RESUMO
5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.
Assuntos
Azacitidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Leucemia/tratamento farmacológico , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , Dano ao DNA/genética , Metilação de DNA/efeitos dos fármacos , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Decitabina , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , DNA Metiltransferase 3BRESUMO
The c-Jun N-terminal kinase (JNK) family, with its three members JNK1, JNK2, and JNK3, is a subfamily of mitogen-activated protein kinases. Involved in many aspects of cellular processes, JNK has been also associated with pathological states such as neurodegenerative diseases, inflammation, and cancers. In oncology, each isoform plays a distinct role depending on the context of the targeted tissue/organ, the tumor stage, and, most likely, the signaling pathway activated upstream. Consequently, the current challenge in finding new successful anti-JNK therapies is to design isoform-selective inhibitors of the JNKs. In this review, a particular focus is given to the JNK inhibitors that have been developed thus far when examining 3D structures of various JNK-inhibitor complexes. Using current data regarding structure-activity relationships and medicinal chemistry approaches, our objective is to provide a better understanding of the design and development of selective JNK inhibitors in the present and future.