RESUMO
Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.
Assuntos
Quinases Ciclina-Dependentes/genética , Reparo do DNA , Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica/efeitos da radiação , Células-Tronco Pluripotentes Induzidas/metabolismo , Apoptose/efeitos da radiação , Biomarcadores/metabolismo , Proteína Quinase CDC2 , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Quebras de DNA de Cadeia Dupla , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Mitose/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Poliploidia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
The role of candida in the aetiology and pathogenesis of periodontal diseases is unclear. In spite of the organism being present in subgingival biofilm samples and in gingival biopsy specimens taken from affected subjects, it has not been possible to demonstrate that it is an active agent in these cases. There is an increase in the prevalence of candida species in the oral cavities and specifically in the subgingival biofilm of HIV-seropositive patients. However, periodontal diseases in HIV-seropositive and HIV-seronegative subjects are similar with regard to the spectrum of periodontopathic bacteria, clinical manifestation, natural course of the disease and response to treatment. Thus, it is safe to assume that candidal micro-organisms play only a minor role, if any, in the aetiology and pathogeneses of periodontal diseases in HIV-seropositive subjects.
