RESUMO
Checkpoint kinase 2 (Chk2) is a significant mediator of diverse responses to DNA damage. The present study was aimed to identify possible interactive proteins of Chk2 and try to clarify the underlying mechanism regarding Chk2 chromatin loading and its phosphorylation to DNA damage response in oral squamous cell carcinoma (OSCC). Differently tagged Chk2 and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were overexpressed into SCC-4 cells. After 48 h of transfection cell fractionation was performed to localize proteins. In addition, immunoreactive species were detected by immunoprecipitation (IP) and immunoblot (IB) analysis, and protein-protein interaction between Chk2 and MCM complex was ensured by glutathione S-transferase (GST) pull-down assay. Expression of MCM2 and MCM6 was downregulated by small interfering RNA (siRNA), and the chromatin and non-chromatin fraction were analyzed. The expression of Chk2 phosphorylation (pT68-Chk2) was measured after administration of different dosages of siMCM2 (0.5 µg, 1 µg, and 2.5 µg) and camptothecin (CPT). Our results showed that Chk2 directly interacts with MCM2, MCM3, MCM5, and MCM6 in SCC-4 cells. Downregulation of MCM2 and MCM6 markedly reduced Chk2 chromatin fraction, and downregulation of MCM2 decreased the expression of pT68-Chk2 to DNA damage response in a dose manner. Our results suggest that the interaction between Chk2 and MCM complex is required for Chk2 chromatin loading and its phosphorylation to DNA damage response in SCC-4 cells.
Assuntos
Quinase do Ponto de Checagem 2/genética , Cromatina/química , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 6 do Complexo de Manutenção de Minicromossomo/genética , Camptotecina/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/efeitos dos fármacos , Imunoprecipitação da Cromatina , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/antagonistas & inibidores , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 6 do Complexo de Manutenção de Minicromossomo/antagonistas & inibidores , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Fosforilação/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , TransfecçãoRESUMO
Six all-hydrocarbon-stapled Cdt1 MBD-derived peptides have been designed and synthesized to perturb the Cdt1-Mcm6 interaction, which is involved in DNA replication. Inconsistency between the helicity of the obtained peptidomimetics and their binding affinity has been observed. The helicity of 13-amino acid stapled peptides increased, while their binding to Mcm6 was decreased. On the other hand, the 30-amino acid stapled peptides exhibited decreased helicity but increased binding affinity.
