitos/metabolismo , Regiões 3' não Traduzidas , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular , Polaridade Celular , Proliferação de Células , Forma Celular , Córnea/anormalidades , Córnea/crescimento & desenvolvimento , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Embrião de Mamíferos , Etilnitrosoureia , Pálpebras/anormalidades , Pálpebras/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutagênicos , Fenótipo , Cultura Primária de CélulasRESUMO
DNA damage activates checkpoint controls in eukaryotic cells. It is not clear, however, whether a certain level of DNA damage is required for the activation of DNA damage checkpoints. We show here that low levels of DNA damage in Chinese hamster ovary (CHO) cells induced by short exposure to hydroxyurea (HU) did not trigger checkpoints, whereas higher levels of DNA damage caused by longer exposure to HU resulted in a cell cycle arrest. Our results argue that a threshold of DNA damage is necessary for activation of DNA damage checkpoints.