RESUMO
Development of chemotherapy has led to a high survival rate of cancer patients; however, the severe side effects of anticancer drugs, including organ hypoplasia, persist. To assume the side effect of anticancer drugs, we established a new ex vivo screening model and described a method for suppressing side effects. Cyclophosphamide (CPA) is a commonly used anticancer drug and causes severe side effects in developing organs with intensive proliferation, including the teeth and hair. Using the organ culture model, we found that treatment with CPA disturbed the growth of tooth germs by inducing DNA damage, apoptosis and suppressing cellular proliferation and differentiation. Furthermore, low temperature suppressed CPA-mediated inhibition of organ development. Our ex vivo and in vitro analysis revealed that low temperature impeded Rb phosphorylation and caused cell cycle arrest at the G1 phase during CPA treatment. This can prevent the CPA-mediated cell damage of DNA replication caused by the cross-linking reaction of CPA. Our findings suggest that the side effects of anticancer drugs on organ development can be avoided by maintaining the internal environment under low temperature.
Assuntos
Antineoplásicos/efeitos adversos , Ciclofosfamida/efeitos adversos , Temperatura , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Modelos Biológicos , Técnicas de Cultura de ÓrgãosRESUMO
Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.
Assuntos
Fenômenos Fisiológicos Dentários/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Odontogênese/genética , Dente/crescimento & desenvolvimento , Animais , Comunicação Celular , Diferenciação Celular/genética , Movimento Celular , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in Nkx2-3, encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that Nkx2-3 is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of Bmp2 and Bmpr2 in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.